Mitochondrial Endocrinology – Mitochondria As Key to Hormones and Metabolism

Molecular and Cellular Endocrinology 379 (2013) 1–1

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Editorial Mitochondrial endocrinology – Mitochondria as key to hormones and metabolism

Mitochondria are puzzling organelles, which provided exciting insights into cellular function as recently reviewed by one of its pio- neers (Schatz, 2013). But still many of their features in (patho)physiologic conditions and in disease states are not fully understood. Despite their critical role for all animal organisms, their impact on several endocrine and metabolic functions is of specific importance. Not do they only host several metabolic pathways, including the tricarboxylic (Krebs) cycle and b-oxidation, mitochondria are also the key to lipid, cholesterol and hormone biosynthesis as well as maintain the cytosolic free calcium concentration. Free cytosolic calcium in turn serves as cellular signal in divergent pathways, such as hormonal signaling (Stark and Roden, 2007). On the other hand, certain hormones exert their central endocrine action directly or indirectly via affecting mitochondrial function in various tissues and divergent cell-types. The growing interest of current research in this field stimulated us to compile contributions to hot topics addressing aspects of so- called mitochondrial endocrinology (Wrutniak-Cabello et al., 2002). First, studies of humans with genetically confirmed mitochondrial abnormalities, commonly called mitochondrial diseases, can serve as nature’s proof of the importance of mitochondria also for endocrine function, which is not limited to the pancreatic ß cell by causing mitochondrial diabetes. These inborn diseases might therefore help to better understand abnormal mitochondrial func- References tion in humans. Reviewing hormone synthesis, with a focus on steroid hormones and vitamin D, and hormone action, particularly Schatz, G., 2013. Getting mitochondria to center stage. Biochem. Biophys. Res. Commun. 434, 407–410. describing the role of thyroid hormones for mitochondrial biogene- Stark, R., Roden, M., 2007. ESCI Award 2006. Mitochondrial function and endocrine sis, is followed by a summary on the complex role of mitochondria diseases. Eur. J. Clin. Invest. 37, 236–248. for sex hormone synthesis but also steroid-independent effects on Wrutniak-Cabello, C., Casas, F., Grandemange, S., Seyer, P., Busson, M., Carazo, A., Cabello, G., 2002. Study of thyroid hormone action on mitochondria opens up a mammalian reproduction. Second, seminal studies in the field of new field of research: mitochondrial endocrinology. Curr. Opin. Endocrinol. metabolism stimulated mitochondrial endocrinology over the last Diab. 9, 387–392. decade by shedding more light on regulation of energy homeostasis Szendroedi, J., Phielix, E., Roden, M., 2011. The role of mitochondria in insulin resistance and type 2 diabetes mellitus. Nat. Rev. Endocrinol. 8, 92–103. or precisely the balance between energy intake and expenditure for alterations associated with ageing, obesity and diabetes mellitus. A Michael Roden relevant proportion of these new insights resulted from the devel- Institute for Clinical Diabetology, opment of novel invasive and noninvasive technologies allowing German Diabetes Center, assessing various aspects of mitochondria, also in humans (Szendr- Leibniz Center for Diabetes Research, Düsseldorf, Germany oedi et al., 2011). The findings range from age-dependent alterations over dynamic changes in mitochondrial function in skeletal Unic. Clinics of Endocrinology and Diabetology, muscle and liver to the chameleon-like behavior of adipose tissue Heinrich-Heine University, Düsseldorf, Germany to adapt heat production and subtle regulation of ß-cell function. E-mail address: [email protected] While the present selection cannot be comprehensive, it was designed to briefly summarize hot topics in mitochondrial endocri- Available online 20 June 2013 nology as of 2013. I would like to thank all the contributors for their excellent cooperation, the team of Mol Cell Endocrinol for their support and my Assistant Mrs. Beate Stodieck for her help with this task.

Contents lists available at ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review Endocrine disorders in mitochondrial disease q

a, b a a, Andrew M. Schaefer ⇑, Mark Walker , Douglass M. Turnbull , Robert W. Taylor ⇑ a Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK b Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK article info abstract

Article history: Endocrine dysfunction in mitochondrial disease is commonplace, but predominantly restricted to disease Available online 13 June 2013 of the endocrine pancreas resulting in diabetes mellitus. Other endocrine manifestations occur, but are relatively rare by comparison. In mitochondrial disease, neuromuscular symptoms often dominate the Keywords: clinical phenotype, but it is of paramount importance to appreciate the multi-system nature of the dis- Mitochondrial disease ease, of which endocrine dysfunction may be a part. The numerous phenotypes attributable to pathogenic Endocrine mutations in both the mitochondrial (mtDNA) and nuclear DNA creates a complex and heterogeneous mtDNA catalogue of disease which can be difficult to navigate for novices and experts alike. In this article we pro- Diabetes vide an overview of the endocrine disorders associated with mitochondrial disease, the way in which the m.3243A > G underlying mitochondrial disorder influences the clinical presentation, and how these factors influence subsequent management. Ó 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 3 2. Mitochondrial biochemistry and genetics...... 3 3. Investigation of mitochondrial disease ...... 3 4. Diabetes mellitus ...... 4 5. Mitochondrial diabetes ...... 4 5.1. Pattern recognition ...... 4 5.2. Age-at-onset ...... 5 5.3. Insulin requirements...... 5 5.4. Body Mass Index (BMI) ...... 5 5.5. End organ disease ...... 5 5.6. Pancreatic pathology...... 6 5.7. Diabetes management...... 6 6. Hypoparathyroidism ...... 6 7. Hypothalamo-pituitary axis...... 7 8. Growth hormone deficiency ...... 7 9. Hypogonadism ...... 7 10. Hypothyroidism ...... 7 11. Hypoadrenalism ...... 7 12. SIADH ...... 7 13. Adipose tissue as an endocrine organ...... 7 14. Autoimmune endocrinopathy ...... 8 15. Conclusion ...... 8 Acknowledgments ...... 8 References ...... 8

q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Corresponding authors. Address: Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health, The Medical School, Newcastle University, ⇑ Framlington Place, Newcastle upon Tyne NE2 4HH, UK. Tel.: +44 1912223685. E-mail addresses: [email protected] (A.M. Schaefer), [email protected] (R.W. Taylor).

0303-7207/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.06.004 A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 3

1. Introduction mitochondrial genotypes, as typified in many pathogenic mtDNA mutations, results in a situation known as heteroplasmy in which The term mitochondrial disease refers to a heterogeneous group the ratio of wild-type to mutated mtDNA determines the onset of of multi-system disorders characterised by mitochondrial respira- clinical symptoms. A minimum critical proportion of mutated tory chain deficiency in which neurological involvement is often mtDNA molecules are required before biochemical deficiency man- prominent (McFarland et al., 2010; Ylikallio and Suomalainen, ifests as a clinical phenotype, with this threshold level varying for 2012). Numerous distinct genotypes give rise to varied and over- different mutations and tissues. Functional consequences are most lapping phenotypes. Endocrine dysfunction is a frequent feature, commonly seen in post-mitotic tissues with high energy require- predominantly due to the prevalence of diabetes mellitus associ- ments (e.g. muscle, brain, and heart) but almost any tissue can ated with the m.3243A > G mutation, the most common hetero- be involved, including the endocrine organs. Individual mtDNA plasmic mtDNA mutation associated with human disease mutations often dictate the pattern of involvement, with some (Schaefer et al., 2008). Other forms of endocrine disease are de- more strongly associated with endocrine disease than others. scribed less frequently, occurring in numerous mitochondrial dis- The exact prevalence of mtDNA disease has proven difficult to orders due to either mutations within mtDNA or associated with define but estimates from our cohort in the North East of England nuclear-driven disorders of mtDNA maintenance. For many muta- suggest that mtDNA mutations of all types cause a point preva- tions, reports of endocrine disease are so rare as to challenge the lence of disease in adults of 9.2/100,000 population, with a further hypothesis that they are mediated by defects of oxidative phos- 16.5/100,000 at risk of developing disease due to carrier status at phorylation at all, and merely represent the background preva- any one time (Schaefer et al., 2008). Birth prevalence studies have lence of endocrine disease in a well studied population. There is reported mutation frequencies of 0.14% for some common mtDNA a danger that associations based on single case reports (sometimes mutations such as the m.3243A > G mutation (Elliott et al., 2008), dating back 20 years and beyond) are repeatedly cited in reviews although most individuals will not manifest clinical disease as such as this, perpetuating an unproven connection with mitochon- the majority of mutations are present at subthreshold levels. drial disease. Analysis of large patient cohorts are likely to be key, Several other factors are important in understanding the behav- and while this dilemma may not be readily resolved for rare muta- iour of pathogenic, heteroplasmic mtDNA mutations in relation to tions, it should be feasible to answer the question in more preva- clinical disease. During mitotic cell division, mitochondria are ran- lent disorders. As ever, further studies are needed in this area. domly segregated to daughter cells and as such the proportion of This review summarises the range of endocrine involvement in mutated mtDNA can shift in the presence of heteroplasmy. The mitochondrial disease and the genotypes and phenotypes in which observation of a rapid segregation in mammalian heteroplasmic these occur. We offer insights from a specialist mitochondrial clinic mtDNA genotypes between generations is evidence for the exis- as to the use of pattern recognition and pedigree analysis in the tence of a mtDNA developmental genetic bottleneck; this involves diagnosis and subsequent management of these complex patients a marked reduction in mtDNA copy number in the germline fol- and their families. lowed by the replication of a subgroup of mtDNA molecules during oogenesis although the precise mechanism remains to be fully determined (Cao et al., 2007; Cree et al., 2008; Wai et al., 2008). 2. Mitochondrial biochemistry and genetics In addition to primary mtDNA mutations, mutations in nuclear genes involved in mtDNA replication or repair (often termed Mitochondria are essential organelles, present in all nucleated mtDNA maintenance) can give rise to secondary qualitative or mammalian cells, whose main role is to produce ATP by the pro- quantitative mtDNA abnormalities. Mutations in nuclear genes cess of oxidative phosphorylation (OXPHOS). The OXPHOS machin- implicated in many other mitochondrial processes including struc- ery is made up of 90 different polypeptides, organised into five tural respiratory chain components, mitochondrial nucleotide sal- transmembrane complexes. The oxidation of foodstuffs generates vage and synthesis and mitochondrial translation are increasingly electrons which are shuttled to oxygen along the first four respira- being described with the advent of next-generation screening tory chain complexes whilst protons are pumped across the inner and mito-exome sequencing (Ylikallio and Suomalainen, 2012; mitochondrial membrane from the matrix to the intermembrane Calvo et al., 2012) highlighting that mitochondrial disease may space forming an electrochemical gradient which is harnessed by be inherited as Mendelian traits, with autosomal dominant (ad-), ATP synthase, to phosphorylate ADP to form ATP. Mitochondrial autosomal recessive (ar-), and even X-linked forms. function and biosynthesis is under the dual genetic control of both the mitochondrial genome – encoding just 13 proteins and 37 gene products in total and the nuclear genome, which encodes for some 3. Investigation of mitochondrial disease 1400–1500 mitochondrial proteins. Whilst mutations within either DNA molecule can cause a respiratory chain defect, the unique ge- The complex interplay between mtDNA heteroplasmy and phe- netic rules which govern the behaviour of the mitochondrial gen- notypic expression, and the potential contribution from both nu- ome provide some insight into the phenotypic heterogeneity clear and mitochondrial genomes, makes mitochondrial disease which particularly characterise mtDNA disorders. one of the most difficult inherited disorders to diagnose. The lack Several recent reviews have detailed the importance of mtDNA of curative treatments for these conditions places greater emphasis mutations in human disease (Greaves et al., 2012; Schon et al., on accurate genetic advice and counselling, which should be 2012). The mitochondrial genome is a highly-organised, 16.6 kb undertaken by a specialist with experience in this area. circular genome whose complete sequence was published over Our own algorithms for the laboratory investigation of mito- 30 years ago (Anderson et al., 1981), prompting the discovery of chondrial disease have been published extensively (Taylor et al., the first pathogenic mutations in 1988 involving either mtDNA 2004; Tuppen et al., 2010; McFarland et al., 2010) and rely on rearrangements or deletions (Holt et al., 1988) or point mutations information from the clinical phenotype and functional data (his- (Wallace et al., 1988). Strictly inherited through the maternal line- tochemistry and biochemistry) to guide genetic studies of both age, it is present within cells in multiple copies, reflecting the de- mtDNA and nuclear genes. Some common mtDNA mutations can mand for OXPHOS-derived energy of that particular tissue. When be reliably detected and screened in blood, but there is a potential all mtDNA molecules within a cell are identical, a situation known for false-negative results in some mutations. This possibility as homoplasmy prevails. The presence of two or more should be highlighted in the case of the m.3243A > G mutation, 4 A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 particularly as it is the most frequently requested analysis for pre- taker et al., 2007). The m.8296A > G MTTK gene mutation was iden- sumed mitochondrial disease and is associated with numerous clin- tified in 0.9% unrelated Japanese patients with diabetes, and 2.3% ical syndromes including mitochondrial encephalomyopathy, lactic with diabetes and deafness (Kameoka et al., 1998). The acidosis and stroke-like episodes (MELAS), maternally-inherited m.14577T > C MTND6 mutation, associated with isolated complex diabetes and deafness (MIDD), and, less commonly, myoclonic epi- I deficiency, was found in 0.79% unrelated Japanese patients with lepsy with ragged red fibres (MERRF). Levels of this mutation within diabetes (Tawata et al., 2000). leucocytes have been shown to decrease by 1.4% per year (Rahman Other mtDNA point mutations have been described but appear et al., 2001) and we have several patients within our own cohort in much rarer. The m.12258T > C MTTS2 gene mutation has been whom the m.3243A > G mutation would not have been detected associated with diabetes (Lynn et al., 1998) but in other mater- from blood alone. The risk of false negative results may be decreased nally-related kindreds, diabetes has been notably absent (Man- by screening alternative mtDNA sources including urinary epithelial sergh et al., 1999). The m.3271T > C MTTL1 mutation has been cells (McDonnell et al., 2004; Blackwood et al., 2010). For some associated with the MIDD, MELAS and MERRF phenotypes (Goto mutations, notably m.3243A > G, the level in this tissue correlates et al., 1991; Suzuki et al., 1996; Tsukuda et al., 1997), whilst the well with clinical severity (Whittaker et al., 2009). Muscle biopsy re- m.3264T > C MTTL1 mutation was observed with MIDD, the pro- mains the gold standard, yet in a minority of patients a complete bio- band having chronic progressive external ophthalmoplegia (CPEO) chemical analysis of muscle tissue can be normal, whilst the and cervical lipomata in addition (Suzuki et al., 1997). demonstration of histological and histochemical hallmarks of mito- In a number of mtDNA mutations, diabetes is not considered chondrial disease still require additional genetic testing. A prag- part of the established phenotype, despite rare reports. This group matic strategy for genetic testing is often best derived from liaison includes the m.8344A > G mutation causing myoclonic epilepsy with a specialist mitochondrial centre. Pattern recognition is key and ragged-red fibres (MERRF) (Austin et al., 1998; Whittaker and specific phenotypes may raise an otherwise rare mutation to et al., 2007), the m.8993T > C mutation which is associated with the forefront of the process (e.g. Alper syndrome due to reces- the maternally-inherited Leigh syndrome (MILS) phenotype (Naga- sively-inherited POLG gene mutations). Pathogenic mtDNA muta- shima et al., 1999) and mtDNA mutations causing Leber hereditary tions and mtDNA rearrangements are now relatively easy to optic atrophy (LHON) (Newman et al., 1991; Du Bois and Feldon, exclude where a muscle biopsy has already been performed, and 1992; Pilz et al., 1994; Cole and Dutton, 2000). in many cases should be screened in both adults and children prior Single, large-scale mtDNA deletions have been reported to to nuclear genetic testing, a process which may require investigating cause diabetes in 11% (6 of 55 patients) of well-defined, clinical co- numerous candidate genes. This once laborious process is being rev- horts of patients with CPEO and Kearns Sayre Syndrome (KSS) olutionised by the next-generation sequencing revolution leading to (Whittaker et al., 2007). An earlier paper reviewing existing case the identification of many new mitochondrial disease genes over the reports of KSS reported the prevalence of diabetes to be 13% (29 last 2–3 years. of 226) but not all cases had genetic confirmation of a deleted mitochondrial genome (Harvey and Barnett, 1992). A single report 4. Diabetes mellitus documents a child who presented with insulin dependent diabetes mellitus (IDDM) and adrenal insufficiency prior to the develop- Diabetes mellitus is well recognised within mitochondrial phe- ment of ophthalmoplegia and a diagnosis of KSS (Mohri et al., notypes and is the most common endocrine manifestation of dis- 1998). Rarely, mtDNA deletions have been reported to cause IDDM ease. This is mainly because of its association with the MIDD in Pearson’s Syndrome, but on the whole pancreatic failure is usu- phenotype which is common in patients carrying the ally exocrine (Superti-Furga et al., 1993; Williams et al., 2012). m.3243A > G MTTL1 mutation (van den Ouweland et al., 1992; Other mtDNA rearrangements, notably maternally-transmitted Whittaker et al., 2007). Diabetes is also a common condition in duplications of the mitochondrial genome, have been reported in its own right, estimated to affect 4.45% of the UK population. It is association with diabetes (Rötig et al., 1992; Ballinger et al., 1994). not surprising, therefore, that it is common for mitochondrial dia- The recognised spectrum of disease due to mutations within betes to be misdiagnosed, even in the presence of other features nuclear maintenance genes is still expanding, but diabetes has that may provide clues as to the underlying genetic disease. The been reported in 11% of adult CPEO phenotypes with ar-POLG importance of pattern recognition in diagnosis is discussed subse- mutations (Horvath et al., 2006). In adult-onset PEO due to RRM2B quently, but for the m.3243A > G mutation, the cardinal features mutations, only 4.5% (1 of 22 in this cohort) had diabetes (Pit- are of maternal inheritance and pre-senile sensorineural hearing ceathly et al., 2012). Late-onset type-2 diabetes is rare (3 of 83) loss. Prevalence of the m.3243A > G mutation in unselected dia- in OPA1 pedigrees (Yu-Wai-Man et al., 2010) and is not a feature betic populations varies between 0% and 2.8% from the larger stud- of ar-PEO1 (Twinkle) gene mutations (Lönnqvist et al., 2009). ies (Vionnet et al., 1993; Katagiri et al., 1994; Otabe et al., 1994; There are other documented mutations associated with diabetes, t’Hart et al., 1994; Kishimoto et al., 1995; Odawara et al., 1995; most of which are sufficiently rare as an individual entity as to not Uchigata et al., 1996; Abad et al., 1997; Saker et al., 1997; Tsukuda warrant lengthy discussion in this review. The role of the 16,189 var- et al., 1997; Holmes-Walker et al., 1998; Lehto et al., 1999; Matsu- iant in causing diabetes remains unclear (Poulton et al., 2002; Das ura et al., 1999; Malecki et al., 2001; Ohkubo et al., 2001; Suzuki et al., 2007) but the numerous reports of pathogenic mtDNA muta- et al., 2003; Maassen et al., 2004; Murphy et al., 2008). Deafness, tions associated with a diabetic phenotype does highlight that the neuromuscular disease, end stage renal disease, and a maternal endocrine pancreas is particularly susceptible to mitochondrial dys- family history all increase the likelihood of mitochondrial disease function. Combined with involvement of other tissues this is a useful (t’Hart et al., 1994; Majamaa et al., 1997; Newkirk et al., 1997; pointer towards mitochondrial disease as a diagnosis. Smith et al., 1999; Ng et al, 2000; Iwasaki et al., 2001; Klemm et al., 2001; Suzuki et al., 2003; Murphy et al., 2008). There are several other mtDNA mutations recognised to consistently express a 5. Mitochondrial diabetes phenotype which includes diabetes. These include the m.14709T > C mutation (Hanna et al., 1995; Vialettes et al., 1997; 5.1. Pattern recognition Choo-Kang et al., 2002) which has been reported to be homoplas- mic in some patients (McFarland et al., 2004) and may cause up to Faced with the fact that mitochondrial diabetes is relatively 13% of mitochondrial diabetes in the North East of England (Whit- scarce in the general diabetes clinic, it is helpful to have a A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 5 structured approach to help decide which patients should be con- present at virtually any age, but typically develops in mid-life with sidered for mtDNA mutation screening. an average age-at-onset of 37 or 38 years (Guillausseau et al., The text book description of the short, deaf patient with diabe- 2004; Whittaker et al., 2007). Only rarely does the diabetes present tes is in reality a rare occurrence and probably represents the tip of in childhood (Guillausseau et al., 2004). the iceberg in terms of mitochondrial diabetes. Historically these descriptions referred to patients with severe disease due to either 5.3. Insulin requirements m.3243A > G or KSS. These ‘textbook’ patients are usually younger and with low Body Mass Index (BMI) as well as height. This prob- Mitochondrial diabetes is felt to occur as a result of insulin defi- ably reflects more extensive multisystem involvement with higher ciency (Reardon et al., 1992; Oka et al., 1993; Kadowaki et al., levels of heteroplasmy in most tissues, and rarely will the diabetic 1993; Kadowaki et al., 1994; Katagiri et al., 1994; Walker et al., clinic be the first medical encounter for these patients. The same is 1995a) rather than insulin resistance (Walker et al., 1995b; Velho true of recessive forms of mitochondrial disease, where clear mul- et al., 1996), but in some patients both mechanisms may play a tisystem disease is likely to have raised the question of mitochon- part (Szendroedi et al., 2009). Approximately 20% of cases of mito- drial disease already. chondrial diabetes may present acutely, with 8% suffering from There are differences between mitochondrial diabetes and type- ketoacidosis (Guillausseau et al., 2001; Guillausseau et al., 2004; 2 diabetes, and these will be discussed in the subsequent para- Maassen et al., 2004), but only 13% of diabetic patients carrying graphs. Within the real world however, of busy clinics filled with the m.3243A > G mutation require insulin at diagnosis (Whittaker common disorders, a physician who diagnoses mitochondrial dia- et al., 2007). Of the remaining 87% of patients, mitochondrial diabetes without additional clues should consider themselves partic- betes develops insidiously but usually progresses rapidly to insulin ularly astute. A maternal family history or sensorineural deafness requirement, another observation unusual in type-2 diabetes; should raise suspicion. While deafness in old age or a family his- 45.2% of such patients made this transition (Whittaker et al., tory of type-2 diabetes is not uncommon, pre-senile sensorineural 2007). Average transition rates to insulin requirement range be- deafness is unusual, and especially so if combined with diabetes tween 2 and 4.2 years (Guillausseau et al., 2001; Guillausseau and a maternal history of either disorder, and/or other conditions et al., 2004; Maassen et al., 2004; Whittaker et al., 2007). Those pa- such as cardiomyopathy, epilepsy, ptosis or unusual sounding tients assessed by Whittaker and colleagues were under stringent strokes. Each additional feature adds weight to the growing suspi- review in a specialist mitochondrial clinic, and early diagnosis of cion of a mitochondrial disorder. Asymptomatic individuals and diabetes through screening programmes may explain the longer apparently ‘skipped’ generations within a pedigree should not les- transition times as compared to other studies. sen the suspicion as this is a common observation in mtDNA mutations due to carriers with low levels of heteroplasmy. In our 5.4. Body Mass Index (BMI) mitochondrial cohort in the North East of England, 82/138 (59%) individuals within m.3243A > G pedigrees showed no signs of dis- One of the clues that appears consistent in mitochondrial diabe- ease at the time of assessment, despite being at risk by virtue of tes is the tendency for patients to have a lower than average BMI maternal inheritance patterns; 82% of relatives opting for predic- (Guillausseau et al., 2004; Whittaker et al., 2007), an unusual tive testing subsequently test positive (Schaefer et al., 2008). This observation in typical type-2 diabetes. Lower BMI tends to corre- frequency remains constant whether testing 1st, 2nd or 3rd degree late with earlier onset of diabetes, earlier insulin requirements, relatives. 30% of these patients go on to develop typical features and higher HbA1C measurements (Guillausseau et al., 2004). This associated with the m.3243A > G mutation over the next 5 years probably reflects a higher overall disease burden as lower BMI (Schaefer et al., 2008) and this figure continues to rise with subse- tends to be associated with an earlier onset of disease and a more quent follow up (Schaefer and colleagues, unpublished data). severe phenotype in general (Schaefer and colleagues, unpublished There is conflicting evidence regarding the predictive value of a data). maternal family history alone (Ng et al., 2000; Choo-Kang et al., 2002). As pure diabetic phenotypes are extremely rare in mtDNA 5.5. End organ disease disease, it is probably true that a family history of diabetes alone is unlikely to suggest mitochondrial disease (Choo-Kang et al., Although neuropathy and renal disease can occur indepen- 2002). The detail of the pedigree analysis is all important, however, dently of diabetes in the m.3243A > G mutation, each complication as often enquiry is restricted to the scope of the clinic being at- has been reported to be significantly more prevalent in those pa- tended, whether diabetic or neurological; and important associa- tients with diabetes. In addition, patients with diabetic retinopathy tions may be easily overlooked. Most mitochondrial patients in or renal impairment demonstrated higher HbA1C levels than those the diabetic clinic will have a more subtle presentation, but deaf- who did not (Whittaker et al., 2007). This suggests that poor gly- ness is usually present when diabetes is diagnosed (Suzuki et al., caemic control plays a major role in their pathogenesis. The risk 1994; Guillausseau et al., 2001; Uimonen et al., 2001). An audio- of neuropathy and renal disease in the same diabetic gram can confirm its presence if it has not been formally assessed. m.3243A > G cohort was reported to be higher than in other forms A variable number of additional features (such as ptosis, proximal of either type-1 or type-2 diabetes, which implies that pre-existent myopathy, cerebellar ataxia, axonal sensorimotor neuropathy, gas- mitochondrial dysfunction within these end organs predisposes to trointestinal dysmotility and pigmentary retinopathy) are com- the microvascular complications of diabetes (Whittaker et al., monly present, but are often subtle and may be missed if not 2007). Prevalence rates for these complications exceed those re- looked for specifically. Regular ophthalmologic assessments in dia- ported in the United Kingdom Prospective Diabetes Study in betic patients afford an opportunity to identify pigmentary retin- type-2 diabetes or the DCCT in type-1 diabetes (The Diabetes Con- opathies, but the pick-up rate is increased if the ophthalmologist trol and Complications Trial Research Group, 1993; Adler et al., is aware of the clinical suspicion. 2003). Interestingly, several studies have reported lower rates of diabetic retinopathy in MIDD (Holmes-Walker et al., 1998; Massin 5.2. Age-at-onset et al., 1999; Smith et al., 1999; Latvala et al., 2002) than would normally be expected in type-1 or type-2 diabetes (Misra et al., 2009; Mitochondrial diabetes usually presents insidiously, much like Thomas et al., 2012). The same appears true of cataracts. This has type-2 diabetes. When due to the m.3243A > G mutation it can been proposed to be due to reduced glucose metabolism by the 6 A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 polyol pathway (Holmes-Walker et al., 1998). Abnormal glucose autoimmune process in mitochondrial diabetes (unlike patients tolerance also appears to increase the clinical expression of the pig- with type 1 diabetes undergoing transplantation), and so the trans- mentary retinopathy typically seen in patients with the planted organs in theory should last longer. m.3243A > G mutation. 15 of 23 patients from MIDD kindreds While discussing management it is worth clarifying the role of had evidence of a pigmentary retinopathy, 13 of which had evi- statins in mitochondrial diabetes. Statins are often recommended dence of glucose intolerance (Holmes-Walker et al., 1998). for diabetic patients to help lessen the risks of atherosclerotic com- m.3243A > G heteroplasmy levels from muscle-derived mtDNA plications. Understandably, use of a drug with the potential to in- did not correlate with the age of onset of diabetes, whilst hetero- duce a myositis often causes concern in relation to pre-existing plasmy levels from blood or muscle did not correlate with the time mitochondrial myopathy. In the majority of cases we do not be- taken to progress to insulin requirement, or the risk of diabetic lieve the risks outweigh the benefits, but do advise caution. As complications; an inverse correlation between age of diabetic on- many patients with mitochondrial disease often have a mildly ele- set and heteroplasmy level in blood derived mtDNA was reported vated creatine kinase (CK < 1000IU) at baseline, we recommend (Whittaker et al., 2007), but the true significance of this was diffi- documentation of pre-treatment CK levels with repeat CK mea- cult to predict as heteroplasmy levels in blood are known to de- surements while on statin therapy and advise to report new myal- crease year by year in patients carrying the m.3243A > G gia or weakness. mutation due to replicative disadvantage (Rahman et al., 2001). A It is evident that mitochondrial diabetes is complicated. The subsequent multicentre study found that correcting for age ne- disease pattern with a high risk of rapid progression to insulin gated the significance of a similar finding in their cohort, but did and the high prevalence of complications means that patients need note a correlation between age-adjusted blood heteroplasmy levels access to a specialist diabetes service offering regular review. The and both HbA1C, levels and low BMI (Laloi-Michelin et al., 2009). situation is further complicated by the multisystem nature of mitochondrial disease. As a consequence, we have just recently estab- 5.6. Pancreatic pathology lished a combined mitochondrial/diabetes clinic run jointly by the neurologists and diabetologists that provides an integrated, Mitochondrial dysfunction within the metabolically active pan- one-stop service for patients with mitochondrial diabetes. We have creatic B-cells is presumed to account for reduced function and also developed Best Practice Guidelines for mitochondrial diabetes ultimately loss of B-cell mass (Kobayashi et al., 1997; Lynn et al., which are available online (http://www.newcastle-mitochon- 2003). It has been difficult to demonstrate apoptosis (Kobayashi dria.com/service/patient-care-guidelines/). et al., 1997) which is the presumed explanation for the surprisingly low levels of heteroplasmy found in remaining B-cell tissue at autopsy (Togashi et al., 2000; Lynn et al., 2003). HLA polymorphisms 6. Hypoparathyroidism associated with type-1 susceptibility have not been found in mitochondrial diabetes (van Essen et al., 2000). Our own m.3243A > G Hypoparathyroidism is best described in KSS which occurs as a cohort do not, in general, carry islet cell or GAD antibodies (unpub- result of a sporadic, single large-scale mtDNA rearrangement. lished data) but these have been found in a minority of patients by Although reports are relatively rare, they seem consistent (Quade other groups (Oka et al., 1993; Murphy et al., 2008). These cases et al., 1992; Isotani et al., 1996; Katsanos et al., 2001; Cassandrini may represent coincidental autoimmune type-1 diabetes but it et al., 2006; Wilichowski et al., 1997). In each case children have has also been hypothesised that antibodies might be produced in multisystem disease, often with renal disease or other endocrinop- direct response to pancreatic B-cell destruction as a result of mito- athies in addition to the classic KSS phenotype. There does not ap- chondrial mechanisms (Oka et al., 1993). Acute or chronic pancre- pear to be an autoimmune basis to the hypoparathyroidoism atitis is rarely described (Kishnani et al., 1996; Schleiffer et al., (Isotani et al., 1996). Autopsy studies suggest absent or atrophic 2000; Verny et al., 2008; Ishiyama et al., 2012). hypoparathyroid glands (Horwitz and Roessmann, 1978; Bordarier et al., 1990). Although some patients with basal ganglia calcifica- 5.7. Diabetes management tion on CT have been found to have hypoparathyroidism (Seigel et al., 1979; Cassandrini et al., 2006) the majority have no abnor- The majority of patients present with non-insulin dependent malities of calcium homeostasis. The genetic basis of KSS was first diabetes. A sulphonylurea is the first agent of choice. Care must confirmed in 1988 (Holt et al., 1988) and reports of hypoparathy- be taken because some patients appear to be particularly sensitive roidism (and other endocrinopathies) in KSS prior to that period to sulphonylurea induced hypoglycaemia. For this reason, we fa- are unsupported by genetic studies ((Horwitz and Roessmann, vour sulphonylureas with a short half-life, and always start with 1978; Seigel et al., 1979; Dewhurst et al., 1986). It is possible that the very lowest dose and titrate up. Metformin is best avoided be- some of these cases may have harboured mtDNA point mutations cause of the risk of exacerbating lactic acidosis. Having said this, (e.g. m.3243A > G) or multiple mtDNA deletions of a nuclear cause. we have used metformin in patients with accompanying obesity Harvey and Barnett’s (1992) review found 14 of 226 patients with and have not experienced any clinical problems. The emergence KSS and CPEO to have hypoparathyroidism, but the relevant mes- of newer agents such as DDP4 inhibitors and GLP-1 analogues offer sage from this study is probably more pertinent to endocrine dis- alternative therapeutic opportunities, and we are using them as ease associated with the phenotype than the genotype, as not all second line treatment in preference to metformin depending upon cases had been genetically confirmed (Harvey and Barnett, 1992). the clinical indications. As detailed above, a minority of patients re- Hypoparathyroidism has been described in patients with ar-RRM2B quire insulin from the time of diagnosis, and those with initial non- mutations, for example, and historically would have been included insulin dependent diabetes often progress rapidly to insulin ther- in this group on the basis of the clinical features (Pitceathly et al., apy. We tailor the insulin regimen to patient’s individual lifestyle 2012). and daily needs. In our experience, hypoparathyroidism is extremely rare in As described above, progression to end stage renal failure and adult forms of mitochondrial disease. The paucity of case reports kidney transplantation is a recognised outcome. This opens up in the literature suggests likewise (Tanaka et al., 2000). It seems the possibility of pancreas transplantation, either as a simulta- most likely to occur in very severely affected patients who present neous kidney and pancreas procedure or as a pancreas after kidney in childhood with multisystem disease. This may suggest that clin- transplant. A key attraction is that there is no on-going ically significant levels of heteroplasmy within the parathyroid A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 7 glands, or alternatively the heteroplasmic threshold for dysfunc- are that it occurs in similar frequency to the background population of the parathyroid glands, is only reached in cases where very tion, and is often associated with thyroid antibodies as might be high levels of heteroplasmy are present in most tissues. expected. Reports in KSS are rare and usually associated with autoimmunity (Berio and Piazzi, 2006; Sanaker et al., 2007); only rare 7. Hypothalamo-pituitary axis reports exist for patients with the m.3243A > G mutation (Balestri and Grosso, 2000; Lau et al., 2007). Hypothyroidism was docu- The most consistent descriptions of endocrine dysfunction mented in 2/18 adult RRM2B patients, but antibody status was due to impairment of the hypothalamopituitary axis appear to not reported (Pitceathly et al., 2012). be in severe mitochondrial phenotypes presenting in childhood. MELAS and KSS appear the most common phenotypes. The endo- 11. Hypoadrenalism crine disorder may precede the neurological features and lactic acidosis is often the clue to a mitochondrial cause. Short stature This is rarely described in mitochondrial disease but impor- is usually present and more classical phenotypic expression tantly has been reported in children prior to the development of develops later if the patient survives. Reports in adults are much typical features of KSS. Patients are often neurologically normal rarer. at presentation but have short stature and may exhibit a lactic acidosis. Adrenocorticotropic hormone (ACTH) is elevated but anti- 8. Growth hormone deficiency bodies negative (Nicolino et al., 1997; Boles et al., 1998). We have recently identified an adult harbouring a single, large-scale Growth hormone (GH) deficiency has been described in KSS, mtDNA deletion and a mild KSS phenotype who developed non- both before and after genetic testing became available (Harvey autoimmune adrenal insufficiency at 51 years of age (personal and Barnett, 1992; Quade et al., 1992; Mohri et al., 1998; Berio observation). and Piazzi, 2000; Cassandrini et al., 2006; Berio and Piazzi, 2007). It has also been reported in MELAS due to the m.3243A > G mutation in children (Yorifuji et al., 1996; Robeck et al., 1996; Balestri 12. SIADH and Grosso, 2000; Matsuzaki et al., 2002) and in very rare adult cases with the MELAS (Ishii et al., 1991) and MIDD phenotypes The syndrome of inappropriate anti-diuretic hormone secretion (Joko et al., 1997). GH deficiency is often declared to be the cause (SIADH) is probably under recognised as an endocrine disorder in of short stature in the mitochondrial myopathies, but the causes mitochondrial disease. Hyponatraemia may also occur as a result are more likely to be complex and multi-factorial for most patients of unrecognised renal disease, adrenal insufficiency, or gastrointes- (Wolny et al., 2009). Publication bias is probably to blame for the tinal losses related to pseudoileus, while in other cases the cause lack of reports documenting short stature and normal GH, but may not be identified (Kubota et al., 2005; Gurrieri et al., 2001). these probably form the majority of cases. In our experience, hyponatraemia is relatively common in patients carrying the m.3243A > G mutation, but not always due to SIADH. When this occurs it is usually transient and associated with stroke- 9. Hypogonadism like episodes and more specifically ongoing seizure activity (Patel et al., 2007). In this respect SIADH can sometimes be an indication Mutations in the POLG gene have been associated with primary of active cerebral dysfunction. MELAS patients appear prone to testicular failure (Filosto et al., 2003), primary ovarian failure (Luo- SIADH, or exacerbation of pre-existing SIADH, with certain anti- ma et al., 2004; Hakonen et al., 2005), and ovarian dysgenesis (Bek- convulsants such as carbamazepine. In most cases this is not severe heirnia et al., 2012). ar-PEO1 mutations are reported to cause enough to prevent the use of clinically useful drugs in the acute female hypergonadotrophic hypogonadism by teen age (Lönnqvist setting, but may affect subsequent treatment choices. et al., 2009). Hypogonadism has been reported in association with mtDNA depletion due to ar-RRM2B mutations whilst cases of both hypergonadotropic and hypogonadotropic hypogonadism have 13. Adipose tissue as an endocrine organ been described in reports of mitochondrial neurogastrointestinal encephalopathy (MNGIE) (Carod-Artal et al., 2007; Kalkan et al., There has been very little work looking at adipose tissue func- 2012). It has been suggested from review of the early literature tion in mitochondrial diabetes. A recent study compared adipose that up to 20% of patients with KSS develop gonadal dysfunction, tissue and liver fat metabolism between patients with the either before or after puberty (Harvey and Barnett, 1992; Quade m.3243A > G mutation and healthy control subjects. They found et al., 1992). Both sexes were affected equally. It is possible how- that patients with the mutation showed evidence of adipose tissue ever that some of the patients diagnosed on clinical and histologi- insulin resistance and a tendency to increased liver fat. However, cal grounds alone may have carried other mtDNA mutations or AR as there were no diabetes controls, it was not clear to what extent forms of disease rather than single deletions of the mtDNA. Genet- the metabolic changes reflect the diabetic state rather than specific ically-confirmed case reports exist but are rare (Barrientos et al., changes related to the mitochondrial disease (Lindroos et al., 1997). Low levels of follicle stimulating hormone (FSH) and lutein- 2011). Further work is needed to determine whether there are adi- izing hormaone (LH) have both been reported in MELAS (Ishii et al., pose tissue abnormalities that are specific to mitochondrial 1991; Robeck et al., 1996; Ohkoshi et al., 1998; Balestri and Grosso, disease. 2000) but are also rare. In both KSS and MELAS it is possible that One such scenario occurs in Ekbom’s Syndrome. Originally de- hypogonadism is underdiagnosed where other forms of multisys- scribed as ‘hereditary ataxia, photomyoclonus, skeletal deformities tem disease dominate the clinical picture. and lipoma’ (Ekbom, 1975), it was subsequently confirmed to occur as a result of the m.8344A > G MTTK mutation associated with 10. Hypothyroidism the MERRF phenotype (Träff et al., 1995). Many MERRF patients develop unusual lipomata around the neck and shoulder region cor- Thyroid disease is not a recognised complication of mitochon- responding to the distribution of brown fat tissue; these lesions drial disease. Observations from our own mitochondrial cohort may be painful, restrict movement, and account for major aesthetic 8 A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 and psychological morbidity, although the mechanisms underlying References this phenomenon are currently undetermined. Abad, M.M., Cotter, P.D., Fodor, F.H., Larson, S., Ginsberg-Fellner, F., Desnick, R.J., et al., 1997. Screening for the mitochondrial DNA A3243G mutation in children with insulin-dependent diabetes mellitus. Metabolism 46, 445–449. 14. Autoimmune endocrinopathy Adler, A.I., Stevens, R.J., Manley, S.E., Bilous, R.W., Cull, C.A., Holman, R.R., 2003. Development and progression of nephropathy in Type 2 diabetes: the United Autoimmune endocrine disease has been reported in several Kingdom Prospective Diabetes Study (UKPDS 64). Kidney Int. 63, 225–232. Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H., Coulson, A.R., Drouin, J., forms of mitochondrial disease. Despite this, overall there is a lack et al., 1981. Sequence and organization of the human mitochondrial genome. of firm evidence to suggest autoimmune disorders are any more Nature 290, 457–465. prevalent in mitochondrial cohorts than in the general population. Austin, S., Vriesendorp, F., Thandroyen, F., Hecht, J., Jones, O., Johns, D., 1998. Expanding the phenotype of the 8344 transfer RNA lysine mitochondrial DNA Most cases of MIDD do not have anti-GAD or islet cell antibodies mutation. Neurology 51, 1447–1450. but these have been reported in small numbers (Oka et al., 1993; Balestri, P., Grosso, S., 2000. Endocrine disorders in two sisters affected by MELAS Murphy et al., 2008) and more frequently in some cohorts (Kobay- syndrome. J. Child Neurol. 15, 755–758. Ballinger, S.W., Shoffner, J.M., Gebhart, S., Koontz, D.A., Wallace, D.C., 1994. ashi et al., 1996). It has been hypothesised that mitochondrial dys- Mitochondrial diabetes revisited. Nat. Genet. 7, 458–459. function may play some role in the development of autoimmunity Barrientos, A., Casademont, J., Genis, D., Cardellach, F., Fernández-Real, J.M., Grau, but this remains unproven. In diabetes, pancreatic B-cell destruc- J.M., et al., 1997. Sporadic heteroplasmic single 5.5 kb mitochondrial DNA tion has been proposed as the catalyst for antibody production deletion associated with cerebellar ataxia, hypogonadotropic hypogonadism, choroidal dystrophy, and mitochondrial respiratory chain complex I deficiency. (Oka et al., 1993). Hum. Mutat. 10, 212–216. Autoimmune hypothyroidism is described rarely in patients Bekheirnia, M.R., Zhang, W., Eble, T., Willis, A., Shaibani, A., Wong, L.J., et al., 2012. with KSS, one such report also having Addison’s disease with adre- POLG mutation in a patient with cataracts, early-onset distal muscle weakness and atrophy, ovarian dysgenesis and 3-methylglutaconic aciduria. Gene 499, nal antibodies (Berio and Piazzi, 2006; Sanaker et al., 2007). 209–212. Autoimmune polyglandular syndrome type II has been reported Berio, A., Piazzi, A., 2000. Kearns–Sayre syndrome with GH deficiency. Pediatr. Med. once in a mild KSS/CPEO phenotype. The endocrine features were Chir. 22, 43–46. Berio, A., Piazzi, A., 2006. A case of Kearns–Sayre sindrome with autoimmune Addison’s disease, IDDM, autoimmune thyroiditis and primary thyroiditis and complete atrio-ventricular block. Min. Cardioangiol. 54, 387– ovarian failure. Interestingly this patient carried both a 2,532-bp 391. deletion of her mtDNA, consistent with KSS, but also a heteroplas- Berio, A., Piazzi, A., 2007. Facial anomalies in a patient with cytochrome-oxidase deficiency and subsequent Kearns–Sayre sindrome with growth hormone mic m.3243A > G mutation which was also present in her mother’s deficiency. Min. Med. 98, 81–85. mtDNA. Whether mitochondrial disease played a role in this is un- Blackwood, J.K., Whittaker, R.G., Blakely, E.L., Alston, C.L., Turnbull, D.M., Taylor, clear (Ohno et al., 1996). R.W., 2010. The investigation and diagnosis of pathogenic mitochondrial DNA mutations in human urothelial cells. Biochem. Biophys. Res. Commun. 393, 740–745. Boles, R.G., Roe, T., Senadheera, D., Mahnovski, V., Wong, L.J.C., 1998. Mitochondrial 15. Conclusion DNA deletion with Kearns Sayre syndrome in a child with Addison disease. Eur. J. Ped. 157, 643–647. Bordarier, C., Duyckaerts, C., Robain, O., Ponsot, G., Laplane, D., 1990. Kearns–Sayre Endocrine dysfunction in mitochondrial disease is common, syndrome: two clinico-pathological cases. Neuropediatrics 21, 106–109. but predominantly due to the prevalence of the m.3243A > G Calvo, S.E., Compton, A.G., Hershman, S.G., Lim, S.C., Lieber, D.S., Tucker, E.J., mutation and its association with diabetes mellitus. Other Laskowski, A., 2012. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci. Transl. Med. 25, 118ra10. mtDNA mutations reliably expressing a diabetic phenotype are Cao, L., Shitara, H., Horii, T., Nagao, Y., Imai, H., Abe, K., et al., 2007. The rare, and other forms of endocrine dysfunction are unusual when mitochondrial bottleneck occurs without reduction of mtDNA content in considering the mitochondrial diseases as a whole. Pattern rec- female mouse germ cells. Nat. Genet. 39, 386–390. Carod-Artal, F.J., Herrero, M.D., Lara, M.C., López-Gallardo, E., Ruiz-Pesini, E., Martí, ognition and detailed pedigee analysis are key when evaluating R., et al., 2007. Cognitive dysfunction and hypogonadotrophic hypogonadism in the likelihood of a mitochondrial disorder, and the presence of a Brazilian patient with mitochondrial neurogastrointestinal endocrine disease may contribute to the diagnostic process. encephalomyopathy and a novel ECGF1 mutation. Eur. J. Neurol. 14, 581–585. Furthermore, appreciation of the endocrine organs at risk in a Cassandrini, D., Savasta, S., Bozzola, M., Tessa, A., Pedemonte, M., Assereto, S., et al., 2006. Mitochondrial DNA deletion in a child with mitochondrial specified genotype/phenotype allows an appropriate level of encephalomyopathy, growth hormone deficiency, and hypoparathyroidism. J. screening to be initiated as part of the patient’s multidisciplinary Child Neurol. 21, 983–985. care strategy. KSS patients are at risk of hypoparathyroidism, and Choo-Kang, A., Lynn, S., Taylor, G., Daly, M.E., Sihota, S.S., Wardell, T.M., et al., 2002. Defining the importance of mitochondrial gene defects in maternally inherited patients with advanced multi-system disease presenting in child- diabetes by sequencing the entire mitochondrial genome. Diabetes 51, 2317– hood, whether due to mtDNA mutations or nuclear mtDNA 2320. maintenance genes, appear at risk of hypothalamopituitary dys- Cole, A., Dutton, G.N., 2000. Leber’s hereditary optic neuropathy and maturity onset diabetes mellitus: is there a metabolic association? Br. J. Ophthalmol. 84, 439– function. Most patients with mitochondrial disease, but most 440. notably those carrying the m.3243A > G mutation, should have Cree, L.M., Samuels, D.C., de Sousa Lopes, S.C., Rajasimha, H.K., Wonnapinij, P., access to annual screening for diabetes. The tendency to multi- Mann, J.R., et al., 2008. A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes. Nat. Genet. 40, system disease in these complex families can make management 249–254. difficult, even in asymptomatic carriers. For this reason we rec- Das, S., Bennett, A.J., Sovio, U., Ruokonen, A., Martikainen, H., Pouta, A., et al., 2007. ommend referral to a specialist mitochondrial centre for disease Detailed analysis of variation at and around mitochondrial position 16189 in a large Finnish cohort reveals no significant associations with early growth or specific advice and management plans. metabolic phenotypes at age 31 years. J. Clin. Endocrinol. Metab. 92, 3219– 3223. Dewhurst, A.G., Hall, D., Schwartz, M.S., McKeran, R.O., 1986. Kearns–Sayre Acknowledgments syndrome, hypoparathyroidism, and basal ganglia calcification. J. Neurol. Neurosurg. Psych. 49, 1323–1324. Du Bois, L.G., Feldon, S.E., 1992. Evidence for a metabolic trigger for Leber’s D.M.T. and R.W.T. are supported by a Strategic Award from the hereditary optic neuropathy. J. Clin. Neuro-Ophthalmol. 12, 15–16. Wellcome Trust (096919/Z/11/Z). A.M.S., D.M.T. and R.W.T. Ekbom, K., 1975. Hereditary ataxia, photomyoclonus, skeletal deformities and acknowledge the support of the UK NHS Specialist Commissioners lipoma. Acta Neurol. Scand. 51, 393–404. Elliott, H.R., Samuels, D.C., Eden, J.A., Relton, C.L., Chinnery, P.F., 2008. Pathogenic which funds the ‘‘Rare Mitochondrial Disorders of Adults and Chil- mitochondrial DNA mutations are common in the general population. Am. J. dren’’ Clinical and Diagnostic Service in Newcastle upon Tyne. Hum. Genet. 83, 254–260. A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 9

Filosto, M., Mancuso, M., Nishigaki, Y., Pancrudo, J., Harati, Y., Gooch, C., et al., 2003. Klemm, T., Neumann, S., Trulzsch, B., Pistrosch, F., Hanefeld, M., Paschke, R., 2001. Clinical and genetic heterogeneity in progressive external ophthalmoplegia due Search for mitochondrial DNA mutation at position 3243 in German patients to mutations in polymerase gamma. Arch. Neurol. 60, 1279–1284. with a positive family history of maternal diabetes mellitus. Exp. Clin. Goto, Y., Nonaka, I., Horai, S., 1991. A new mtDNA mutation associated with Endocrinol. Diabet. 109, 283–287. mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like Kobayashi, T., Oka, Y., Katagiri, H., Falorni, A., Kasuga, A., Takei, I., et al., 1996. episodes (MELAS). Biochim. Biophys. Acta 1097, 238–240. Association between HLA and islet cell antibodies in diabetic patients with a Greaves, L.C., Reeve, A.K., Taylor, R.W., Turnbull, D.M., 2012. Mitochondrial DNA and mitochondrial DNA mutation at base pair 3243. Diabetologia 39, 1196–1200. disease. J. Pathol. 226, 274–286. Kobayashi, T., Nakanishi, K., Nakase, H., Kajio, H., Okubo, M., Murase, T., et al., 1997. Guillausseau, P.J., Massin, P., Dubois-Laforge, D., Timsit, J., Virally, M., Gin, H., et al., In situ characterization of islets in diabetes with a mitochondrial DNA mutation 2001. Maternally inherited diabetes and deafness: a multicenter study. Ann. Int. at nucleotide position 3243. Diabetes 46, 1567–1572. Med. 134, 721–728. Kubota, H., Tanabe, Y., Takanashi, J.I., Kohno, Y., 2005. Episodic hyponatremia in Guillausseau, P.J., Dubois-Laforge, D., Massin, P., Laloi-Michelin, M., Bellanne- mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes Chantelot, C., Gin, H., et al., 2004. Heterogeneity of diabetes phenotype in (MELAS). J. Child Neurol. 20, 116–119. patients with 3243-bp mutation of mitochondrial DNA (maternally inherited Laloi-Michelin, M., Meas, T., Ambonville, C., Bellanné-Chantelot, C., Beaufils, S., diabetes and deafness or MIDD). Diabet. Metab. 30, 181–186. Massin, P., et al., 2009. Mitochondrial Diabetes French Study Group. The clinical Gurrieri, C., Kivela, J.E., Bojanic´, K., Gavrilova, R.H., Flick, R.P., Sprung, J., et al., 2001. variability of maternally inherited diabetes and deafness is associated with the Anesthetic considerations in mitochondrial encephalomyopathy, lactic acidosis, degree of heteroplasmy in blood leukocytes. J. Clin. Endocrinol. Metab. 94, and stroke-like episodes syndrome: a case series. Can. J. Anaesth. 58, 751–763. 3025–3030. Hakonen, A.H., Heiskanen, S., Juvonen, V., Lappalainen, I., Luoma, P.T., Rantamaki, Latvala, T., Mustonen, E., Uusitalo, R., Majamaa, K., 2002. Pigmentary retinopathy in M., et al., 2005. Mitochondrial DNA polymerase W748S mutation: a common patients with the MELAS mutation 3243A > G in mitochondrial DNA. Arch. Clin. cause of autosomal recessive ataxia with ancient European origin. Am. J. Hum. Exp. Ophthalmol. 240, 795–801. Genet. 77, 430–441. Lau, K.K., Yang, S.P., Haddad, M.N., Butani, L., Makker, S.P., 2007. Mitochondrial Hanna, M.G., Nelson, I., Sweeney, M.G., Cooper, J.M., Watkins, P.J., Morgan-Hughes, encephalopathy with lactic acidosis and stroke-like episodes syndrome with J.A., et al., 1995. Congenital encephalomyopathy and adult-onset myopathy and hypothyroidism and focal segmental glomerulosclerosis in a paediatric patient. diabetes mellitus: different phenotypic associations of a new heteroplasmic Int. J. Urol. Nephrol. 39, 941–946. mtDNA tRNA glutamic acid mutation. Am. J. Hum. Genet. 56, 1026–1033. Lehto, M., Wipemo, C., Ivarsson, S., Lindgren, C., Lipsanen-Nyman, M., Weng, J., et al., Harvey, J.N., Barnett, D., 1992. Endocrine dysfunction in Kearns–Sayre syndrome. 1999. High frequency of mutations in MODY and mitochondrial genes in Clin. Endocrinol. 37, 97–103. Scandinavian patients with familial early-onset diabetes. Diabetologia 42, Holmes-Walker, D., Mitchell, P., Boyages, S., 1998. Does mitochondrial genome 1131–1137. mutation in subjects with maternally inherited diabetes and deafness decrease Lindroos, B., Suuronen, R., Miettinen, S., 2011. The potential of adipose stem cells in severity of diabetic retinopathy. Diabet. Med. 15, 946–952. regenerative medicine. Stem Cell Rev. 7, 269–291. Holt, I.J., Harding, A.E., Morgan-Hughes, J.A., 1988. Deletions of muscle Lönnqvist, T., Paetau, A., Valanne, L., Pihko, H., 2009. Recessive twinkle mutations mitochondrial DNA in patients with mitochondrial myopathies. Nature 331, cause severe epileptic encephalopathy. Brain 132, 1553–1562. 717–719. Luoma, P., Melberg, A., Rinne, J.O., Kaukonen, J.A., Nupponen, N.N., Chalmers, R.M., Horvath, R., Hudson, G., Ferrari, G., Fütterer, N., Ahola, S., Lamantea, E., et al., 2006. et al., 2004. Parkinsonism, premature menopause, and mitochondrial DNA Phenotypic spectrum associated with mutations of the mitochondrial polymerase gamma mutations: clinical and molecular genetic study. Lancet polymerase c gene. Brain 129, 1674–1684. 364, 875–882. Horwitz, S.J., Roessmann, U., 1978. Kearns Sayre syndrome with Lynn, S., Wardell, T., Johnson, M.A., Chinnery, P.F., Daly, M.E., Walker, M., et al., 1998. hypoparathyroidism. Ann. Neurol. 3, 513–518. Mitochondrial diabetes: investigation and identification of a novel mutation. Ishii, A., Watanabe, S., Ohkoshi, N., Mizusawa, H., Kanazawa, I., 1991. Mitochondrial Diabetes 47, 1800–1802. encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) Lynn, S., Borthwick, G.M., Charnley, R.M., Walker, M., Turnbull, D.M., 2003. associated with hypothalamo-pituitary hypofunction: a case report. Clin. Heteroplasmic ratio of the A3243G mitochondrial DNA mutation in single Neurol. 31, 179–183. pancreatic beta cells. Diabetologia 46, 296–299. Ishiyama, A., Komaki, H., Saito, T., Saito, Y., Nakagawa, E., Sugai, K., et al., 2012. Maassen, J., t’Hart, L., van Essen, E., Heine, R., Nijpels, G., Jahangir Tafrechi, R., et al., Unusual exocrine complication of pancreatitis in mitochondrial disease. Brain 2004. Mitochondrial diabetes: molecular mechanisms and clinical presentation. Dev., pii:S0387-7604(12)00273-2. Diabetes 53, S103–S109. Isotani, H., Fukumoto, Y., Kawamura, H., Furukawa, K., Ohsawa, N., Goto, Y.I., et al., Majamaa, K., Turkka, J., Karppa, M., Winqvist, S., Hassinen, I.E., 1997. The common 1996. Hypoparathyroidism and insulin-dependent diabetes mellitus in a MELAS mutation A3243G in mitochondrial DNA among young patients with an patient with Kearns–Sayre syndrome harbouring a mitochondrial DNA occipital brain infarct. Neurology 49, 1331–1334. deletion. Clin. Endocrin. 45, 637–641. Malecki, M.T., Klupa, T., Wanic, K., Frey, J., Cyganek, K., Sieradzki, J., 2001. Search for Iwasaki, N., Babazono, T., Tsuchiya, K., Tomonaga, O., Suzuki, A., Togashi, M., et al., mitochondrial A3243G tRNA(Leu) mutation in Polish patients with Type 2 2001. Prevalence of A-to-G mutation at nucleotide 3243 of the mitochondrial diabetes mellitus. Med. Sci. Monit. 7, 246–250. tRNALeu(UUR) gene in Japanese patients with diabetes mellitus and end-stage Mansergh, F.C., Millington-Ward, S., Kennan, A., Kiang, A.-S., Humphries, M., Farrar, renal disease. J. Hum. Genet. 46, 330–334. G.J., et al., 1999. Retinitis pigmentosa and progressive sensorineural hearing loss Joko, T., Iwashige, K., Hashimoto, T., Ono, Y., Kobayashi, K., Sekiguchi, N., et al., 1997. caused by a C12258A mutation in the mitochondrial MTTS2 gene. Am. J. Hum. A case of mitochondrial encephalomyopathy, lactic acidosis and stroke-like Genet. 64, 971–985. episodes associated with diabetes mellitus and hypothalamo-pituitary Massin, P., Virally-Monod, M., Vialettes, B., Paques, M., Gin, H., Porokhov, B., et al., dysfunction. Endocr. J. 44, 805–809. 1999. Prevalence of macular pattern dystrophy in maternally inherited diabetes Kadowaki, H., Tobe, K., Mori, Y., Sakura, H., Sakuta, R., Nonaka, I., et al., 1993. and deafness. GEDIAM group. Ophthalmology 106, 1821–1827. Mitochondrial gene mutation and insulin-deficient type of diabetes mellitus. Matsuura, N., Suzuki, S., Yokota, Y., Kazahari, K., Kazahari, M., Toyota, T., et al., 1999. Lancet 341, 893–894. The prevalence of mitochondrial gene mutations in childhood diabetes in Japan. Kadowaki, T., Kadowaki, H., Mori, Y., Tobe, K., Sakuta, R., Suzuki, Y., et al., 1994. A J. Pediatr. Endocrinol. Metab. 12, 27–30. subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. Matsuzaki, M., Izumi, T., Shishikura, K., Suzuki, H., Hirayama, Y., 2002. N. Eng. J. Med. 330, 962–968. Hypothalamic growth hormone deficiency and supplementary GH therapy in Kalkan, I.H., Tayfur, O., Oztasß, E., Beyazit, Y., Yildiz, H., Tunç, B., 2012. A novel finding two patients with mitochondrial myopathy, encephalopathy, lactic acidosis and in MNGIE (Mitochondrial Neurogastrointestinal Encephalomyopathy): stroke-like episodes. Neuropediatrics 33, 271–273. hypergonadotropic hypogonadism. Hormones (Athens) 11, 377–379. McDonnell, M.T., Blakely, E.L., Schaefer, A.M., McFarland, R., Turnbull, D.M., Taylor, Kameoka, K., Isotani, H., Tanaka, K., Kitaoka, H., Ohsawa, N., 1998. Impaired insulin R.W., 2004. Non-invasive diagnosis of the 3243A > G mitochondrial DNA secretion in Japanese diabetic subjects with an A-to-G mutation at nuceotide mutation using urinary epithelial cells. Eur. J. Hum. Genet. 12, 778–781. 8296 of the mitochondrial DNA in tRNALys. Diabet. Care 21, 2034–2035. McFarland, R., Schaefer, A.M., Gardner, J.L., Lynn, S., Hayes, C.M., Barron, M.J., et al., Katagiri, H., Asano, T., Ishihara, H., Inukai, K., Anai, M., Yamanouchi, T., et al., 1994. 2004. Familial myopathy: new insights into the T14709C mitochondrial tRNA Mitochondrial diabetes mellitus: prevalence and clinical characterization of mutation. Ann. Neurol. 55, 478–484. diabetes due to mitochondrial tRNALeu(UUR) gene mutation in Japanese patients. McFarland, R., Taylor, R.W., Turnbull, D.M., 2010. A neurological perspective on Diabetologia 37, 504–510. mitochondrial disease. Lancet Neurol. 9, 829–840. Katsanos, K.H., Elisaf, M., Bairaktari, E., Tsianos, E.V., 2001. Severe hypomagnesemia Misra, A., Bachmann, M.O., Greenwood, R.H., Jenkins, C., Shaw, A., Barakat, O., et al., and hypoparathyroidism in Kearns–Sayre syndrome. Am. J. Nephrol. 21, 150– 2009. Trends in yield and effects of screening intervals during 17 years of a large 153. UK community-based diabetic retinopathy screening programme. Diabet. Med. Kishimoto, M., Hashiramoto, M., Araki, S., Ishida, Y., Kazumi, T., Kand, E., et al., 1995. 26, 1040–1047. Diabetes mellitus carrying a mutation in the mitochondrial tRNA Leu(UUR) gene. Mohri, I., Taniike, M., Fujimura, H., Matsuoka, T., Inui, K., Nagai, T., et al., 1998. A case Diabetologia 38, 193–200. of Kearns–Sayre syndrome showing a constant proportion of deleted Kishnani, P.S., Van Hove, J.L.K., Shoffner, J.S., Kaufman, A., Bossen, E.H., Kahler, S.G., mitochondrial DNA in blood cells during 6 years of follow-up. J. Neurol. Sci. 1996. Acute pancreatitis in an infant with lactic acidosis and a mutation at 158, 106–109. nucleotide 3243 in the mitochondrial DNA tRNALeu(UUR) gene. Eur. J. Pediatr. Murphy, R., Turnbull, D.M., Walker, M., Hattersley, A.T., 2008. Clinical features, 155, 898–903. diagnosis and management of maternally inherited diabetes and deafness 10 A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11

(MIDD) associated with the 3243A > G mitochondrial point mutation. Diabet. Smith, P., Bain, S., Good, P., Hattersley, A., Barnett, A., Gibson, J., et al., 1999. Med. 25, 383–399. Pigmentary retinal dystrophy and the syndrome of maternally inherited Nagashima, T., Mori, M., Katayama, K., Nunomura, M., Nishihara, H., Hiraga, H., et al., diabetes and deafness caused by the mitochondrial DNA 3243 tRNA Leu A to 1999. Adult Leigh syndrome with mitochondrial DNA mutation at 8993. Acta G mutation. Opthalmology 106, 1101–1108. Neuropathol. 97, 416–422. Superti-Furga, A., Schoenle, E., Tuchschmid, P., Caduff, R., Sabato, V., DeMattia, D., Newkirk, J.E., Taylor, R.W., Howell, N., Bindoff, L.A., Chinnery, P.F., Alberti, K.G., et al., et al., 1993. Pearson bone marrow-pancreas syndrome with insulin-dependent 1997. Maternally inherited diabetes and deafness: prevalence in a hospital diabetes, progressive renal tubulopathy, organic aciduria and elevated fetal diabetic population. Diabet. Med. 14, 457–460. haemoglobin caused by deletion and duplication of mitochondrial DNA. Eur. J. Newman, N.J., Lott, M.T., Wallace, D.C., 1991. The clinical characteristics of Pediatr. 152, 44–50. pedigrees of Leber’s hereditary optic neuropathy with the 11778 mutation. Suzuki, S., Hinokio, Y., Hirai, S., Onoda, M., Matsumoto, M., Ohtomo, M., et al., 1994. Am. J. Ophthalmol. 111, 750–762. Pancreatic beta-cell defect associated with mitochondrial point mutation of the Ng, M., Yeung, V., Chow, C., Li, J., Smith, P., Mijovic, C., et al., 2000. Mitochondrial tRNALeu(UUR) gene: a study in seven families with mitochondrial DNA A3243G mutation in patients with early or late-onset Type 2 diabetes encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). mellitus in Hong Kong Chinese. Clin. Endocrinol. 52, 557–564. Diabetologia 37, 818–825. Nicolino, M., Ferlin, T., Forest, M., Godinot, C., Carrier, H., David, M., et al., 1997. Suzuki, Y., Tsukuda, K., Atsumi, Y., Goto, Y., Hosokawa, K., Asahina, T., et al., 1996. Identiﬁcation of a large-scale mitochondrial deoxyribonucleic acid deletion in Clinical picture of a case of diabetes with mitochondrial tRNA mutation at endocrinopathies and deafness: report of two unrelated cases with diabetes position 3271. Diabet. Care 19, 1304–1305. mellitus and adrenal insufﬁciency, respectively. J. Clin Endocrin. Metab. 82, Suzuki, Y., Suzuki, S., Hinokio, Y., Chiba, M., Atsumi, Y., Hosokawa, K., et al., 1997. 3063–3067. Diabetes associated with a novel 3264 mitochondrial tRNALeu(UUR) mutation. Odawara, M., Sasaki, K., Yamashita, K., 1995. Prevalence and clinical Diabet. Care 20, 1138–1140. characterisation of Japanese diabetes mellitus with an A to G mutation at Suzuki, Y., Taniyama, M., Muramatsu, T., Ohta, S., Murata, C., Atsumi, Y., et al., 2003. nucleotide 3243 of the mitochondrial tRNALeu(UUR) gene. J. Clin. Endocrinol. Mitochondrial tRNALeu(UUR) mutation at position 3243 and symptomatic Metab. 80, 1290–1294. polyneuropathy in Type 2 diabetes. Diabet. Care 26, 1315–1316. Ohkoshi, N., Ishii, A., Shiraiwa, N., Shoji, S.I., Yoshizawa, K., 1998. Dysfunction of the Szendroedi, J., Schmid, A.I., Meyerspeer, M., Cervin, C., Kacerovsky, M., Smekal, G., hypothalamic-pituitary system in mitochondrial encephalomyopathies. J. Med. et al., 2009. Impaired mitochondrial function and insulin resistance of skeletal 29, 13–29. muscle in mitochondrial diabetes. Diabet. Care 32, 677–679. Ohkubo, K., Yamano, A., Nagashima, M., Mori, Y., Anzai, K., Akehi, Y., et al., 2001. Tanaka, K., Takaday, Y., Matsunaka, T., Yuyama, S., Fujino, S., Maguchi, M., et al., Mitochondrial gene mutations in the tRNALeu(UUR) region and diabetes: 2000. Diabetes mellitus, deafness, muscle weakness and hypocalcemia in a prevalence and clinical phenotypes in Japan. Clin. Chem. 47, 1641–1648. patient with an A3243G mutation of the mitochondrial DNA. Int. Med. 39, 249– Ohno, K., Yamamoto, M., Engel, A.G., Harper, C.M., Roberts, L.R., Tan, G.H., 1996. 252. MELAS- and Kearns–Sayre-type co-mutation [corrected] with myopathy and Tawata, M., Hayashi, J., Isobe, K., Ohkubo, E., Ohtaka, M., Chen, J., et al., 2000. A new autoimmune polyendocrinopathy. Ann. Neurol. 39, 761–766. mitochondrial DNA mutation at 14577 T/C is probably a major pathogenic Oka, Y., Katagiri, H., Yazaki, Y., Murase, T., Kobayashi, T., 1993. Mitochondrial gene factor for maternally inherited Type 2 diabetes. Diabetes 49, 1269–1272. mutation in islet-cell-antibody-positive patients who were initially non-insulin Taylor, R.W., Schaefer, A.M., Barron, M.J., McFarland, R., Turnbull, D.M., 2004. The dependent diabetics. Lancet 342, 527–528. diagnosis of mitochondrial muscle disease. Neuromuscul. Disord. 14, 237–245. Otabe, S., Sakura, H., Shimokawa, K., Mori, Y., Kadowaki, H., Yasuda, K., et al., 1994. t’Hart, L.M., Lemkes, H.H., Heine, R.J., Stolk, R.P., Feskens, E.J., Jansen, J.J., et al., 1994. The high prevalence of diabetic patients with a mutation in the mitochondrial Prevalence of maternally inherited diabetes and deafness in diabetic gene in Japan. J. Clin. Endocrinol. Metab. 79, 768–771. populations in The Netherlands. Diabetologia 37, 1169–1170. Patel, I.B., Sidani, M., Zoorob, R., 2007. Mitochondrial encephalopathy, lactic acidosis The Diabetes Control and Complications Trial Research Group, 1993. The effect of and stroke-like syndrome (MELAS): a case report, presentation, and intensive treatment of diabetes on the development and progression of long- management. Southern Med. J. 100, 70–72. term complications in insulin dependent diabetes mellitus. N. Engl. J. Med. 329, Pilz, D., Quarrell, O.W., Jones, E.W., 1994. Mitochondrial mutation commonly 977–986. associated with Leber’s hereditary optic neuropathy observed in a patient with Thomas, R.L., Dunstan, F., Luzio, S.D., Roy Chowdury, S., Hale, S.L., North, R.V., et al., Wolfram syndrome (DIDMOAD). J. Med. Genet. 31, 328–330. 2012. Incidence of diabetic retinopathy in people with Type 2 diabetes mellitus Pitceathly, R.D., Smith, C., Fratter, C., Alston, C.L., He, L., Craig, K., et al., 2012. Adults attending the diabetic retinopathy screening service for Wales: retrospective with RRM2B-related mitochondrial disease have distinct clinical and molecular analysis. BMJ 344, e874. characteristics. Brain 135, 3392–3403. Togashi, M., Yanada, H., Iwasaki, N., et al., 2000. Selective loss of pancreatic B-cells Poulton, J., Luan, J., Macaulay, V., Hennings, S., Mitchell, J., Wareham, N.J., 2002. Type in a diabetic patient with a mitochondrial 3243 mutation. J. Jpn. Diabet. Soc. 43, 2 diabetes is associated with a common mitochondrial variant: evidence from a 455–458. population-based case-control study. Hum. Mol. Genet. 11, 1581–1583. Träff, J., Holme, E., Ekbom, K., Nilsson, B.Y., 1995. Ekbom’s syndrome of Quade, A., Zierz, S., Klingmüller, D., 1992. Endocrine abnormalities in mitochondrial photomyoclonus, cerebellar ataxia and cervical lipoma is associated with the myopathy with external ophthalmoplegia. Clin. Invest. 70, 396–402. tRNALys A8344G mutation in mitochondrial DNA. Acta Neurol. Scand. 92, 394– Rahman, S., Poulton, J., Marchington, D., Suomalainen, A., 2001. Decrease of 3243 397. A ? G mtDNA mutation from blood in MELAS syndrome: a longitudinal study. Tsukuda, K., Suzuki, Y., Kameoka, K., Osawa, N., Goto, Y.-I., Katagiri, H., et al., 1997. Am. J. Hum. Genet. 68, 238–240. Screening of patients with maternally transmitted diabetes for mitochondrial Reardon, W., Ross, R., Sweeney, M., Luxon, L., Pembrey, M., Harding, A., et al., 1992. gene mutations in the tRNALeu(UUR) region. Diabet. Med. 14, 1032–1037. Diabetes mellitus associated with a pathogenic point mutation in mitochondrial Tuppen, H.A., Blakely, E.L., Turnbull, D.M., Taylor, R.W., 2010. Mitochondrial DNA DNA. Lancet 340, 1376–1379. mutations and human disease. Biochim. Biophys. Acta 1797, 113–128. Robeck, S., Stefan, H., Engelhardt, A., Neundörfer, B., 1996. Follow-up studies and Uchigata, M., Mizota, M., Yangisawa, Y., Nakagawa, Y., Otani, T., Ikegami, H., et al., disorders of endocrinologic function in MELAS syndrome. Nervenarzt 67, 465– 1996. Large-scale study of an A-to-G transition at position 3243 of the 470. mitochondrial gene and IDDM in Japanese patients. Diabetologia 39, 245–246. Rötig, A., Bessis, J.L., Romero, N., Cormier, V., Saudubray, J.M., Narcy, P., et al., 1992. Uimonen, S., Moilanen, J.S., Sorri, M., Hassinen, I.E., Majamaa, K., 2001. Hearing Maternally inherited duplication of the mitochondrial genome in a syndrome of impairment in patients with 3243A > G mtDNA mutation: phenotype and rate proximal tubulopathy, diabetes mellitus, and cerebellar ataxia. Am. J. Hum. of progression. Hum. Genet. 108, 284–289. Genet. 50, 364–370. van den Ouweland, J., Lemkes, H., Ruitenbeek, W., Sandkujl, L., de Vijlder, M., Saker, P., Hattersley, A., Barrow, B., Hammersley, M., Horton, V., Gillmer, M., et al., Struyvenberg, P., et al., 1992. Mutation in mitochondrial tRNALeu(UUR) gene in a 1997. UKPDS 21: low prevalence of the mitochondrial transfer RNA gene large pedigree with maternally transmitted Type 2 diabetes and deafness. Nat. (tRNALeu(UUR)) mutation at position 3243 bp in UK Caucasian Type 2 diabetic Genet. 1, 368–371. patients. Diabet. Med. 14, 42–45. van Essen, E., Roep, B., t’Hart, L., Jansen, J., van den Ouweland, J., Lemkes, H., et al., Sanaker, P.S., Husebye, E.S., Fondenes, O., Bindoff, L.A., 2007. Clinical evolution of 2000. HLA-DQ polymorphism and degree of heteroplasmy of the A3243G Kearns–Sayre syndrome with polyendocrinopathy and respiratory failure. Acta mitochondrial DNA mutation in maternally inherited diabetes and deafness. Neurol. Scand. 115, 64–67. Diabet. Med. 17, 841–847. Schaefer, A.M., McFarland, R., Blakely, E.L., He, L., Whittaker, R.G., Taylor, R.W., et al., Velho, G., Byrne, M.M., Clement, K., Sturis, J., Pueyo, M.E., Blanche, H., et al., 1996. 2008. Prevalence of mitochondrial DNA disease in adults. Ann. Neurol. 63, 35– Clinical phenotypes, insulin secretion, and insulin sensitivity in kindreds with 39. maternally inherited diabetes and deafness due to mitochondrial tRNALeu(UUR) Schleiffer, T., t’Hart, L.M., Schurfeld, C., Kraatz, K., Riemann, J.F., 2000. Maternally gene mutation. Diabetes 45, 478–487. inherited diabetes and deafness (MIDD): unusual occult exocrine pancreatic Verny, C., Amati-Bonneau, P., Letournel, F., Person, B., Dib, N., Malinge, M.C., et al., manifestation in an affected German family. Exp. Clin. Endocrinol. Diabet. 108, 2008. Mitochondrial DNA A3243G mutation involved in familial diabetes, 81–85. chronic intestinal pseudo-obstruction and recurrent pancreatitis. Diabet. Schon, E.A., DiMauro, S., Hirano, M., 2012. Human mitochondrial DNA: roles of Metab. 34, 620–626. inherited and somatic mutations. Nat. Rev. Genet. 13, 878–890. Vialettes, B., Paquis-Fluckinger, V., Pelissier, J.-F., Bendahan, D., Narbonne, H., Seigel, R.S., Seeger, J.F., Gabrielsen, T.O., Allen, R.J., 1979. Computerised tomography Silvestre-Aillaud, P., et al., 1997. Phenotypic expression of diabetes secondary to in oculocraniosomatic disease (Kearns–Sayre syndrome). Radiology 130, 159– a T14709C mutation of mitochondrial DNA. Comparison with MIDD syndrome 164. (A3243G mutation): a case report. Diabet. Care 20, 1731–1737. A.M. Schaefer et al. / Molecular and Cellular Endocrinology 379 (2013) 2–11 11

Vionnet, N., Passa, P., Froguel, P., 1993. Prevalence of mitochondrial gene mutations Wilichowski, E., Grüters, A., Kruse, K., Rating, D., Beetz, R., Korenke, G.C., et al., 1997. in families with diabetes mellitus. Lancet 342, 1429–1430. Hypoparathyroidism and deafness associated with pleioplasmic large scale Wai, T., Teoli, D., Shoubridge, E.A., 2008. The mitochondrial DNA genetic bottleneck rearrangements of the mitochondrial DNA: a clinical and molecular genetic results from replication of a subpopulation of genomes. Nat. Genet. 40, 1484– study of four children with Kearns-Sayre syndrome. Pediatr. Res. 41, 193–200. 1488. Williams, T.B., Daniels, M., Puthenveetil, G., Chang, R., Wang, R.Y., Abdenur, J.E., Walker, M., Taylor, R.W., Stewart, M., Bindoff, L.A., Shearing, P., Anyaoka, V., et al., 2012. Pearson syndrome: unique endocrine manifestations including neonatal 1995a. Insulin and proinsulin secretion in subjects with abnormal glucose diabetes and adrenal insufficiency. Mol. Genet. Metab. 106, 104–107. tolerance and a mitochondrial tRNALeu(UUR) mutation. Diabet. Care 18, 1507– Wolny, S., McFarland, R., Chinnery, P., Cheetham, T., 2009. Abnormal growth in 1509. mitochondrial disease. Acta Paediatr. 98, 553–554. Walker, M., Taylor, R.W., Stewart, M., Bindoff, L., Jackson, M., Alberti, K.G., et al., Ylikallio, E., Suomalainen, A., 2012. Mechanisms of mitochondrial diseases. Ann. 1995b. Insulin sensitivity and mitochondrial gene mutation. Diabet. Care 18, Med. 44, 41–59. 273–275. Yorifuji, T., Kawai, M., Momoi, T., Sasaki, H., Furusho, K., Muroi, J., et al., 1996. Wallace, D.C., Singh, G., Lott, M.T., Hodge, J.A., Schurr, T.G., Lezza, A.M., et al., 1988. Nephropathy and growth hormone deficiency in a patient with mitochondrial Mitochondrial DNA mutation associated with Leber’s hereditary optic tRNALeu(UUR) mutation. J. Med. Genet. 33, 621–622. neuropathy. Science 242, 1427–1430. Yu-Wai-Man, P., Griffiths, P.G., Gorman, G.S., Lourenco, C.M., Wright, A.F., Auer- Whittaker, R.G., Schaefer, A.M., McFarland, R., Taylor, R.W., Walker, M., Turnbull, Grumbach, M., et al., 2010. Multi-system neurological disease is common in D.M., 2007. Prevalence and progression of diabetes in mitochondrial disease. patients with OPA1 mutations. Brain 133, 771–786. Diabetologia 50, 2085–2089. Whittaker, R.G., Blackwood, J.K., Alston, C., Blakely, E.L., Elson, J.L., McFarland, R., et al., 2009. Urine heteroplasmy level is the best predictor of clinical outcome in patients with the m.3243A > G mtDNA mutation. Neurology 72, 568–569. Molecular and Cellular Endocrinology 379 (2013) 12–18

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Mitochondrial function and insulin secretion

Pierre Maechler ⇑

Department of Cell Physiology and Metabolism, Geneva University Medical Centre, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland article info abstract

Article history: In the endocrine fraction of the pancreas, the b-cell rapidly reacts to fluctuations in blood glucose concen- Available online 20 June 2013 trations by adjusting the rate of insulin secretion. Glucose-sensing coupled to insulin exocytosis depends on transduction of metabolic signals into intracellular messengers recognized by the secretory machin- Keywords: ery. Mitochondria play a central role in this process by connecting glucose metabolism to insulin release. b-cell Mitochondrial activity is primarily regulated by metabolic fluxes, but also by dynamic morphology Pancreatic islets changes and free Ca2+ concentrations. Recent advances of mitochondrial Ca2+ homeostasis are discussed; Insulin secretion in particular the roles of the newly-identified mitochondrial Ca2+ uniporter MCU and its regulatory part- Mitochondria ner MICU1, as well as the mitochondrial Na+–Ca2+ exchanger. This review describes how mitochondria Diabetes function both as sensors and generators of metabolic signals; such as NADPH, long chain acyl-CoA, glutamate. The coupling factors are additive to the Ca2+ signal and participate to the amplifying pathway of glucose-stimulated insulin secretion. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction are classified as type 2 diabetes, or non-insulin dependent diabetes mellitus. The patients display dysregulation of insulin secretion, of- 1.1. The pancreatic b-cell ten combined with insulin resistance of target tissues. The aetiol- ogy of type 2 diabetes is still poorly understood and has been Glucose homeostasis depends on optimal regulation of insulin elucidated in only a limited number of subtypes. Among these, secretion from the b-cells and the action of insulin on its target tis- maturity onset diabetes of the young (MODY) and mitochondrial sues; in particular muscles, liver, and adipose tissue. The b-cells are diabetes have been linked to specific gene mutations and primary located in the endocrine fraction of the pancreas, i.e., the islets of b-cell dysfunction (Froguel et al., 1993; Byrne et al., 1996; Clocquet Langerhans. In humans, b-cells constitute about 70% of the islets et al., 2000; Maassen et al., 2004). The impact of such mutations on of Langerhans, which are spread throughout the pancreas and the b-cell highlights the importance of the mitochondria in the compose 1–2% of this organ (Rahier et al., 1983). It means that control of insulin secretion. Other endocrine tissues play an impor- among the 1013–1014 of cells composing our body, the 109 b-cells tant role in metabolic dysregulation and the reader is referred to contribute to less than 0.01% of this count. In other words, one sin- the other articles of this special issue of Molecular and Cellular gle drop of blood contains as much red blood cells as our whole Endocrinology for corresponding information. body contains b-cells. Nevertheless, this minute amount of endocrine tissue is essential for life since there is no alternative hormone to insulin, as dramatically illustrated by patients suffering 1.3. Metabolic activation of the b-cell from type 1 diabetes. The cytoplasm of each b-cell contains about 13,000 secretory granules filled with crystallized insulin (Dean, Both the secretion and the action of insulin contribute to glu- 1973). During glucose stimulation only a small proportion of the cose homeostasis. Regulated insulin release requires tight coupling granule pool undergoes exocytosis. in the b-cell between glucose metabolism and insulin secretory response. The exocytotic process is closely controlled by signals gen- 1.2. Diabetes and the b-cell erated by nutrient metabolism (Fig. 1), as well as by neurotransmitters and circulating hormones (Huypens et al., The initial stages of type 1 diabetes, before b-cell destruction, 2000; Schuit et al., 2001; Rubi and Maechler, 2010). The b-cell rap- are characterized by defects in the function of b-cells (O’Sullivan- idly reacts to fluctuations in the blood glucose concentration by Murphy and Urano, 2012). The large majority of diabetic patients adjusting the rate of insulin secretion. This review describes the molecular basis of metabolism–secretion coupling. In particular, Tel.: +41 22 379 55 54. it will be discussed how mitochondria function both as sensors ⇑ E-mail address: [email protected] and generators of metabolic signals.

Fig. 1. Model for coupling of glucose metabolism to insulin secretion in the b-cell. Glucose equilibrates across the plasma membrane and is phosphorylated by glucokinase (GK). Further, glycolysis produces pyruvate, which preferentially enters the mitochondria and is metabolized by the TCA cycle. The TCA cycle generates reducing equivalents transferred by NADH and FADH2 to the electron transport chain (ETC), leading to hyperpolarization of the mitochondrial membrane (Dwm) and generation of ATP. ATP is then 2+ transferred to the cytosol, raising the ATP/ADP ratio. Subsequently, closure of KATP-channels depolarizes the cell membrane (Dwc). This opens voltage-dependent Ca 2+ 2+ channels, increasing cytosolic Ca concentration ([Ca ]c), which triggers insulin exocytosis. The amplifying pathway of metabolism–secretion coupling is contributed by additive coupling factors.

2. Overview of metabolism–secretion coupling in b-cells system illustrates the tight coupling between glycolysis and mitochondrial activation in b-cells, optimizing transfer of pyruvate into 2.1. Pathways upstream of mitochondria mitochondria through the recently identified mitochondrial pyruvate carrier (Herzig et al., 2012). Subsequently, catabolism of glu- In b-cells, metabolism–secretion coupling refers to both the cose-derived pyruvate induces mitochondrial activation resulting consensus model and the contribution of additional coupling fac- in ATP generation. Although mitochondria, and the Krebs cycle in tors, i.e., the trigger and amplifying pathways of glucose-stimu- particular, also oxidize fatty acids and amino acids, carbohydrates lated insulin secretion (Fig. 1). This process is initiated by the are the most important fuel under physiological conditions for the passive entry of glucose within the b-cell across the plasma mem- b-cell. brane through GLUT2 and its subsequent phosphorylation by glucokinase, thereby promoting glycolysis (Iynedjian, 2009). In the 2.3. Pathways downstream of mitochondria cytosolic compartment, glycolysis extracts reducing equivalents transmitted to NADH. Maintenance of glycolytic flux requires reox- Export of ATP to the cytosolic compartment promotes the clo- + + idation of NADH to NAD to avoid arrest of glycolysis. sure of ATP-sensitive K -channels (KATP-channel) on the plasma In most tissues, cytosolic conversion of pyruvate to lactate by membrane and, as a consequence, depolarization of the cell (Ash- the lactate dehydrogenase ensures NADH oxidation to NAD+, while croft, 2006). This leads to Ca2+ influx through voltage-gated Ca2+ in b-cells this task is devoted mainly to mitochondrial NADH shut- channels and a rise in cytosolic Ca2+ concentrations (Fig. 1), which tles, transferring glycolysis-derived electrons to mitochondria. is the main and necessary signal for exocytosis of insulin (Eliasson et al., 2008). Additional signals are required to sustain the secretion 2+ 2.2. The mitochondrial NADH shuttle system elicited by glucose-induced Ca rise. They participate in the amplifying pathway (Maechler et al., 2006), formerly referred to as the The mitochondrial NADH shuttle system is composed of the KATP-channel independent stimulation of insulin secretion. Effi- glycerolphosphate and the malate/aspartate shuttles (MacDonald, cient coupling of glucose recognition to insulin secretion is ensured 1982), with its respective key members the mitochondrial glycerol by the mitochondrion, an organelle that integrates and generates phosphate dehydrogenase (mGPDH) and the aspartate–glutamate metabolic signals (Maechler et al., 2006). This role is additive to carrier (AGC). The aspartate–glutamate carrier 1 (AGC1, also the generation of ATP necessary for the elevation of cytosolic 2+ 2+ named Aralar1) is a Ca2+-sensitive member of the malate/aspartate Ca . The list of additive factors proposed to amplify the Ca sig- shuttle (del Arco and Satrustegui, 1998). Overexpression of AGC1 nals comprises cAMP, NADPH, long chain acyl-CoA derivatives, glu- increases glucose-induced mitochondrial activation and secretory tamate, and superoxides. As opposed to the recognized primary 2+ response, both in insulinoma INS-1E cells and rat islets (Rubi role of Ca as a necessary signal, the roles of most of these additive et al., 2004). This is accompanied by enhanced glucose oxidation factors are still under debate. and reduced lactate production. In insulinoma INS-1E b-cells, the mirror experiment consisting in silencing AGC1 reduces glucose 3. Metabolic activation of mitochondria oxidation and the secretory response, although primary rat b-cells are not sensitive to such a manoeuvre (Casimir et al., 2009). There- 3.1. Activation of the Krebs cycle fore, aspartate–glutamate carrier capacity appears to set a limit for NADH shuttle function and mitochondrial metabolism, exhibiting Pyruvate entry within the mitochondria induces metabolic acti- cell type-specific dependence. The importance of the NADH shuttle vation of this organelle. There, pyruvate either loses one carbon to 14 P. Maechler / Molecular and Cellular Endocrinology 379 (2013) 12–18 generate acetyl-CoA or gains one carbon to form oxaloacetate; electron transport chain. However, as complex I activity is limited reactions catalyzed by pyruvate dehydrogenase (PDH) and pyru- by the inherent thermodynamic constraints of proton gradient for- vate carboxylase (PC), respectively (Fig. 2). PDH is an important mation, excess of NADH contributed by the high TCA cycle activity site of regulation as, among other effectors, the enzyme is activated must be reoxidized by alternative dehydrogenases, i.e., through by an elevation of mitochondrial [Ca2+](Duchen, 1999; Rutter cataplerotic reactions. et al., 1996). PDH is also regulated by reversible phosphorylation of its E1a subunit, activity of the PDH kinases inhibiting the en- 3.2. Activation of the electron transport chain zyme (Sugden and Holness, 2003). Increasing the expression of either the PDH phosphatase or the PDH kinase 3 does not change TCA cycle activation induces transfer of reducing equivalents to glucose-stimulated insulin secretion (Nicholls, 2002). Regarding the electron transport chain resulting in hyperpolarization of the down-regulation of PDH kinases, silencing of PDH kinase 1 in mitochondrial membrane, respiration, and generation of ATP INS-1 832/13 cells has been reported to increase the secretory re- (Fig. 2). Electron transport chain activity promotes proton export sponse to glucose (Krus et al., 2010), whereas knockdown of both from the mitochondrial matrix across the inner membrane, estab- PDH kinase 1 and kinase 3 in INS-1E cells does not affect metabo- lishing a strong mitochondrial membrane potential, which is neg- lism–secretion coupling (Akhmedov et al., 2012). Therefore, the ative on the inside. The respiratory chain comprises five importance of the phosphorylation state of PDH for the regulation complexes, the subunits of which are encoded by both the nuclear of b-cell function remains unclear. and mitochondrial genomes (Wallace, 1999). Complex I is the Condensation of the 2-carbon acetyl group carried by coen- acceptor of electrons from NADH in the inner mitochondrial mem- zyme-A with the 4-carbon oxaloacetate yields citrate, thereby acti- brane and complex II (succinate dehydrogenase) transfers elec- vating the tricarboxylic acid (TCA) cycle (or Krebs cycle). The trons to coenzyme-Q from FADH2, the latter being generated pyruvate carboxylase enzyme ensures the provision of carbon skel- both by the oxidative activity of the TCA cycle and the glycerol- eton (i.e., anaplerosis) to the TCA cycle, a key pathway in b-cells phosphate shuttle. Complex V (ATP synthase) promotes ATP for- (Fransson et al., 2006). The remarkably high anaplerotic activity mation from ADP and inorganic phosphate. The synthesized ATP in b-cells indicates important loss of TCA cycle intermediates is translocated to the cytosol in exchange for ADP by the adenine (i.e., cataplerosis), which is compensated for by de novo oxaloace- nucleotide translocator (ANT). Thus, the actions of the separate tate synthesis by pyruvate carboxylation. In the control of glu- complexes of the electron transport chain and the adenine nucleo- cose-stimulated insulin secretion, TCA cycle intermediates are tide translocator couple respiration to ATP supply. recruited to serve as substrates leading to the formation of mitochondrion-derived coupling factors (Maechler et al., 2006). 3.3. Regulation of mitochondrial activity by Ca2+ Through its oxidative activity, the TCA cycle extracts reducing equivalents from metabolic intermediates, which are then trans- Mitochondrial activity can be modulated according to the nat- ported mainly by NADH and, quantitatively less important, by ure of the nutrients, although glucose is the chief secretagogue as

FADH2. In order to maintain input of pyruvate products into the compared to amino acid catabolism (Newsholme et al., 2005) and TCA cycle upon glucose stimulation, this reduced redox state re- fatty acid b-oxidation (Rubi et al., 2002). Additional factors regulat- quires continues reoxidation of mitochondrial NADH to NAD+. This ing mitochondrial activation include mitochondrial Ca2+ concen- 2+ is achieved primarily by complex I NADH dehydrogenase on the trations ([Ca ]m)(Duchen, 1999; McCormack et al., 1990),

Fig. 2. In the mitochondria, pyruvate (Pyr) is a substrate both for pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), forming respectively acetyl-CoA and oxaloacetate (OA). Condensation acetyl-CoA with OA generates citrate that is either processed by the TCA cycle or exported out of the mitochondrion as a precursor for long chain acyl-CoA (LC-CoA) synthesis. The glycerophosphate and malate/aspartate shuttles, as well as the TCA cycle, generate reducing equivalents in the form of NADH and

FADH2, which are transferred to the electron transport chain (ETC). This leads to the hyperpolarization of the mitochondrial membrane (wm) and the synthesis of ATP, then transported to the cytosol by the adenine nucleotide translocator (ANT). Upon glucose stimulation, glutamate (Glut) is produced from a-ketoglutarate (aKG) by glutamate dehydrogenase (GDH) and exported out of mitochondria through the glutamate carrier (GC1). Ca2+ enters into mitochondria via MCU (regulated by MICU1) and gets out via NCLX. P. Maechler / Molecular and Cellular Endocrinology 379 (2013) 12–18 15 mitochondrial protein tyrosine phosphatase (Pagliarini et al., fusion events under the control of specific mitochondrial mem- 2005), mitochondrial GTP (Kibbey et al., 2007), and matrix alkalin- brane anchor proteins (Westermann, 2008). Over the last years, ization (Wiederkehr et al., 2009). Among these factors, mitochon- mitochondrial fission/fusion state was investigated in insulin drial Ca2+ regulation has recently been highlighted, thanks to a secreting cells. Altering fission by down regulation of fission-pro- series of discoveries related to Ca2+ transport through the mito- moting Fis1 protein impairs respiratory function and glucose-stim- 2+ chondrial membrane. Elevation of [Ca ]m enhances mitochondrial ulated insulin secretion (Twig et al., 2008). Intriguingly, a similar oxidative activity (Maechler et al., 1998) and promotes generation phenotype, i.e., reduced energy metabolism and secretory defects, of coupling factors for insulin exocytosis (Maechler et al., 1997). is caused by the mirror experiment consisting in mitochondrial 2+ 2+ Conversely, buffering mitochondrial free Ca limits [Ca ]m peaks fragmentation by overexpression of Fis1 (Park et al., 2008). Adding induced by glucose stimulation in INS-1E b-cells. Such limitation puzzlement to our comprehension of mitochondrial dynamics in b- 2+ in [Ca ]m amplitude reduces mitochondrial respiration and ATP cell function, fragmented pattern obtained by dominant-negative generation with corresponding effects on insulin secretion (Wie- expression of fusion-promoting Mfn1 protein does not affect derkehr et al., 2011). metabolism–secretion coupling (Park et al., 2008). Recently, it In the recent years, significant advances in mitochondrial Ca2+ was reported that glucose stimulation of INS-1E cells induces channels have been made. In 2011, the mitochondrial Ca2+ uni- reversible shortening of mitochondrial tubules (Jhun et al., 2013). porter (MCU) has been identified as the channel responsible for Expression of a dominant-negative mutant of fission-promoting mitochondrial Ca2+ uptake (Baughman et al., 2011; De Stefani Drp1 prevents glucose-induced mitochondria shortening and insu- et al., 2011). MCU is part of a complex located in the inner mito- lin secretion (Jhun et al., 2013). Therefore, mitochondrial fragmen- chondrial membrane and its activity is modulated by another re- tation per se seems not to alter insulin secreting cells, at least not cently identified protein, the mitochondrial Ca2+ uptake 1 in vitro. Regarding Ca2+ homeostasis, mitochondrial fragmentation (MICU1). MICU1 holds Ca2+ sensing subunits, in other words two in mouse b-cells lacking prohibitin-2 is associated with blunted canonical EF hands, which are essential for its activity (Perocchi glucose-induced Ca2+ rise but preserved KCl response; indicating et al., 2010). If Ca2+ gets in mitochondria, it should also get out at that ATP generation rather than Ca2+ channels is defective in these some point, although the vast majority of mitochondrial Ca2+ is cells (S. Supale and P. Maechler, unpublished observation). In vivo, buffered as chelated ion. Mitochondrial Ca2+ efflux is thought to transgenic mice with b-cell-specific ablation of fusion-promoting be mediated by the Na2+/Ca2+ exchanger (NCLX) identified in Opa1 are hyperglycaemic. Islets from these mice exhibit disman- 2010 (Palty et al., 2010). Therefore, MCU and MICU1 would be tled mitochondrial architecture, reduced ATP generation and insu- implicated in mitochondrial Ca2+ uptake, whereas NCLX would be lin release (Zhang et al., 2011). Additionally, b-cell proliferation is responsible for Ca2+ efflux (Fig. 2). reduced in b-cell-specific Opa1-deficient mice (Zhang et al., 2011). In pancreatic b-cells, silencing of NCLX extends elevations of 2+ [Ca ]m evoked by cell depolarization and also accelerates the rise 4. Mitochondria as the source of additional coupling factors for in ATP/ADP ratio in response to glucose stimulation (Tarasov et al., 2+ insulin exocytosis 2012). Consistently, the rise in [Ca ]m evoked by glucose is enhanced in b-cells when NCLX is silenced or expressed in a domi- 4.1. Mitochondria as a source of coupling factors nant negative form (Nita et al., 2012). These recent data are in agreement with a previous study showing increased mitochondrial Glucose metabolism induces the triggering and the amplifying metabolism and enhanced glucose-stimulated insulin secretion pathways, in other words the necessary Ca2+ rise and generation when the Na2+/Ca2+ exchanger was pharmacologically inhibited of additional coupling factors, respectively (Henquin, 2000). The by CGP37157 (Lee et al., 2003). Regarding the role of NCLX in amplifying pathway can be experimentally uncovered when glu- ATP production, the inhibitor CGP37157 increases glucose-induced cose stimulation occurs whilst cytosolic Ca2+ is clamped at permis- ATP generation, whereas knockdown of NCLX using siRNA does not sive levels (Gembal et al., 1992). This suggests the existence of (Nita et al., 2012), suggesting additional effects of CGP37157. metabolic coupling factors, generated by glucose, participating to Ca2+ import into mitochondria is regulated by the recently iden- the amplifying pathway. Mitochondria have been identified as a tified MCU (Baughman et al., 2011; De Stefani et al., 2011) and source of additional coupling factors for insulin exocytosis. For in- MICU1 (Perocchi et al., 2010). Silencing of MCU in b-cells impairs 2+ stance, the demonstration has been done using permeabilized the rise in [Ca ]m evoked by cell depolarization and reduces the insulin-secreting cells clamped with permissive Ca2+ concentra- plateau phase of ATP/ADP ratio upon glucose stimulation (Tarasov tions and stimulated with mitochondrial substrates (Maechler et al., 2012). Accordingly, knockdown of MCU in mouse b-cells et al., 1997). inhibits glucose-induced exocytosis (Tarasov et al., 2013). Such 2+ manipulation of [Ca ]m does not affect mitochondrial membrane potential, either at basal or stimulatory glucose concentrations 4.2. Mitochondria as a source of nucleotides serving as coupling factors (Tarasov et al., 2012). Regarding MICU1, its silencing in insulinoma cells reduces mitochondrial Ca2+ uptake, ATP levels, and insulin ATP is undoubtedly the primary metabolic factor produced by secretion upon glucose stimulation (Alam et al., 2012). In the same mitochondria during glucose-stimulated insulin secretion. ATP 2+ study, knockdown of MCU provoked similar inhibitory effects closes the KATP-channel leading to the obligatory Ca elevation (Alam et al., 2012). Collectively, these recent findings indicate that promoting insulin exocytosis (Miki et al., 1999). Moreover, ATP is both the channel and its regulatory partner, i.e., MCU and MICU1 implicated in secretory granule movement (Yu et al., 2000; Varadi 2+ respectively, are necessary for proper regulation of [Ca ]m in b- et al., 2002), priming of the granules prior to exocytosis (Eliasson cells and participate to glucose-stimulated insulin secretion. et al., 1997), and in the process of insulin exocytosis per se (Vallar et al., 1987; Rorsman et al., 2000). 3.4. Regulation of mitochondrial dynamics The purine nucleoside GTP is also implicated to some extent in the process of metabolism–secretion coupling. In the cytosol, GTP Mitochondrial activation also involves changes in organelle is mainly generated through the action of nucleoside diphosphate morphology and contacts, in particular with the Ca2+-rich endo- kinase from ATP-dependent phosphorylation of GDP. Glucose stim- plasmic reticulum (de Brito and Scorrano, 2010). Mitochondria ulation raises GTP levels (Detimary et al., 1996), promoting insulin form dynamic networks, continuously modified by fission and exocytosis via the activity of GTPases (Vallar et al., 1987). In 16 P. Maechler / Molecular and Cellular Endocrinology 379 (2013) 12–18 mitochondria, GTP acts as a positive regulator of the TCA cycle ity of lipid signals implicated in exocytosis (Brun et al., 1996). In (Kibbey et al., 2007). the cytosol, this process promotes the accumulation of long chain NADH and its phosphorylated form NADPH are responsible for acyl-CoAs such as palmitoyl-CoA (Liang and Matschinsky, 1991; transfer of reducing equivalents in biochemical pathways. NADPH Prentki et al., 1992), enhancing Ca2+-evoked insulin exocytosis is mostly found in the cytosolic compartment, whilst NADH is par- (Deeney et al., 2000). Accordingly, LCPTI overexpression in INS- ticularly abundant in mitochondria. Glucose stimulation modifies 1E b-cells increases oxidation of fatty acids, whilst it reduces glu- the redox state of these pyridine nucleotides, raising the cose-stimulated insulin secretion (Rubi et al., 2002). To date, the NAD(P)H/NAD(P)+ ratio (Capito et al., 1984), first in the cytosol exact role of long chain acyl-CoA derivatives is still debated, sev- and then in mitochondria (Patterson et al., 2000). Consistent with eral studies indicating that malonyl-CoA acts as a factor regulating the model of metabolism–secretion coupling, increase in NAD(P)H the partitioning of fatty acids into effectors in insulin exocytosis precedes the elevation in cytosolic Ca2+ concentrations (Pralong (Prentki et al., 2002). Fatty acids derived from triglyceride stores et al., 1990). Beside the rapid changes in NAD(P)H/NAD(P)+ ratio, may also play a permissive role in the secretory response (Frigerio the total pool of NADPH is elevated upon glucose stimulation et al., 2010; Peyot et al., 2009). through the phosphorylating activity of NAD-kinase (Gray et al., 2012). 4.4. Mitochondria as a source of glutamate serving as coupling factor Cytosolic NADPH is generated by glucose metabolism via the pentose phosphate shunt (Verspohl et al., 1979), although in b- The observation of direct stimulation of insulin exocytosis by cells mitochondrial shuttles appear to play an important role in mitochondrial activation in permeabilized b-cells (Maechler this process (Farfari et al., 2000). The export of citrate out of the et al., 1997) led to the identification of glutamate as a putative mitochondria might serve as a signal of fuel abundance, participat- intracellular messenger (Maechler and Wollheim, 1999; Hoy ing in metabolism–secretion coupling (Farfari et al., 2000). Once in et al., 2002; Maechler et al., 2002). Collectively, work from our lab- the cytosolic compartment, citrate metabolism contributes to the oratory and others indicate that permissive levels of glutamate are formation of NADPH and malonyl-CoA, both candidate molecules necessary for the full development of the secretory response to on the list of metabolic coupling factors. glucose stimulation. The cytosolic target of glutamate might be NADPH has been proposed as a coupling factor in glucose-stim- the insulin granule itself, as several studies by different groups ulated insulin secretion, originally by using toadfish islets (Watkins have shown requirement of glutamate uptake by secretory vesicles et al., 1968) indicating a direct effect of NADPH on the release of for insulin exocytosis (Maechler and Wollheim, 1999; Hoy et al., insulin (Watkins, 1972) secondary to the uptake of NADPH by 2002; Eto et al., 2003; Gammelsaeter et al., 2011; Storto et al., granules (Watkins and Moore, 1977). Subsequently, the role of 2006). NADPH as a coupling factor has been substantiated by experiments If intracellular glutamate renders insulin granules exocytosis- showing direct stimulation of insulin exocytosis upon intracellular competent, concentrations of this amino acid should raise in re- addition of NADPH (Ivarsson et al., 2005). It has also been reported sponse to glucose stimulation. Indeed, during glucose stimulation that the NADPH/NADP+ ratio mediates a fast-inactivating K+ cur- total cellular glutamate levels have been shown to increase in hu- rent through regulation of Kv2.1 channels (MacDonald et al., 2003). man, mouse and rat islets as well as in clonal b-cells (Maechler and Finally, the second messenger cAMP robustly potentiates glu- Wollheim, 1999; Rubi et al., 2001; Brennan et al., 2002; Bertrand cose-stimulated insulin secretion (Ahren, 2000). Glucose stimula- et al., 2002; Broca et al., 2003; Carobbio et al., 2004; Lehtihet tion can promote elevation of cAMP (Charles et al., 1975) that is et al., 2005), When b-cells are forced to express an enzyme that generated by adenylyl cyclase at the plasma membrane using decarboxylates intracellular glutamate, the glucose-induced gluta- ATP. The cAMP levels are negatively modulated by superoxide, an mate rise is impaired as well as the secretory response (Rubi et al., effect mediated by NADPH oxidases (Li et al., 2012). In particular, 2001). The mitochondrial enzyme glutamate dehydrogenase the glucose response of islets deficient in NOX2 is characterized (GDH), encoded by Glud1, plays a key role in glucose-induced glu- by lower superoxide, higher cAMP levels, and increased insulin tamate generation (Fig. 2). Abrogation of GDH specifically in the b- / secretion (Li et al., 2012). Among other hormones, glucagon and cells of bGlud1À À mice reduces the secretory response (Carobbio GLP-1 (glucagon-like peptide 1) increase cAMP concentrations in et al., 2009). Moreover, measurements of carbon fluxes in mouse b-cells (Schuit et al., 2001), resulting in the amplification of the islets revealed that, upon glucose stimulation, GDH contributes secretory response to glucose in human islets (Huypens et al., to the net synthesis of glutamate from the TCA cycle intermediate 2000). In addition to its effects on insulin release, GLP-1 might pre- a-ketoglutarate (Vetterli et al., 2012). In b-cells lacking GDH, glu- serve b-cell mass, rendering this hormone and biologically active cose-stimulated insulin secretion is reduced by half, correlating related molecules of interest for the treatment of diabetes (Drucker with impaired glutamate formation while the Ca2+ rise is preserved and Nauck, 2006). (Vetterli et al., 2012). Importantly, the amplifying pathway charac- terizing the full development of the glucose response fails to devel- / 4.3. Mitochondria as a source of precursors for fatty acids serving as op in the absence of GDH, as demonstrated in bGlud1À À islets coupling factors (Vetterli et al., 2012). Regarding export of the newly synthesized glutamate out of The relative contribution of glucose versus lipid products for mitochondria, the glutamate carrier GC1 seems to play an impor- oxidative catabolism shapes the metabolic profile of mitochondria. tant role. Silencing of GC1 reduces glucose-stimulated insulin The rate-limiting step for transport and oxidation of fatty acids into secretion, an effect rescued by the provision of exogenous gluta- mitochondria is catalyzed by carnitine palmitoyltransferase (the li- mate to the b-cell (Casimir et al., 2009). Finally, prevention of glu- ver isoform LCPTI in the b-cell). Upon glucose stimulation, citrate tamate release from b-cells results in concomitant elevations of derived from mitochondria reacts with coenzyme-A (CoA) to gen- intracellular glutamate levels and glucose-evoked insulin secretion erate cytosolic acetyl-CoA necessary for the synthesis malonyl- (Feldmann et al., 2011). Collectively, data indicate that permissive CoA and then long-chain acyl-CoA. The malonyl-CoA thus formed levels of glutamate are required in the amplifying pathway of the reduces fatty acid oxidation by inhibiting LCPTI. The hypothesis b-cell. Permissive concentrations of glutamate are also important that malonyl-CoA/long-chain acyl-CoA act as coupling factors in for proper function of the malate–aspartate shuttle, an key player the secretory response was originally based on the inhibition of in insulin secreting cells (Rubi et al., 2004; Casimir et al, 2009), fatty acid oxidation by malonyl-CoA, which increases the availabil- as discussed above. P. Maechler / Molecular and Cellular Endocrinology 379 (2013) 12–18 17

5. Conclusion control of glucose-stimulated insulin secretion. J. Biol. Chem. 284, 25004– 25014. Charles, M.A., Lawecki, J., Pictet, R., Grodsky, G.M., 1975. Insulin secretion. In pancreatic b-cells, mitochondrial activity translates glucose Interrelationships of glucose, cyclic adenosine 3:5-monophosphate, and metabolism into signals controlling the rate of insulin exocytosis. calcium. J. Biol. Chem. 250, 6134–6140. Consequently, mitochondrial function can adjust insulin secretion Clocquet, A.R., Egan, J.M., Stoffers, D.A., Muller, D.C., Wideman, L., Chin, G.A., Clarke, W.L., Hanks, J.B., Habener, J.F., Elahi, D., 2000. Impaired insulin secretion and to the actual glycemia. This role is specific for b-cells, since in most increased insulin sensitivity in familial maturity-onset diabetes of the young 4 cell types mitochondrial metabolism is triggered by specific needs (insulin promoter factor 1 gene). Diabetes 49, 1856–1864. of the cells, in terms of energy and building blocks. In b-cells, mito- de Brito, O.M., Scorrano, L., 2010. An intimate liaison: spatial organization of the endoplasmic reticulum-mitochondria relationship. EMBO J. 29, 2715–2723. chondrial metabolism is primarily dictated by the glycolytic flux. De Stefani, D., Raffaello, A., Teardo, E., Szabo, I., Rizzuto, R., 2011. A forty-kilodalton The concept of metabolism–secretion coupling that characterizes protein of the inner membrane is the mitochondrial calcium uniporter. Nature the b-cell is tightly controlled by on and off signals, most of them 476, 336–340. Dean, P.M., 1973. Ultrastructural morphometry of the pancreatic-cell. Diabetologia requiring mitochondrial function. Future studies should better de- 9, 115–119. fine the molecular targets and mechanism of action of coupling Deeney, J.T., Gromada, J., Hoy, M., Olsen, H.L., Rhodes, C.J., Prentki, M., Berggren, P.O., factors controlling insulin secretion. Corkey, B.E., 2000. Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells). J. Biol. Chem. 275, 9363–9368. Acknowledgments del Arco, A., Satrustegui, J., 1998. Molecular cloning of Aralar, a new member of the mitochondrial carrier superfamily that binds calcium and is present in human muscle and brain. J. Biol. Chem. 273, 23327–23334. The author’s laboratory benefits from continuous support by Detimary, P., Van den Berghe, G., Henquin, J.C., 1996. Concentration dependence the Swiss National Science Foundation and the State of Geneva. and time course of the effects of glucose on adenine and guanine nucleotides in mouse pancreatic islets. J. Biol. Chem. 271, 20559–20565. The most precious contribution of present and past members of Drucker, D.J., Nauck, M.A., 2006. The incretin system: glucagon-like peptide-1 the laboratory is acknowledged. receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 368, 1696–1705. Duchen, M.R., 1999. Contributions of mitochondria to animal physiology: from References homeostatic sensor to calcium signalling and cell death. J. Physiol. 516, 1–17. Eliasson, L., Renstrom, E., Ding, W.G., Proks, P., Rorsman, P., 1997. Rapid ATP- dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in Ahren, B., 2000. Autonomic regulation of islet hormone secretion–implications for mouse pancreatic B-cells. J. Physiol. 503, 399–412. health and disease. Diabetologia 43, 393–410. Eliasson, L., Abdulkader, F., Braun, M., Galvanovskis, J., Hoppa, M.B., Rorsman, P., Akhmedov, D., De Marchi, U., Wollheim, C.B., Wiederkehr, A., 2012. Pyruvate 2008. Novel aspects of the molecular mechanisms controlling insulin secretion. dehydrogenase E1alpha phosphorylation is induced by glucose but does not J. Physiol. 586, 3313–3324. control metabolism–secretion coupling in INS-1E clonal beta-cells. Biochim. Eto, K., Yamashita, T., Hirose, K., Tsubamoto, Y., Ainscow, E.K., Rutter, G.A., Kimura, Biophys. Acta 1823, 1815–1824. S., Noda, M., Iino, M., Kadowaki, T., 2003. Glucose metabolism and glutamate Alam, M.R., Groschner, L.N., Parichatikanond, W., Kuo, L., Bondarenko, A.I., Rost, R., analog acutely alkalinize pH of insulin secretory vesicles of pancreatic {beta}- Waldeck-Weiermair, M., Malli, R., Graier, W.F., 2012. Mitochondrial Ca2+ uptake cells. Am. J. Physiol. Endocrinol. Metab. 285, E262–E271. 1 (MICU1) and mitochondrial Ca2+ uniporter (MCU) contribute to metabolism– Farfari, S., Schulz, V., Corkey, B., Prentki, M., 2000. Glucose-regulated anaplerosis secretion coupling in clonal pancreatic beta-cells. J. Biol. Chem. 287, 34445– and cataplerosis in pancreatic beta-cells: possible implication of a pyruvate/ 34454. citrate shuttle in insulin secretion. Diabetes 49, 718–726. Ashcroft, F.M., 2006. K(ATP) channels and insulin secretion: a key role in health and Feldmann, N., Del Rio, R.M., Gjinovci, A., Tamarit-Rodriguez, J., Wollheim, C.B., disease. Biochem. Soc. Trans. 34, 243–246. Wiederkehr, A., 2011. Reduction of plasma membrane glutamate transport Baughman, J.M., Perocchi, F., Girgis, H.S., Plovanich, M., Belcher-Timme, C.A., Sancak, potentiates insulin but not glucagon secretion in pancreatic islet cells. Mol. Cell Y., Bao, X.R., Strittmatter, L., Goldberger, O., Bogorad, R.L., Koteliansky, V., Endocrinol. 338, 46–57. Mootha, V.K., 2011. Integrative genomics identifies MCU as an essential Fransson, U., Rosengren, A.H., Schuit, F.C., Renstrom, E., Mulder, H., 2006. component of the mitochondrial calcium uniporter. Nature 476, 341–345. Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in Bertrand, G., Ishiyama, N., Nenquin, M., Ravier, M.A., Henquin, J.C., 2002. The the ATP:ADP ratio in rat pancreatic islets. Diabetologia 49, 1578–1586. elevation of glutamate content and the amplification of insulin secretion in Frigerio, F., Brun, T., Bartley, C., Usardi, A., Bosco, D., Ravnskjaer, K., Mandrup, S., glucose-stimulated pancreatic islets are not causally related. J. Biol. Chem. 277, Maechler, P., 2010. Peroxisome proliferator-activated receptor alpha 32883–32891. (PPARalpha) protects against oleate-induced INS-1E beta cell dysfunction by Brennan, L., Shine, A., Hewage, C., Malthouse, J.P., Brindle, K.M., McClenaghan, N., preserving carbohydrate metabolism. Diabetologia 53, 331–340. Flatt, P.R., Newsholme, P., 2002. A nuclear magnetic resonance-based Froguel, P., Zouali, H., Vionnet, N., Velho, G., Vaxillaire, M., Sun, F., Lesage, S., Stoffel, demonstration of substantial oxidative L-alanine metabolism and L-alanine- M., Takeda, J., Passa, P., 1993. Familial hyperglycemia due to mutations in enhanced glucose metabolism in a clonal pancreatic beta-cell line: metabolism glucokinase. Definition of a subtype of diabetes mellitus. N. Engl. J. Med. 328, of L-alanine is important to the regulation of insulin secretion. Diabetes 51, 697–702. 1714–1721. Gammelsaeter, R., Coppola, T., Marcaggi, P., Storm-Mathisen, J., Chaudhry, F.A., Broca, C., Brennan, L., Petit, P., Newsholme, P., Maechler, P., 2003. Mitochondria- Attwell, D., Regazzi, R., Gundersen, V., 2011. A role for glutamate transporters in derived glutamate at the interplay between branched-chain amino acid and the regulation of insulin secretion. PLoS One 6, e22960. glucose-induced insulin secretion. FEBS Lett. 545, 167–172. Gembal, M., Gilon, P., Henquin, J.C., 1992. Evidence that glucose can control insulin Brun, T., Roche, E., Assimacopoulos-Jeannet, F., Corkey, B.E., Kim, K.H., Prentki, M., release independently from its action on ATP-sensitive K+ channels in mouse B 1996. Evidence for an anaplerotic/malonyl-CoA pathway in pancreatic beta-cell cells. J. Clin. Invest. 89, 1288–1295. nutrient signaling. Diabetes 45, 190–198. Gray, J.P., Alavian, K.N., Jonas, E.A., Heart, E.A., 2012. NAD kinase regulates the size of Byrne, M.M., Sturis, J., Menzel, S., Yamagata, K., Fajans, S.S., Dronsfield, M.J., Bain, the NADPH pool and insulin secretion in pancreatic beta-cells. Am. J. Physiol. S.C., Hattersley, A.T., Velho, G., Froguel, P., Bell, G.I., Polonsky, K.S., 1996. Altered Endocrinol. Metab. 303, E191–E199. insulin secretory responses to glucose in diabetic and nondiabetic subjects with Henquin, J.C., 2000. Triggering and amplifying pathways of regulation of insulin mutations in the diabetes susceptibility gene MODY3 on chromosome 12. secretion by glucose. Diabetes 49, 1751–1760. Diabetes 45, 1503–1510. Herzig, S., Raemy, E., Montessuit, S., Veuthey, J.L., Zamboni, N., Westermann, B., Capito, K., Hedeskov, C.J., Landt, J., Thams, P., 1984. Pancreatic islet metabolism and Kunji, E.R., Martinou, J.C., 2012. Identification and functional expression of the redox state during stimulation of insulin secretion with glucose and fructose. mitochondrial pyruvate carrier. Science 337, 93–96. Acta Diabetol. Lat. 21, 365–374. Hoy, M., Maechler, P., Efanov, A.M., Wollheim, C.B., Berggren, P.O., Gromada, J., 2002. Carobbio, S., Ishihara, H., Fernandez-Pascual, S., Bartley, C., Martin-Del-Rio, R., Increase in cellular glutamate levels stimulates exocytosis in pancreatic beta- Maechler, P., 2004. Insulin secretion profiles are modified by overexpression of cells. FEBS Lett. 531, 199–203. glutamate dehydrogenase in pancreatic islets. Diabetologia 47, 266–276. Huypens, P., Ling, Z., Pipeleers, D., Schuit, F., 2000. Glucagon receptors on human Carobbio, S., Frigerio, F., Rubi, B., Vetterli, L., Bloksgaard, M., Gjinovci, A., islet cells contribute to glucose competence of insulin release. Diabetologia 43, Pournourmohammadi, S., Herrera, P.L., Reith, W., Mandrup, S., Maechler, P., 1012–1019. 2009. Deletion of glutamate dehydrogenase in beta-cells abolishes part of the Ivarsson, R., Quintens, R., Dejonghe, S., Tsukamoto, K., In ‘t Veld, P., Renstrom, E., insulin secretory response not required for glucose homeostasis. J. Biol. Chem. Schuit, F.C., 2005. Redox control of exocytosis: regulatory role of NADPH, 284, 921–929. thioredoxin, and glutaredoxin. Diabetes 54, 2132–2142. Casimir, M., Rubi, B., Frigerio, F., Chaffard, G., Maechler, P., 2009. Silencing of the Iynedjian, P.B., 2009. Molecular physiology of mammalian glucokinase. Cell Mol. mitochondrial NADH shuttle component aspartate–glutamate carrier AGC1/ Life Sci. 66, 27–42. Aralar1 in INS-1E cells and rat islets. Biochem. J. 424, 459–466. Jhun, B.S., Lee, H., Jin, Z.G., Yoon, Y., 2013. Glucose stimulation induces dynamic Casimir, M., Lasorsa, F.M., Rubi, B., Caille, D., Palmieri, F., Meda, P., Maechler, P., change of mitochondrial morphology to promote insulin secretion in the 2009. Mitochondrial glutamate carrier GC1 as a newly identified player in the insulinoma cell line INS-1E. PLoS One 8, e60810. 18 P. Maechler / Molecular and Cellular Endocrinology 379 (2013) 12–18

Kibbey, R.G., Pongratz, R.L., Romanelli, A.J., Wollheim, C.B., Cline, G.W., Shulman, Prentki, M., Vischer, S., Glennon, M.C., Regazzi, R., Deeney, J.T., Corkey, B.E., 1992. G.I., 2007. Mitochondrial GTP regulates glucose-stimulated insulin secretion. Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in Cell Metab. 5, 253–264. nutrient-induced insulin secretion. J. Biol. Chem. 267, 5802–5810. Krus, U., Kotova, O., Spegel, P., Hallgard, E., Sharoyko, V.V., Vedin, A., Moritz, T., Prentki, M., Joly, E., El-Assaad, W., Roduit, R., 2002. Malonyl-CoA signaling, lipid Sugden, M.C., Koeck, T., Mulder, H., 2010. Pyruvate dehydrogenase kinase 1 partitioning, and glucolipotoxicity: role in beta-cell adaptation and failure in controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal the etiology of diabetes. Diabetes 51 (Suppl. 3), S405–S413. beta-cells. Biochem. J. 429, 205–213. Rahier, J., Goebbels, R.M., Henquin, J.C., 1983. Cellular composition of the human Lee, B., Miles, P.D., Vargas, L., Luan, P., Glasco, S., Kushnareva, Y., Kornbrust, E.S., diabetic pancreas. Diabetologia 24, 366–371. Grako, K.A., Wollheim, C.B., Maechler, P., Olefsky, J.M., Anderson, C.M., 2003. Rorsman, P., Eliasson, L., Renstrom, E., Gromada, J., Barg, S., Gopel, S., 2000. The cell Inhibition of mitochondrial Na(+)–Ca(2+) exchanger increases mitochondrial physiology of biphasic insulin secretion. News Physiol. Sci. 15, 72–77. metabolism and potentiates glucose-stimulated insulin secretion in rat Rubi, B., Maechler, P., 2010. Minireview: new roles for peripheral dopamine on pancreatic islets. Diabetes 52, 965–973. metabolic control and tumor growth: let’s seek the balance. Endocrinology 151, Lehtihet, M., Honkanen, R.E., Sjoholm, A., 2005. Glutamate inhibits protein 5570–5581. phosphatases and promotes insulin exocytosis in pancreatic beta-cells. Rubi, B., Ishihara, H., Hegardt, F.G., Wollheim, C.B., Maechler, P., 2001. GAD65- Biochem. Biophys. Res. Commun. 328, 601–607. mediated glutamate decarboxylation reduces glucose-stimulated insulin Li, N., Li, B., Brun, T., Deffert-Delbouille, C., Mahiout, Z., Daali, Y., Ma, X.J., Krause, secretion in pancreatic beta cells. J. Biol. Chem. 276, 36391–36396. K.H., Maechler, P., 2012. NADPH oxidase NOX2 defines a new antagonistic role Rubi, B., Antinozzi, P.A., Herrero, L., Ishihara, H., Asins, G., Serra, D., Wollheim, C.B., for reactive oxygen species and cAMP/PKA in the regulation of insulin secretion. Maechler, P., Hegardt, F.G., 2002. Adenovirus-mediated overexpression of liver Diabetes 61, 2842–2850. carnitine palmitoyltransferase I in INS1E cells: effects on cell metabolism and Liang, Y., Matschinsky, F.M., 1991. Content of CoA-esters in perifused rat islets insulin secretion. Biochem. J. 364, 219–226. stimulated by glucose and other fuels. Diabetes 40, 327–333. Rubi, B., del Arco, A., Bartley, C., Satrustegui, J., Maechler, P., 2004. The malate– Maassen, J.A., LM, T.H., Van Essen, E., Heine, R.J., Nijpels, G., Jahangir Tafrechi, R.S., aspartate NADH shuttle member Aralar1 determines glucose metabolic fate, Raap, A.K., Janssen, G.M., Lemkes, H.H., 2004. Mitochondrial diabetes: molecular mitochondrial activity, and insulin secretion in beta cells. J. Biol. Chem. 279, mechanisms and clinical presentation. Diabetes 53 (Suppl. 1), S103–S109. 55659–55666. MacDonald, M.J., 1982. Evidence for the malate aspartate shuttle in pancreatic Rutter, G.A., Burnett, P., Rizzuto, R., Brini, M., Murgia, M., Pozzan, T., Tavare, J.M., islets. Arch. Biochem. Biophys. 213, 643–649. Denton, R.M., 1996. Subcellular imaging of intramitochondrial Ca2+ with MacDonald, P.E., Salapatek, A.M., Wheeler, M.B., 2003. Temperature and redox state recombinant targeted aequorin: significance for the regulation of pyruvate dependence of native Kv2.1 currents in rat pancreatic beta-cells. J. Physiol. 546, dehydrogenase activity. Proc. Natl. Acad. Sci. USA 93, 5489–5494. 647–653. Schuit, F.C., Huypens, P., Heimberg, H., Pipeleers, D.G., 2001. Glucose sensing in Maechler, P., Wollheim, C.B., 1999. Mitochondrial glutamate acts as a messenger in pancreatic beta-cells: a model for the study of other glucose-regulated cells in glucose-induced insulin exocytosis. Nature 402, 685–689. gut, pancreas, and hypothalamus. Diabetes 50, 1–11. Maechler, P., Kennedy, E.D., Pozzan, T., Wollheim, C.B., 1997. Mitochondrial Storto, M., Capobianco, L., Battaglia, G., Molinaro, G., Gradini, R., Riozzi, B., Di activation directly triggers the exocytosis of insulin in permeabilized Mambro, A., Mitchell, K.J., Bruno, V., Vairetti, M.P., Rutter, G.A., Nicoletti, F., pancreatic beta-cells. EMBO J. 16, 3833–3841. 2006. Insulin secretion is controlled by mGlu5 metabotropic glutamate Maechler, P., Kennedy, E.D., Wang, H., Wollheim, C.B., 1998. Desensitization of receptors. Mol. Pharmacol. 69, 1234–1241. mitochondrial Ca2+ and insulin secretion responses in the beta cell. J. Biol. Sugden, M.C., Holness, M.J., 2003. Recent advances in mechanisms regulating Chem. 273, 20770–20778. glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs. Maechler, P., Gjinovci, A., Wollheim, C.B., 2002. Implication of glutamate in the Am. J. Physiol. Endocrinol. Metab. 284, E855–E862. kinetics of insulin secretion in rat and mouse perfused pancreas. Diabetes 51 Tarasov, A.I., Semplici, F., Ravier, M.A., Bellomo, E.A., Pullen, T.J., Gilon, P., Sekler, I., (S1), S99–S102. Rizzuto, R., Rutter, G.A., 2012. The mitochondrial Ca2+ uniporter MCU is Maechler, P., Carobbio, S., Rubi, B., 2006. In beta-cells, mitochondria integrate and essential for glucose-induced ATP increases in pancreatic beta-cells. PLoS One 7, generate metabolic signals controlling insulin secretion. Int. J. Biochem. Cell. e39722. Biol. 38, 696–709. Tarasov, A.I., Semplici, F., Li, D., Rizzuto, R., Ravier, M.A., Gilon, P., Rutter, G.A., 2013. McCormack, J.G., Halestrap, A.P., Denton, R.M., 1990. Role of calcium ions in Frequency-dependent mitochondrial Ca2+ accumulation regulates ATP regulation of mammalian intramitochondrial metabolism. Physiol. Rev. 70, synthesis in pancreatic b cells. Pflugers Arch 465, 543–554. 391–425. Twig, G., Elorza, A., Molina, A.J., Mohamed, H., Wikstrom, J.D., Walzer, G., Stiles, L., Miki, T., Nagashima, K., Seino, S., 1999. The structure and function of the ATP- Haigh, S.E., Katz, S., Las, G., Alroy, J., Wu, M., Py, B.F., Yuan, J., Deeney, J.T., Corkey, sensitive K+ channel in insulin-secreting pancreatic beta-cells. J. Mol. B.E., Shirihai, O.S., 2008. Fission and selective fusion govern mitochondrial Endocrinol. 22, 113–123. segregation and elimination by autophagy. EMBO J. 27, 433–446. Newsholme, P., Brennan, L., Rubi, B., Maechler, P., 2005. New insights into amino Vallar, L., Biden, T.J., Wollheim, C.B., 1987. Guanine nucleotides induce Ca2+- acid metabolism, beta-cell function and diabetes. Clin. Sci. (London) 108, 185– independent insulin secretion from permeabilized RINm5F cells. J. Biol. Chem. 194. 262, 5049–5056. Nicholls, D.G., 2002. Mitochondrial function and dysfunction in the cell: its Varadi, A., Ainscow, E.K., Allan, V.J., Rutter, G.A., 2002. Involvement of conventional relevance to aging and aging-related disease. Int. J. Biochem. Cell. Biol. 34, kinesin in glucose-stimulated secretory granule movements and exocytosis in 1372–1381. clonal pancreatic beta-cells. J. Cell. Sci. 115, 4177–4189. Nita, I.I., Hershfinkel, M., Fishman, D., Ozeri, E., Rutter, G.A., Sensi, S.L., Khananshvili, Verspohl, E.J., Handel, M., Ammon, H.P., 1979. Pentosephosphate shunt activity of D., Lewis, E.C., Sekler, I., 2012. The mitochondrial Na+/Ca2+ exchanger rat pancreatic islets: its dependence on glucose concentration. Endocrinology upregulates glucose dependent Ca2+ signalling linked to insulin secretion. 105, 1269–1274. PLoS One 7, e46649. Vetterli, L., Carobbio, S., Pournourmohammadi, S., Martin-Del-Rio, R., Skytt, D.M., O’Sullivan-Murphy, B., Urano, F., 2012. ER stress as a trigger for beta-cell Waagepetersen, H.S., Tamarit-Rodriguez, J., Maechler, P., 2012. Delineation of dysfunction and autoimmunity in type 1 diabetes. Diabetes 61, 780–781. glutamate pathways and secretory responses in pancreatic islets with beta-cell Pagliarini, D.J., Wiley, S.E., Kimple, M.E., Dixon, J.R., Kelly, P., Worby, C.A., Casey, P.J., specific abrogation of the glutamate dehydrogenase. Mol. Biol. Cell. 23, 3851–3862. Dixon, J.E., 2005. Involvement of a mitochondrial phosphatase in the regulation Wallace, D.C., 1999. Mitochondrial diseases in man and mouse. Science 283, 1482– of ATP production and insulin secretion in pancreatic beta cells. Mol. Cell. 19, 1488. 197–207. Watkins, D.T., 1972. Pyridine nucleotide stimulation of insulin release from isolated Palty, R., Silverman, W.F., Hershfinkel, M., Caporale, T., Sensi, S.L., Parnis, J., Nolte, C., toadfish insulin secretion granules. Endocrinology 90, 272–276. Fishman, D., Shoshan-Barmatz, V., Herrmann, S., Khananshvili, D., Sekler, I., Watkins, D.T., Moore, M., 1977. Uptake of NADPH by islet secretion granule 2010. NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. membranes. Endocrinology 100, 1461–1467. Proc. Natl. Acad. Sci. USA 107, 436–441. Watkins, D., Cooperstein, S.J., Dixit, P.K., Lazarow, A., 1968. Insulin secretion from Park, K.S., Wiederkehr, A., Kirkpatrick, C., Mattenberger, Y., Martinou, J.C., Marchetti, toadfish islet tissue stimulated by pyridine nucleotides. Science 162, 283–284. P., Demaurex, N., Wollheim, C.B., 2008. Selective actions of mitochondrial Westermann, B., 2008. Molecular machinery of mitochondrial fusion and fission. J. fission/fusion genes on metabolism–secretion coupling in insulin-releasing Biol. Chem. 283, 13501–13505. cells. J. Biol. Chem. 283, 33347–33356. Wiederkehr, A., Park, K.S., Dupont, O., Demaurex, N., Pozzan, T., Cline, G.W., Patterson, G.H., Knobel, S.M., Arkhammar, P., Thastrup, O., Piston, D.W., 2000. Wollheim, C.B., 2009. Matrix alkalinization: a novel mitochondrial signal for Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H sustained pancreatic beta-cell activation. EMBO J. 28, 417–428. responses in pancreatic islet beta cells. Proc. Natl. Acad. Sci. USA 97, 5203–5207. Wiederkehr, A., Szanda, G., Akhmedov, D., Mataki, C., Heizmann, C.W., Schoonjans, Perocchi, F., Gohil, V.M., Girgis, H.S., Bao, X.R., McCombs, J.E., Palmer, A.E., Mootha, K., Pozzan, T., Spat, A., Wollheim, C.B., 2011. Mitochondrial matrix calcium is an V.K., 2010. MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) activating signal for hormone secretion. Cell Metab. 13, 601–611. uptake. Nature 467, 291–296. Yu, W., Niwa, T., Fukasawa, T., Hidaka, H., Senda, T., Sasaki, Y., Niki, I., 2000. Peyot, M.L., Guay, C., Latour, M.G., Lamontagne, J., Lussier, R., Pineda, M., Ruderman, Synergism of protein kinase A, protein kinase C, and myosin light-chain kinase N.B., Haemmerle, G., Zechner, R., Joly, E., Madiraju, S.R., Poitout, V., Prentki, M., in the secretory cascade of the pancreatic beta-cell. Diabetes 49, 945–952. 2009. Adipose triglyceride lipase is implicated in fuel- and non-fuel-stimulated Zhang, Z., Wakabayashi, N., Wakabayashi, J., Tamura, Y., Song, W.J., Sereda, S., Clerc, insulin secretion. J. Biol. Chem. 284, 16848–16859. P., Polster, B.M., Aja, S.M., Pletnikov, M.V., Kensler, T.W., Shirihai, O.S., Iijima, M., Pralong, W.F., Bartley, C., Wollheim, C.B., 1990. Single islet beta-cell stimulation by Hussain, M.A., Sesaki, H., 2011. The dynamin-related GTPase Opa1 is required nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and for glucose-stimulated ATP production in pancreatic beta cells. Mol. Biol. Cell secretion. EMBO J. 9, 53–60. 22, 2235–2245. Molecular and Cellular Endocrinology 379 (2013) 19–29

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Mitochondrial and skeletal muscle health with advancing age

Adam R. Konopka, K. Sreekumaran Nair ⇑

Endocrine Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota, United States article info abstract

Article history: With increasing age there is a temporal relationship between the decline of mitochondrial and skeletal Available online 16 May 2013 muscle volume, quality and function (i.e., health). Reduced mitochondrial mRNA expression, protein abundance, and protein synthesis rates appear to promote the decline of mitochondrial protein quality Keywords: and function. Decreased mitochondrial function is suspected to impede energy demanding processes Aging such as skeletal muscle protein turnover, which is critical for maintaining protein quality and thus skel- Sarcopenia etal muscle health with advancing age. The focus of this review was to discuss promising human phys- Mitochondria iological systems underpinning the decline of mitochondrial and skeletal muscle health with advancing Protein metabolism age while highlighting therapeutic strategies such as aerobic exercise and caloric restriction for combat- ing age-related functional impairments. Ó 2013 Published by Elsevier Ireland Ltd.

1. Introduction mitochondrial function. A reduction in mitochondrial abundance and function with age has been observed across various species Reports of skeletal muscle atrophy that accompany advancing (c elegans, drosphilla, mice, humans) and tissues (skin, nerve, age (i.e., sarcopenia) and the associated reductions in skeletal mus- brain, skeletal muscle). Moreover, perturbations in skeletal muscle cle function and quality have been observed for several decades mitochondrial energetics have been correlated with reduced aero- (Critchley, 1931; Rosenberg, 1989, 1997). Recently, panels of lead- bic capacity (Short et al., 2005a), walking capacity (Coen et al., ing scientists and physicians associated with large-scale epidemio- 2012) and skeletal muscle function (Safdar et al., 2010) in older logical studies have created specific, objective criteria based on adults. The mechanisms of age-related changes in skeletal muscle lean tissue mass and functional capacity to improve the diagnosis are multifactorial but the purpose of this review is to highlight and treatment of sarcopenia (Delmonico et al., 2007; Fielding et al., the apparent temporal and functional connection between the de- 2011; Goodpaster et al., 2006; Morley et al., 2011; Newman et al., cline of mitochondrial and skeletal muscle health (Fig. 1). 2003). Human aging starts after the third decade and the progression of skeletal muscle atrophy with age is a slow process ( 1% per 2. Reduced mitochondrial content and function with age year), but accelerates as humans approach 80 years of age (Baum- gartner et al., 1998). With expansion in human lifespan, the ele- Electron microscopic assessment of skeletal muscle biopsy sam- vated rate of muscle loss becomes more problematic since ples revealed lower mitochondrial volume density in older adults skeletal muscle is critical for functionality and substrate metabo- (Conley et al., 2000). A decline in mitochondrial content, as repre- lism. When the substrate reservoir deteriorates with age, the asso- sented by mitochondrial DNA copy number, has also been demon- ciated cardiometabolic disease states (i.e. insulin resistance, strated in rodents (Barazzoni et al., 2000) and humans (Short et al., diabetes, cardiovascular disease, obesity) become more prevalent 2005a). These findings, coupled with investigations that observed (Atlantis et al., 2009). Many studies have observed reduced skeletal reduced levels of mitochondrial protein synthesis (Rooyackers muscle mass and infiltration of adipose tissue depots within or be- et al., 1996) and expression of proteins encoded by both mitochon- tween skeletal muscle groups that are associated with reduced drial and nuclear DNA (Lanza et al., 2008; Short et al., 2005a), are muscle function, insulin resistance and obesity (Delmonico et al., expected to alter mitochondrial function. Semi-quantitative analy- 2009; Goodpaster et al., 2005, 2000). A key link between a reduc- ses, such as immunoblotting or maximal enzyme activity, support tion in skeletal muscle health and prevalence of metabolic disor- the notion that aging skeletal muscle contains less abundance of ders with advancing age may be related to impaired enzymes in oxidative metabolism (i.e. Krebs Cycle, beta-oxidation) and/or proteins involved in the electron transport chain (ETC) (Coo-

Corresponding author. Address: Mayo Clinic, 200 First St. SW, Joseph 5-194, per et al., 1992; Ghosh et al., 2011; Lanza et al., 2008; Rooyackers ⇑ Rochester, MN, United States. Tel.: +1 507 255 2415; fax: +1 507 255 4828. et al., 1996; Tonkonogi et al., 2003; Trounce et al., 1989). Collec- E-mail address: [email protected] (K. Sreekumaran Nair). tively, reductions in mitochondrial proteins and volume may limit

0303-7207/$ - see front matter Ó 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.mce.2013.05.008 20 A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29

Sedentary Aging

? Mitochondrial: Skeletal Muscle: Volume Volume Quality Quality Function Function

Functional Capacity

Morbidity

Healthcare Costs

Fig. 1. Reduced mitochondrial and skeletal volume, quality and function with sedentary aging. Sedentary aging is associated with the decline of mitochondrial and skeletal muscle volume, quality and function. The casual link between the loss of mitochondrial homeostasis and sarcopenia is unknown, however, both appear with advancing age and are associated with the loss of functional capacity and corresponding increases in comorbidities and annual healthcare costs. Exercise and physical activity are effective prescriptions to attenuate the negative consequences of sedentary aging illustrated in Fig. 1.

ATP production for energy demanding processes such as myocellu- patterns and fiber type composition can create conflicting results lar remodeling to maintain protein quality. between studies. These variables need to be recognized and ad- Advancements of in vitro and ex vivo measures of mitochondrial dressed to properly assess the true age-related phenotype. Glob- energetics have detected diminished capacity for basal (Petersen ally, when investigations utilize large sample sizes and rigorous et al., 2003) and maximal (Conley et al., 2000; Kent-Braun and control to avoid many of the confounding variables there appears Ng, 2000; Short et al., 2005a) mitochondrial ATP synthesis in older to be an age-related decline in mitochondrial protein content, adults. When expressing the rate of mitochondrial ATP synthesis quality and function in the quadriceps femoris muscles. These data relative to mitochondrial content there remains a deficit in older provide well-founded evidence for perturbations in mitochondrial adults suggesting that there is not only a reduction in mitochon- health and connections to impaired functional capacity during sed- drial protein content but also mitochondrial protein quality. These entary aging. findings appear to be related to physical activity, as sedentary indi- Aerobic training is an effective exercise prescription to stimu- viduals had lower in vivo mitochondrial function compared to ac- late markers of oxidative capacity as established in the 1960s (Hol- tive individuals (Kent-Braun and Ng, 2000; Larsen et al., 2012). It loszy, 1967), when it was revealed that aerobic exercise of is important to acknowledge that in aging human skeletal muscle, sufficient intensity increased mitochondrial enzyme activity in ani- findings of mitochondrial dysfunction are highly equivocal and the mal models. Numerous other investigations have confirmed these disparity between studies is not well discussed. In Table 1 we pro- results, however, few studies in humans have directly investigated vide potential confounding variables related to the characteristics if age influences exercise induced mitochondrial adaptations after of research participants (column A) as well as the use of various the same exercise training program. From the few available stud- measurements of mitochondrial abundance or function (column ies, it appears that mitochondrial molecular regulation and protein B). Key differences exist when interpreting data since each mea- content are increased after 12–16 weeks of exercise training, inde- surement in Table 1 assesses different constituents of mitochon- pendent of age, suggesting older individuals (<80 y) adapt favor- drial abundance or function and each method presents key ably to exercise training (Ghosh et al., 2011; Short et al., 2003). strengths and weaknesses as has been reviewed in detail previ- However, the influence of various exercise training programs (i.e., ously (Lanza and Nair, 2010; Perry et al., 2013). One difference aerobic vs. resistance vs. concurrent training) on mitochondrial could be comparisons between content or maximal activities of en- and skeletal muscle function (ex vivo or in vivo) has yet to be deter- zymes in the mitochondrial matrix (e.g., citrate synthase, bHAD) mined and warrants investigation. Collectively, these data suggest which are completely encoded by nuclear DNA vs. proteins in- that exercise can improve or prevent the loss of mitochondrial volved in oxidative phosphorylation (e.g., cytochrome c oxidase, health during sedentary aging (Fig. 2). NADH) that are encoded by both nuclear and mitochondrial genomes. Although analysis of maximal mitochondrial energetics in vivo (i.e., 31P-MRS) and ex vivo (i.e., high-resolution respirome- 3. Molecular Regulation of Aging Mitochondria try) are highly correlated (Lanza et al., 2011), subtle discrepancies still exist between different approaches for measuring mitochon- The mitochondria consist of proteins encoded from both mito- drial function in vivo (basal vs. maximally stimulated) and ex vivo chondrial (mtDNA) and nuclear DNA (nDNA). Although mtDNA (ATP production vs. oxygen respiration; permeabilized fibers vs. contains just 27 genes that encode 13 proteins (all within the elec- isolated mitochondria). Also, sampling of human muscle tissue tron transport chain), 2 ribosomal and 22 translational RNA, proper from various muscle groups consisting of different recruitment organelle biogenesis and function require input from both gen- A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29 21

Table 1 Divergent subject characteristics and analytical techniques may contribute to discrepancies regarding aging mitochondrial health.

A. Subject characteristics References B. Analytical techniques References Age Bua et al. (2006), Chabi et al. (2005) Maximal enzyme activity Barrientos et al. (1996), Coggan et al. (1992), Holloszy (1967), Holloszy and Coyle (1984), Zucchini et al. (1995) Sample size Chretien et al. (1998), Short et al. Protein abundance Lanza et al. (2008) (2005a) Mobility or orthopedic Boffoli et al. (1994), Joseph et al. mtDNA copy number and Aiken et al. (2002), Short et al. (2005a) limitations (2012), Lezza et al. (1994), Safdar et al. mutations (2010) Exercise/physical activity Barrientos et al. (1996), Lanza et al. mRNA expression Short et al. (2003), Wright et al. (2007) history (2008), Proctor et al. (1995) Sarcopenia/frailty Moore et al. (2010), Waters et al. Electron microscopy Conley et al. (2000), Hoppeler et al. (1985), Howald et al. (1985) (2009) Adiposity Karakelides et al. (2010) In vivo function (31P-MRS) Amara et al. (2007), Conley et al. (2007), Kent-Braun and Ng (2000), Lanza et al. (2011), Lanza and Nair (2010), Petersen et al. (2003) Comorbidities (i.e., insulin Barreiro et al. (2009), Ghosh et al. Ex vivo function Lanza et al. (2011), Lanza and Nair (2010), Picard et al. resistance, COPD, etc.) (2011), Petersen et al. (2003) (2010,2011), Rasmussen et al. (2003a,b) Diet and medications Fisher-Wellman and Neufer (2012), Skeletal muscle investigated Amara et al. (2007), Houmard et al. (1998), Larsen et al. (2012) Lanza et al. (2012), Robinson et al. (2009)

The study of aging mitochondria presents inconsistent findings potentially associated with (A) variability in subject characteristics and (B) different analytical techniques to assess mitochondrial abundance or function within skeletal muscle. To examine the true age-related decline in mitochondrial health, descriptive characteristics listed in (A) need to be comprehensively discussed as these variables may each independently affect mitochondrial health. Moreover, the different analytical techniques listed in (B) are all highly utilized but each approach studies different components of the mitochondria (i.e., citrate synthase enzyme activity vs. ex vivo ATP production in isolated mitochondria) and therefore may contribute the equivocal findings between studies. Collectively, both subject characteristics and analytical techniques need to be considered when interpreting data describing the aging mitochondrial phenotype. References provided utilize or discuss the associated subject characteristic or analytical technique. omes. Several transcription factors and molecular regulators have ble of maintaining SIRT3 compared to there younger counterparts been highlighted in orchestrating mitochondrial biogenesis and (Lanza et al., 2008). Indeed, SIRT3 abundance and activity increase substrate metabolism. The exploration of the molecular regulation after contractile activity and may be a potential mechanism for im- of mitochondria has received much attention to gain insight into proved ATP synthesis, ROS production and insulin action (Gurd the etiology of aging mitochondria and associated disease condi- et al., 2012). Exploring the upstream regulation of the sirtuin fam- tions. The family of sirtuins, NAMPT, PGC-1a, NRF 1, NRF 2, TFAM ily is essential to fully appreciate how various interventions (i.e., as well as metabolite sensors such as AMPK, CAMK and calcium exercise, caloric restriction, medications) mediate the intracellular flux play an integral role in maintaining mitochondrial homeosta- signaling pathways associated with mitochondrial biogenesis and sis as illustrated in Fig. 2. protein quality.

3.1. Sirtuins 3.2. Peroxisome proliferator-activated receptor-c coactivator (PGC)- 1a The Sirtuin family (SIRT 1–7) is an NAD-dependent histone/protein deacetylase that interacts with transcription factors and cofac- In aging human skeletal muscle, there have been observations tors influencing many metabolic pathways (for review see of either reduced or unaltered levels of PGC-1a (Lanza et al., (Guarente, 2011; Gurd, 2011; Westphal et al., 2007; White and 2008; Ling et al., 2004). Since PGC-1a is considered the master reg- Schenk, 2012)). SIRT1 is the most well described Sirtuin due to ulator of mitochondrial biogenesis, lower levels may partially re- the favorable impact on targets associated with cellular growth, duce downstream transcription factors as well as mitochondrial chromatin remodeling, substrate metabolism and mitochondrial content and function. This hypothesis is supported by a diminished biogenesis. Specifically, the capability of SIRT1 to deacetylate capacity to stimulate mitochondrial biogenesis or maintain mito- PGC-1a, relaying signal transduction for mitochondrial biogenesis, chondrial content during active-aging in animals with genetically improved substrate utilization and insulin action is particularly altered PGC-1a (Leick et al., 2010). Additionally, animals with relevant in improving the aging phenotype. In addition, SIRT3, overexpressed PGC-1a demonstrate mitochondrial biogenesis and which is localized to the mitochondria, is associated with mito- reversal of many age-related diseases including sarcopenia (Wenz, chondrial efficiency and ROS production. SIRT3 knockout animals 2011; Wenz et al., 2009). Acute and chronic aerobic exercise in- demonstrate hyperacetylation interfering with proper mRNA tran- crease PGC-1a mRNA expression similarly in young and old indi- scription, elevated levels of ROS and concomitant reduction in ATP viduals (Cobley et al., 2012; Short et al., 2003). Older people who synthesis much like many aging models (Ahn et al., 2008; Kim have maintained a high level of aerobic exercise for several years et al., 2010). These data are substantiated from mechanistic studies had greater protein expression of PGC-1a than sedentary young displaying the ability of SIRT3 to deacetylate and activate MnSOD people yet failed to achieve similar levels as younger people who and glutathione-scavenging pathway enzymes to protect from also maintained high levels of aerobic training (Lanza et al., reactive oxygen species (ROS) during SIRT3 overexpression and 2008) suggesting that exercise can mitigate some age-related caloric restriction (Someya et al., 2010). Elevated ROS emissions losses but cannot fully protect the molecular regulation of mito- from the mitochondria have been implicated in the progression chondrial biogenesis. Skeletal muscle biopsy samples obtained of mitochondrial dysfunction and development of insulin resis- from a unique group of adults over 80 y of age revealed that those tance (Fisher-Wellman and Neufer, 2012). Therefore, SIRT 1 and who engaged in vigorous life-long endurance exercise have greater 3 may play a role in mitochondrial health, insulin action and func- PGC-1a mRNA expression compared to their healthy counterparts tional capacity with advancing age. who performed normal activities of daily living (Trappe et al., Sedentary older adults contain less SIRT3 content, however, it 2012). Although PGC-1a is not obligatory for exercise induced appears those who perform vigorous endurance exercise are capa- mitochondrial biogenesis (Leick et al., 2008) it still appears to be 22 A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29

Aerobic Exercise Training

Metabolites NAD AMP

PGC-1α

Mitochondrial Transcription Mitochondrial Dynamics Fusion NRF-1 TFAM nDNA mtDNA FIS1 MFN DRP1 1 & 2 Transcription Transcription OXPHOS mRNA 2 rRNAs 13 OXPHOS mRNA Fission

Mitochondrial Protein Synthesis Mitochondrial Protein Breakdown

Protein nDNA Synthesis

Nucleus FissionPhagophore Autophagosome Bulk Protein Breakdown

Protein Synthesis LON mtDNA Targeted Protein Breakdown Mitochondrion

Mitochondrial Content & Function

+ + H H H+ H+ Intermembrane space

I III IV V Inner membrane II Matrix

+ NADH NAD O2 H2O ADP+Pi ATP

Fig. 2. Mitochondrial adaptations to aerobic exercise training. With contractile activity, elevated levels of metabolic byproducts (i.e., NAD, AMP, Ca++, ROS, etc.) provide a stimulus for increased molecular regulators of mitochondrial transcription, replication and dynamics (i.e., NAMPT, SIRT-1, PGC-1a, NRF-1, -2, TFAM, MFN-1, -2, FIS1, DRP1). Collectively, these alterations promote the increase in mitochondrial protein turnover allowing for the degradation of damaged proteins and de novo synthesis of new functional proteins. Overall, the elevated rate of mitochondrial protein turnover suggests an improvement in the quality of mitochondrial proteins for enhanced ATP production and lower reactive oxygen species (ROS) emission. Enhanced mitochondrial function may augment myocellular remodeling, skeletal muscle anabolism and functional capacity in older adults.

a valuable component in the effects of exercise on skeletal muscle strated that aerobic (Konopka et al., 2010) and resistance (Kim and metabolic health with advancing age. Interestingly, an isoform et al., 2005; Roth et al., 2003; Ryan et al., 2011; Williamson of PGC-1a (PGC-1a4) has been comprehensively demonstrated to et al., 2010) exercise training reduced catabolic mRNA expression be involved in skeletal muscle hypertrophy in vitro and in vivo (i.e., FOXO3a, MuRF-1, Atrogin-1 and/or myostatin) with concomi- (Ruas et al., 2012). PGC-1a4 appears to be the primary isoform tant skeletal muscle hypertrophy. These adaptations could be asso- associated with skeletal muscle growth through the stimulation ciated with elevated levels of PGC-1a4 as recently revealed but of IGF-1 and suppression of myostatin, MuRF-1 and Atrogin-1 further examination is needed. Elevated PGC-1a isoforms with mRNA expression. Interestingly, previous studies have demon- exercise training in sedentary humans occurs concomitantly with A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29 23 increased mitochondrial protein content, mixed muscle protein 4.1. Mitochondrial protein synthesis synthesis, skeletal muscle hypertrophy and aerobic capacity (Har- ber et al., 2009b, 2012; Ruas et al., 2012; Short et al., 2004, 2003). Infusion of stable isotope tracer into young and older adults Collectively, these investigations highlight the advantages of PGC- demonstrated decreased rates of in vivo mitochondrial protein syn- 1a in promoting a healthy aging, skeletal muscle phenotype. thesis rate in skeletal muscle of older adults and is accompanied with diminished mitochondrial enzyme activity (Rooyackers et al., 1996). These data provide a feasible connection between 3.3. Mitochondrial transcription factor A (TFAM) and Nuclear the decreased ability to replace mitochondrial proteins in aging respiratory factor (NRF)-1 and -2 skeletal muscle leading to mitochondrial enzymatic dysfunction. Furthermore, mitochondrial protein synthesis rates are initially re- Recent research has revealed that PGC-1a relocates to both the duced during middle age, which may be a precursor to the progres- mitochondrial and nuclear compartments after a physiological sive loss of mitochondrial protein abundance and function with stimulus, such as aerobic exercise, to coordinate mitochondrial older age. Other investigations have confirmed the deficit in mito- biogenesis from both nuclear and mitochondrial genomes (Safdar chondrial protein synthesis rate in older adults (Guillet et al., 2004) et al., 2011). Most reports describe PGC-1a by directly acting on while advancements of innovative methodology has allowed for NRF-1 and -2 within the nucleus to stimulate increased levels of the analysis of individual mitochondrial protein synthesis rates mitochondrial transcription factor A (TFAM) followed by import to elucidate specific proteins that may participate in the etiology into the mitochondria. However, it appears that PGC-1a can also of aging mitochondrial dysfunction (Jaleel et al., 2008; Lanza act independently on NRF-1 and TFAM by binding to the NRF-1 et al., 2012). promoter region within the nucleus as well as being complexed While increased mitochondrial content is an established adap- with TFAM and the mtDNA D-loop region within the mitochondrial tation of aerobic exercise, the impact of exercise on mitochondrial matrix to transcriptionally coordinate nuclear- and mitochondrial- protein turnover is not well characterized. One group has demon- encoded proteins. These studies established an updated paradigm strated that acute and chronic aerobic exercise increases mito- into the mechanisms of how PGC-1a orchestrates mitochondrial chondrial protein synthesis rates in younger individuals biogenesis through key transcriptional regulators NRF-1 and TFAM. (Wilkinson et al., 2008). However, the effects of exercise on aging In addition to binding to mtDNA for transcriptional induction of mitochondrial protein turnover have not yet been examined. Due mitochondrial biogenesis, TFAM also has a strong affinity for to the clear associations with mitochondrial biogenesis and func- mtDNA to stabilize and package the genome into a nucleoid struc- tion, studies are needed to comprehensively elucidate the impact ture (for detailed review see (Campbell et al., 2012)). Stabilizing of exercise training on overcoming diminished mitochondrial pro- mtDNA appears to be a protective mechanism to prevent damage tein synthesis in aging humans. and/or loss of mtDNA copy number as observed in aging skeletal muscle (Short et al., 2005a). However, in aging brain tissue, TFAM 4.2. Mitochondrial protein degradation was elevated with a concomitant reduction in mtDNA most likely due to impaired binding of TFAM to mtDNA regions (Picca et al., Due to difficulties of properly assessing protein degradation in 2012). Mitochondrial protein turnover via Lon protease, discussed human skeletal muscle, the literature is equivocal and largely un- in the next section, is thought to regulate the TFAM:mtDNA ratio to known. However, global assessments indicate whole-body protein enhance stability and transcription (Matsushima et al., 2010). degradation is reduced in older adults (Balagopal et al., 1997; Hen- Exercise is known to increase TFAM and mtDNA number highlight- derson et al., 2009). These data, in conjunction with lower rates of ing the potential for improved TFAM-mtDNA binding with chronic mixed muscle (Short et al., 2004), myosin heavy chain (Balagopal exercise. The regulation of transcription, translation and mitochon- et al., 1997) and mitochondrial protein synthesis (Rooyackers drial biogenesis is still not completely understood and further re- et al., 1996), suggest that a low protein turnover in older adults search is warranted. may allow for a reduction in protein quality by accumulation of modified proteins in organelles (i.e., mitochondria) and tissues 4. Skeletal muscle and mitochondrial protein turnover (i.e., skeletal muscle) creating further dysfunction with age (Fig. 3). It is important to note that different skeletal muscle sub- In addition to transcriptional regulation, the accretion of new fractions (Balagopal et al., 1997; Rooyackers et al., 1996; Short proteins and degradation of older proteins may have a large impact et al., 2004; Trappe et al., 2004), fiber types (Dickinson et al., on mitochondrial morphology and function. It is important to note 2010), and individual proteins (Jaleel et al., 2008) have diverse that changes in mitochondrial protein turnover may not always be rates of protein turnover and warrant further examination as a reflected by expression of mitochondrial proteins. For example, component of the age-related loss of mitochondrial and skeletal when the rates of de novo synthesized proteins are elevated while muscle health. Currently, methods to determine skeletal muscle being matched by the breakdown of older irreversibly modified and mitochondrial protein degradation are not well developed. proteins, no apparent changes in protein abundance can be de- These key limitations highlight the need for advancement of novel tected. More importantly, elevated protein turnover (i.e., the techniques to properly assess protein degradation and propel our replacement of modified and presumably dysfunctional proteins understanding of human skeletal muscle biology. by de novo synthesized proteins) may be a strategic mechanism to maintain mitochondrial protein quality and function (Fig. 3). 4.2.1. Mitochondrial dynamics This notion is supported by a study demonstrating that lifelong cal- One particular area of interest regarding mitochondrial turn- orie restricted mice maintained mitochondrial function with age over is the collaboration of mitochondrial fusion, fission and but did not increase mitochondrial abundance (Lanza et al., autophagy (i.e. mitochondrial dynamics) to regulate organelle 2012). Instead of mitochondrial proliferation, calorie restricted morphology. Mitochondrial fusion is the combination of outer mice improved mitochondrial protein quality compared to their mitochondrial membrane and subsequent mixing of intramito- ad libitum fed counterparts. These concepts strongly emphasize chondrial components to dilute any damaged mitochondrial DNA the need to measure mitochondrial protein synthesis and break- or proteins. Additionally, mitofusion proteins also assist in molding down as a process to increase or maintain mitochondrial function the inner mitochondrial membrane cristae, making the collective with age. purpose of mitofusion to prevent the dissipation of mitochondrial 24 A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29

Aging

Protein Synthesis

New Proteins Amino Protein Acids Protein Quality Turnover Old Proteins

Protein Degradation

Exercise Training

Fig. 3. Effect of aging and exercise on protein damage and quality. With aging, excess reactive oxygen species (ROS) are produced from mitochondria with a concomitant reduction of protein degradation and synthesis (i.e., protein turnover). The combination of elevated ROS and reduced protein turnover can lead to the accumulation of damage to proteins resulting in reduced protein quality and function. We hypothesis that exercise training can attenuate age-related production of ROS and stimulate protein turnover, in turn, degrading oxidatively damaged proteins and replacing with newly synthesized, functional proteins. membrane potential and thus ATP synthesis. Investigations utiliz- 2002). In aging models, Lon protease is reduced and therefore ing animal knockout models of mitofusion proteins have demon- hypothesized to play a role in the development of mitochondrial strated diminished mitochondrial function and biogenesis as well dysfunction in older tissues (Bota et al., 2002; Lee et al., 1999). An- as muscle atrophy (Chen et al., 2010). other mitochondrial quality control pathway is autophagy, as evi- Conversely, when fusion is no longer possible due to the loss of denced by the maintenance of mitochondrial function in the liver mitochondrial membrane integrity, fission is responsible for the of older transgenic mice compared to wild type mice (Zhang and fragmentation and excision of any altered or damaged mitochon- Cuervo, 2008). Similarly, overexpression of autophagy proteins in drial components that are subsequently degraded by mitochon- human umbilical vein endothelial cells appears to remove dam- drial specific autophagy (i.e. mitophagy) (Seo et al., 2010). aged mitochondrial proteins when challenged with reactive oxy- Mitochondrial dynamics are essential to maintain normal mitogen species in vitro (Mai et al., 2012). Data in human skeletal chondrial metabolism, morphology and homeostasis in highly oxi- muscle are limited but recent studies have observed no measur- dative tissues such as skeletal muscle (Masiero et al., 2009). able differences between young and older individuals at the mRNA Therefore, from recent research revealing that select mitofusion level for markers of UPP or autophagy (Fry et al., 2012a). It is inter- and mitofission markers (i.e., mRNA) are reduced in aging human esting to note that mRNA of UPP was elevated and/or autophagy skeletal muscle (Crane et al., 2010), we can infer that mitochon- reduced in humans undergoing accelerated atrophy (i.e. >80 y old drial turnover is compromised which could partially mediate (Raue et al., 2007; Williamson et al., 2010), para- (Fry et al., age-related mitochondrial dysfunction and impaired skeletal mus- 2012b) and hemiplegia (von Walden et al., 2012)). Development cle health. Interestingly, markers of mitochondrial dynamics are of dynamic assays to measure protein degradation specific to the elevated with acute exercise in young individuals (Cartoni et al., mitochondrial and myofibrillar proteins are needed to provide a di- 2005; Slivka et al., 2012) suggesting that exercise can increase rect functional connection to protein metabolism and age. mitochondrial turnover, which may lead to favorable mitochon- Acute aerobic exercise appears to increase mRNA expression of drial functional improvements. proteolytic pathways within mixed muscle homogenates (Harber et al., 2009a, 2010; Louis et al., 2007; Pasiakos et al., 2010). These 4.2.2. Proteolytic pathways data suggest that the molecular induction of protein degradation is The autophagy-lysosome and ubiquitin–proteasome (UPP) elevated by acute exercise, most likely providing amino acids for de pathways are two major systems that mediate protein degradation novo synthesis (Balagopal et al., 2001) and myocellular remodeling and maintain cellular homeostasis. Autophagy appears to be asso- that leads to improved contractile function after chronic exercise ciated with more bulk protein degradation of large areas and/or (Harber et al., 2004, 2009b, 2012; Trappe et al., 2001) Interestingly, organelles, such as mitochondria, that are encapsulated by the exercise training programs that improve skeletal muscle size and phagophore, fused with the lysosome and subsequently broken function in adults (<80 y) observed reductions in proteolytic mark- down to amino acids (Fig. 2). Conversely, the UPP is responsible ers (Konopka et al., 2010; Williamson et al., 2010), most likely for marking select proteins that are damaged or misfolded with shifting protein balance in favor of skeletal muscle protein accre- an ubiquitin tail for degradation via the proteasome. Recent re- tion. Collectively, these data reveal the differences between tran- search suggests that the UPP may interact with autophagy by sient alterations and chronic adaptations in proteolytic assisting the regulation of mitochondrial dynamics and disposal machinery while highlighting the need for additional investiga- of damaged mitochondrial proteins. Additionally, evidence indi- tions examining protein turnover to substantiate the link to myo- cates the role of UPP in regulating cellular homeostasis may be dis- cellular and mitochondrial function after acute and chronic tinct in various skeletal muscle organelles or sub-fractions (i.e. exercise. sarcoplasmic, myofibrillar, mitochondrial). One mitochondrial quality control mechanism is the ATP-stim- 5. Insufficient antioxidant capacity and oxidative damage ulated Lon protease located in the mitochondrial matrix (Fig. 2). Lon protease is believed to be an integral factor in the degradation Reactive oxygen species (ROS) are molecules containing one or of oxidatively damaged mitochondrial proteins (Bota and Davies, more unpaired electrons mainly produced from complex I and III in A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29 25 the ETC. These byproducts are normally detoxified by antioxidants affect action potential propagation, ETC complexes, calcium trans- (e.g. MnSOD, CuZnSOD, catalase) but when they are generated in port/regulation, and myosin and actin interaction; collectively excess of antioxidant capacity they can irreversibly modify (i.e. reducing skeletal muscle function in rodent models (Fulle et al., damage) lipids, proteins, and DNA. Due to inconsistent findings 2004; Rossi et al., 2008). In a unique model of muscle dysfunction, on the antioxidant capacity in older adults (Ghosh et al., 2011; individuals with chronic obstructive pulmonary disease were char- Gianni et al., 2004; Leeuwenburgh et al., 1994; Pansarasa et al., acterized with increased oxidative damage that was negatively 2000) there is a strong need to determine antioxidant capacity at correlated with aerobic capacity and isometric skeletal muscle the systemic, skeletal muscle and mitochondrial compartments force production (Barreiro et al., 2009). These data are supported to fully identify where these defense mechanisms fail and where in older adults as elevated oxidative damage has been correlated development of therapies should be focused to ameliorate oxidant with diminished functional capacity (Howard et al., 2007; Semba damage and cellular dysfunction with age. et al., 2007a) and increased risk of mortality (Semba et al., Damage to mtDNA is suspected to occur easily due to the prox- 2007b). Therefore, there are implications that excess ROS may be imity near the ETC and lack of protection by histones. The com- an underlying characteristic in the progression of muscle atrophy bined effects of reduced protein turnover and excess ROS by introducing oxidative damage that can negatively affect the emission create an environment conducive in aging skeletal mus- functional capabilities of older adults. cle for the accumulation of oxidatively damaged mtDNA and contractile proteins (Fig. 3). Accrual of mtDNA damage may then allow production of dysfunctional proteins within the ETC, further exag- 6. A role for epigenetic regulation of mitochondrial and gerating ROS emission and oxidative damage. This scenario is con- contractile proteins sidered the mitochondrial theory of aging first hypothesized by (Harman, 1972). However, it is important to note that the appear- The age-related decline in transcription (i.e., mRNA expression) ance of mtDNA deletion/mutations in human skeletal muscle is rel- and translation (i.e., protein synthesis) are apparent in the loss of atively small, with data indicating that mtDNA deletions may reach mitochondrial and contractile protein quality. The underlying levels of physiological significance only in adults greater than 80 y mechanisms for reduced transcription include epigenetics, which old (Bua et al., 2006; Chabi et al., 2005; Kopsidas et al., 1998; Kov- have a responsibility in shaping the aging phenotype in response alenko et al., 1997) although many age-related changes including to environmental influences like physical activity and diet. Com- sarcopenia begin much earlier. When mtDNA deletions are present mon epigenetic mechanisms are modifications to DNA and/or his- in single muscle fibers they appear red and ragged, leading to fiber tones (i.e., acetylation, methylation). Changes in the methylation atrophy and eventually fiber loss (Bua et al., 2006). Moreover, and acetylation status can modify chromatin structure, which in mtDNA deletions and fiber atrophy are more prevalent in fast turn alters the binding capability of transcription factors to create MHC II fibers, which is consistent with age-related loss of fast fiber mRNA (Fig. 4). Epigenomic research has large implications in composition and contractile properties. The pattern of mtDNA improving our understanding of the aging mitochondrial and con- deletion mutations, fiber atrophy and fiber loss provides a clear tractile dysfunction but is currently at its nascent stages. relationship between mitochondria and skeletal muscle atrophy. It appears with older age there is an increase in DNA methyla- Data from mitochondrial bioenergetics supports the role of al- tion of promoter regions for genes involved in oxidative phosphor- tered mitochondrial membrane potential in mediating excess lev- ylation and the level of DNA methylation is inversely correlated els of oxidant production (Fisher-Wellman and Neufer, 2012). with gene expression (Ling et al., 2007; Ronn et al., 2008). These The concept is based on redox biology of mitochondria where over data suggest that altered methylation could be involved in reduced nutrition (i.e. increased supply) and/or reduced physical activity mitochondrial mRNA expression and protein synthesis observed in (i.e. reduced demand) appears to create a buildup of protons thus older adults. More expansive investigation is necessary to deter- creating a high membrane potential that could cause a cessation mine key promoter regions that may be differentially methylated. of electron flow through the ETC. The disruption in electron flow Furthermore, alterations in mitochondrial function may provide is thought to increase oxidant production by acting as a release feedback to influence the epigenetic regulation of the mitochon- valve to dissipate the elevated membrane potential. Any mecha- drial and nuclear genome. nism to allow protons to flow back to the mitochondrial matrix In addition to mitochondrial regulation, epigenetic alterations (ATP synthesis or uncoupling) will also help relieve membrane po- also appear to influence key skeletal muscle contractile proteins. tential and therefore oxidant production. This notion is supported The balance between histone acetylation and deacetylation by findings that lifelong caloric restriction attenuates H2O2 emis- changes in concordance with shifts in myosin heavy chain compo- sion by eliminating excess energy intake (i.e. supply) and mitigates sition during unloading (Pandorf et al., 2009). This is of particular the accumulation of post-translational modifications, especially interest when exploring the mechanisms mediating changes in oxidation and deamidation, while concomitantly maintaining MHC composition of aging skeletal muscle that occurs with con- mitochondrial energetics with age (Lanza et al., 2012). These novel comitant reductions in size and contractile performance of fast, data are available due to recent advancements where proteome MHC IIa fibers (Lexell et al., 1983; Trappe et al., 2003). Fast myosin quality can be assessed by the evaluation of post-translational light chain isoforms, thought to influence contractile function, modifications using tandem mass spectrometry. Not only can this were found to be negatively correlated with promoter methylation innovative approach comprehensively detect global protein modi- (Donoghue et al., 1991) suggesting that methylation may inhibit fications, it can also determine which amino acid residues are most fast fiber performance as observed in older adults. commonly modified. Implementing proteome analysis of post- Currently, exercise seems to be one of the most impactful stim- translational modifications may provide a platform to develop uli that alters skeletal muscle physiology (i.e., MHC and mitochon- and test innovative strategies to reduce the accumulation of mod- drial protein abundance and function (Balagopal et al., 1997; ified and functionally impaired proteins. Coggan et al., 1992; Ghosh et al., 2011; Harber et al., 2009b, In addition to caloric restriction, physical activity levels play a 2012; Konopka et al., 2011; Short et al., 2005b, 2003)) and serves strong role in modulating skeletal muscle quality as sedentary old- as a countermeasure to many negative consequences of sedentary er individuals have a noticeable decline in antioxidant capacity aging. However, the effects of exercise and age on epigenetic regu- with concomitant oxidative damage compared to age-matched ac- lation of mRNA expression and skeletal muscle adaptations are rel- tive individuals (Safdar et al., 2010). Oxidative modifications can atively unknown. Acute aerobic exercise can induce histone 26 A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29

Mitochondrial Proteins Contractile Proteins

Condensed Chromatin During Sedentary Aging

Mediated by DNMT/HAT/HMTs & HDACs/HDMs

Open Chromatin Induced by Exercise

PGC-1α, SIRT1, TFAM MHC, MLC, Actin, Troponin

Fig. 4. Epigenetic regulation of mitochondrial and contractile proteins. Epigenetic control of mitochondrial and contractile proteins may improve our knowledge on the development of the aging phenotype. During sedentary aging, chromatin is condensed by altered levels of acetylation and/or methylation that prevent binding of transcription factors. With exercise, modifications of histones (indirect modification) and/or DNA (direct modification) may allow an open chromatin structure where transcription factors can bind to DNA promoter regions for mRNA transcription to occur. The shifts between condensed chromatin during sedentary aging and open chromatin with exercise are partially mediated by DNA and histone modifying enzymes: DNA methyl transfereases (DNMT), histone acetyl transferases (HATs), histone methyl transferases (HMTs), histone deacetylases (HDACs) and histone demethylases (HDMs). The schematic was adopted from (Zwetsloot et al., 2009) and altered to reflect our perspective on epigenetic control of mitochondrial and contractile protein quality. modifications that may mediate chromatin remodeling and disas- proteins and DNA. In many aging populations, adherence or ability sociations with transcription factors implicated in substrate to participate in exercise is limited due to orthopedic or disease metabolism (McGee et al., 2009; McGee and Hargreaves, 2004). limitations (i.e. heart failure, chronic obstructive pulmonary dis- Moreover, acute and chronic contractile activity increased SIRT1 ease) which warrants exploration of other therapies (i.e., caloric deacylate activity within the nucleus which is most likely related restriction, diet composition, non-traditional exercise, etc.) to help to increased PGC-1a mRNA (Gurd, 2011; Gurd et al., 2011). An elo- prevent the negative health consequences and elevated healthcare quent study (Barres et al., 2012) recently revealed that acute aero- costs related to sedentary aging. bic exercise of sufficient intensity can alter global and promoter specific methylation leading to changes in mRNA expression re- Acknowledgment lated to mitochondrial and substrate regulation in young adults. More investigations like Barres et al., are needed to clearly deter- The authors are grateful for the skillful and diligent assistance mine the influence of age and chronic exercise on the epigenetic of Katherine Klaus, Dawn Morse, Jill Schimke, Maureen Bigelow, regulation of global and specific promoter regions related to mito- Daniel Jakaitis, Roberta Soderberg, Beth Will, Deborah Sheldon chondrial (e.g., PGC-1a, SIRT1, TFAM) and contractile proteins (e.g. and Melissa Aakre. This research was supported by National Insti- MHC, MLC, actin, troponin) (Fig. 4). Discovering the epigenetic tutes of Health grants UL1-RR-024150-01 and AG09531, R01- modulation of skeletal muscle has vast potential to facilitate the DK41973 (KSN) and T32 DK007352 (ARK). Additional support development of novel therapies to improve protein quality with was provided by Mayo Foundation and the Murdock-Dole Profes- age. sorship (KSN).

6.1. Perspectives on aging skeletal muscle and mitochondrial health References

The common loss of mitochondrial and skeletal muscle volume, Ahn, B.H., Kim, H.S., Song, S., Lee, I.H., Liu, J., Vassilopoulos, A., Deng, C.X., Finkel, T., 2008. A role for the mitochondrial deacetylase Sirt3 in regulating energy function and quality is an attractive relationship to help explain homeostasis. Proc. Natl. Acad. Sci. USA 105, 14447–14452. the comorbidities associated with aging. More research is needed Aiken, J., Bua, E., Cao, Z., Lopez, M., Wanagat, J., McKenzie, D., McKiernan, S., 2002. to conﬁrm that the loss of mitochondria health undermines sarco- Mitochondrial DNA deletion mutations and sarcopenia. Ann. NY. Acad. Sci. 959, 412–423. penia, especially due to the variability within human studies. Some Amara, C.E., Shankland, E.G., Jubrias, S.A., Marcinek, D.J., Kushmerick, M.J., Conley, inconsistency seems to be related to different physical activity and K.E., 2007. Mild mitochondrial uncoupling impacts cellular aging in human lifestyle choices between subjects. This variability may partially be muscles in vivo. Proc. Natl. Acad. Sci. USA 104, 1057–1062. explained by the implementation of epigenetic research to gain Atlantis, E., Martin, S.A., Haren, M.T., Taylor, A.W., Wittert, G.A., 2009. Inverse associations between muscle mass, strength, and the metabolic syndrome. comprehensive insight into the molecular regulation of aging in Metabolism 58, 1013–1022. sedentary and physically active individuals. Exercise training and Balagopal, P., Rooyackers, O.E., Adey, D.B., Ades, P.A., Nair, K.S., 1997. Effects of aging physical activity remain effective strategies to attenuate the devel- on in vivo synthesis of skeletal muscle myosin heavy-chain and sarcoplasmic protein in humans. Am. J. Physiol. 273, E790–800. opment of mitochondrial and skeletal muscle dysfunction but Balagopal, P., Schimke, J.C., Ades, P., Adey, D., Nair, K.S., 2001. Age effect on many questions remain unanswered. Moreover, the role of chronic transcript levels and synthesis rate of muscle MHC and response to resistance caloric restriction requires controlled studies that balance the po- exercise. Am. J. Physiol. Endocrinol. Metab. 280, E203–8. Barazzoni, R., Short, K.R., Nair, K.S., 2000. Effects of aging on mitochondrial DNA tential adverse effects due to loss of lean mass and energy balance copy number and cytochrome c oxidase gene expression in rat skeletal muscle, vs. beneﬁcial effects associated with reduced oxidative damage to liver, and heart. J. Biol. Chem. 275, 3343–3347. A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29 27

Barreiro, E., Rabinovich, R., Marin-Corral, J., Barbera, J.A., Gea, J., Roca, J., 2009. Papanicolaou, D., Rolland, Y., Rooks, D., Sieber, C., Souhami, E., Verlaan, S., Chronic endurance exercise induces quadriceps nitrosative stress in patients Zamboni, M., 2011. Sarcopenia: an undiagnosed condition in older adults. with severe COPD. Thorax 64, 13–19. Current consensus definition: prevalence, etiology, and consequences. Barres, R., Yan, J., Egan, B., Treebak, J.T., Rasmussen, M., Fritz, T., Caidahl, K., Krook, International working group on sarcopenia. J. Am. Med. Dir. Assoc. 12, 249–256. A., O’Gorman, D.J., Zierath, J.R., 2012. Acute exercise remodels promoter Fisher-Wellman, K.H., Neufer, P.D., 2012. Linking mitochondrial bioenergetics to methylation in human skeletal muscle. Cell Metab. 15, 405–411. insulin resistance via redox biology. Trends Endocrinol. Metab. 23, 142–153. Barrientos, A., Casademont, J., Rotig, A., Miro, O., Urbano-Marquez, A., Rustin, P., Fry, C.S., Drummond, M.J., Glynn, E.L., Dickinson, J.M., Gundermann, D.M., Cardellach, F., 1996. Absence of relationship between the level of electron Timmerman, K.L., Walker, D.K., Volpi, E., Rasmussen, B.B., 2012a. Skeletal transport chain activities and aging in human skeletal muscle. Biochem. Muscle Autophagy and Protein Breakdown Following Resistance Exercise are Biophys. Res. Commun. 229, 536–539. Similar in Younger and Older Adults. J. Gerontol. A Biol. Sci. Med. Sci.. Baumgartner, R.N., Koehler, K.M., Gallagher, D., Romero, L., Heymsfield, S.B., Ross, Fry, C.S., Drummond, M.J., Lujan, H.L., Dicarlo, S.E., Rasmussen, B.B., 2012b. R.R., Garry, P.J., Lindeman, R.D., 1998. Epidemiology of sarcopenia among the Paraplegia increases skeletal muscle autophagy. Muscle Nerve 46, 793–798. elderly in New Mexico. Am. J. Epidemiol. 147, 755–763. Fulle, S., Protasi, F., Di Tano, G., Pietrangelo, T., Beltramin, A., Boncompagni, S., Boffoli, D., Scacco, S.C., Vergari, R., Solarino, G., Santacroce, G., Papa, S., 1994. Decline Vecchiet, L., Fano, G., 2004. The contribution of reactive oxygen species to with age of the respiratory chain activity in human skeletal muscle. Biochim. sarcopenia and muscle ageing. Exp. Gerontol. 39, 17–24. Biophys. Acta. 1226, 73–82. Ghosh, S., Lertwattanarak, R., Lefort, N., Molina-Carrion, M., Joya-Galeana, J., Bowen, Bota, D.A., Davies, K.J., 2002. Lon protease preferentially degrades oxidized B.P., Garduno-Garcia Jde, J., Abdul-Ghani, M., Richardson, A., DeFronzo, R.A., mitochondrial aconitase by an ATP-stimulated mechanism. Nat. Cell Biol. 4, Mandarino, L., Van Remmen, H., Musi, N., 2011. Reduction in reactive oxygen 674–680. species production by mitochondria from elderly subjects with normal and Bota, D.A., Van Remmen, H., Davies, K.J., 2002. Modulation of Lon protease activity impaired glucose tolerance. Diabetes 60, 2051–2060. and aconitase turnover during aging and oxidative stress. FEBS Lett. 532, 103– Gianni, P., Jan, K.J., Douglas, M.J., Stuart, P.M., Tarnopolsky, M.A., 2004. Oxidative 106. stress and the mitochondrial theory of aging in human skeletal muscle. Exp. Bua, E., Johnson, J., Herbst, A., Delong, B., McKenzie, D., Salamat, S., Aiken, J.M., 2006. Gerontol. 39, 1391–1400. Mitochondrial DNA-deletion mutations accumulate intracellularly to Goodpaster, B.H., Thaete, F.L., Kelley, D.E., 2000. Thigh adipose tissue distribution is detrimental levels in aged human skeletal muscle fibers. Am. J. Hum. Genet. associated with insulin resistance in obesity and in type 2 diabetes mellitus. 79, 469–480. Am. J. Clin. Nutr. 71, 885–892. Campbell, C.T., Kolesar, J.E., Kaufman, B.A., 2012. Mitochondrial transcription factor Goodpaster, B.H., Krishnaswami, S., Harris, T.B., Katsiaras, A., Kritchevsky, S.B., A regulates mitochondrial transcription initiation, DNA packaging, and genome Simonsick, E.M., Nevitt, M., Holvoet, P., Newman, A.B., 2005. Obesity, regional copy number. Biochim. Biophys. Acta 1819, 921–929. body fat distribution, and the metabolic syndrome in older men and women. Cartoni, R., Leger, B., Hock, M.B., Praz, M., Crettenand, A., Pich, S., Ziltener, J.L., Luthi, Arch. Int. Med. 165, 777–783. F., Deriaz, O., Zorzano, A., Gobelet, C., Kralli, A., Russell, A.P., 2005. Mitofusins 1/2 Goodpaster, B.H., Park, S.W., Harris, T.B., Kritchevsky, S.B., Nevitt, M., Schwartz, A.V., and ERRalpha expression are increased in human skeletal muscle after physical Simonsick, E.M., Tylavsky, F.A., Visser, M., Newman, A.B., 2006. The loss of exercise. J. Physiol. 567, 349–358. skeletal muscle strength, mass, and quality in older adults: the health, aging Chabi, B., Mousson de Camaret, B., Chevrollier, A., Boisgard, S., Stepien, G., 2005. and body composition study. J. Gerontol. A. Biol. Sci. Med. Sci. 61, 1059–1064. Random mtDNA deletions and functional consequence in aged human skeletal Guarente, L., 2011. Franklin H. Epstein Lecture: sirtuins, aging, and medicine. N muscle. Biochem. Biophys. Res. Commun. 332, 542–549. Engl. J. Med. 364, 2235–2244. Chen, H., Vermulst, M., Wang, Y.E., Chomyn, A., Prolla, T.A., McCaffery, J.M., Chan, Guillet, C., Prod’homme, M., Balage, M., Gachon, P., Giraudet, C., Morin, L., Grizard, J., D.C., 2010. Mitochondrial fusion is required for mtDNA stability in skeletal Boirie, Y., 2004. Impaired anabolic response of muscle protein synthesis is muscle and tolerance of mtDNA mutations. Cell 141, 280–289. associated with S6K1 dysregulation in elderly humans. FASEB J. 18, 1586–1587. Chretien, D., Gallego, J., Barrientos, A., Casademont, J., Cardellach, F., Munnich, A., Gurd, B.J., 2011. Deacetylation of PGC-1alpha by SIRT1: importance for skeletal Rotig, A., Rustin, P., 1998. Biochemical parameters for the diagnosis of muscle function and exercise-induced mitochondrial biogenesis. Appl. Physiol. mitochondrial respiratory chain deficiency in humans, and their lack of age- Nutr. Metab. 36, 589–597. related changes. Biochem. J. 329 (Pt 2), 249–254. Gurd, B.J., Yoshida, Y., McFarlan, J.T., Holloway, G.P., Moyes, C.D., Heigenhauser, G.J., Cobley, J.N., Bartlett, J.D., Kayani, A., Murray, S.W., Louhelainen, J., Donovan, T., Spriet, L., Bonen, A., 2011. Nuclear SIRT1 activity, but not protein content, Waldron, S., Gregson, W., Burniston, J.G., Morton, J.P., Close, G.L., 2012. PGC- regulates mitochondrial biogenesis in rat and human skeletal muscle. Am. J. 1alpha transcriptional response and mitochondrial adaptation to acute exercise Physiol. Regul. Integr. Compos. Physiol. 301, R67–75. is maintained in skeletal muscle of sedentary elderly males. Biogerontology 13, Gurd, B.J., Holloway, G.P., Yoshida, Y., Bonen, A., 2012. In mammalian muscle, SIRT3 621–631. is present in mitochondria and not in the nucleus; and SIRT3 is upregulated by Coen, P.M., Jubrias, S.A., Distefano, G., Amati, F., Mackey, D.C., Glynn, N.W., Manini, chronic muscle contraction in an adenosine monophosphate-activated protein T.M., Wohlgemuth, S.E., Leeuwenburgh, C., Cummings, S.R., Newman, A.B., kinase-independent manner. Metabolism 61, 733–741. Ferrucci, L., Toledo, F.G., Shankland, E., Conley, K.E., Goodpaster, B.H., 2012. Harber, M.P., Gallagher, P.M., Creer, A.R., Minchev, K.M., Trappe, S.W., 2004. Single Skeletal muscle mitochondrial energetics are associated with maximal aerobic muscle fiber contractile properties during a competitive season in male runners. capacity and walking speed in older adults. J. Gerontol. A. Biol. Sci. Med. Sci.. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R1124–31. Coggan, A.R., Spina, R.J., King, D.S., Rogers, M.A., Brown, M., Nemeth, P.M., Holloszy, Harber, M.P., Crane, J.D., Dickinson, J.M., Jemiolo, B., Raue, U., Trappe, T.A., Trappe, J.O., 1992. Skeletal muscle adaptations to endurance training in 60- to 70-yr-old S.W., 2009a. Protein synthesis and the expression of growth-related genes are men and women. J. Appl. Physiol. 72, 1780–1786. altered by running in human vastus lateralis and soleus muscles. Am. J. Physiol. Conley, K.E., Jubrias, S.A., Esselman, P.C., 2000. Oxidative capacity and ageing in Regul. Integr. Comp. Physiol. 296, R708–14. human muscle. J. Physiol. 526 (Pt 1), 203–210. Harber, M.P., Konopka, A.R., Douglass, M.D., Minchev, K., Kaminsky, L.A., Trappe, Conley, K.E., Jubrias, S.A., Amara, C.E., Marcinek, D.J., 2007. Mitochondrial T.A., Trappe, S., 2009b. Aerobic exercise training improves whole muscle and dysfunction: impact on exercise performance and cellular aging. Exerc. Sport. single myofiber size and function in older women. Am. J. Physiol. Regul. Integr. Sci. Rev. 35, 43–49. Comp. Physiol. 297, R1452–9. Cooper, J.M., Mann, V.M., Schapira, A.H., 1992. Analyses of mitochondrial Harber, M.P., Konopka, A.R., Jemiolo, B., Trappe, S.W., Trappe, T.A., Reidy, P.T., 2010. respiratory chain function and mitochondrial DNA deletion in human skeletal Muscle protein synthesis and gene expression during recovery from aerobic muscle: effect of ageing. J. Neurol. Sci. 113, 91–98. exercise in the fasted and fed states. Am. J. Physiol. Regul. Integr. Comp. Physiol. Crane, J.D., Devries, M.C., Safdar, A., Hamadeh, M.J., Tarnopolsky, M.A., 2010. The 299, R1254–62. effect of aging on human skeletal muscle mitochondrial and intramyocellular Harber, M.P., Konopka, A.R., Undem, M.K., Hinkley, J.M., Minchev, K., Kaminsky, L.A., lipid ultrastructure. J. Gerontol. A. Biol. Sci. Med. Sci. 65, 119–128. Trappe, T.A., Trappe, S., 2012. Aerobic exercise training induces skeletal muscle Critchley, M., 1931. The neurology of old age. Lancet 1, 1221–1231. hypertrophy and age-dependent adaptations in myofiber function in young and Delmonico, M.J., Harris, T.B., Lee, J.S., Visser, M., Nevitt, M., Kritchevsky, S.B., older men. J. Appl. Physiol. 113, 1495–1504. Tylavsky, F.A., Newman, A.B., 2007. Alternative definitions of sarcopenia, lower Harman, D., 1972. The biologic clock: the mitochondria? J. Am. Geriatr. Soc. 20, extremity performance, and functional impairment with aging in older men and 145–147. women. J. Am. Geriatr. Soc. 55, 769–774. Henderson, G.C., Dhatariya, K., Ford, G.C., Klaus, K.A., Basu, R., Rizza, R.A., Jensen, Delmonico, M.J., Harris, T.B., Visser, M., Park, S.W., Conroy, M.B., Velasquez-Mieyer, M.D., Khosla, S., O’Brien, P., Nair, K.S., 2009. Higher muscle protein synthesis in P., Boudreau, R., Manini, T.M., Nevitt, M., Newman, A.B., Goodpaster, B.H., 2009. women than men across the lifespan, and failure of androgen administration to Longitudinal study of muscle strength, quality, and adipose tissue infiltration. amend age-related decrements. FASEB J. 23, 631–641. Am. J. Clin. Nutr. 90, 1579–1585. Holloszy, J.O., 1967. Biochemical adaptations in muscle. Effects of exercise on Dickinson, J.M., Lee, J.D., Sullivan, B.E., Harber, M.P., Trappe, S.W., Trappe, T.A., 2010. mitochondrial oxygen uptake and respiratory enzyme activity in skeletal A new method to study in vivo protein synthesis in slow- and fast-twitch muscle. J. Biol. Chem. 242, 2278–2282. muscle fibers and initial measurements in humans. J. Appl. Physiol. 108, 1410– Holloszy, J.O., Coyle, E.F., 1984. Adaptations of skeletal muscle to endurance 1416. exercise and their metabolic consequences. J. Appl. Physiol. 56, 831–838. Donoghue, M.J., Alvarez, J.D., Merlie, J.P., Sanes, J.R., 1991. Fiber type- and position- Hoppeler, H., Howald, H., Conley, K., Lindstedt, S.L., Claassen, H., Vock, P., Weibel, dependent expression of a myosin light chain-CAT transgene detected with a E.R., 1985. Endurance training in humans: aerobic capacity and structure of novel histochemical stain for CAT. J. Cell Biol. 115, 423–434. skeletal muscle. J. Appl. Physiol. 59, 320–327. Fielding, R.A., Vellas, B., Evans, W.J., Bhasin, S., Morley, J.E., Newman, A.B., Abellan Houmard, J.A., Weidner, M.L., Gavigan, K.E., Tyndall, G.L., Hickey, M.S., Alshami, A., van Kan, G., Andrieu, S., Bauer, J., Breuille, D., Cederholm, T., Chandler, J., De 1998. Fiber type and citrate synthase activity in the human gastrocnemius and Meynard, C., Donini, L., Harris, T., Kannt, A., Keime Guibert, F., Onder, G., vastus lateralis with aging. J. Appl. Physiol. 85, 1337–1341. 28 A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29

Howald, H., Hoppeler, H., Claassen, H., Mathieu, O., Straub, R., 1985. Influences of Mai, S., Muster, B., Bereiter-Hahn, J., Jendrach, M., 2012. Autophagy proteins LC3B, endurance training on the ultrastructural composition of the different muscle ATG5 and ATG12 participate in quality control after mitochondrial damage and fiber types in humans. Pflug. Arch 403, 369–376. influence lifespan. Autophagy 8, 47–62. Howard, C., Ferrucci, L., Sun, K., Fried, L.P., Walston, J., Varadhan, R., Guralnik, J.M., Masiero, E., Agatea, L., Mammucari, C., Blaauw, B., Loro, E., Komatsu, M., Metzger, D., Semba, R.D., 2007. Oxidative protein damage is associated with poor grip Reggiani, C., Schiaffino, S., Sandri, M., 2009. Autophagy is required to maintain strength among older women living in the community. J. Appl. Physiol. 103, 17– muscle mass. Cell Metab. 10, 507–515. 20. Matsushima, Y., Goto, Y., Kaguni, L.S., 2010. Mitochondrial Lon protease regulates Jaleel, A., Short, K.R., Asmann, Y.W., Klaus, K.A., Morse, D.M., Ford, G.C., Nair, K.S., mitochondrial DNA copy number and transcription by selective degradation of 2008. In vivo measurement of synthesis rate of individual skeletal muscle mitochondrial transcription factor A (TFAM). Proc. Natl. Acad. Sci. USA 107, mitochondrial proteins. Am. J. Physiol. Endocrinol. Metab. 295, E1255–68. 18410–18415. Joseph, A.M., Adhihetty, P.J., Buford, T.W., Wohlgemuth, S.E., Lees, H.A., Nguyen, McGee, S.L., Hargreaves, M., 2004. Exercise and myocyte enhancer factor 2 L.M., Aranda, J.M., Sandesara, B.D., Pahor, M., Manini, T.M., Marzetti, E., regulation in human skeletal muscle. Diabetes 53, 1208–1214. Leeuwenburgh, C., 2012. The impact of aging on mitochondrial function and McGee, S.L., Fairlie, E., Garnham, A.P., Hargreaves, M., 2009. Exercise-induced biogenesis pathways in skeletal muscle of sedentary high- and low-functioning histone modifications in human skeletal muscle. J. Physiol. 587, 5951–5958. elderly individuals. Aging Cell.. Moore, A.Z., Biggs, M.L., Matteini, A., O’Connor, A., McGuire, S., Beamer, B.A., Fallin, Karakelides, H., Irving, B.A., Short, K.R., O’Brien, P., Nair, K.S., 2010. Age, obesity, and M.D., Fried, L.P., Walston, J., Chakravarti, A., Arking, D.E., 2010. Polymorphisms sex effects on insulin sensitivity and skeletal muscle mitochondrial function. in the mitochondrial DNA control region and frailty in older adults. PLoS One 5, Diabetes 59, 89–97. e11069. Kent-Braun, J.A., Ng, A.V., 2000. Skeletal muscle oxidative capacity in young and Morley, J.E., Abbatecola, A.M., Argiles, J.M., Baracos, V., Bauer, J., Bhasin, S., older women and men. J. Appl. Physiol. 89, 1072–1078. Cederholm, T., Coats, A.J., Cummings, S.R., Evans, W.J., Fearon, K., Ferrucci, L., Kim, J.S., Cross, J.M., Bamman, M.M., 2005. Impact of resistance loading on Fielding, R.A., Guralnik, J.M., Harris, T.B., Inui, A., Kalantar-Zadeh, K., Kirwan, myostatin expression and cell cycle regulation in young and older men and B.A., Mantovani, G., Muscaritoli, M., Newman, A.B., Rossi-Fanelli, F., Rosano, women. Am. J. Physiol. Endocrinol. Metab. 288, E1110–9. G.M., Roubenoff, R., Schambelan, M., Sokol, G.H., Storer, T.W., Vellas, B., von Kim, H.S., Patel, K., Muldoon-Jacobs, K., Bisht, K.S., Aykin-Burns, N., Pennington, J.D., Haehling, S., Yeh, S.S., Anker, S.D., 2011. Sarcopenia with limited mobility: an van der Meer, R., Nguyen, P., Savage, J., Owens, K.M., Vassilopoulos, A., Ozden, international consensus. J. Am. Med. Dir. Assoc. 12, 403–409. O., Park, S.H., Singh, K.K., Abdulkadir, S.A., Spitz, D.R., Deng, C.X., Gius, D., 2010. Newman, A.B., Kupelian, V., Visser, M., Simonsick, E., Goodpaster, B., Nevitt, M., SIRT3 is a mitochondria-localized tumor suppressor required for maintenance Kritchevsky, S.B., Tylavsky, F.A., Rubin, S.M., Harris, T.B., 2003. Sarcopenia: of mitochondrial integrity and metabolism during stress. Cancer Cell 17, 41–52. alternative definitions and associations with lower extremity function. J. Am. Konopka, A.R., Douglass, M.D., Kaminsky, L.A., Jemiolo, B., Trappe, T.A., Trappe, S., Geriatr. Soc. 51, 1602–1609. Harber, M.P., 2010. Molecular adaptations to aerobic exercise training in Pandorf, C.E., Haddad, F., Wright, C., Bodell, P.W., Baldwin, K.M., 2009. Differential skeletal muscle of older women. J. Gerontol. A Biol. Sci. Med. Sci. 65, 1201–1207. epigenetic modifications of histones at the myosin heavy chain genes in fast and Konopka, A.R., Trappe, T.A., Jemiolo, B., Trappe, S.W., Harber, M.P., 2011. Myosin slow skeletal muscle fibers and in response to muscle unloading. Am. J. Physiol. heavy chain plasticity in aging skeletal muscle with aerobic exercise training. J. Cell Physiol. 297, C6–16. Gerontol. A Biol. Sci. Med. Sci. 66, 835–841. Pansarasa, O., Castagna, L., Colombi, B., Vecchiet, J., Felzani, G., Marzatico, F., 2000. Kopsidas, G., Kovalenko, S.A., Kelso, J.M., Linnane, A.W., 1998. An age-associated Age and sex differences in human skeletal muscle: role of reactive oxygen correlation between cellular bioenergy decline and mtDNA rearrangements in species. Free Radical Res. 33, 287–293. human skeletal muscle. Mutat. Res. 421, 27–36. Pasiakos, S.M., McClung, H.L., McClung, J.P., Urso, M.L., Pikosky, M.A., Cloutier, G.J., Kovalenko, S.A., Kopsidas, G., Kelso, J.M., Linnane, A.W., 1997. Deltoid human Fielding, R.A., Young, A.J., 2010. Molecular responses to moderate endurance muscle mtDNA is extensively rearranged in old age subjects. Biochem. Biophys. exercise in skeletal muscle. Int. J. Sport Nutr. Exerc. Metab. 20, 282–290. Res. Commun. 232, 147–152. Perry, C.G., Kane, D.A., Lanza, I.R., Neufer, P.D., 2013. Methods for assessing Lanza, I.R., Nair, K.S., 2010. Mitochondrial metabolic function assessed in vivo and mitochondrial function in diabetes. Diabetes 62, 1041–1053. in vitro. Curr. Opin. Clin. Nutr. Metab. Care 13, 511–517. Petersen, K.F., Befroy, D., Dufour, S., Dziura, J., Ariyan, C., Rothman, D.L., DiPietro, L., Lanza, I.R., Short, D.K., Short, K.R., Raghavakaimal, S., Basu, R., Joyner, M.J., Cline, G.W., Shulman, G.I., 2003. Mitochondrial dysfunction in the elderly: McConnell, J.P., Nair, K.S., 2008. Endurance exercise as a countermeasure for possible role in insulin resistance. Science 300, 1140–1142. aging. Diabetes 57, 2933–2942. Picard, M., Ritchie, D., Wright, K.J., Romestaing, C., Thomas, M.M., Rowan, S.L., Lanza, I.R., Bhagra, S., Nair, K.S., Port, J.D., 2011. Measurement of human skeletal Taivassalo, T., Hepple, R.T., 2010. Mitochondrial functional impairment with muscle oxidative capacity by 31P-MR spectroscopy: a cross-validation with aging is exaggerated in isolated mitochondria compared to permeabilized in vitro measurements. J. Magn. Reson Imag. 34, 1143–1150. myofibers. Aging Cell 9, 1032–1046. Lanza, I.R., Zabielski, P., Klaus, K.A., Morse, D.M., Heppelmann, C.J., Bergen 3rd, H.R., Picard, M., Taivassalo, T., Ritchie, D., Wright, K.J., Thomas, M.M., Romestaing, C., Dasari, S., Walrand, S., Short, K.R., Johnson, M.L., Robinson, M.M., Schimke, J.M., Hepple, R.T., 2011. Mitochondrial structure and function are disrupted by Jakaitis, D.R., Asmann, Y.W., Sun, Z., Nair, K.S., 2012. Chronic caloric restriction standard isolation methods. PLoS One 6, e18317. preserves mitochondrial function in senescence without increasing Picca, A., Fracasso, F., Pesce, V., Cantatore, P., Joseph, A.M., Leeuwenburgh, C., mitochondrial biogenesis. Cell Metab. 16, 777–788. Gadaleta, M.N., Lezza, A.M., 2012. Age- and calorie restriction-related changes Larsen, R.G., Callahan, D.M., Foulis, S.A., Kent-Braun, J.A., 2012. Age-related changes in rat brain mitochondrial DNA and TFAM binding. Age (Dordr).. in oxidative capacity differ between locomotory muscles and are associated Proctor, D.N., Sinning, W.E., Walro, J.M., Sieck, G.C., Lemon, P.W., 1995. Oxidative with physical activity behavior. Appl. Physiol. Nutr. Metab. 37, 88–99. capacity of human muscle fiber types: effects of age and training status. J. Appl. Lee, C.K., Klopp, R.G., Weindruch, R., Prolla, T.A., 1999. Gene expression profile of Physiol. 78, 2033–2038. aging and its retardation by caloric restriction. Science 285, 1390–1393. Rasmussen, U.F., Krustrup, P., Kjaer, M., Rasmussen, H.N., 2003a. Experimental Leeuwenburgh, C., Fiebig, R., Chandwaney, R., Ji, L.L., 1994. Aging and exercise evidence against the mitochondrial theory of aging. A study of isolated human training in skeletal muscle: responses of glutathione and antioxidant enzyme skeletal muscle mitochondria. Exp. Gerontol. 38, 877–886. systems. Am. J. Physiol. 267, R439–45. Rasmussen, U.F., Krustrup, P., Kjaer, M., Rasmussen, H.N., 2003b. Human skeletal Leick, L., Wojtaszewski, J.F., Johansen, S.T., Kiilerich, K., Comes, G., Hellsten, Y., muscle mitochondrial metabolism in youth and senescence. no signs of Hidalgo, J., Pilegaard, H., 2008. PGC-1alpha is not mandatory for exercise- and functional changes in ATP formation and mitochondrial oxidative capacity. training-induced adaptive gene responses in mouse skeletal muscle. Am. J. Pflug. Arch 446, 270–278. Physiol. Endocrinol. Metab. 294, E463–74. Raue, U., Slivka, D., Jemiolo, B., Hollon, C., Trappe, S., 2007. Proteolytic gene Leick, L., Lyngby, S.S., Wojtaszewski, J.F., Pilegaard, H., 2010. PGC-1alpha is required expression differs at rest and after resistance exercise between young and old for training-induced prevention of age-associated decline in mitochondrial women. J. Gerontol. A Biol. Sci. Med. Sci. 62, 1407–1412. enzymes in mouse skeletal muscle. Exp. Gerontol. 45, 336–342. Robinson, M.M., Hamilton, K.L., Miller, B.F., 2009. The interactions of some Lexell, J., Henriksson-Larsen, K., Winblad, B., Sjostrom, M., 1983. Distribution of commonly consumed drugs with mitochondrial adaptations to exercise. J. different fiber types in human skeletal muscles: effects of aging studied in Appl. Physiol. 107, 8–16. whole muscle cross sections. Muscle Nerve 6, 588–595. Ronn, T., Poulsen, P., Hansson, O., Holmkvist, J., Almgren, P., Nilsson, P., Tuomi, T., Lezza, A.M., Boffoli, D., Scacco, S., Cantatore, P., Gadaleta, M.N., 1994. Correlation Isomaa, B., Groop, L., Vaag, A., Ling, C., 2008. Age influences DNA methylation between mitochondrial DNA 4977-bp deletion and respiratory chain enzyme and gene expression of COX7A1 in human skeletal muscle. Diabetologia 51, activities in aging human skeletal muscles. Biochem. Biophys. Res. Commun. 1159–1168. 205, 772–779. Rooyackers, O.E., Adey, D.B., Ades, P.A., Nair, K.S., 1996. Effect of age on in vivo rates Ling, C., Poulsen, P., Carlsson, E., Ridderstrale, M., Almgren, P., Wojtaszewski, J., of mitochondrial protein synthesis in human skeletal muscle. Proc. Natl. Acad. Beck-Nielsen, H., Groop, L., Vaag, A., 2004. Multiple environmental and genetic Sci. USA 93, 15364–15369. factors influence skeletal muscle PGC-1alpha and PGC-1beta gene expression in Rosenberg, I., 1989. Summary comments: epidemiological and methodological twins. J. Clin. Invest. 114, 1518–1526. problems in determinig nutritional status of older persons. Am. J. Clin. Nutr. 50, Ling, C., Poulsen, P., Simonsson, S., Ronn, T., Holmkvist, J., Almgren, P., Hagert, P., 1231–1233. Nilsson, E., Mabey, A.G., Nilsson, P., Vaag, A., Groop, L., 2007. Genetic and Rosenberg, I.H., 1997. Sarcopenia: origins and clinical relevance. J. Nutr. 127, 990S– epigenetic factors are associated with expression of respiratory chain 991S. component NDUFB6 in human skeletal muscle. J. Clin. Invest. 117, 3427–3435. Rossi, P., Marzani, B., Giardina, S., Negro, M., Marzatico, F., 2008. Human skeletal Louis, E., Raue, U., Yang, Y., Jemiolo, B., Trappe, S., 2007. Time course of proteolytic, muscle aging and the oxidative system: cellular events. Curr. Aging Sci. 1, 182– cytokine, and myostatin gene expression after acute exercise in human skeletal 191. muscle. J. Appl. Physiol. 103, 1744–1751. A.R. Konopka, K. Sreekumaran Nair / Molecular and Cellular Endocrinology 379 (2013) 19–29 29

Roth, S.M., Martel, G.F., Ferrell, R.E., Metter, E.J., Hurley, B.F., Rogers, M.A., 2003. Trappe, S., Godard, M., Gallagher, P., Carroll, C., Rowden, G., Porter, D., 2001. Myostatin gene expression is reduced in humans with heavy-resistance Resistance training improves single muscle fiber contractile function in older strength training: a brief communication. Exp. Biol. Med. (Maywood) 228, women. Am. J. Physiol. Cell. Physiol. 281, C398–406. 706–709. Trappe, S., Gallagher, P., Harber, M., Carrithers, J., Fluckey, J., Trappe, T., 2003. Single Ruas, J.L., White, J.P., Rao, R.R., Kleiner, S., Brannan, K.T., Harrison, B.C., Greene, N.P., muscle fibre contractile properties in young and old men and women. J. Physiol Wu, J., Estall, J.L., Irving, B.A., Lanza, I.R., Rasbach, K.A., Okutsu, M., Nair, K.S., Yan, 552, 47–58. Z., Leinwand, L.A., Spiegelman, B.M., 2012. A PGC-1alpha isoform induced by Trappe, T., Williams, R., Carrithers, J., Raue, U., Esmarck, B., Kjaer, M., Hickner, R., resistance training regulates skeletal muscle hypertrophy. Cell 151, 1319–1331. 2004. Influence of age and resistance exercise on human skeletal muscle Ryan, A.S., Ivey, F.M., Prior, S., Li, G., Hafer-Macko, C., 2011. Skeletal muscle proteolysis: a microdialysis approach. J. Physiol. 554, 803–813. hypertrophy and muscle myostatin reduction after resistive training in stroke Trappe, S., Hayes, E., Galpin, A.J., Kaminsky, L.A., Jemiolo, B., Fink, W.J., Trappe, T.A., survivors. Stroke 42, 416–420. Jansson, A., Gustafsson, T., Tesch, P.A., 2012. New records in aerobic power Safdar, A., Hamadeh, M.J., Kaczor, J.J., Raha, S., Debeer, J., Tarnopolsky, M.A., 2010. among octogenarian lifelong endurance athletes. J. Appl. Physiol.. Aberrant mitochondrial homeostasis in the skeletal muscle of sedentary older Trounce, I., Byrne, E., Marzuki, S., 1989. Decline in skeletal muscle mitochondrial adults. PLoS One 5, e10778. respiratory chain function: possible factor in ageing. Lancet 1, 637–639. Safdar, A., Little, J.P., Stokl, A.J., Hettinga, B.P., Akhtar, M., Tarnopolsky, M.A., 2011. von Walden, F., Jakobsson, F., Edstrom, L., Nader, G.A., 2012. Altered autophagy gene Exercise increases mitochondrial PGC-1alpha content and promotes nuclear- expression and persistent atrophy suggest impaired remodeling in chronic mitochondrial cross-talk to coordinate mitochondrial biogenesis. J. Biol. Chem. hemiplegic human skeletal muscle. Muscle Nerve 46, 785–792. 286, 10605–10617. Waters, D.L., Mullins, P.G., Qualls, C.R., Raj, D.S., Gasparovic, C., Baumgartner, R.N., Semba, R.D., Ferrucci, L., Sun, K., Walston, J., Varadhan, R., Guralnik, J.M., Fried, L.P., 2009. Mitochondrial function in physically active elders with sarcopenia. Mech. 2007a. Oxidative stress and severe walking disability among older women. Am. Ageing Dev. 130, 315–319. J. Med. 120, 1084–1089. Wenz, T., 2011. Mitochondria and PGC-1alpha in aging and age-associated diseases. Semba, R.D., Ferrucci, L., Sun, K., Walston, J., Varadhan, R., Guralnik, J.M., Fried, L.P., J. Aging Res. 2011, 810619. 2007b. Oxidative stress is associated with greater mortality in older women Wenz, T., Rossi, S.G., Rotundo, R.L., Spiegelman, B.M., Moraes, C.T., 2009. Increased living in the community. J. Am. Geriatr. Soc. 55, 1421–1425. muscle PGC-1alpha expression protects from sarcopenia and metabolic disease Seo, A.Y., Joseph, A.M., Dutta, D., Hwang, J.C., Aris, J.P., Leeuwenburgh, C., 2010. New during aging. Proc. Natl. Acad. Sci. USA 106, 20405–20410. insights into the role of mitochondria in aging: mitochondrial dynamics and Westphal, C.H., Dipp, M.A., Guarente, L., 2007. A therapeutic role for sirtuins in more. J. Cell Sci. 123, 2533–2542. diseases of aging? Trends Biochem. Sci. 32, 555–560. Short, K.R., Vittone, J.L., Bigelow, M.L., Proctor, D.N., Rizza, R.A., Coenen-Schimke, White, A.T., Schenk, S., 2012. NAD(+)/NADH and skeletal muscle mitochondrial J.M., Nair, K.S., 2003. Impact of aerobic exercise training on age-related changes adaptations to exercise. Am. J. Physiol. Endocrinol. Metab. 303, E308–21. in insulin sensitivity and muscle oxidative capacity. Diabetes 52, 1888– Wilkinson, S.B., Phillips, S.M., Atherton, P.J., Patel, R., Yarasheski, K.E., Tarnopolsky, 1896. M.A., Rennie, M.J., 2008. Differential effects of resistance and endurance Short, K.R., Vittone, J.L., Bigelow, M.L., Proctor, D.N., Nair, K.S., 2004. Age and aerobic exercise in the fed state on signalling molecule phosphorylation and protein exercise training effects on whole body and muscle protein metabolism. Am. J. synthesis in human muscle. J. Physiol. 586, 3701–3717. Physiol. Endocrinol. Metab. 286, E92–101. Williamson, D.L., Raue, U., Slivka, D.R., Trappe, S., 2010. Resistance exercise, skeletal Short, K.R., Bigelow, M.L., Kahl, J., Singh, R., Coenen-Schimke, J., Raghavakaimal, S., muscle FOXO3A, and 85-year-old women. J. Gerontol. A Biol. Sci. Med. Sci. 65, Nair, K.S., 2005a. Decline in skeletal muscle mitochondrial function with aging 335–343. in humans. Proc. Natl. Acad. Sci. USA 102, 5618–5623. Wright, D.C., Han, D.H., Garcia-Roves, P.M., Geiger, P.C., Jones, T.E., Holloszy, J.O., Short, K.R., Vittone, J.L., Bigelow, M.L., Proctor, D.N., Coenen-Schimke, J.M., Rys, P., 2007. Exercise-induced mitochondrial biogenesis begins before the increase in Nair, K.S., 2005b. Changes in myosin heavy chain mRNA and protein expression muscle PGC-1alpha expression. J. Biol. Chem. 282, 194–199. in human skeletal muscle with age and endurance exercise training. J. Appl. Zhang, C., Cuervo, A.M., 2008. Restoration of chaperone-mediated autophagy in Physiol. 99, 95–102. aging liver improves cellular maintenance and hepatic function. Nat. Med. 14, Slivka, D.R., Dumke, C.L., Tucker, T.J., Cuddy, J.S., Ruby, B., 2012. Human mRNA 959–965. response to exercise and temperature. Int. J. Sports Med. 33, 94–100. Zucchini, C., Pugnaloni, A., Pallotti, F., Solmi, R., Crimi, M., Castaldini, C., Biagini, G., Someya, S., Yu, W., Hallows, W.C., Xu, J., Vann, J.M., Leeuwenburgh, C., Tanokura, M., Lenaz, G., 1995. Human skeletal muscle mitochondria in aging: lack of Denu, J.M., Prolla, T.A., 2010. Sirt3 mediates reduction of oxidative damage and detectable morphological and enzymic defects. Biochem. Mol. Biol. Int. 37, prevention of age-related hearing loss under caloric restriction. Cell 143, 802– 607–616. 812. Zwetsloot, K.A., Laye, M.J., Booth, F.W., 2009. Novel epigenetic regulation of skeletal Tonkonogi, M., Fernstrom, M., Walsh, B., Ji, L.L., Rooyackers, O., Hammarqvist, F., muscle myosin heavy chain genes. Focus on ‘‘Differential epigenetic Wernerman, J., Sahlin, K., 2003. Reduced oxidative power but unchanged modifications of histones at the myosin heavy chain genes in fast and slow antioxidative capacity in skeletal muscle from aged humans. Pflug. Arch 446, skeletal muscle fibers and in response to muscle unloading’’. Am. J. Physiol. Cell 261–269. Physiol. 297, C1–3. Molecular and Cellular Endocrinology 379 (2013) 30–34

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

The role of weight loss and exercise in correcting skeletal muscle mitochondrial abnormalities in obesity, diabetes and aging

Frederico G.S. Toledo, Bret H. Goodpaster ⇑

Division of Endocrinology and Metabolism, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States article info abstract

Article history: Mitochondria within skeletal muscle have been implicated in insulin resistance of obesity and type 2 dia- Available online 20 June 2013 betes mellitus as well as impaired muscle function with normal aging. Evaluating the potential of interventions to improve mitochondria is clearly relevant to the prevention or treatment of metabolic diseases Keywords: and age-related dysfunction. This review provides an overview and critical evaluation of the effects of Mitochondria weight loss and exercise interventions on skeletal muscle mitochondria, along with implications for insu- Weight loss lin resistance, obesity, type 2 diabetes and aging. The available literature strongly suggests that the lower Exercise mitochondrial capacity associated with obesity, type 2 diabetes and aging is not an irreversible lesion. Physical activity However, weight loss does not appear to affect this response, even when the weight loss is extreme. In Skeletal muscle Obesity contrast, increasing physical activity improves mitochondrial content and perhaps the function of individual mitochondrion. Despite the consistent effect of exercise to improve mitochondrial capacity, studies mechanistically linking mitochondria to insulin resistance, reductions in intramyocellular lipid or improvement in muscle function remain inconclusive. In summary, studies of diet and exercise training have advanced our understanding of the link between mitochondrial oxidative capacity and insulin resistance in obesity, type 2 diabetes and aging. Nevertheless, additional inquiry is necessary to establish the signiﬁcance and clinical relevance of those perturbations, which could lead to targeted therapies for a myriad of conditions and diseases involving mitochondria. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 2. A potential role for mitochondria in the pathophysiology of skeletal muscle insulin resistance Mitochondria within skeletal muscle (SkM) provide energy required for contraction, and by extension, mobility and the ability Despite a great deal of progress that has been made in unravel- to perform physical work. To support this primary role of skeletal ing the mechanisms behind the etiology of IR, its pathophysiology muscle, mitochondria are also critical to other essential myocellu- remains far from clear. This is due in part to the recognized multi- lar functions, including storage and utilization of fuel, primarily plicity of factors that contribute to the development of IR. No single glucose and fatty acids, as well as cell signaling and modulating disturbance exclusively explains the pathophysiology of this con- oxidative stress. Therefore, it is not surprising that mitochondria dition. For instance, both genetic and a number of acquired factors within skeletal muscle have been implicated in aging as well as (e.g., obesity, sedentary lifestyle, and neurohormonal inﬂuences) insulin resistance (IR) of obesity and type 2 diabetes mellitus work in concert to modulate insulin sensitivity in organs such as (T2DM). Targeting mitochondria with interventions to prevent, liver and muscle, which are responsible for partitioning glucose attenuate, or treat aging, age-related diseases and metabolic dis- and maintaining normal plasma glucose homeostasis. Therefore, eases and dysfunction could then have important public health several players participate in the pathophysiology of IR. In the past implications. The primary purpose of this review is to provide an decade, mitochondria have emerged as an additional player in IR objective overview and critical evaluation of studies examining associated with obesity, T2DM, and perhaps aging. the effects of weight loss and exercise interventions on skeletal Although mitochondria have been reported as abnormal or muscle mitochondria, along with implications for deranged energy ‘‘dysfunctional’’ in insulin-resistant states and aging, their exact metabolism, most notably IR, obesity, T2DM and aging. role in glucose homeostasis and fuel metabolism is not completely understood. Nevertheless, in the context of IR of obesity, T2DM and Corresponding author. Address: University of Pittsburgh, 3459 Fifth Avenue, aging, there have been great advances in our understanding of how ⇑ MUH N810, Pittsburgh, PA 15213, United States. Tel.: +1 412 692 2848; fax: +1 412 mitochondria react to weight loss and to physical activity. These 692 2165. E-mail address: [email protected] (B.H. Goodpaster).

0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.06.018 F.G.S. Toledo, B.H. Goodpaster / Molecular and Cellular Endocrinology 379 (2013) 30–34 31 advances have shed light on the relationship between mitochon- ditions are much less clear. Since lipids are oxidized in mitochondria and fuel metabolism. dria, one such factor could theoretically be the overall The objective of this review is to give the reader an overview of mitochondrial capacity of SkM. It has been theorized that if deﬁ- what has been learned from human studies that have investigated cient, a reduced mitochondrial capacity might predispose to lipid how skeletal muscle mitochondria adapt and react to changes in accumulation and thus aggravate IR (Lowell and Shulman, 2005). metabolism induced by diet and exercise in the context of glucose Since the pioneering studies by Randle and colleagues in the metabolism. This review will focus predominantly on human SkM 1960s (Randle et al., 1963) supporting a model of fuel competition studies for two reasons: (a) Reports of abnormal mitochondrial and IR, there have been numerous investigations into the role of capacity in SkM have direct relevance to human disease; and (b) fuel selection in the etiology of muscle IR (for a review see Kelley While the association between mitochondria and obesity-induced and Mandarino, 2000). Many of these earlier studies revealed IR has been replicated numerous times in humans, animal data impairments in fatty acid oxidation, lower mitochondrial enzyme has been less consistent and probably reﬂective of interspecies dif- activities and lower expression of genes related to oxidative capac- ferences between animals and humans. ity within skeletal muscle in obesity and T2DM (Hulver et al., 2003; Kelley et al., 1999; Kim et al., 2000; McGarry, 1995; Simoneau 2.1. Terminology et al., 1999; Mootha et al., 2003; Patti et al., 2003).

Terms such as ‘‘mitochondrial dysfunction’’ (Petersen et al., 2003; Petersen et al., 2004) and ‘‘mitochondrial impairments’’ (Kel- 3. Effects of weight loss and exercise training on mitochondrial ley et al., 2002) have been commonly employed in the obesity, dia- oxidative capacity in insulin-resistant obesity betes, and physiology of aging literature. However, this review favors avoiding those terminologies, because they are potentially While a lower mitochondrial oxidative capacity observed in confusing for a couple of reasons. First, ‘‘dysfunction’’ implies insulin-resistant subjects has been consistently demonstrated, its pathology in SkM, but it is far from established whether altered pathophysiological significance remains unsettled. To date, it is mitochondria reflect a physiological adaptation, pathological mal- not clear whether it represents a pathological defect or a physio- adaptation, or primarily a pathological phenomenon. Second, there logical adaptation. Favoring the notion that it represents a patho- is a multiplicity of mitochondrial functions in cell biology. Mito- logical state, the term ‘‘mitochondrial dysfunction’’ has been chondrial function can be measured in terms of ATP generation, proposed (Petersen et al., 2003; Kelley et al., 2002). In support of substrate oxidation, intracellular calcium buffering, induction of this hypothesis, signs of mitochondrial abnormalities were re- apoptosis, to name a few. Therefore, the term mitochondrial ‘‘func- ported in lean healthy subjects with a parental history of type 2 tion’’ can be imprecise unless clearly defined. In this review, mito- diabetes, raising the possibility of an inheritable etiology (Petersen chondrial function specifically refers to the function of fuel et al., 2004). However, others have proposed that the lower oxida- oxidation. Furthermore and equally important, the total mitochon- tive capacity in IR is not necessarily a form of mitochondrial drial oxidative capacity of a cell depends on not only its total mito- pathology and may represent a consequence of the insulin-resis- chondrial content, but also on the functional capacity of each tant state or stress induced by nutrient overload (Stump et al., mitochondrion (i.e. ‘‘intrinsic mitochondrial function’’). When 2003; Anderson et al., 2009). While it remains unclear whether referring to mitochondrial ‘‘function’’ or ‘‘dysfunction’’, it may not the reduction in oxidative capacity represents either a defect or be always obvious whether it refers to either a deficit in total oxi- an adaptation, the extent to which it is either reversible or an dative capacity, or to the intrinsic function of each mitochondrion. enduring defect has now been well characterized by interventional Since the total oxidative capacity of a cell depends on both content studies that are discussed in this review. Examining how mito- and function of mitochondria, we prefer to employ the term mitochondria respond to weight loss, exercise training, and insulin sen- chondrial capacity, as adopted in previous studies (Toledo et al., sitizers has broadened our understanding of mitochondrial biology 2006; Toledo et al., 2007; Toledo et al., 2008), to denote the global in IR. integrated components of content and function, which is a more One of the earliest studies to suggest that significant mitochon- relevant parameter for cell metabolism. Thus, whenever appropri- drial plasticity is preserved in the obese/insulin-resistant state ate, mitochondrial function per individual mitochondrion or mito- showed that overall oxidative capacity in SkM can be improved chondrial content will be specified. It should also be noted that the by lifestyle modifications typically recommended for obesity term ‘mitochondrial capacity’ reflects the maximal cellular capacity (Menshikova et al., 2005). In a 16-week lifestyle modification pro- for oxidation in SkM. Therefore, the term distinctively does not ap- gram consisting of reduced calorie intake and weekly moderate- ply to situations where measurements occur in sub-maximal con- intensity aerobic training, obese non-diabetic volunteers experi- ditions of metabolic demand (e.g. muscle at rest); in those enced a 9.7% weight loss and significant improvements in whole- instances mitochondrial activity for a given condition, rather than body aerobic capacity, insulin sensitivity and whole-body fat oxi- maximal capacity, is being quantified. dation. In parallel with these changes, total NADH oxidase and succinate oxidase activities were increased after the intervention, 2.2. Are mitochondria abnormal in insulin-resistant skeletal muscle? denoting improved electron-transport chain oxidative capacity and remodeling of mitochondrial oxidative capacity. However, At a cellular level, a common feature of IR is the presence of in- mtDNA content was not significantly increased in response to the creased lipid accumulation in insulin-responsive tissues such as li- intervention, suggesting impaired biogenesis with intact functional ver and SkM. This ectopic fat accumulation helps explain why remodeling of mitochondria. In line with the notion of subnormal obesity and lipodystrophy are associated with IR: when adipose biogenesis, citrate synthase (a surrogate marker of mitochondrial tissue capacity to store lipids is sub-optimal, lipid is ectopically content) was unchanged in half of the participants in that study. stored in SkM and liver, but this ectopic fat disrupts substrate uti- Similarly, citrate synthase was also unchanged in the CALERIE lization in these tissues, leading to IR. While excess intramyocellu- study, in which overweight volunteers underwent either caloric lar lipid (IMCL) has been clearly associated with IR, and excess lipid restriction or caloric restriction with exercise. In both instances, supply to muscle certainly plays a role in conditions of energy ex- citrate synthase activity did not increase (Civitarese et al., 2007). cess, high-fat diet and obesity, the underlying cellular mechanisms The uncertainty surrounding the effect of those interventions for inappropriate accumulation of lipids during physiological con- on mitochondrial content expansion may be in part attributable 32 F.G.S. Toledo, B.H. Goodpaster / Molecular and Cellular Endocrinology 379 (2013) 30–34 to methodological reasons. The tight cytoskeleton apparatus of whether weight loss and exercise training have independent ef- SkM represents a technical challenge for isolation of mitochondria fects upon mitochondrial plasticity. and pure mitochondrial preparations without contaminants are not easy to obtain. Furthermore, mitochondria can be functionally 3.2. Separate effects of weight loss and exercise on mitochondria harmed during isolation methods resulting in potentially mislead- ing results (Picard et al., 2010; Picard et al., 2011), For instance, In normal healthy SkM, exercise training is a potent stimulus for mtDNA has been shown to be a poor marker of mitochondrial con- mitochondrial biogenesis. Therefore, it stands to reason that exer- tent (Larsen et al., 2012). Finally, at least in some instances citrate cise training may be the key factor necessary for stimulating mito- synthase can be regulated by exercise in a manner disconnected to chondrial biogenesis in IR. On the other hand, the distinct mitochondrial content (Tonkonogi et al., 1997). The first unequiv- possibility that mitochondrial dysfunction may be secondary to ex- ocal demonstration that mitochondrial content expands in re- cess lipid accumulation in SkM has also been proposed and thus sponse to lifestyle modifications in obesity/IR was made possible weight loss per se might also conceivably influence mitochondrial by studies employing transmission electron microscopy with dysfunction. In a study that compared the relative contribution of quantitative stereologic analysis (Toledo et al., 2006). The advan- exercise training versus that of weight loss, overweight and obese tage of that approach is that mitochondrial content is measured subjects with insulin resistance were randomized to either diet-in- in situ, therefore overcoming the issues of preparation purity, func- duced weight loss, or diet-induced weight loss combined with tional damage, and modulation of enzyme activity independent of exercise training (Toledo et al., 2008). Both groups experienced enzyme content. Using that approach, it was found that SkM mito- comparable degrees of total body weight loss, fat mass loss, and chondrial content increased on average by 42% after weight loss comparable improvements in SkM insulin sensitivity. However, and exercise training (Toledo et al., 2006). Mitochondrial morphol- only the group that combined training with caloric restriction ogy was also affected, noted by a 19% increase in mitochondrial experienced an improvement in mitochondrial content and respi- size. In that study, both mitochondrial content and size strongly ratory chain enzymatic activity, demonstrating that exercise train- correlated with the degree of change in insulin sensitivity ing, not weight loss, is the key factor responsible for mitochondrial (r = 0.72 and r = 0.68, respectively). While not necessarily suggest- plasticity. Subsequent studies have confirmed that exercise training causality, it reinforced the notion of a tight link between mito- ing can increase SkM mitochondrial content in insulin-resistant chondria and IR. However, as will be discussed later in this review, subjects with and without T2DM (Meex et al., 2010; Phielix the link between mitochondria and insulin resistance can some- et al., 2010). times be dissociated. Although it has now been well established that exercise training per se improves mitochondrial content in IR, it is less clear 3.1. Intervention effects in T2DM whether intrinsic mitochondrial function improves too. In studies from our group, exercise training for 16–20 weeks resulted in en- In the spectrum of obesity and IR, the most severe impairments hanced electron transport chain activity that exceeded changes in in mitochondrial content seem to occur in T2DM (Chomentowski mitochondrial mtDNA content and citrate synthase activity, sug- et al., 2011). Subjects with T2DM also display the greatest degrees gesting that training results in improvements in total oxidative of derangements in insulin sensitivity in SkM and the greatest capacity that exceed that expected from an increase in mitochon- IMCL lipid accumulation. They also have hyperglycemia, which drial content alone (Toledo et al., 2007; Menshikova et al., 2005; might conceivably be another insult to mitochondria. But as dem- Toledo et al., 2008). However, at least one study concluded that onstrated by interventional studies, even subjects with T2DM re- intrinsic function per mitochondrion does not improve with train- tain a substantial capacity for increasing mitochondrial ing: subjects with T2DM and BMI-matched control individuals biogenesis in response to simple lifestyle modifications. In a study enrolled in a 12-week exercise program and mitochondrial func- involving subjects with T2DM, a 4-month intervention consisting tion was assessed by high-resolution respirometry in permeabili- of diet to achieve weight loss and daily exercise training resulted zed muscle fibers (Phielix et al., 2010). When mitochondrial in robust improvements in SkM mitochondrial oxidative capacity respiratory rates were adjusted for mtDNA content, no improve- (Toledo et al., 2007). Mitochondrial content measured by quantita- ments in mitochondrial function after exercise training were ob- tive TEM, citrate synthase, and the activity of NADH oxidase were served. However, compared to earlier studies (Toledo et al., all increased by the intervention. While many factors unrelated to 2006; Toledo et al., 2007; Menshikova et al., 2005; Toledo et al., mitochondria are responsible for improvements in blood glucose 2008), this study had a relative shorter duration of training levels in diabetic subjects after that type of intervention, that study (12 weeks versus 16–20 weeks), a lower exercise training dose, also reported a relationship between changes in SkM mitochondria and included a combination of aerobic and resistance exercise. and hyperglycemia as measured by HbA1c. In line with this obser- It is likely that a certain threshold of duration of aerobic training, vation, Fritz et al. reported that skeletal muscle adaptations of in- exercise frequency and intensity, or a combination of these, must creased oxidative gene expression induced by low-intensity be achieved in order to enhance intrinsic mitochondrial function, exercise are a correlate of systemic metabolic improvements in and may explain differences in the training response among stud- type 2 diabetes, including insulin sensitivity (Fritz et al., 2006). ies. Therefore, a conservative interpretation of the current studies While causality is difficult to establish with those types of observa- published so far is that intrinsic mitochondrial function does not tions, those studies reveal a still poorly-understood relationship always improve, but may do so in certain circumstances depend- between mitochondria in SkM and systemic metabolism. ing on the type and duration of training, and the frequency and The aforementioned studies have collectively demonstrated intensity of exercise bouts. Clearly, more research is needed in that in IR mitochondria are not irreversibly impaired, and that at this field in order to establish what the minimum exercise train- least partial amelioration of this abnormality can be achieved by ing prescription should be in order to achieve enhancement of lifestyle modifications that are typically recommended to adults intrinsic mitochondrial function. with IR. However, it is also important to consider that both dietary The effects of weight loss in improving obesity-related insulin caloric restriction and exercise are stimuli for a negative caloric resistance are well established, but less is known about the impact balance and that while weight loss may reduce IMCL lipid content, of weight loss on SkM mitochondria. This relatively paucity of data chronic exercise increases it (Dube et al., 2008; Goodpaster et al., is not surprising since mitochondrial abnormalities in obesity/IR 2001; Pruchnic et al., 2004). Therefore, it is important to consider have only been appreciated relatively recently. The majority of F.G.S. Toledo, B.H. Goodpaster / Molecular and Cellular Endocrinology 379 (2013) 30–34 33 studies published so far show that weight loss by dietary caloric 4. Effects of weight loss and exercise on mitochondria in aging restriction does not reverse the lower mitochondrial capacity observed in IR, including in response to degrees of weight loss Regardless of whether or not mitochondria are mechanistically deemed clinically significant for glucose and lipid metabolism. In linked with IR, there is little argument that mitochondria likely an older study, Kern et al. suggested an increase in succinate dehy- play an important role in altered energy metabolism with aging. drogenase (SDH) activity in SkM of obese women who had under- The effects of exercise to increase mitochondria content and capac- gone moderate weight loss (Kern et al., 1999). However, it is not ity in older humans are also generally very consistent (Pruchnic entirely clear if the SDH activity increased in response to concom- et al., 2004; Jubrias et al., 2001; Menshikova et al., 2006; Orlander itant changes in physical activity since the study did not control for and Aniansson, 1980; Rimbert et al., 2004; Short et al., 2003; physical activity and no objective metric of aerobic capacity was Waters et al., 2003). However, the separate effect of weight loss reported. Simoneau et al. studied muscle biopsy samples from ob- and exercise on mitochondria content and performance in older ese volunteers who experienced significant weight loss (14–16% of humans is not known. Indeed, there is some evidence to suggest baseline weight) and observed no improvements in enzymatic that aging is associated with a blunted response to interventions markers of mitochondria, namely citrate synthase and CPT-I, sug- known to increase mitochondria (Reznick et al., 2007). gesting no effect on mitochondria (Simoneau et al., 1999). Cyto- A decline in mitochondria content and/or function often paral- chrome oxidase and beta-hydroxyacyl CoA dehydrogenase lels the loss of muscle mass and function with age. Only until re- activities decreased significantly in women, but not in men, leaving cently, however, have studies been conducted – in cell systems some doubt about the uniformity of mitochondrial response to and animal models – to provide mechanistic evidence linking weight loss. mitochondria with muscle growth/atrophy signaling, autophagy We have conducted a more comprehensive assessment of the and sarcopenia (Masiero et al., 2009; Romanello et al., 2010; Wenz impact of weight loss per se on both mitochondrial content and et al., 2009). A conundrum for older obese men and women is that function in the insulin resistant state associated with obesity (To- traditional diet-induced weight loss programs can result in not ledo et al., 2008). Non-diabetic insulin resistant obese subjects lost only loss of adipose tissue but also a significant loss of muscle 10.8% of their initial weight with moderate caloric restriction, (Chomentowski et al., 2009). How this potentially translates into which was associated with a nearly 30% improvement in insulin further decrements in muscle function is not clear. sensitivity. Subjects were instructed not to change their levels of The CALERIE study reported that energy restriction-induced physical activity and, accordingly, maximal aerobic capacity did weight loss increased expression of oxidative phosphorylation not change. Despite marked weight loss and improved insulin sen- genes in SkM (Civitarese et al., 2007), although the related enzyme sitivity, mitochondrial content did not change as assessed by quan- activities were unchanged. Moreover, the average age of their sub- titative TEM. Other markers of mitochondrial content, such as jects was <60 years old (39–41). Subjects in the CALERIE study mtDNA copy number and cardiolipin content (a marker of inner- were not obese, and thus not representative of the typical obese mitochondrial membrane mass), were not changed either. NADH- insulin-resistant phenotype in which a lower mitochondrial oxida- oxidase was also unaffected, indicating no changes in mitochon- tive capacity has been mostly reported. We have reported in a ran- drial function. The lack of changes in mitochondrial content and domized trial that diet-induced weight loss fails to increase function occurred in spite of a robust decrease in IR in SkM. These markers of mitochondrial oxidative capacity in overweight to ob- observations suggest that the lower oxidative capacity in obesity/ ese older men and women (Dube et al., 2011). This was in contrast IR cannot be explained solely as consequence of IR. This observa- to an improvement in mitochondrial enzyme activity in those who tion is relevant because experimental evidence suggests that acute completed an exercise-training program without significant insulin signaling in SkM lowers mitochondrial oxidative capacity weight loss. Although further evidence is clearly needed to deter- (Asmann et al., 2006). mine the effects of weight loss on mitochondria in the elderly, Another important finding from this study was that mitochon- the limited evidence is consistent with that observed for middle- drial oxidative capacity did not change in spite of a reduction in aged men adults. Based on the high prevalence of older adults IMCL content, and therefore suggests that the lower oxidative attempting to lose weight with dieting, it is imperative to provide capacity in obesity/IR is unlikely to be a consequence of excess li- additional objective evidence concerning the pros and cons of pid accumulation per se. Alternatively, the same result could be weight loss in this population. interpreted as an indication that perhaps greater degrees of caloric restriction are necessary for mitochondrial recovery. This does not, however, seem to the case. In a study by Berggren and colleagues, 5. Conclusions mitochondrial fatty acid oxidation was measured by incubation of skeletal muscle homogenates with [1-14C]palmitate and measuring In summary, the lower mitochondrial capacity associated with 14 CO2 production (Berggren et al., 2008). Mitochondrial fatty acid obesity and T2DM is not an irreversible lesion: substantial plastic- oxidation in obese women who had marked weight loss (about ity of mitochondrial biogenesis is retained in the insulin resistant 50 kg) was similar to that of extremely obese subjects. Moreover, state. However, weight loss does not appear to significantly affect SkM fatty acid oxidation did not change in extremely obese women this response, even when the weight loss is extreme. In contrast, after 1 year of weight loss (mean 55 kg lost). There were also no increasing physical activity improves mitochondrial content and changes in mRNA content for PDK4, CPT I, and PGC-1a, suggesting perhaps oxidative function of individual mitochondrion as well. that the transcriptional program for mitochondrial biogenesis is These biological responses are not triggered by amelioration of not activated by weight loss. Therefore, even significantly profound the insulin-resistant state or a reduction in IMCL content, but likely weight loss does not seem to result in amelioration of mitochon- a reflection of a concerted biological response to chronic contrac- drial oxidative capacity. In fact, some studies suggest that oxidative tile activity induced by exercise. pathways may even be reduced by weight loss. After biliopancreat- Studies of diet and exercise training have advanced our under- ic diversion surgery for morbid obesity, weight loss reduced the standing of the link between perturbations in mitochondrial oxida- expression of peroxisome proliferator-activated receptor-a tive capacity and the insulin resistant state seen in obesity, T2DM, ( 46.7%), carnitine palmitoyltransferase-1B ( 43.1%), acyl-CoA and aging. Nevertheless, a complete picture of the role of mito- À À oxidase 1 ( 37.8%), and acetyl-CoA carboxylase B ( 48.7%) (Fabris chondria in T2DM and aging remains a work in progress. More À À et al., 2004). studies are necessary to establish the relative impact and 34 F.G.S. Toledo, B.H. Goodpaster / Molecular and Cellular Endocrinology 379 (2013) 30–34 significance of those mitochondrial perturbations, which could Menshikova, E.V., Ritov, V.B., Toledo, F.G., Ferrell, R.E., Goodpaster, B.H., Kelley, D.E., lead to targeted therapies for these conditions. 2005. Effects of weight loss and physical activity on skeletal muscle mitochondrial function in obesity. Am. J. Physiol. Endocrinol. Metab. 288, E818. Menshikova, E.V., Ritov, V.B., Fairfull, L., Ferrell, R.E., Kelley, D.E., Goodpaster, B.H., References 2006. Effects of exercise on mitochondrial content and function in aging human skeletal muscle. J. Gerontol. Ser. A – Biol. Sci. Med. Sci. 61, 534–540. Mootha, V.K., Lindgren, C.M., Eriksson, K.F., Subramanian, A., Sihag, S., Lehar, J., Anderson, E.J., Lustig, M.E., Boyle, K.E., Woodlief, T.L., Kane, D.A., Lin, C.T., Price 3rd, Puigserver, P., Carlsson, E., Ridderstrale, M., Laurila, E., Houstis, N., Daly, M.J., J.W., Kang, L., Rabinovitch, P.S., Szeto, H.H., Houmard, J.A., Cortright, R.N., Patterson, N., Mesirov, J.P., Golub, T.R., Tamayo, P., Spiegelman, B., Lander, E.S., Wasserman, D.H., Neufer, P.D., 2009. Mitochondrial H O emission and cellular 2 2 Hirschhorn, J.N., Altshuler, D., Groop, L.C., 2003. PGC-1alpha-responsive genes redox state link excess fat intake to insulin resistance in both rodents and involved in oxidative phosphorylation are coordinately downregulated in humans. J. Clin. Invest. 119, 573–581. human diabetes. Nat. Genet. 34, 267–273. Asmann, Y.W., Stump, C.S., Short, K.R., Coenen-Schimke, J.M., Guo, Z., Bigelow, M.L., Orlander, J., Aniansson, A., 1980. Effect of physical training on skeletal muscle metabolism Nair, K.S., 2006. Skeletal muscle mitochondrial functions, mitochondrial DNA and ultrastructure in 70 to 75-year-old men. Acta Physiol. Scand. 109, 149–154. copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic Patti, M.E., Butte, A.J., Crunkhorn, S., Cusi, K., Berria, R., Kashyap, S., Miyazaki, Y., Kohane, I., subjects at equal levels of low or high insulin and euglycemia. Diabetes 55, Costello, M., Saccone, R., Landaker, E.J., Goldfine, A.B., Mun, E., DeFronzo, R., Finlayson, 3309–3319. J., Kahn, C.R., Mandarino, L.J., 2003. Coordinated reduction of genes of oxidative Berggren, J.R., Boyle, K.E., Chapman, W.H., Houmard, J.A., 2008. Skeletal muscle lipid metabolism in humans with insulin resistance and diabetes: potential role of PGC1 oxidation and obesity: influence of weight loss and exercise. Am. J. Physiol. and NRF1. Proc. Nat. Acad. Sci. USA 100, 8466–8471. Endocrinol. Metab. 294, E726–E732. Petersen, K.F., Befroy, D., Dufour, S., Dziura, J., Ariyan, C., Rothman, D.L., DiPietro, Chomentowski, P., Dube, J.J., Amati, F., Stefanovic-Racic, M., Zhu, S., Toledo, F.G.S., L., Cline, G.W., Shulman, G.I., 2003. Mitochondrial dysfunction in the elderly: Goodpaster, B.H., 2009. Moderate exercise attenuates the loss of skeletal muscle possible role in insulin resistance. Science 300, 1140–1142. mass that occurs with intentional caloric restriction-induced weight loss in older, Petersen, K.F., Dufour, S., Befroy, D., Garcia, R., Shulman, G.I., 2004. Impaired overweight to obese adults. J. Gerontol. Ser. A – Biol. Sci. Med. Sci. 64, 575–580. mitochondrial activity in the insulin-resistant offspring of patients with type 2 Chomentowski, P., Coen, P.M., Radikova, Z., Goodpaster, B.H., Toledo, F.G., 2011. diabetes. N. Engl. J. Med. 350, 664–671. Skeletal muscle mitochondria in insulin resistance: differences in Phielix, E., Meex, R., Moonen-Kornips, E., Hesselink, M.K., Schrauwen, P., 2010. intermyofibrillar versus subsarcolemmal subpopulations and relationship to Exercise training increases mitochondrial content and ex vivo mitochondrial metabolic flexibility. J. Clin. Endocrinol. Metab. 96, 494–503. function similarly in patients with type 2 diabetes and in control individuals. Civitarese, A.E., Carling, S., Heilbronn, L.K., Hulver, M.H., Ukropcova, B., Deutsch, Diabetologia 53, 1714–1721. W.A., Smith, S.R., Ravussin, E., Team, C.P., 2007. Calorie restriction increases Picard, M., Ritchie, D., Wright, K.J., Romestaing, C., Thomas, M.M., Rowan, S.L., muscle mitochondrial biogenesis in healthy humans. PLoS Med. 4, e76. Taivassalo, T., Hepple, R.T., 2010. Mitochondrial functional impairment with Dube, J.J., Amati, F., Stefanovic-Racic, M., Toledo, F.G.S., Sauers, S.E., Goodpaster, B.H., aging is exaggerated in isolated mitochondria compared to permeabilized 2008. Exercise-induced alterations in intramyocellular lipids and insulin myofibers. Aging Cell 9, 1032–1046. resistance: the athlete’s paradox revisited. Am. J. Physiol. Endocrinol. Metab. Picard, M., Taivassalo, T., Ritchie, D., Wright, K.J., Thomas, M.M., Romestaing, C., 294, E882. Hepple, R.T., 2011. Mitochondrial structure and function are disrupted by Dube, J.J., Amati, F., Toledo, F.G., Stefanovic-Racic, M., Rossi, A., Coen, P., Goodpaster, standard isolation methods. PLoS ONE 6, e18317. B.H., 2011. Effects of weight loss and exercise on insulin resistance, and Pruchnic, R., Katsiaras, A., He, J., Kelley, D.E., Winters, C., Goodpaster, B.H., 2004. intramyocellular triacylglycerol, diacylglycerol and ceramide. Diabetologia 54, Exercise training increases intramyocellular lipid and oxidative capacity in 1147–1156. older adults. Am. J. Physiol. Endocrinol. Metab. 287, E857–E862. Fabris, R., Mingrone, G., Milan, G., Manco, M., Granzotto, M., Pozza, A.D., Scarda, A., Randle, P.J., Garland, P.B., Hales, C.N., Newsholme, E.A., 1963. The glucose fatty acid Serra, R., Greco, A.V., Federspil, G., Vettor, R., 2004. Further lowering of muscle cycle: its role in insulin sensitivity and the metabolic didturbances of diabetes lipid oxidative capacity in obese subjects after biliopancreatic diversion. J. Clin. mellitus. Lancet 1, 785–789. Endocrinol. Metab. 89, 1753–1759. Reznick, R.M., Zong, H., Li, J., Morino, K., Moore, I.K., Yu, H.J., Liu, Z.X., Dong, J., Fritz, T., Kramer, D.K., Karlsson, H.K., Galuska, D., Engfeldt, P., Zierath, J.R., Krook, A., Mustard, K.J., Hawley, S.A., Befroy, D., Pypaert, M., Hardie, D.G., Young, L.H., 2006. Low-intensity exercise increases skeletal muscle protein expression of Shulman, G.I., 2007. Aging-associated reductions in AMP-activated protein PPARdelta and UCP3 in type 2 diabetic patients. Diabetes Metab. Res. Rev. 22, kinase activity and mitochondrial biogenesis. Cell Metab. 5, 151–156. 492–498. Rimbert, V., Boirie, Y., Bedu, M., Hocquette, J.-F., Ritz, P., Morio, B., 2004. Muscle fat Goodpaster, B.H., He, J., Watkins, S., Kelley, D.E., 2001. Skeletal muscle lipid content oxidative capacity is not impaired by age but by physical inactivity: association and insulin resistance: evidence for a paradox in endurance-trained athletes. J. with insulin sensitivity. FASEB J. 18, 737–739. Clin. Endocrinol. Metab. 86, 5755–5761. Romanello, V., Guadagnin, E., Gomes, L., Roder, I., Sandri, C., Petersen, Y., Milan, G., Hulver, M.W., Berggren, J.R., Cortright, R.N., Dudek, R.W., Thompson, R.P., Pories, Masiero, E., Del Piccolo, P., Foretz, M., Scorrano, L., Rudolf, R., Sandri, M., 2010. W.J., MacDonald, K.G., Cline, G.W., Shulman, G.I., Dohm, G.L., Houmard, J.A., Mitochondrial fission and remodelling contributes to muscle atrophy. EMBO J. 2003. Skeletal muscle lipid metabolism with obesity. Am. J. Physiol. Endocrinol. 29, 1774–1785. Metab. 284, E741. Short, K.R., Vittone, J.L., Bigelow, M.L., Proctor, D.N., Rizza, R.A., Coenen-Schimke, Jubrias, S.A., Esselman, P.C., Price, L.B., Cress, M.E., Conley, K.E., 2001. Large energetic J.M., Nair, K.S., 2003. Impact of aerobic exercise training on age-related changes adaptations of elderly muscle to resistance and endurance training. J. Appl. in insulin sensitivity and muscle oxidative capacity. Diabetes 52, 1888–1896. Physiol. 90, 1663–1670. Simoneau, J.A., Veerkamp, J.H., Turcotte, L.P., Kelley, D.E., 1999. Markers of capacity Kelley, D.E., Mandarino, L.J., 2000. Fuel selection in human skeletal muscle in insulin to utilize fatty acids in human skeletal muscle: relation to insulin resistance and resistance: a reexamination. Diabetes 49, 677–683. obesity and effects of weight loss. FASEB J. 13, 2051–2060. Kelley, D.E., Goodpaster, B., Wing, R.R., Simoneau, J.A., 1999. Skeletal muscle fatty Stump, C.S., Short, K.R., Bigelow, M.L., Schimke, J.M., Nair, K.S., 2003. Effect of insulin acid metabolism in association with insulin resistance, obesity, and weight loss. on human skeletal muscle mitochondrial ATP production, protein synthesis, Am. J. Physiol. 277, E1130. and mRNA transcripts. Proc. Natl. Acad. Sci. USA 100, 7996–8001. Kelley, D., He, J., Menshikova, E., Ritov, V., 2002. Dysfunction of mitochondria in Toledo, F.G., Watkins, S., Kelley, D.E., 2006. Changes induced by physical activity and human skeletal muscle in type 2 diabetes mellitus. Diabetes 51, 2944–2950. weight loss in the morphology of intermyofibrillar mitochondria in obese men Kern, P.A., Simsolo, R.B., Fournier, M., 1999. Effect of weight loss on muscle fiber and women. J. Clin. Endocrinol. Metab. 91, 3224–3227. type, fiber size capillarity, and succinate dehydrogenase activity in humans. J. Toledo, F.G., Menshikova, E.V., Ritov, V.B., Azuma, K., Radikova, Z., DeLany, J., Kelley, Clin. Endocrinol. Metab. 84, 4185–4190. D.E., 2007. Effects of physical activity and weight loss on skeletal muscle Kim, J.Y., Hickner, R.C., Cortright, R.L., Dohm, G.L., Houmard, J.A., 2000. Lipid mitochondria and relationship with glucose control in type 2 diabetes. Diabetes oxidation is reduced in obese human skeletal muscle. Am. J. Physiol. Endocrinol. 56, 2142–2147. Metab. 279, E1039. Toledo, F.G., Menshikova, E.V., Azuma, K., Radikova, Z., Kelley, C.A., Ritov, V.B., Larsen, S., Nielsen, J., Hansen, C.N., Nielsen, L.B., Wibrand, F., Stride, N., Schroder, Kelley, D.E., 2008. Mitochondrial capacity in skeletal muscle is not stimulated H.D., Boushel, R., Helge, J.W., Dela, F., Hey-Mogensen, M., 2012. Biomarkers of by weight loss despite increases in insulin action and decreases in mitochondrial content in skeletal muscle of healthy young human subjects. J. intramyocellular lipid content. Diabetes 57, 987–994. Physiol. 590, 3349–3360. Toledo, F.G.S., Menshikova, E.V., Azuma, K., Radikova, Z., Kelley, C.A., Ritov, V.B., Lowell, B.B., Shulman, G.I., 2005. Mitochondrial dysfunction and type 2 diabetes. Kelley, D.E., 2008. Mitochondrial capacity in skeletal muscle is not stimulated Science 307, 384–387. by weight loss despite increases in insulin action and decreases in Masiero, E., Agatea, L., Mammucari, C., Blaauw, B., Loro, E., Komatsu, M., Metzger, D., intramyocellular lipid content. Diabetes 57, 987–994. Reggiani, C., Schiaffino, S., Sandri, M., 2009. Autophagy is required to maintain Tonkonogi, M., Harris, B., Sahlin, K., 1997. Increased activity of citrate synthase in muscle mass. Cell Metab. 10, 507–515. human skeletal muscle after a single bout of prolonged exercise. Acta Physiol. McGarry, J.D., 1995. The mitochondrial carnitine palmitoyl transerase system: its Scand. 161, 435–436. broadening role in fuel homeostasis and new insights into its molecular Waters, D.L., Brooks, W.M., Qualls, C.R., Baumgartner, R.N., 2003. Skeletal muscle features. Biochem. Soc. Trans. 23, 321–324. mitochondrial function and lean body mass in healthy exercising elderly. Mech. Meex, R.C., Schrauwen-Hinderling, V.B., Moonen-Kornips, E., Schaart, G., Mensink, Ageing Dev. 124, 301–309. M., Phielix, E., van de Weijer, T., Sels, J.P., Schrauwen, P., Hesselink, M.K., 2010. Wenz, T., Rossi, S.G., Rotundo, R.L., Spiegelman, B.M., Moraes, C.T., 2009. Restoration of muscle mitochondrial function and metabolic flexibility in type 2 Increased muscle PGC-1alpha expression protects from sarcopenia and diabetes by exercise training is paralleled by increased myocellular fat storage metabolic disease during aging. Proc. Natl. Acad. Sci. USA 106, 20405–20410. and improved insulin sensitivity. Diabetes 59, 572–579. Molecular and Cellular Endocrinology 379 (2013) 35–42

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review Hepatic energy metabolism in human diabetes mellitus, obesity and non-alcoholic fatty liver disease

a a,b, Chrysi Koliaki , Michael Roden ⇑ a Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research, Heinrich Heine University, Düsseldorf, Germany b Division of Endocrinology and Diabetology and Metabolic Diseases, University Clinics Düsseldorf, Düsseldorf, Germany article info abstract

Article history: Alterations of hepatic mitochondrial function have been observed in states of insulin resistance and non- Available online 12 June 2013 alcoholic fatty liver disease (NAFLD). Patients with overt type 2 diabetes mellitus (T2DM) can exhibit reduction in hepatic adenosine triphosphate (ATP) synthesis and impaired repletion of their hepatic Keywords: ATP stores upon ATP depletion by fructose. Obesity and NAFLD may also associate with impaired ATP Mitochondrion recovery after ATP-depleting challenges and augmented oxidative stress in the liver. On the other hand, Steatosis patients with obesity or NAFLD can present with upregulated hepatic anaplerotic and oxidative ﬂuxes, Non-alcoholic steatohepatitis (NASH) including b-oxidation and tricarboxylic cycle activity. The present review focuses on the methods and Lipotoxicity data on hepatic energy metabolism in various states of human insulin resistance. We propose that the liver can adapt to increased lipid exposition by greater lipid storing and oxidative capacity, resulting in increased oxidative stress, which in turn could deteriorate hepatic mitochondrial function in chronic insulin resistance and NAFLD. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 35 2. Literature search...... 36 3. Assessment of hepatic energy metabolism...... 36 3.1. In vitro methods ...... 36 3.2. In vivo methods...... 36 3.3. Ex vivo methods ...... 37 4. The physiological role of liver energy metabolism and mitochondrial function ...... 37 5. Liver mitochondrial function in healthy humans...... 38 6. Liver mitochondrial function in T2DM ...... 39 7. Liver mitochondrial function in obesity and steatosis ...... 39 8. Liver mitochondrial function in advanced NAFLD ...... 40 9. Conclusions...... 40 Acknowledgements ...... 41 References ...... 41

1. Introduction hepatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, and finally liver cirrhosis and hepatocellular carcinoma (Angulo, Non-alcoholic fatty liver disease (NAFLD) comprises a broad 2002; Smith and Adams, 2011). Hepatic steatosis is defined as spectrum of chronic liver diseases, ranging from uncomplicated intrahepatic fat content above 5.5% (Browning et al., 2004; Roden, 2006) and represents a clinical finding typically coexisting with obesity, while NASH and other forms of advanced NAFLD are char- Corresponding author. Address: Institute for Clinical Diabetology, German ⇑ Diabetes Center, Leibniz Center for Diabetes Research, Heinrich Heine University, acterized by histological signs of inflammation and fibrosis (Roden, Auf’m Hennekamp 65, 40225 Düsseldorf, Germany. Tel.: +49 211 3382 201. 2006; Smith and Adams, 2011). As liver biopsies are not routinely E-mail addresses: [email protected] (C. Koliaki), Michael.- performed, only rough estimates of the prevalence of NAFLD are [email protected] (M. Roden).

0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.06.002 36 C. Koliaki, M. Roden / Molecular and Cellular Endocrinology 379 (2013) 35–42 available, which range from 3% to 30%, with NASH being present in ergy metabolism and insulin resistance’’, ‘‘hepatic energy metabo- approximately one third of all cases (Clark, 2006; Smith and lism and NAFLD’’, ‘‘hepatic energy metabolism and T2DM’’, Adams, 2011). Even higher estimates are suggested for insulin ‘‘hepatic energy metabolism and obesity’’, ‘‘hepatic energy metab- resistant cohorts such as patients with type 2 diabetes mellitus olism and liver steatosis’’, ‘‘hepatic mitochondrial function and (T2DM) or severe obesity, suggesting that these entities and NAFLD insulin resistance’’, ‘‘hepatic mitochondrial function and NAFLD’’. share common pathogenic mechanisms (Roden, 2006). NAFLD re- Full-text articles and reference lists of selected review papers were sults from the dynamic interplay of increased lipid influx into the critically reviewed. Our literature search strategy was restricted to liver, increased de novo hepatic lipogenesis and defective lipid uti- studies in humans, but a limited number of mechanistic studies lization, which will stimulate hepatic lipid accumulation. Chronic using animal models of insulin resistance and NAFLD were also re- dietary overload with fructose and saturated fatty acids, will also viewed and are briefly discussed herein. enhance accumulation of lipid metabolites along with oxidative and endoplasmic reticulum stress and release of cytokines, and 3. Assessment of hepatic energy metabolism thereby foster NAFLD progression (Krebs and Roden, 2004; Roden, 2006; Smith and Adams, 2011). Liver mitochondrial function can be evaluated directly or indi- Insulin resistance is tightly associated with ectopic fat accumu- rectly by in vitro, in vivo and ex vivo techniques. lation in peripheral tissues, including skeletal muscle and liver as the most important sites (Roden, 2005; Szendroedi and Roden, 2009). For skeletal muscle, increased intramyocellular fat content, 3.1. In vitro methods specifically increased lipid availability, promotes insulin resistance through several mechanisms including diacylglycerol (DAG) acti- The in vitro methods include measurements of mitochondrial vation of novel protein kinase C (PKC) isoforms, leading to im- mass and functionality in biopsy-derived liver samples. They com- paired insulin-stimulated glucose transport and muscle glycogen prise mitochondrial membrane potential and proton leak kinetics, synthesis (Roden, 2004). Insulin resistant humans such as patients assessment of mitochondrial content by ultrastructural observa- with T2DM and first-degree relatives of T2DM patients, may fur- tions, citrate synthase activity and ratio of mitochondrial relative ther show impaired mitochondrial function in muscle, character- to nuclear DNA, polarographic determination of oxygen consump- ized by lower flux through adenosine triphosphate (ATP) tion rates, enzyme activities of mitochondrial respiratory com- synthase under basal and insulin-stimulated conditions (Petersen plexes I–V, markers of oxidative stress such as mitochondrial et al., 2005; Szendroedi et al., 2007). These abnormalities have production of superoxide anion and lipid peroxidation products, been mainly attributed to decreased mitochondrial content rather and anti-oxidant capacity such as superoxide dismutase specific than to an inherent impairment of mitochondrial functionality activity and reduced to oxidized glutathione ratio (Bouderba (Boushel et al., 2007; Morino et al., 2005). Whether impaired mito- et al., 2012; García-Ruiz et al., 1995; Pérez-Carreras et al., 2003; chondrial function is causally associated with insulin resistance Raffaella et al., 2008; Vendemiale et al., 2001; Vial et al., 2011). and how intramyocellular lipids modulate mitochondrial substrate oxidation remains a matter of debate, because recent data support 3.2. In vivo methods a dissociation of muscle mitochondrial function from insulin sensitivity (Asmann et al., 2006; Boushel et al., 2007; De Feyter et al., Most studies assessed hepatic energy metabolism in vivo by 2008; Holloszy, 2009). using non-invasive, phosphorous magnetic resonance spectros- Elevated hepatocellular lipid content can promote hepatic insu- copy (31P MRS) techniques to quantify ATP concentrations or syn- lin resistance in the setting of NAFLD, through mechanisms similar thesis in human liver (Bourdel-Marchasson et al., 1996; Chmelík to those involved in lipid-induced muscle insulin resistance, such et al., 2008; Cortez-Pinto et al., 1999; Nair et al., 2003; Schmid as hepatic accumulation of DAG and DAG-induced activation of et al., 2008; Sharma et al., 2009; Szendroedi et al., 2009). 31P- PKCe (Jornayvaz and Shulman, 2012; Kumashiro et al., 2011; Sam- MRS allows for quantification of hepatic phosphorous metabolites uel et al., 2004). Although alterations in mitochondrial function such as gamma nucleotide triphosphate (c-NTP), alpha NTP (a- could contribute to hepatic insulin resistance and NAFLD, the exact NTP), beta NTP (b-NTP), inorganic phosphate (Pi), phosphomono- nature of this relationship remains a hot topic of metabolic re- esters and phosphodiesters. Fig. 1 depicts a typical liver 31P MRS search. Recent data from both humans and animal models showed spectrum of one healthy subject with all the peaks corresponding either decreased, unchanged or even increased hepatic mitochon- to hepatocellular phosphorous metabolites that are resolved with drial function and oxidative phosphorylation capacity in insulin this technique. This technique can now be also applied on clinical resistant states such as T2DM, obesity and NAFLD (Lockman and scanners (Laufs et al., 2013). High resolution three dimensional Nyirenda, 2010; Vial et al., 2010). It cannot be precluded that tis- (3D) magnetic spectroscopy imaging is the most recent develop- sue-specific differences exist in the association between mitochon- ment providing absolute concentrations of phosphorus com- drial function and insulin resistance or intracellular lipid content, pounds, corrected for hepatocellular fat content, as well as their but this requires confirmation by adequately controlled human regional distribution within the liver (Chmelík et al., 2008). 31P studies, examining both liver and muscle mitochondrial function MRS can also allow for assessing hepatic ATP synthesis, yielding in these metabolic states. a direct estimate of the unidirectional flux through ATP synthase The present review aims to provide a concise update of the (Schmid et al., 2008). Intravenous fructose challenge with monitor- available data on hepatic energy metabolism in several phenotypes ing of the degree of ATP depletion and the extent of ATP recovery of insulin resistance (T2DM, obesity, NAFLD), and analyzes patho- yields a measure of the flexibility of hepatic energy homeostasis genetic concepts possibly underlying alterations of energy homeo- (Abdelmalek et al., 2012). Fructose induces transient decrease of stasis in human liver. hepatic ATP as a result of its rapid phosphorylation by fructokinase after entering hepatocytes. Since hepatic fructose metabolism also 2. Literature search causes a rapid intracellular uric acid elevation, serum uric acid concentrations have been proposed as a surrogate marker of hepatic The PubMed electronic database was searched repeatedly over ATP repletion upon ATP-depleting challenges (Abdelmalek et al., three months for all types of articles published in English language 2012). Another non-invasive molecular imaging tool is positron until January 2013, using the following search terms: ‘‘hepatic en- emission tomography (PET) combined with intravenous adminis- C. Koliaki, M. Roden / Molecular and Cellular Endocrinology 379 (2013) 35–42 37

Fig. 1. Liver 31P MRS spectrum of a representative healthy person obtained with the 3-Tesla whole-body magnetic resonance spectrometer (Philips Achieva, Best, The Netherlands) at the German Diabetes Center, Düsseldorf, Germany. The eleven peaks correspond to the phosphorous metabolites of liver cells. Upper right panel: transversal image with a voxel of interest (VOI) placement, using a 14 cm linear polarized surface coil, positioned over the lateral aspect of liver. ppm: parts per million; NTP: triphosphate nucleoside; NADPH: nicotinamide adenine dinucleotide phosphate; UDPG: uridine diphosphoglucose; PEP: phosphoenol-pyruvate; GPC: glycerol phosphocholine; GPE: glycerol phosphoethanolamine; Pi: inorganic phosphate; PC: phosphocholine; PE: phosphoethanolamine Phosphomonoesters (PME) include PE and PC, phosphodiesters (PDE) include GPE and GPC. tration of lipid radiotracers such as 18-fluoro-6-thia-heptadeca- tains blood glucose within a narrow concentration range, by its noic acid and 11C-palmitate, which enables the quantification of ability to store glucose as glycogen and produce glucose after hepatic fatty acid uptake, oxidation and esterification (Iozzo either glycogen breakdown (glycogenolysis) or de novo glucose et al., 2010; Viljanen et al., 2009). Additional in vivo methods in- production from gluconeogenic precursors (gluconeogenesis) (Ro- clude stable isotope tracer techniques, such as the oral administra- den and Bernroider, 2003). In healthy humans, hepatic glycogenol- 13 2 tion of [U- C]propionate and deuterated water ( H2O), and the ysis and gluconeogenesis are stimulated in the fasted state and 13 13 intravenous infusion of [3,4- C2]glucose, [1,2- C2]b-hydroxybu- immediately inhibited in the postprandial state as a result of rapid 13 13 tyrate, [3,4- C2]acetoacetate and [ C4]palmitate, which help proinsulin action (Tappy, 1995). On the contrary, patients with T2DM file hepatic glucose and mitochondrial metabolism and assess exhibit reduced postprandial hepatic glycogen synthesis and in- various systemic and hepatic pathways including lipolysis, gluco- creased hepatic glucose output in both fasting and postprandial neogenesis, tricarboxylic acid cycle function (TCA), non-oxidative conditions, mainly driven by enhanced hepatic gluconeogenesis pathways replenishing TCA cycle intermediates (anaplerosis) and (Krssak et al., 2004). The rise in the portal glucagon:insulin ratio ketogenesis (Sunny et al., 2011). Stable isotopes, such as 13C-octa- and the increased hepatic free fatty acid oxidation are held mainly noate, 13C-methionine and 13C-ketoisocaproate, have been also ap- responsible for enhanced gluconeogenesis in T2DM (Roden and plied in non-invasive carbon-labeled breath tests, to assess hepatic Bernroider, 2003). mitochondrial b-oxidation and the severity of NAFLD, by measur- Liver mitochondria represent the major orchestrator of hepato- 13 ing the cumulative percentage of isotope exhalation or the CO2 cellular energy metabolism, since they are the site of fatty acid oxi- enrichment in exhaled air (Banasch et al., 2011; Miele et al., dation and ATP synthesis (Pessayre et al., 2002). Three different 2003; Portincasa et al., 2006). Finally, plasma concentrations of sources contribute to the hepatic levels of free fatty acids: de novo 3-hydroxybutyrate have been used as a simple and less informa- lipogenesis within hepatocytes from acetyl-CoA, uptake of circulat- tive, but organ-specific biochemical surrogate marker of hepatic li- ing plasma free fatty acids released by adipose tissue with lipolysis pid oxidation (Kotronen et al., 2009). and hydrolysis of intestinal chylomicrons (McGarry and Foster, 1980). Hepatic free fatty acids can either enter mitochondria to undergo -oxidation or be esterified into triglycerides. Hepatic tri- 3.3. Ex vivo methods b glycerides in turn either accumulate within hepatocytes as cytoplasmic lipid droplets, or are secreted as very low density lipo- High resolution respirometry by oxygraphs has been frequently protein (VLDL) particles into blood circulation (Lavoie and Gauthi- applied to skeletal muscle, but could also be performed in liver tis- er, 2006; McGarry and Foster, 1980; Nguyen et al., 2008; Pessayre sue and isolated mitochondria. This method will provide useful et al., 2001). The entry of long-chain free fatty acids into hepatic information on coupled and uncoupled maximal respiratory capac- mitochondria is regulated by carnitine palmitoyl-transferase type ity of liver tissue or hepatic mitochondria after addition of various I (CPT-I), which is located in the outer mitochondrial membrane mitochondrial substrates such as octanoyl-coenzyme A, malate, and sensitive to inhibition by malonyl-CoA, the first substrate of pyruvate, glutamate, succinate. To our knowledge, there are no hepatic de novo lipogenesis (Pessayre et al., 2001). Successive cy- published data with this technique in humans so far, but prelimin- cles of -oxidation split hepatic free fatty acids into subunits of ary data in mice are promising and lay the ground for applying b acetyl-CoA, which are either completely degraded to carbon diox- high resolution respirometry to quantify human liver mitochon- ide in the TCA (or Krebs) cycle, or condensed to ketone bodies drial function in several disease states (Benard et al., 2006; Kozlov (ketogenesis), which are then secreted by hepatic cells into circula- et al., 2006; Kuznetsov et al., 2002). tion (Lavoie and Gauthier, 2006). Fatty acid oxidation in hepatic mitochondria is associated with the reduction of oxidized coen- 4. The physiological role of liver energy metabolism and zymes, which are in turn re-oxidized by the mitochondrial respira- mitochondrial function tory chain (Pessayre et al., 2002, 2001). During their re-oxidation, they transfer their electrons to the polypeptide complexes of the The human liver plays a critical role in regulating glucose and mitochondrial respiratory chain. The electron transfer along the lipid metabolism and whole-body energy homeostasis. Liver main- respiratory chain is coupled with an export of protons from 38 C. Koliaki, M. Roden / Molecular and Cellular Endocrinology 379 (2013) 35–42 mitochondrial matrix to intermembrane space, creating a large electrochemical gradient across the inner mitochondrial membrane, which acts as an energy reservoir. When energy is needed, protons can re-enter matrix through ATP synthase (complex V), causing the conversion of adenosine diphosphate (ADP) into ATP. The adenine dinucleotide translocator proteins can then export mitochondrial ATP in exchange for cytosolic ADP, and the cytoplasmic ATP can be used to power all hepatocellular energy-requiring metabolic processes (Pessayre et al., 2002, 2001).

5. Liver mitochondrial function in healthy humans

Despite the limited data on the normal range of mitochondrial function in human liver, some information can be derived from Fig. 2. Hypothetical changes in hepatic energy metabolism in states of obesity, clinical studies, which have compared direct or indirect measures steatosis, non-alcoholic steatohepatitis (NASH) and type 2 diabetes mellitus of hepatic mitochondrial function between insulin resistant and (T2DM). Different features of hepatic energy metabolism such as ATP, b-oxidation and respiratory complex activities were obtained from studies including healthy insulin sensitive humans. Table 1 summarizes the key data of these control groups. The respective percent changes are compared to the data of the studies, while Fig. 2 depicts in a graphical way the percentage dif- respective healthy control group, which were set as 100%. Data are derived from the ferences of liver mitochondrial function between healthy humans following references: Cortez-Pinto et al. (1999); Iozzo et al. (2010); Miele et al. and several insulin resistant phenotypes. (2003); Pérez-Carreras et al. (2003); Schmid et al. (2011); Sunny et al. (2011); Szendroedi et al. (2009). Employing 31P MRS to assess liver ATP turnover revealed that young healthy humans display hepatic concentrations of c-ATP

Table 1 Studies in humans on hepatic energy metabolism under conditions of type 2 diabetes mellitus (T2DM), insulin resistance and non-alcoholic fatty liver disease (NAFLD).

Reference Cohort Methods Results

Szendroedi et al. (2009) 9 T2DM in vivo 31P-MRS ;cATP and Pi contents 9 matcheda controls in T2DM 9 young lean controls Schmid et al. (2011) 9 T2DM in vivo 31P MRS ;ﬂux through ATP synthase 8 matcheda controls in T2DM Abdelmalek et al. (2012) 25 obese T2DM in vivo 31P MRS ;ATP recovery with high or low fructose fructose challenge in high fructose consumers consumption

Nair et al. (2003) 7 overweight in vivo 31P MRS ;ATP content 7 obese fructose challenge in overweight and obese 5 lean controls unchanged ATP recovery Viljanen et al. (2009) 34 healthy obese 18-ﬂuoro-6-thia- ;hepatic fatty acid uptake heptadecanoic acid PET after VLCD for 6 weeks imaging

Iozzo et al. (2010) 8 obese 11C-palmitate PET imaging hepatic fatty acid oxidation " 7 lean controls in obese Kotronen et al. (2009) 29 NAFLD plasma levels of no differences in hepatic lipid oxidation 29 controls 3-OHB Sharma et al. (2009) 20 obese + NAFLD in vivo 31P MRS PME/Pi and PME/cATP ratios " 20 non-obese + NAFLD in obese + NAFLD 20 non-obese -NAFLD Sunny et al. (2011) 8 high HCL in vivo stable isotope tracers TCA cycle, anaplerosis, lipolysis and gluconeogenesis " 8 low HCL in high HCL

Cortez-Pinto et al. (1999) 8 NASH in vivo 31P MRS ;ATP recovery 7 controls fructose challenge in NASH Sanyal et al. (2001) 6-10 NASH in vitro hepatic ß-oxidation and ox. stress " 6 steatosis lipid peroxidation in NASH and steatosis 6 controls serum b-OHB mitochondrial defects in NASH Miele et al. (2003) 10 NASH 13C-octanoate breath test hepatic mitochondrial " 20 controls ß-oxidation in NASH Pérez-Carreras et al. 43 NASH in vitro ETC ;activity of ETC CI-V correlation with insulin resistance and (2003) inﬂammation 16 controls enzyme activity

Serviddio et al. (2008b) 10 NASH in vitro proton leak, UCP-2, proton leak and oxidative stress " 8 controls UCP-2 expression, ROS production, ATP content

ATP: adenosine triphosphate; ß-OHB: ß-hydroxybutyrate; 3-OHB: 3-hydroxybutyrate; CI-V: complexes I–V; ETC: electron transport chain; HCL: hepatocellular lipids; 31P MRS: phosphorous magnetic resonance spectroscopy; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PET: positron emission tomography; Pi: inorganic phosphate; PME: phosphomonoesters; ROS: reactive oxygen species; TCA: tricarboxylic acid cycle; T2DM: type 2 diabetes mellitus; UCP-2: uncoupling protein 2; VLCD: very low calorie diet. A Matched for age and body mass index. C. Koliaki, M. Roden / Molecular and Cellular Endocrinology 379 (2013) 35–42 39 ranging from 2.0 to 2.6 mmol/l and hepatic Pi levels ranging from ods might impair hepatic mitochondrial function (Schmid et al., 1.2 to 1.7 mmol/l (Szendroedi et al., 2009). Elderly non-diabetic, 2011). but slightly insulin resistant humans, display absolute c-ATP levels Likewise, high dietary fructose consumption (>15 g per day) can in the range of 1.9–3.1 mmol/l, Pi concentrations in the range of 1– severely deplete hepatic ATP stores and impair ATP re-synthesis 1.9 mmol/l and flux through ATP synthase in the range of 15– after intravenous fructose challenge in obese patients with T2DM 41 mmol/l/min (Schmid et al., 2011; Szendroedi et al., 2009). Com- (Abdelmalek et al., 2012)(Table 1). This was particularly true for bining 31P MRS with an ATP-depleting fructose challenge identified patients with serum uric acid concentrations of 5.5 mg/dl or more. that in healthy subjects, hepatic b-ATP levels fall by 50% to their This study suggests that high dietary fructose intake could contrib- nadir at 12 min, and recover fully at 60 min after fructose adminis- ute to the worsening of abnormal hepatic energy homeostasis in tration (Cortez-Pinto et al., 1999). T2DM patients, and may further predispose them to the develop- Furthermore, enzyme activities of mitochondrial respiratory ment and/or progression of NAFLD (Abdelmalek et al., 2010; Ouy- chain proteins have been measured in biopsy-derived liver speci- ang et al., 2008). mens obtained from healthy humans. Relative to citrate synthase Studies in animal models of T2DM such as the diabetes-prone activity, complex I activity was found to range between 30 and Psammomys obesus model, are in the same direction with the clin- 43 nmol/min/mg protein, complex II activity between 28 and ical studies, showing an impaired hepatic energy metabolism in rat 46 nmol/min/mg protein, complex III activity between 38 and liver tissue after a hypercaloric diabetogenic diet (Bouderba et al., 60 nmol/min/mg protein, complex IV activity between 24 and 2012). In this rat model, liver mitochondrial function was assessed 46 nmol/min/mg protein, and complex V activity between 99 and with respirometry in isolated mitochondria and respiratory com- 167 nmol/min/mg protein (Pérez-Carreras et al., 2003). In the same plex enzyme activities, and was found to be significantly declined study, citrate synthase specific activity, reflecting mitochondrial in diabetic animals. protein mass, was found to range between 112 and 168 nmol/ In conclusion, patients with T2DM display lower hepatic energy min/mg protein in healthy controls. metabolism compared to both young and elderly non-diabetic hu- Measuring biochemical surrogates of hepatic lipid oxidation re- mans, which is expressed as reduced hepatic flux through ATP syn- vealed that fasting b-hydroxybutyrate levels range between 81 and thase and reduced hepatic ATP and Pi concentrations. Of note, 99 lmol/l and are suppressed by 50% under conditions of hyperin- parameters of hepatic mitochondrial metabolism other than hepa- sulinemia in healthy humans (Sanyal et al., 2001). Furthermore, it tic ATP homeostasis have not yet been evaluated in T2DM patients. has been shown that hepatic fatty acid oxidation accounts for 40% of liver fat uptake and fatty acid esterification for 60%, as assessed with 11C-palmitate kinetics and PET imaging in healthy humans 7. Liver mitochondrial function in obesity and steatosis (Iozzo et al., 2010). Of note, the rates of ATP synthesis in human liver are approxi- Similar to T2DM, some studies found that overweight and obese mately 50% lower than those in isolated perfused rat liver, indicat- humans have lower hepatic ATP levels compared to normal-weight ing species-specific differences in hepatic energy metabolism humans (Table 1). Hepatic ATP content related inversely to BMI not under normal conditions (Schmid et al., 2008). only in the obese, but also in normal-weight subjects (Cortez-Pinto et al., 1999; Nair et al., 2003). However, obese persons can feature impaired (Cortez-Pinto et al., 1999) or normal (Nair et al., 2003) repletion of hepatic ATP upon fructose challenging. Of note, liver 6. Liver mitochondrial function in T2DM mitochondria of rats with high fat diet-induced obesity and insulin resistance exhibit an elevated rate of b-oxidation and TCA cycle Only recently, two studies reported that patients with T2DM activity, which is however combined with an impaired respiratory have lower hepatic ATP turnover measured by 31P MRS, when com- capacity and greater oxidative stress (Raffaella et al., 2008; Satapati pared with age- and BMI-matched non-diabetic subjects (Schmid et al., 2012). et al., 2011; Szendroedi et al., 2009) and young lean controls Hepatic fatty acid oxidation measured by 11C-palmitate com- (Szendroedi et al., 2009)(Table 1). In detail, the absolute hepatic bined with PET imaging is 50% higher (Fig. 2), while fatty acid up- concentrations of c-ATP and Pi were 23–26% and 28–31% lower take and esterification are not different in obese compared to lean in T2DM (Szendroedi et al., 2009)(Fig. 2). Furthermore, c-ATP persons (Iozzo et al., 2010). Another PET study found that a very and Pi absolute concentrations were negatively correlated with low calorie diet decreases by 26% hepatic fatty acid uptake and insulin-mediated endogenous glucose production (r = 0.67, ameliorates hepatic insulin resistance in healthy obese persons À p = 0.01), even after adjusting for hepatocellular fat content, but (Viljanen et al., 2009), without however providing any data about they were not significantly associated with peripheral insulin sen- other aspects of hepatic mitochondrial metabolism such as mito- sitivity (M-value) (Szendroedi et al., 2009). Endogenous glucose chondrial respiration or fat oxidation. production was the only significant independent predictor of c- A very frequent comorbidity of obesity is liver steatosis, since ATP levels, explaining 57% of variance of hepatic ATP concentra- 60–90% of persons with steatosis (based on liver biopsies) are over- tions (Szendroedi et al., 2009). A similar group of T2DM patients weight or obese (Choudhury and Sanyal, 2004). Considering that featured a 42% reduction in the hepatic flux through ATP synthase, there are only limited data for obese humans, the few studies con- which was mainly driven by decreased hepatic Pi levels (Schmid ducted in steatotic humans complement those in obese, and shed et al., 2011). In this study, flux through ATP synthase was positively more light into the association of obesity with liver mitochondrial correlated with both peripheral and hepatic insulin sensitivity function. From plasma 3-hydroxybutyrate concentrations, hepatic (r = 0.66–0.72, p < 0.05), independently of hepatic lipid content, lipid oxidation was rated similar in overweight patients with stea- and was negatively correlated with waist circumference and BMI tosis and healthy humans, under both basal and insulin-stimulated (r = 0.52 to 0.81, p 0:001). Although the cohort of T2DM pa- conditions (Kotronen et al., 2009)(Table 1). However, significant À À tients displayed adequate glycemic control under oral glucose low- differences not only in fat oxidation, but also in other aspects of he- ering treatment, hepatic flux through ATP synthase correlated patic mitochondrial function were observed in another study negatively with measures of short- and long-term glycemic control employing non-invasive in vivo tracer techniques. Sunny et al. re- such as fasting glucose and glycosylated hemoglobin. This indi- ported that persons with steatosis, as defined by liver fat content cates that even minor degree of hyperglycemia for long time peri- of more than 6%, have two-fold greater hepatic mitochondrial oxi- 40 C. Koliaki, M. Roden / Molecular and Cellular Endocrinology 379 (2013) 35–42 dative metabolism than those with liver fat content less than 6% et al., 2003)(Table 1 and Fig. 2). It has been also reported that pa- (Sunny et al., 2011). Both groups had similar age and comparable tients with NASH display an increased proton leak across the elec- degree of obesity, elevation of liver enzymes and whole-body insu- tron transport chain due to a 2-fold increased hepatic expression of lin resistance, whereas hepatic insulin resistance was more pro- uncoupling protein 2 (UCP-2) (Serviddio et al., 2008b)(Table 1). nounced in those with steatosis. In detail, subjects with steatosis The upregulation of UCP-2 in human NASH induces an uncoupling exhibited 100% higher TCA cycle flux, 50% higher rates of lipolysis between oxidative phosphorylation and ATP production, reduces and anaplerosis, and 25% higher rates of gluconeogenesis com- the redox pressure on mitochondrial respiratory chain, and acts pared to those without steatosis (Table 1 and Fig. 2). Of note, hepa- as a potential protective mechanism against further liver damage. tic fat content correlated positively with measures of both UCP-2-dependent mitochondrial uncoupling can be perceived as a oxidative and non-oxidative mitochondrial metabolism. These protective mechanism to halt damage progression but compro- findings support the concept that mitochondrial pathways could mises on the other hand the liver capacity to respond to acute be upregulated in steatosis as an adaptive mechanism in response high-energy demands, such as ischaemia–reperfusion injury. Addi- to chronic fat overload. Recent animal studies are in absolute tional abnormalities of hepatic energy metabolism that have been accordance with this contention, by showing elevated TCA cycle reported in human and animal NASH (rat models of high fat, function and mitochondrial b-oxidation in states of diet-induced methionine and choline-deficient diet) include increased ROS pro- hepatic insulin resistance and liver steatosis (Satapati et al., 2012). duction, abnormal cellular and mitochondrial redox homeostasis, Taken together, all the above data in humans with obesity and oxidative stress-mediated depletions of mitochondrial DNA encod- steatosis illustrate the importance of the homeostasis of hepatic ing some of the polypeptide components of mitochondrial respira- fatty acid handling. Any increase in hepatic fatty acid uptake would tory chain and increased rate of b-oxidation (Miele et al., 2003; be balanced by an up-regulation of b-oxidation, which – in the set- Morris et al., 2011; Serviddio et al., 2008a; Romestaing et al., ting of chronic lipid overloading – would stimulate ATP production 2008)(Table 1). and generation of ROS. This would in turn lead to increased triglyc- The underlying pathophysiology of perturbed hepatic energy eride synthesis and export as VLDL, as well as to hepatic oxidative metabolism in NASH can be described by a vicious circle, involving stress with promotion of NAFLD and ultimately impairment of free fatty acids, lipid peroxidation products and inflammatory mitochondrial functionality. markers (Fromenty et al., 2004; Pessayre, 2007; Pessayre et al., 2002, 2001). This ominous cascade begins with a mild respiratory dysfunction induced by lipid oversupply in hepatocytes. In addi- 8. Liver mitochondrial function in advanced NAFLD tion, the increased availability of hepatic free fatty acids results in an increased import of free fatty acids into hepatic mitochondria Advanced NAFLD typically associates with insulin resistance and an elevated rate of mitochondrial fatty acid b-oxidation (Miele and unfavorable hepatic adaptations of hepatocellular energy et al., 2003). Due to the increased rate of b-oxidation, an imbalance homeostasis, which render the liver more vulnerable to oxidative occurs between a high electron input and a restricted electron out- injury and cell death, and are reflected by ultrastructural mito- flow, leading to accumulation of electrons within respiratory com- chondrial defects (Sanyal et al., 2001). It has been suggested that plexes I and III and a subsequent reaction of these electrons with impaired mitochondrial respiratory capacity plays a key role in oxygen to form ROS. ROS can promote lipid peroxidation and lipid the pathogenesis of advanced NAFLD, particularly in the progres- peroxidation products can in turn alter mitochondrial DNA and sion to NASH and cirrhosis (Begriche et al., 2006; Serviddio et al., cause severe oxidative damage to critical mitochondrial proteins 2011, 2008a). Studies in humans have failed to support a concept such as cytochrome c oxidase and adenine nucleotide translocator of generalized defects in mitochondrial b-oxidation in biopsy-pro- proteins, resulting in impaired electron flow along the respiratory ven NAFLD (Sanyal et al., 2001). In contrast, patients with NAFLD chain and establishing a vicious circle between impaired respira- and insulin resistance show greater liver mitochondrial b-oxida- tory chain capacity, ROS formation, lipid peroxidation and mito- tion, while those with drug-induced NAFLD have diminished mitochondrial damage (Fromenty et al., 2004; Pessayre, 2007; chondrial oxidation (Fromenty et al., 2004; Miele et al., 2003). An Pessayre et al., 2002, 2001). ROS and lipid peroxidation products augmented hepatic b-oxidation and oxidative stress seem thus to can also promote hepatic inflammation, fibrosis and cell death, accompany peripheral insulin resistance as the most prominent leading to the characteristic necroinflammatory and fibrotic alter- characteristics of advanced NAFLD (Sanyal et al., 2001). ations of hepatic tissue observed in advanced stages of NAFLD. Due NASH is recognized as the most common subtype of advanced to excessive ROS production, the anti-oxidant defense systems of NAFLD (Clark, 2006). In a pilotic study in eight patients with mitochondria (enzymes and vitamins) are rapidly consumed, and biopsy-proven NASH, it was found that the ability of these patients this depletion of anti-oxidant capacity hampers the inactivation to regenerate their hepatic ATP reserve after a transient ATP deple- of ROS and may further augment ROS-mediated damage. tion induced by fructose ingestion was reduced by 30% compared to age- and sex-matched controls (Cortez-Pinto et al., 1999) (Fig. 2). Of note, in human NASH, functional mitochondrial abnor- 9. Conclusions malities are furthermore combined with a number of structural defects such as loss of mitochondrial cristae and paracrystalline The liver is a central player in the physiological regulation of inclusions, and presence of linear crystalline inclusions in swollen whole-body energy homeostasis as well as the pathogenesis of mitochondria (Sanyal et al., 2001). Furthermore, it has been shown the epidemiologically relevant endocrine disorders obesity and that patients with NASH have a severely defective hepatic mito- diabetes. Patients with long-standing T2DM can have lower hepa- chondrial respiratory chain, and this dysfunction correlates posi- tic ATP synthesis during fasting and after fructose administration. tively with inflammation and peripheral insulin resistance In advanced NAFLD, abnormal hepatic mitochondrial function (Pérez-Carreras et al., 2003). More specifically, patients with NASH and morphology may occur, possibly due to local lipotoxic, inflam- were found to exhibit significantly decreased activity of all respira- matory and oxidative stress pathways. On the other hand, non-dia- tory chain complexes compared to control subjects (42.4–70.6% betic obese humans can show normal or even greater hepatic reduction for complexes I–V), and increased availability of hepatic mitochondrial function than lean humans. Such increases in b-oxi- free fatty acids, expressed as an increased ratio of long-chain dation, ketogenesis and anaplerotic fluxes can be interpreted as a acylcarnitine esters relative to free carnitine (Pérez-Carreras response to lipid oversupply and may correlate with steatosis. C. Koliaki, M. Roden / Molecular and Cellular Endocrinology 379 (2013) 35–42 41

Thus, we propose that hepatic energy metabolism transitionally De Feyter, H.M., van den Broek, N.M., Praet, S.F., Nicolay, K., van Loon, L.J., Prompers, adapts to chronic lipid overload in states of obesity and steatosis J.J., 2008. Early or advanced stage type 2 diabetes is not accompanied by in vivo skeletal muscle mitochondrial dysfunction. Eur. J. Endocrinol. 158, 643–653. by upregulated oxidative capacity, which can be followed by pro- Fromenty, B., Robin, M.A., Igoudjil, A., Mansouri, A., Pessayre, D., 2004. The ins and gressive decline in liver mitochondrial function during prolonged outs of mitochondrial dysfunction in NASH. Diabetes Metab. 30, 121–138. chronic insulin resistance, associated with T2DM and NASH García-Ruiz, C., Colell, A., Morales, A., Kaplowitz, N., Fernández-Checa, J.C., 1995. Role of oxidative stress generated from the mitochondrial electron transport (Fig. 2). chain and mitochondrial glutathione status in loss of mitochondrial function Of note, these conclusions are based on a limited number of and activation of transcription factor nuclear factor-kappa B: studies with small-scale human studies. More clinical studies combining isolated mitochondria and rat hepatocytes. Mol. Pharmacol. 48, 825–834. Holloszy, J.O., 2009. Skeletal muscle ‘‘mitochondrial deficiency’’ does not mediate in vivo and ex vivo state-of-the art methods are needed to address insulin resistance. Am. J. Clin. Nutr. 89, 463S–466S. hepatic energy metabolism in well-phenotyped and matched co- Iozzo, P., Bucci, M., Roivainen, A., Någren, K., Järvisalo, MJ., Kiss, J., Guiducci, L., horts. Such an approach could help to identify novel markers of Fielding, B., Naum, AG., Borra, R., Virtanen, K., Savunen, T., Salvadori, PA., Ferrannini, E., Knuuti, J., Nuutila, P., 2010. Fatty acid metabolism in the liver, the deterioration of hepatic energy metabolism and predictors of measured by positron emission tomography, is increased in obese individuals. progression of NAFLD in obesity and diabetes mellitus. Gastroenterology 139, 846–856. Jornayvaz, F.R., Shulman, G.I., 2012. Diacylglycerol activation of protein kinase Ce and hepatic insulin resistance. Cell Metab. 15, 574–584. Acknowledgements Kotronen, A., Seppälä-Lindroos, A., Vehkavaara, S., Bergholm, R., Frayn, K.N., Fielding, B.A., Yki-Järvinen, H., 2009. Liver fat and lipid oxidation in humans. Liver Int. 29, 1439–1446. C.K. is on leave of absence from the Endocrine Unit and Diabetes Kozlov, A.V., Staniek, K., Haindl, S., Piskernik, C., Ohlinger, W., Gille, L., Nohl, H., Research Center of Attikon University Hospital, Athens University Bahrami, S., Redl, H., 2006. Different effects of endotoxic shock on the Medical School, Greece, and was supported by the National Foun- respiratory function of liver and heart mitochondria in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 290, G543–G549. dation of State Scholarships of Greece (IKY). The work of M.R. is Krebs, M., Roden, M., 2004. Nutrient-induced insulin resistance in human skeletal or has been supported in part by the European Foundation for muscle. Curr. Med. Chem. 11, 901–908. the Study of Diabetes, German Research Foundation, Schmutzler- Krssak, M., Brehm, A., Bernroider, E., Anderwald, C., Nowotny, P., Dalla Man, C., Cobelli, C., Cline, G.W., Shulman, G.I., Waldhäusl, W., Roden, M., 2004. Stiftung, Skröder-Stiftung, and the German Center for Diabetes Re- Alterations in postprandial hepatic glycogen metabolism in type 2 diabetes. search (DZD e.V.). Diabetes 53, 3048–3056. Kumashiro, N., Erion, D.M., Zhang, D., Kahn, M., Beddow, S.A., Chu, X., Still, C.D., Gerhard, G.S., Han, X., Dziura, J., Petersen, K.F., Samuel, V.T., Shulman, G.I., 2011. References Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease. Proc. Nat. Acad Sci. USA 108, 16381–16385. Kuznetsov, A.V., Strobl, D., Ruttmann, E., Königsrainer, A., Margreiter, R., Gnaiger, E., Abdelmalek, M.F., Suzuki, A., Guy, C., Unalp-Arida, A., Colvin, R., Johnson, R.J., Diehl, 2002. Evaluation of mitochondrial respiratory function in small biopsies of liver. A.M., 2010. Increased fructose consumption is associated with fibrosis severity Anal. Biochem. 305, 186–194. in patients with nonalcoholic fatty liver disease. Hepatology 51, 1961–1971. Laufs, A., Livingstone, R., Nowotny, B., Nowotny, P., Wickrath, F., Giani, G., Bunke, J., Abdelmalek, M.F., Lazo, M., Horska, A., Bonekamp, S., Lipkin, E.W., Roden, M., Hwang, J.H., 2013. Quantitative liver 31P MR spectroscopy at 3T on a Balasubramanyam, A., Bantle, J.P., Johnson, R.J., Diehl, A.M., Clark, J.M., 2012. clinical scanner. Magn. Res. Med. (in press). Higher dietary fructose is associated with impaired hepatic adenosine Lavoie, J.M., Gauthier, M.S., 2006. Regulation of fat metabolism in the liver: link to triphosphate homeostasis in obese individuals with type 2 diabetes. non-alcoholic hepatic steatosis and impact of physical exercise. Cell Mol. Life Hepatology 56, 952–960. Sci. 63, 1393–1409. Angulo, P., 2002. Nonalcoholic fatty liver disease. N. Engl. J. Med. 346, 1221–1231. Lockman, K.A., Nyirenda, M.J., 2010. Interrelationships between hepatic fat and Asmann, Y.W., Stump, C.S., Short, K.R., Coenen-Schimke, J.M., Guo, Z., Bigelow, M.L., insulin resistance in non-alcoholic fatty liver disease. Curr. Diabetes Rev. 6, Nair, K.S., 2006. Skeletal muscle mitochondrial functions, mitochondrial DNA 341–347. copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic McGarry, J.D., Foster, D.W., 1980. Regulation of hepatic fatty acid oxidation and subjects at equal levels of low or high insulin and euglycemia. Diabetes 55, ketone body production. Annu. Rev. Biochem. 49, 395–420. 3309–3319. Miele, L., Grieco, A., Armuzzi, A., Candelli, M., Forgione, A., Gasbarrini, A., Gasbarrini, Banasch, M., Ellrichmann, M., Tannapfel, A., Schmidt, W.E., Goetze, O., 2011. The G., 2003. Hepatic mitochondrial beta-oxidation in patients with nonalcoholic non-invasive (13)C-methionine breath test detects hepatic mitochondrial steatohepatitis assessed by 13C-octanoate breath test. Am. J. Gastroenterol. 98, dysfunction as a marker of disease activity in non-alcoholic steatohepatitis. 2335–2336. Eur. J. Med. Res. 16, 258–264. Morino, K., Petersen, K.F., Dufour, S., Befroy, D., Frattini, J., Shatzkes, N., Neschen, S., Begriche, K., Igoudjil, A., Pessayre, D., Fromenty, B., 2006. Mitochondrial dysfunction White, M.F., Bilz, S., Sono, S., Pypaert, M., Shulman, G.I., 2005. Reduced in NASH: causes, consequences and possible means to prevent it. mitochondrial density and increased IRS-1 serine phosphorylation in muscle Mitochondrion 6, 1–28. of insulin-resistant offspring of type 2 diabetic parents. J. Clin. Invest. 115, Benard, G., Faustin, B., Passerieux, E., Galinier, A., Rocher, C., Bellance, N., Delage, J.P., 3587–3593. Casteilla, L., Letellier, T., Rossignol, R., 2006. Physiological diversity of Morris, E.M., Rector, R.S., Thyfault, J.P., Ibdah, J.A., 2011. Mitochondria and redox mitochondrial oxidative phosphorylation. Am. J. Physiol. Cell Physiol. 291, signaling in steatohepatitis. Antioxid Redox Signal 15, 485–504. C1172–C1182. Nair, S., P Chacko, V., Arnold, C., Diehl, A.M., 2003. Hepatic ATP reserve and Bouderba, S., Sanz, M.N., Sánchez-Martín, C., El-Mir, M.Y., Villanueva, G.R., Detaille, efficiency of replenishing: comparison between obese and nonobese normal D., Koceïr, E.A., 2012. Hepatic mitochondrial alterations and increased oxidative individuals. Am. J. Gastroenterol 98, 466–470. stress in nutritional diabetes-prone Psammomys obesus model. Exp. Diabetes Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., Dumon, H., 2008. Res. 2012, 430176. Liver lipid metabolism. J. Anim Physiol. Anim Nutr. (Berl.) 92, 272–283. Bourdel-Marchasson, I., Biran, M., Thiaudière, E., Delalande, C., Decamps, A., Ouyang, X., Cirillo, P., Sautin, Y., McCall, S., Bruchette, J.L., Diehl, A.M., Johnson, R.J., Manciet, G., Canioni, P., 1996. 31P magnetic resonance spectroscopy of human Abdelmalek, M.F., 2008. Fructose consumption as a risk factor for non-alcoholic liver in elderly patients: changes according to nutritional status and fatty liver disease. J. Hepatol. 48, 993–999. inflammatory state. Metabolism 45, 1059–1061. Pérez-Carreras, M., Del Hoyo, P., Martín, M.A., Rubio, J.C., Martín, A., Castellano, G., Boushel, R., Gnaiger, E., Schjerling, P., Skovbro, M., Kraunsøe, R., Dela, F., 2007. Colina, F., Arenas, J., Solis-Herruzo, J.A., 2003. Defective hepatic mitochondrial Patients with type 2 diabetes have normal mitochondrial function in skeletal respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology 38, muscle. Diabetologia 50, 790–796. 999–1007. Browning, J.D., Szczepaniak, L.S., Dobbins, R., Nuremberg, P., Horton, J.D., Cohen, J.C., Pessayre, D., 2007. Role of mitochondria in non-alcoholic fatty liver disease. J. Grundy, S.M., Hobbs, H.H., 2004. Prevalence of hepatic steatosis in an urban Gastroenterol. Hepatol. 1, 20–27. population in the United States: impact of ethnicity. Hepatology 40, 1387–1395. Pessayre, D., Berson, A., Fromenty, B., Mansouri, A., 2001. Mitochondria in Chmelík, M., Schmid, A.I., Gruber, S., Szendroedi, J., Krssák, M., Trattnig, S., Moser, E., steatohepatitis. Semin. Liver Dis. 21, 57–69. Roden, M., 2008. Three-dimensional high-resolution magnetic resonance Pessayre, D., Mansouri, A., Fromenty, B., 2002. Nonalcoholic steatosis and spectroscopic imaging for absolute quantification of 31P metabolites in human steatohepatitis. V. Mitochondrial dysfunction in steatohepatitis. Am. J. liver. Magn. Reson. Med. 60, 796–802. Physiol. Gastrointest Liver Physiol. 282, 193–199. Choudhury, J., Sanyal, A.J., 2004. Clinical aspects of fatty liver disease. Sem. Liver Dis. Petersen, K.F., Dufour, S., Shulman, G.I., 2005. Decreased insulin-stimulated ATP 24, 349–362. synthesis and phosphate transport in muscle of insulin-resistant offspring of Clark, J.M., 2006. The epidemiology of nonalcoholic fatty liver disease in adults. J. type 2 diabetic parents. PLoS Med. 2, e233. Clin. Gastroenterol 40 (Suppl 1), S5-10. Portincasa, P., Grattagliano, I., Lauterburg, B.H., Palmieri, V.O., Palasciano, G., Cortez-Pinto, H., Chatham, J., Chacko, V.P., Arnold, C., Rashid, A., Diehl, A.M., 1999. Stellaard, F., 2006. Liver breath tests non-invasively predict higher stages of Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a non-alcoholic steatohepatitis. Clin. Sci. (Lond.). 111, 135–143. pilot study. JAMA 282, 1659–1664. 42 C. Koliaki, M. Roden / Molecular and Cellular Endocrinology 379 (2013) 35–42

Raffaella, C., Francesca, B., Italia, F., Marina, P., Giovanna, L., Susanna, I., 2008. mitochondrial proton leak and increases susceptibility of non-alcoholic Alterations in hepatic mitochondrial compartment in a model of obesity and steatohepatitis (NASH) liver to ischaemia-reperfusion injury. Gut 57, 957–965. insulin resistance. Obesity 16, 958–964. Serviddio, G., Bellanti, F., Vendemiale, G., Altomare, E., 2011. Mitochondrial Roden, M., 2004. How free fatty acids inhibit glucose utilization in human skeletal dysfunction in nonalcoholic steatohepatitis. Expert Rev. Gastroenterol. muscle. News Physiol. Sci. 19, 92–96. Hepatol. 5, 233–244. Roden, M., 2005. Muscle triglycerides and mitochondrial function: possible Sharma, R., Sinha, S., Danishad, K.A., Vikram, N.K., Gupta, A., Ahuja, V., Jagannathan, mechanisms for the development of type 2 diabetes. Int. J. Obes. (Lond.) 29, N.R., Pandey, R.M., Misra, A., 2009. Investigation of hepatic gluconeogenesis 111–115. pathway in non-diabetic Asian Indians with non-alcoholic fatty liver disease Roden, M., 2006. Mechanisms of Disease: hepatic steatosis in type 2 diabetes– using in vivo ((31)P) phosphorus magnetic resonance spectroscopy. pathogenesis and clinical relevance. Nat. Clin. Pract. Endocrinol. Metab. 2, 335– Atherosclerosis 203, 291–297. 448. Smith, B.W., Adams, L.A., 2011. Non-alcoholic fatty liver disease. Crit. Rev. Clin. Lab. Roden, M., Bernroider, E., 2003. Hepatic glucose metabolism in humans–its role in Sci. 48, 97–113. health and disease. Best Pract. Res. Clin. Endocrinol. Metab. 17, 365–383. Sunny, N.E., Parks, E.J., Browning, J.D., Burgess, S.C., 2011. Excessive hepatic Romestaing, C., Piquet, M.A., Letexier, D., Rey, B., Mourier, A., Servais, S., Belouze, M., mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic Rouleau, V., Dautresme, M., Ollivier, I., Favier, R., Rigoulet, M., Duchamp, C., fatty liver disease. Cell Metab. 14, 804–810. Sibille, B., 2008. Mitochondrial adaptations to steatohepatitis induced by a Szendroedi, J., Roden, M., 2009. Ectopic lipids and organ function. Curr. Opin. methionine- and choline-deﬁcient diet. Am. J. Physiol. Endocrinol. Metab. 294, Lipidol. 20, 50–56. 110–119. Szendroedi, J., Schmid, A.I., Chmelik, M., Toth, C., Brehm, A., Krssak, M., Nowotny, P., Samuel, V.T., Liu, Z.X., Qu, X., Elder, B.D., Bilz, S., Befroy, D., Romanelli, A.J., Shulman, Wolzt, M., Waldhausl, W., Roden, M., 2007. Muscle mitochondrial ATP synthesis G.I., 2004. Mechanism of hepatic insulin resistance in non-alcoholic fatty liver and glucose transport/phosphorylation in type 2 diabetes. PLoS Med. 4, 154. disease. J. Biol. Chem. 279, 32345–32353. Szendroedi, J., Chmelik, M., Schmid, A.I., Nowotny, P., Brehm, A., Krssak, M., Moser, Sanyal, A.J., Campbell-Sargent, C., Mirshahi, F., Rizzo, W.B., Contos, M.J., Sterling, E., Roden, M., 2009. Abnormal hepatic energy homeostasis in type 2 diabetes. R.K., Luketic, V.A., Shiffman, M.L., Clore, J.N., 2001. Nonalcoholic steatohepatitis: Hepatology 50, 1079–1086. association of insulin resistance and mitochondrial abnormalities. Tappy, L., 1995. Regulation of hepatic glucose production in healthy subjects and Gastroenterology 120, 1183–1192. patients with non-insulin-dependent diabetes mellitus. Diabetes Metab. 21, Satapati, S., Sunny, N.E., Kucejova, B., Fu, X., He, T.T., Méndez-Lucas, A., Shelton, J.M., 233–240. Perales, J.C., Browning, J.D., Burgess, S.C., 2012. Elevated TCA cycle function in Vendemiale, G., Grattagliano, I., Caraceni, P., Caraccio, G., Domenicali, M., Dall’Agata, the pathology of diet-induced hepatic insulin resistance and fatty liver. J. Lipid. M., Trevisani, F., Guerrieri, F., Bernardi, M., Altomare, E., 2001. Mitochondrial Res. 53, 1080–1092. oxidative injury and energy metabolism alteration in rat fatty liver: effect of the Schmid, A.I., Chmelík, M., Szendroedi, J., Krssák, M., Brehm, A., Moser, E., Roden, M., nutritional status. Hepatology 33, 808–815. 2008. Quantitative ATP synthesis in human liver measured by localized 31P Vial, G., Dubouchaud, H., Leverve, X.M., 2010. Liver mitochondria and insulin spectroscopy using the magnetization transfer experiment. NMR Biomed. 21, resistance. Acta Biochim. Pol. 57, 389–392. 437–443. Vial, G., Dubouchaud, H., Couturier, K., Cottet-Rousselle, C., Taleux, N., Athias, A., Schmid, A.I., Szendroedi, J., Chmelik, M., Krssák, M., Moser, E., Roden, M., 2011. Liver Galinier, A., Casteilla, L., Leverve, X.M., 2011. Effects of a high-fat diet on energy ATP synthesis is lower and relates to insulin sensitivity in patients with type 2 metabolism and ROS production in rat liver. J. Hepatol. 54, 348–356. diabetes. Diabetes Care. 34, 448–453. Viljanen, A.P., Iozzo, P., Borra, R., Kankaanpää, M., Karmi, A., Lautamäki, R., Järvisalo, Serviddio, G., Sastre, J., Bellanti, F., Viña, J., Vendemiale, G., Altomare, E., 2008a. M., Parkkola, R., Rönnemaa, T., Guiducci, L., Lehtimäki, T., Raitakari, O.T., Mari, Mitochondrial involvement in non-alcoholic steatohepatitis. Mol. Aspects Med. A., Nuutila, P., 2009. Effect of weight loss on liver free fatty acid uptake and 29, 22–35. hepatic insulin resistance. J. Clin. Endocrinol. Metab. 94, 50–55. Serviddio, G., Bellanti, F., Tamborra, R., Rollo, T., Capitanio, N., Romano, A.D., Sastre, J., Vendemiale, G., Altomare, E., 2008b. Uncoupling protein-2 (UCP2) induces Molecular and Cellular Endocrinology 379 (2013) 43–50

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Energy dissipation in brown adipose tissue: From mice to men

Maarten J. Vosselman, Wouter D. van Marken Lichtenbelt, Patrick Schrauwen ⇑

Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Center+, Maastricht, The Netherlands article info abstract

Article history: In rodents, brown adipose tissue (BAT) is a metabolic organ that produces heat in response to cold and Available online 28 April 2013 dietary intake through mitochondrial uncoupling. For long time, BAT was considered to be solely important in small mammals and infants, however recent studies have shown that BAT is also functional in Keywords: adult humans. Interestingly, the presence and/or functionality of this thermogenic tissue is diminished Brown adipose tissue in obese people, suggesting a link between human BAT and body weight regulation. In the last years, evi- Beige adipocytes dence has also emerged for the existence of adipocytes that may have an intermediate thermogenic phe- UCP-1 notype between white and brown adipocytes, so called brite or beige adipocytes. Together, these ﬁndings Thermogenesis have resulted in a renewed interested in (human) brown adipose tissue and pathways to increase the Mitochondria Obesity activity and recruitment of these thermogenic cells. Stimulating BAT hypertrophy and hyperplasia in humans could be a potential strategy to target obesity. Here we will review suggested pathways leading to BAT activation in humans, and discuss novel putative BAT activators in rodents into human perspective. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 2011). Importantly, an inverse relationship has been shown between adiposity and BAT activity, indicating a relationship be- Brown adipose tissue (BAT) is a crucial organ in facultative tween BAT and obesity (Cypess et al., 2009; Saito et al., 2009; thermogenesis (acute response) and has a great plasticity to re- van Marken Lichtenbelt et al., 2009; Vijgen et al., 2011). In addi- spond to long-term changes (e.g. cold acclimation), known as tion, there is evidence that BAT contributes to nonshivering ther- adaptive thermogenesis. In addition to its important role in main- mogenesis (Orava et al., 2011; Ouellet et al., 2012; Vijgen et al., taining thermal homeostasis, BAT is likely to be involved in en- 2011; Yoneshiro et al., 2011), although this relationship has not ergy homeostasis as well, since ablation of the essential protein always been found (van Marken Lichtenbelt et al., 2009; Vossel- for heat production in BAT, uncoupling protein-1 (UCP-1), leads man et al., 2012). It has been estimated that fully activated BAT to an obese phenotype in mice housed at a thermoneutral tem- in humans can contribute to 5% of the basal metabolic rate (van perature (Feldmann et al., 2009). Furthermore, it has been shown Marken Lichtenbelt and Schrauwen, 2011). This means that stim- in mice that BAT is involved in plasma triglyceride clearance (Bar- ulation of BAT can have an impact on long-term energy balance telt et al., 2011) and glucose homeostasis (Guerra et al., 2001; and thus body weight, however only when other factors (e.g. food Gunawardana and Piston, 2012). This implies the important role intake) remain stable (Christiansen and Garby, 2002). Maybe of BAT in rodents to combat obesity and its related metabolic dis- more important, stimulation of BAT could be supportive in body eases, such as diabetes and cardiovascular disease. Interestingly, weight maintenance. Therefore, finding strategies to increase prospective studies have now demonstrated BAT to be present BAT activity and recruitment in humans could be important to and functional in most (prevalence varying from 40% to 100%) combat obesity and its related chronic metabolic diseases. Cur- young lean human adults by exposing them to cold (Cypess rently, much effort is being put in finding ways to increase BAT et al., 2012; Orava et al., 2011; Ouellet et al., 2012; Vijgen thermogenesis and the recruitment of brown adipocytes in ro- et al., 2011; Vosselman et al., 2012; Yoneshiro et al., 2012, dents. The recent discovery of the so-called brown-in-white (brite) or beige adipocytes has further increased the interest in Corresponding author. Address: Department of Human Biology, NUTRIM School BAT. Increased ‘‘browning’’ of WAT could be an attractive way ⇑ for Nutrition, Toxicology and Metabolism, Maastricht University Medical Center, to induce weight loss. It is therefore important to find strategies P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel.: +31 (0) 43 3881502; fax: to increase the thermogenic machinery of BAT and brown-like tis- +31 (0) 43 3670976. sues in humans. This review will provide an overview of the most E-mail addresses: [email protected] (M.J. Vosselman), promising pathways to increase BAT activity and recruitment in [email protected] (W.D. van Marken Lichtenbelt), [email protected] (P. Schrauwen). humans.

0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.04.017 44 M.J. Vosselman et al. / Molecular and Cellular Endocrinology 379 (2013) 43–50

2. Brown, beige and white adipose tissue (suprarenal and perihepatic), and sometimes in areas such as the abdominal wall and acromial–clavicular area. The most prominent 2.1. Brown versus white adipose tissue and most reported BAT depot in humans is supraclavicular BAT, which has not been found as a distinct depot in mice. In rodents, brown adipose tissue is clearly distinguishable from white adipose tissue, since it is richly innervated by the sympa- 2.2. Brown like cells in white adipose tissue thetic nervous system (SNS), is highly vascularized, and contains brown adipocytes with several small lipid vacuoles and many large In addition to classical BAT, a distinct type of adipocytes has mitochondria (Frontini and Cinti, 2010). Unique for the brown adi- been found within WAT depots, the so-called brite (Petrovic pocyte is the protein UCP-1 located in the inner mitochondrial et al., 2010) or beige adipocytes (Wu et al., 2012). However, this membrane, which allows protons in the intermembrane space to is still under dispute as white fat cells may differentiate into brown re-enter the mitochondrial matrix without generating ATP, ulti- fat cells (Cinti, 2002). At present, no consensus on the terminology mately resulting in heat production. of these brown-like white adipocytes has been reached, and is ur- White and brown adipose tissue in mice can be found in distinc- gently awaited. However, for sake of clarity we will refer to these tive or classical (i.e. pure white or pure brown) depots (Fig. 1A). All cells as beige adipocytes in the remainder of this review. These these depots have been characterized by genetic markers, and have beige adipocytes can appear within WAT depots after long-term a distinct genetic profile that probably determines its function adrenergic stimulation and cold exposure, and especially appear (Waldén et al., 2012). Note that these adipose tissue depots some- in the inguinal depot. In the basal state, these beige cells resemble times are also viewed as one organ, known as the adipose organ the unilocular white adipocytes, whereas upon stimulation these (Cinti, 2001). cells obtain a more brown like phenotype. Furthermore, these cells The largest BAT depot found in mice, iBAT, is predominantly do not express the myogenic markers nor the brown adipocyte found in human neonates and infants, and then gradually disap- specific markers Zic1, Lhx8, Meox2, and PRDM16 (Petrovic et al., pears after childhood (Heaton, 1972) and is rarely seen in human 2010), but express specific markers as well (e.g. Hoxc9) (Waldén 18 adults (Fig. 1B). In adult humans, BAT ([ F]FDG-uptake) is often et al., 2012). Interestingly, Wu et al. (2012) were able to isolate found in the neck, the mediastinum (para-aortic), and above the brown-like cells from the subcutaneous (inguinal) adipose depot kidney (suprarenal), which is comparable to some BAT depots and found a distinct pool of progenitors giving rise to these so- (cBAT, mBAT, prBAT) in mice. Furthermore, BAT in humans is lo- called beige cell lines. Linkage of the expressed genes after micro- cated along the spinal cord, in the axillary and abdominal region array analysis in these cell lines revealed that beige adipocytes are

A B

C D

Fig. 1. Overview of brown adipose tissue and beige adipose depots in rodents and humans. (A) Image of a mouse demonstrating classical BAT depots and WAT depots that are susceptible to browning (beige depots). (B) The upper part shows a PET image of a lean human adult demonstrating 18F-FDG-uptake in BAT locations during cold exposure and the lower image a transversal PET/CT fusion slice, which demonstrates a rare example of interscapular BAT in a lean human adult. (C) Overview of depot specific markers for both BAT and beige depots in mice. (D) Overview of depot specific markers determined in human BAT based on marker found in beige depots from mice. BAT = Brown adipose tissue, WAT = White adipose tissue. ÃMeasured in children (Sharp et al., 2012), ÃÃMeasured in human adults (Wu et al., 2012). M.J. Vosselman et al. / Molecular and Cellular Endocrinology 379 (2013) 43–50 45 identical, but not similar, to classical brown adipocytes. These cytes. This leads to increased cAMP levels activating protein kinase beige adipocytes were characterized by unique genetic markers, A (PKA), which has both acute and chronic effects on BAT. The such as CD137, Tmem26, and Tbx1, and did not contain unique acute response of PKA is to increase lipolysis leading to increased BAT markers such as Ebf3, Eva1 and Fbxo31 (Fig. 1C). Furthermore, cytosolic FFA levels. These FFA’s are essential for mitochondrial en- they found that unstimulated beige cells resemble white adipo- ergy dissipation, as they are both used as substrates and to activate cytes since they have similar low expression of the brown adipo- UCP-1 in the inner mitochondrial membrane. This acute effect in- cyte markers UCP-1, Cidea, and Cox7a1. However, upon cAMP creases UCP-1 activity within seconds. On the other hand, pro- stimulation absolute levels of UCP-1 mRNA and respiratory capac- longed stimulation of BAT for hours and days will result in ity were comparable to classical brown adipocytes. In coherence increased amounts of UCP-1 protein, mitochondrial biogenesis, with this, Petrovic et al. (2010) showed beige cell recruitment in and both hyperplasia and hypertrophy of BAT (Lowell and Spiegel- the inguinal depot, and that even the purest WAT depot (epidydi- man, 2000). The thyroid hormone works synergistic with NE and is mal) contains preadicpocytes that can be induced to functional required to generate the full thermogenic response (Silva, 2006). beige adipocytes. Again, a low basal UCP-1 expression was found Crucial is the enzyme type II iodothyronine deiodinase (D2, which during baseline conditions, however, after rosiglitazone (PPARc can transform the inactive prohormone thyroxine (T4) into the bio- activator) treatment beige cells increased UCP-1 expression and active hormone triiodothyronine (T3). demonstrated many features of the classical brown adipocytes. In contrast to the extensive knowledge in rodents, the regula- Moreover, oxygen consumption increased after treating these cells tion and activation of human BAT is still fairly unknown. However, with norepinephrine. In conclusion, it is clear that beige adipocytes both the SNS and the thyroid axis are thought to be crucial in acti- are molecular and developmentally different from classical brown vation of BAT in humans too. For instance, patients with pheochro- adipocytes, however, after stimulation these cells show compara- mocytoma, who have elevated plasma catecholamine levels due to ble thermogenic potential. a catecholamine-secreting tumor in de adrenal gland, had high rates of [18F]FDG-uptake in BAT in the basal state (Hadi et al., 2007; Joshi and Lele, 2012; Kuji et al., 2008; Yamaga et al., 2008), 2.3. Type of brown adipocytes in humans which disappeared after resection of the tumor (Hadi et al., 2007; Yamaga et al., 2008). Thus, high systemic levels of NE can Do humans have brown, beige or both types of adipocytes? activate human BAT, indicating a role for the adrenergic part of Studies measuring BAT via FDG-PET/CT cannot determine the type the SNS. In support, inhibition of -adrenergic receptors with proof BAT, as it merely measures glucose uptake. However, biopsies b pranolol diminished [18F]FDG-uptake in the supraclavicular BAT from the supraclavicular region (Virtanen et al., 2009) and the peri- during room temperature (Parysow et al., 2007; Soderlund et al., thyroid region (Zingaretti et al., 2009) have already demonstrated a 2007). In 2009, anatomical evidence for the SNS involvement in mixture of both white and brown adipocytes, and showed that BAT was found, as BAT biopsies from the perithyroid (Zingaretti brown adipocytes occur as islands within WAT. The clearly brown et al., 2009) and supraclavicular region (Virtanen et al., 2009) dem- colored depots as found in mice, have not (yet) been found in hu- onstrated sympathetic innervation and mRNA expression of the - mans. Interestingly, a recent study has examined several adipose b3 adrenergic receptor respectively. Finally, disruption of the sympa- depots in 13 post-mortem children (aged between 3 days and thetic fibers completely abolished [18F]FDG-uptake in BAT in a pa- 18 years) and also found the brown like cells dispersed within tient with the Horner Syndrome (deficiency of sympathetic WAT (Sharp et al., 2012). These brown adipocyte islands were lo- activity) (Lebron et al., 2010). With respect to the thyroid hormone cated in the subcutaneous supraclavicular areas, posterior medias- axis, it is known from patients that thyroid hormone replacement tinum, retroperitoneal, intraabdominal, and mesenteric depots. has significant effects on resting energy expenditure (al-Adsani In the study by Wu et al. (2012), human adipose tissue biopsies et al., 1997). It has also been shown that elevated levels of thyroid (both WAT and BAT) from the supraclavicular area were analyzed hormones are able to increase mitochondrial uncoupling in skele- for gene expression. Interestingly, we found increased mRNA tal muscle (Lebon, 2001; Mitchell et al., 2010). Although these re- expression of the genes that were characteristic for beige cells sults may suggest that thyroid hormone also plays a role in (CD137, Tmem26, Tbx1) and not classical murine BAT (Ebf3, activation of BAT, evidence for such a role in humans has so far Eva1, Fbxo31) (Fig. 1D). In addition, we also performed immuno- not been demonstrated. histochemistry to identify the beige marker proteins (CD137 and Tmem26), and found positive staining for both proteins in the UCP-1 positive cells. Thus, human BAT from the supraclavicular 4. Known BAT activators in humans area in adults has beige characteristics. In the study by Sharp et al. (2012), total RNA was isolated from the BAT depots from Since the discovery of functional BAT in adult humans, some post-mortem children, and interestingly, all BAT depots expressed studies have examined possible routes to activate BAT in humans, beige-cell selective genes and no classical brown fat-selective which will be discussed here. genes (Fig. 1D). They found that these BAT depots correlated well with the expression of brown fat genes such as PGC1a and 4.1. Cold exposure and acclimatization PRDM16, and with beige-cell genes (e.g. Cited1), however not with classical BAT genes (e.g. Zic1) (Sharp et al., 2012). These findings Currently, the strategy for which most evidence has been gath- thus indicate that human BAT may be composed of mainly beige ered to activate BAT in humans is cold exposure. In our cold expo- adipocytes. sure experiments we applied a personalized cooling protocol in which we decreased ambient temperature until shivering occurred 3. BAT physiology (16–17 °C) and then increased temperature slightly again. At these mild cold temperatures, cold-induced thermogenesis levels in Crucial in activating BAT thermogenesis is the sympathetic ner- young lean male adults were observed between 5% and 30% (van vous system (SNS). Furthermore, the thermogenic capacity of BAT Marken Lichtenbelt et al., 2009; Vosselman et al., 2012). Brown is dependent on the thyroid hormone axis as well. After sympa- adipose tissue was present in all individuals, however the activity thetic stimulation, norepinephrine (NE) is released at the nerve level of BAT did not correlate to cold-induced thermogenesis. Other endings activating the adrenergic receptors on the brown adipo- studies did find a relationship between BAT presence and/or activ- 46 M.J. Vosselman et al. / Molecular and Cellular Endocrinology 379 (2013) 43–50 ity and cold-induced thermogenesis (Orava et al., 2011; Ouellet PET-CT) showed a higher increase in energy expenditure upon cap- et al., 2012; Vijgen et al., 2012, 2011; Yoneshiro et al., 2011). Cold sinoid intake compared to subjects without BAT. The proposed exposure likely increased BAT activity via the SNS as shown by in- mechanism is that capsinoids activate the transient receptor po- creased plasma NE levels during cold exposure (Orava et al., 2011; tential channel 1 (TRPV1) located in the upper digestive tract lead- Vosselman et al., 2012). ing to increased sympathetic nerve activity to BAT, as It is well known from animal studies that long-term cold expo- demonstrated in interscapular BAT in rats (Ono et al., 2011). Direct sure leads to BAT recruitment in both BAT and WAT depots (Young evidence for the effect of capsinoids on BAT in humans is needed to et al., 1984). A cold acclimation study in humans in 1961 demon- draw definite conclusions. Furthermore, whether long-term strated that long-term cold exposure was effective to increase administration of these bioactive compounds can induce weight cold-induced thermogenesis in man, with a decrease in shivering, loss via BAT remains to be studied. indicating a potential role for BAT (Davis, 1961). Preliminary results from our lab confirm this adaptive response to cold acclima- 4.4. Insulin tion as both cold-induced (non-shivering) thermogenesis and BAT activity increase (unpublished results). Another way to induce glucose uptake in BAT is via insulin (Orava et al., 2011). In this study, insulin infusion (hyperinsuline- 4.2. Isoprenaline and ephedrine mic euglycemia) was compared with cold exposure on BAT glucose uptake and perfusion. They showed that insulin increased Rodent studies have clearly demonstrated that BAT can be acti- [18F]FDG-uptake in BAT to similar levels as in skeletal muscle, vated (acute) and recruited (chronic) upon adrenergic receptor and much higher than in WAT. This was most likely due to the high agonist treatment. Recently, three studies measured the effect of expression of GLUT4 in BAT compared to WAT. However, the in- adrenergic stimulation on BAT activity in human adults. We mea- creased [18F]FDG-uptake in BAT was not accompanied by increased sured BAT activity in lean human adults during infusion of the non- perfusion, suggesting that glucose is solely transported in BAT selective b-adrenergic agonist isoprenaline, and compared this to without concomitant thermogenesis taking place. Since insulin cold exposure (Vosselman et al., 2012). Isoprenaline infusion in- leads to high glucose uptake in BAT, it would be interesting to mea- creased energy expenditure with 20% comparable to levels during sure whether BAT is involved in glucose uptake, and possibly ther- cold exposure (17%). Surprisingly, nine out of ten subjects showed mogenesis, in the postprandial state as well. It is known from no BAT activity during isoprenaline infusion, whereas cold expo- rodent studies that single meals can activate BAT, which could be sure increased BAT activity in all subjects. Cypess et al. (2012) due to insulin release (Cannon and Nedergaard, 2004). studied the effects of ephedrine (1 mg/kg), which is a sympathomimetic drug activating b-adrenergic receptors directly and indirectly by enhancing NE release from the sympathetic terminals, 5. Novel putative BAT activators on BAT activity and compared this with cold exposure and placebo. In coherence with our results (Vosselman et al., 2012), ephedrine Next to the known activators of BAT as described above, novel did not result in BAT activity measured by FDG-PET/CT measure- findings in mainly rodent studies hint towards other potentially ments, whereas cold exposure activated BAT in all subjects (Cypess important activators of BAT. The focus of this paragraph will be et al., 2012). However, a recent study using higher dosages of on the hormones irisin (released by muscle) and the natriuretic ephedrine (2.5 mg/kg) did find increased BAT activity in six out peptides (released by heart). We have chosen for these hormones of nine lean human adults, however not in obese subjects (Carey as they are released under physiological conditions (exercise and et al., 2013). This is the first study that showed that pharmacolog- cardiac stress), and because there is already indirect evidence of ical treatment of BAT in humans is possible. The activity level of these activators in humans. For information on the other potential BAT was still 4-fold lower than observed during cold exposure. BAT activators we would like to refer to other reviews (Whittle, The explanation for the lack of effect of systemic adrenergic stim- 2012; Whittle and Vidal-Puig, 2012). ulation on BAT activation is likely that the concentrations of NE reached at the brown adipocyte cell surface during central stimu- 5.1. Exercise and brown adipose tissue: irisin lation (e.g. cold exposure) are higher than systemically reached by the sympathomimetics used in these studies. Thus, pharmaco- Exercise is well known for its beneficial effects on systemic logical stimulation of BAT is possible, however high dosages are re- metabolism. In the past decades, several animal studies have quired. A major burden of very high levels of sympathomimetic investigated whether exercise has beneficial effects on BAT activity drugs is the cardiovascular load induced by adrenergic agonists. and recruitment. It was hypothesized that exercise could affect All three studies observed increased blood pressure and heart rate BAT function via the SNS, as exercise is known to increase general levels, with the highest increase in systolic blood pressure SNS activity (Wickler et al., 1987). However, most studies did not (45 mmHg) measured in the study by Carey et al. (2013). find any stimulatory effect of exercise on BAT activity (Scarpace et al., 1994; Segawa et al., 1998; Shibata and Nagasaka, 1987; 4.3. Capsinoids Wickler et al., 1987), except for studies using swimming exercise protocols (Hirata, 1982a,b; Oh-ishi et al., 1996), which probably in- Another route to activate BAT via the SNS is via ingestion of cer- duced BAT activity to compensate for the heat loss to the water. tain food components. One such component are the capsinoids However, recent studies in rodents did observe stimulating ef- (non-pungent capsaicin analogs), the active compound found in fects of exercise on brown and beige adipocytes (Boström et al., chili pepper, which is known to increase BAT activity in rodents 2012; Seebacher and Glanville, 2010; Slocum et al., 2012; Xu (Kawabata et al., 2009), and RMR in humans (Whiting et al., et al., 2011). A study in rats demonstrated that a low level of exer- 2012), although this has not been consistenly shown (among oth- cise training is beneficial for the metabolic response upon cold ers: Galgani and Ravussin, 2010). A recent study showed that cap- exposure in classical BAT (Seebacher and Glanville, 2010). It was sinoids could be effective in activating BAT in humans (Yoneshiro shown that exercise in combination with cold exposure led to an et al., 2012), without inducing unwanted side-effects. Although increase in UCP-1 expression in BAT, whereas cold exposure they did not directly measure the effect of capsinoid intake on (12 °C) alone did not, suggesting that physical activity is required BAT activity, they found that subjects with BAT (based on cold for an optimal heat producing BAT machinery. Xu et al. (2011) M.J. Vosselman et al. / Molecular and Cellular Endocrinology 379 (2013) 43–50 47 found that exercise in mice led to increased recruitment of adipo- sectional study of 117 middle-aged women (BMI range 20–47.7 kg/ genic progenitor cells in interscapular BAT and increased UCP-1 m2) was found. Interestingly, acute anaerobic exercise (sprint exer- expression ( twofold). Interestingly, in addition to these stimulat- cise) in young healthy subjects increased irisin levels, whereas ing effects of exercise on classical BAT, they found an increased chronic exercise of 8 weeks (three times sprint exercise per week) expression of the thermogenic gene program in epidydimal (vis- did not. It is important to note that this study solely looked at ceral) adipose tissue, including increased UCP-1 levels ( twofold). anaerobic exercise; it is known that aerobic exercise increases Another study showed the occurrence of beige cells within the ret- PGC1a to a greater extent than anaerobic exercise (Handschin roperitoneal depot (visceral) of rats already after 1 week of exer- and Spiegelman, 2008), and this (aerobic) type of exercise would cise (De Matteis et al., 2012). Quantification of UCP-1 positive thus be more effective in irisin production. adipocytes within the retriperitoneal WAT showed a 8-fold in- These first human studies question the potential beneficial ef- crease in the number of brown cells in the exercise group versus fects of irisin on metabolic status. However, prospective studies controls. that measure the direct effects of exercise on browning are re- Interestingly, a recent study demonstrated that endurance exer- quired, and prospective studies should focus on aerobic exercise cise predominantly results in browning of subcutaneous WAT protocols. In addition to the physiological release of irisin by exer- (Boström et al., 2012). In this study, Boström et al. found that mice cise, the therapeutic use of irisin in human clinical trials should be overexpressing the transcriptional coactivator PGC1-a showed in- investigated. creased browning in inguinal WAT. Since exercise also increases PGC1-a, they examined the effect of endurance exercise on mark- 5.2. Natriuretic peptides and brown adipose tissue ers of browning and observed similar effects. In further studies, they identified a new hormone called irisin, which is released by A recent study showed that the cardiac natriuretic peptides skeletal muscle after proteolysis of the membrane protein FNDC5. (NPs) are capable of browning white adipocytes from mice and hu- Boström et al. found that the irisin precursor FNDC5 induced mans (Bordicchia et al., 2012). The cardiac peptides, atrial NP (ANP) browning in primary subcutaneous white adipocytes demon- and the ventricular form (BNP), are predominantly known for their strated by increased UCP1 mRNA (7–500-fold) and the upregula- role in the homeostatic control of blood pressure, by promoting tion of thermogenic genes (Ucp1, Elovl3, Cox7a1 and Otop1). vasodilatation, natriuresis and diuresis, and inhibiting renin and Adenoviral-mediated overexpression of FNDC5 in mouse liver re- aldosterone release (Levin et al., 1998). Later, these hormones were sulted in plasma irisin levels to be increased 3–4-fold, which in- also found to regulate lipolysis as demonstrated both in vitro as creased UCP1 mRNA 13-fold in subcutaneous WAT. Furthermore, in vivo (Sengenès et al., 2000). Natriuretic peptides mediate these the same technique was used in C557BL/6 mice, which are prone lipolytic effects predominantly via the NP receptor A (NPRA), to diet-induced obesity and diabetes, and increased irisin levels whereas the clearance receptor (NPRC) removes the peptides from leading to improved glucose tolerance, decreased fasting insulin, the circulation. Binding of the NPs to the guanylyl cyclase receptor increased oxygen consumption and reduced body weight. Together NPRA leads to increased cellular cGMP, which stimulates lipolysis these results demonstrate the possible beneficial effects of endur- by acting on HSL (Sengenès et al., 2000). These lipolytic effects of ance exercise on the recruitment of beige cells within subcutane- the NPs were only observed in human WAT, and were thought to ous white adipose tissue, and moreover, the potential of irisin to be primate specific due to the high expression of clearance recep- induce a more healthy metabolic phenotype. tors and a low expression of ‘‘biologically active’’ receptors in other Are these stimulating effects of exercise on the recruitment of species (Sengenès et al., 2002). beige cells translatable to humans? Importantly, the authors This was confirmed in the study by Bordicchia et al. (2012), in showed that irisin in mice and humans are 100% identical and that which primary adipocyte cultures from wildtype mice showed no plasma irisin levels were increased twofold after 10 weeks of lipolytic response upon ANP infusion. However, in NPRC knockout endurance training in human subjects (Boström et al., 2012). Inter- mice they did find increased lipolysis in these adipocytes, indicat- estingly, a study in patients with heart failure demonstrated in- ing the inhibitory effects of this clearance receptor. Interestingly, creased FNDC5 expression in skeletal muscle in the patient group these knockout mice had reduced adipose tissue mass and a more with a better aerobic performance (Lecker et al., 2012). Conversely, brownish adipose tissue phenotype. In support, brown adipocyte a recent study by Timmons et al. (2012) demonstrated that the marker genes, such as PRDM16, were elevated in both BAT and stimulating effect of exercise on FNDC5 is limited. They analyzed WAT (inguinal and epididymal). These results indicated the brown- FNDC5 induction by means of gene expression arrays in muscle ing effects on WAT via the NPs. It was then shown that exposing biopsies from 200 subjects from different exercise programs mice to cold (4° for 6 h) significantly increased plasma BNP levels, (both endurance and resistance straining) from earlier published and ANP and BNP mRNA expression in the heart. Furthermore, BNP studies. They found that endurance exercise (6 weeks of endurance infusion in mice increased UCP-1 and PGC-1a mRNA expression in cycling) in young adults as well as resistance training in 20– both WAT and BAT (Bordicchia et al., 2012). Altogether, these data 80 year old men did not increase FNDC5 mRNA. However, only demonstrate that the NPs have the capacity to enhance BAT activ- highly active elderly subjects did show increased (30%) FNDC5 ity and recruitment in mice in vitro and in vivo. compared to sedentary controls. The authors therefore conclude Do these NPs exert similar effects in humans? Administering that the stimulatory effect of exercise on irisin production is lim- ANP systemically and via a microdialysis probe increased lipolysis ited and that irisin probably has little contribution to the overall in healthy men (Birkenfeld et al., 2005). One functional role for the broad benefits of exercise on metabolic status. lipolytic effects of NPs could be substrate supply of fatty acids to Another recent paper on irisin presented data from cross-sec- the heart and muscle during aerobic exercise (Moro et al., 2006). tional and interventional studies on the physiological role of irisin In addition, it is thought that the NPs are important regulators in in humans looking at correlations with anthropometric, metabolic, postprandial fatty acid oxidation in humans (Birkenfeld et al., and hormonal parameters (Huh et al., 2012). The presence of 2008). Interestingly, it is known in humans that low NP levels FNDC5 in human tissues, in skeletal muscle but also in the pericar- are associated with hypertension, obesity, insulin resistance and dium, intracranial artery, and rectum (cardiac and smooth mus- diabetes (Khan et al., 2011; Magnusson et al., 2012). Furthermore, cles) was confirmed and muscle mass was found to be the weight loss in obese subjects by lifestyle intervention (Chainani- primary predictor of circulating irisin in humans. However no sup- Wu et al., 2010) and bariatric surgery (Changchien et al., 2011; port for a beneficial role for irisin in metabolic regulation in a cross Chen-Tournoux et al., 2010; St Peter et al., 2006) showed that 48 M.J. Vosselman et al. / Molecular and Cellular Endocrinology 379 (2013) 43–50

BNP levels are increased after weight loss. Interestingly patients sure is the most effective in stimulating BAT in humans. Adjusting with heart failure who suffer from severe weight loss (cachexia) ambient temperature in public buildings to the lower range of our have increased levels of both forms of NPs (de Lemos et al., thermoneutral zone could therefore be a sensible and physiological 2003; Tikkanen et al., 1985), and elevated energy expenditure lev- way to increase thermogenesis by increasing the thermogenic po- els, and it could be suggested that elevated NP levels increase tential of BAT. Adrenergic agonists (isoprenaline and ephedrine) brown adipocyte recruitment and activity leading to elevated EE. have not shown to be effective in BAT activation as high dosages Birkenfeld et al. (2005, 2008) showed that ANP infusion increased are required. This indicates that pharmacological activation of postprandial energy expenditure, however energy expenditure in BAT via the adrenergic part of the SNS is difficult. Furthermore, a the fasted state was not affected. The dosage (25 ng/kg/min) of major drawback of adrenergic agonists and sympathomimetics is ANP used by Birkenfeld et al. (2008) increased plasma ANP concen- the associated cardiovascular stress. Sympathetic activation via trations fourfold (approximately 300 pg/mL), which is lower than capsinoids could be a way to increase energy expenditure and pos- found in heart failure patients (>500 pg/mL). This relative low dose sibly weight loss (with low risks of adverse events), and the indi- already affected lipid mobilization and postprandial thermogenesis rect evidence of BAT being a mediator is promising. Insulin has (and possibly BAT) without causing any adverse effects. Currently, been shown to induce glucose uptake in BAT to higher levels than therapeutic use of NPs (carperitide and nesiritide) in patients with WAT, and comparable to skeletal muscle. However, since perfusion acute heart failure and acutely decompensated heart failure is only of BAT was absent, it remains unclear whether actual thermogen- possible by means of infusion and not orally (Saito, 2010). esis takes place after insulin stimulation. The potential effect of NPs on browning in humans has been Interestingly, studies in rodents have shown additional demonstrated in the study of Bordicchia et al., where they tested pathways to activate BAT and recruit beige adipocytes. Two of whether NPs could induce a thermogenic gene program in differ- them – irisin and NPs – have recently attracted much attention, entiated human multipotent adipose-derived stem (hMADS) cells but definitive answers in humans are so far lacking. Therefore, and subcutaneous adipocytes. Interestingly, both ANP and BNP the coming years are crucial in finding and testing novel activators activated PGC-1a and UCP-1 expression, induced mitochondrial of BAT in human clinical trials, but most of all to test the hypoth- biogenesis, and increased uncoupled and total respiration. These esis that activation of BAT may indeed be of importance in the findings imply the potential role of the NPs in increasing acute treatment of human obesity. Furthermore, future studies should thermogenesis and brown adipocyte recruitment in humans. They also reveal if continuous activation of mitochondrial uncoupling demonstrated that the mechanism of action of the NP’s share a in BAT could lead to hyperthermia, as has previously been shown common downstream target with the adrenergic pathway, namely to occur when dinitrophenol was used in humans to obtain weight p38 MAPK. Activation of the p38 MAPK pathway ultimately leads loss. to increased transcription of UCP-1 and PGC-1a (Bordicchia et al., 2012). Moreover, it was shown that ANP treatment of hMADS led Acknowledgements to a similar increase in UCP-1, PGC-1a, and cytochrome c protein levels as shown during b-adrenergic treatment. The authors also We would like to thank Boudewijn Brans for his helpful sugges- found that both the adrenergic and NP’s signaling pathways work tions and Anouk van der Lans for providing the PET-CT image. additive at very low (physiological) concentrations. The activation pathway of the NPs could therefore play a prominent role in addi- References tion to the well-known adrenergic pathway in inducing both short- term as long-term effects on BAT. Currently, this is the only direct al-Adsani, H., Hoffer, L., Silva, J., 1997. Resting energy expenditure is sensitive to small dose changes in patients on chronic thyroid hormone replacement. J. Clin. evidence of browning effects via NPs in humans and future studies Endocrinol. Metab. 82, 1118–1125. are warranted. Bartelt, A., Bruns, O.T., Reimer, R., Hohenberg, H., Ittrich, H., Peldschus, K., Kaul, M.G., Tromsdorf, U.I., Weller, H., Waurisch, C., Eychmüller, A., Gordts, P.L., Rinninger, F., Bruegelmann, K., Freund, B., Nielsen, P., Merkel, M., Heeren, J., 2011. Brown adipose tissue activity controls triglyceride clearance. Nat. Med. 17, 200–205. 6. Conclusions and perspectives Birkenfeld, A., Boschmann, M., Moro, C., Adams, F., Heusser, K., Franke, G., Berlan, M., Luft, F., Lafontan, M., Jordan, J., 2005. Lipid mobilization with physiological atrial The current global obesity problem is affecting more than 1.4 natriuretic peptide concentrations in humans. J. Clin. Endocrinol. Metab. 90, 3622–3628. billion adults of 20 years and older, and strikingly, more than 40 Birkenfeld, A., Budziarek, P., Boschmann, M., Moro, C., Adams, F., Franke, G., Berlan, million children under the age of five were overweight in 2010 M., Marques, M., Sweep, F., Luft, F., Lafontan, M., Jordan, J., 2008. Atrial (WHO). Obesity goes along with increased risk on developing dis- natriuretic peptide induces postprandial lipid oxidation in humans. Diabetes 57, 3199–3204. eases such as type 2 diabetes and cardiovascular diseases. Finding Bordicchia, M., Liu, D., Amri, E.-Z., Ailhaud, G., Dessì-Fulgheri, P., Zhang, C., strategies to induce weight loss are therefore necessary. Currently, Takahashi, N., Sarzani, R., Collins, S., 2012. Cardiac natriuretic peptides act via brown adipose tissue is regarded as a potential tissue to tackle p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes. J. Clin. Invest. 122, 1022–1036. obesity due to its great capacity to increase energy expenditure Boström, P., Wu, J., Jedrychowski, M., Korde, A., Ye, L., Lo, J., Rasbach, K., Boström, E., and thereby stimulating weight loss. The rediscovery of functional Choi, J., Long, J., Kajimura, S., Zingaretti, M.C., Vind, B.F., Tu, H., Cinti, S., Højlund, BAT in humans has resulted in an explosion of BAT studies, espe- K., Gygi, S.P., Spiegelman, B.M., 2012. A PGC1-a-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481, 463– cially in rodents, to find potential molecules that could lead to 468. BAT hypertrophy and hyperplasia. It is now clear that a third type Cannon, B., Nedergaard, J., 2004. Brown adipose tissue: function and physiological of adipocyte exists, the beige adipocyte, which can be recruited significance. Physiol. Rev. 84, 277–359. within WAT after cold acclimation and long-term adrenergic Carey, A.L., Formosa, M.F., Van Every, B., Bertovic, D., Eikelis, N., Labert, G.W., Kalff, V., Duffy, S.J., Cherk, M.H., Kingwell, B.A., 2013. Ephedrine activates brown receptor stimulation. This distinct type of adipocyte has shown adipose tissue in lean but not obese humans. Diabetologia 56, 147–155. to arise from a different lineage as the other two types, although Chainani-Wu, N., Weidner, G., Purnell, D., Frenda, S., Merritt-Worden, T., Kemp, C., functionally and metabolically seen it is similar to the brown adi- Kersh, E., Ornish, D., 2010. Relation of B-type natriuretic peptide levels to body mass index after comprehensive lifestyle changes. Am. J. Cardiol. 105, 1570– pocyte. Current evidence shows that human BAT is likely com- 1576. posed of mainly beige adipocytes. Changchien, E., Ahmed, S., Betti, F., Higa, J., Kiely, K., Hernandez-Boussard, T., Prospective studies in humans are scarce, mostly because of the Morton, J., 2011. B-type natriuretic peptide increases after gastric bypass surgery and correlates with weight loss. Surg. Endosc. 25, 2338–2343. difficulties associated with the technique to measure BAT activity Chen-Tournoux, A., Khan, A., Baggish, A., Castro, V., Semigran, M., McCabe, E., (PET-CT). Nevertheless, current studies have shown that cold expo- Moukarbel, G., Reingold, J., Durrani, S., Lewis, G., Newton-Cheh, C., Scherrer- M.J. Vosselman et al. / Molecular and Cellular Endocrinology 379 (2013) 43–50 49

Crosbie, Kaplan, L.M., Wang, T.J., 2010. Effect of weight loss after weight loss the prospective Malmo Diet and Cancer study. J. Clin. Endocrinol. Metab. 97, surgery on plasma N-terminal pro-B-type natriuretic peptide levels. Am. J. 638–645. Cardiol. 106, 1450–1455. Mitchell, C., Savage, D., Dufour, S., Schoenmakers, N., Murgatroyd, P., Befroy, D., Christiansen, E., Garby, L., 2002. Prediction of body weight changes caused by Halsall, D., Northcott, S., Raymond-Barker, P., Curran, S., Henning, E., Keogh, J., changes in energy balance. Eur. J. Clin. Invest. 32, 826–830. Owen, P., Lazarus, J., Rothman, D.L., Farooqi, I.S., Shulman, G.I., Chatterjee, K., Cinti, S., 2001. The adipose organ: morphological perspectives of adipose tissues. Petersen, K.F., 2010. Resistance to thyroid hormone is associated with raised Proc. Nutr. Soc. 60, 319–328. energy expenditure, muscle mitochondrial uncoupling, and hyperphagia. J. Clin. Cinti, S., 2002. Adipocyte differentiation and transdifferentiation: Plasticity of the Invest. 120, 1345–1354. adipose organ. J. Endicrinol. Invest. 25, 823–825. Moro, C., Polak, J., Hejnova, J., Klimcakova, E., Crampes, F., Stich, V., Lafontan, M., Cypess, A.M., Lehman, S., Williams, G., Tal, I., Rodman, D., Goldfine, A.B., Kuo, F.C., Berlan, M., 2006. Atrial natriuretic peptide stimulates lipid mobilization during Palmer, E.L., Tseng, Y.H., Doria, A., Kolodny, G.M., Kahn, C.R., 2009. Identification repeated bouts of endurance exercise. Am. J. Physiol. Endocrinol. Met. 290, and importance of brown adipose tissue in adult humans. N. Engl. J. Med. 360, E864–869. 1509–1517. Oh-ishi, S., Kizaki, T., Toshinai, K., Haga, S., Fukuda, K., Nagata, N., Ohno, H., 1996. Cypess, A.M., Chen, Y.C., Sze, C., Wang, K., English, J., Chan, O., Holman, A.R., Tal, I., Swimming training improves brown-adipose-tissue activity in young and old Palmer, M.R., Kolodny, G.M., Kahn, C.R., 2012. Cold but not sympathomimetics mice. Mech. Ageing Dev. 89, 67–78. activates human brown adipose tissue in vivo. Proc. Natl. Acad. Sci. USA 109, Ono, K., Tsukamoto-Yasui, M., Hara-Kimura, Y., Inoue, N., Nogusa, Y., Okabe, Y., 10001–10005. Nagashima, K., Kato, F., 2011. Intragastric administration of capsiate, a transient Davis, T., 1961. Chamber cold acclimatization in man. J. Appl. Physiol. 16, 1011– receptor potential channel agonist, triggers thermogenic sympathetic 1015. responses. J. Appl. Physiol. 110, 789–798. de Lemos, J., McGuire, D., Drazner, M., 2003. B-type natriuretic peptide in Orava, J., Nuutila, P., Lidell, M., Oikonen, V., Noponen, T., Viljanen, T., Scheinin, M., cardiovascular disease. Lancet 362, 316–322. Taittonen, M., Niemi, T., Enerbäck, S., Virtanen, K.A., 2011. Different metabolic De Matteis, R., Lucertini, F., Guescini, M., Polidori, E., Zeppa, S., Stocchi, V., Cinti, S., responses of human brown adipose tissue to activation by cold and insulin. Cell Cuppini, R., 2012. Exercise as a new physiological stimulus for brown adipose Metab. 14, 272–279. tissue activity. Nutr. Metab. Cardiovac. Dis. (Epub ahead of print). Ouellet, V., Labbé, S., Blondin, D., Phoenix, S., Guérin, B., Haman, F., Turcotte, E., Feldmann, H.M., Golozoubova, V., Cannon, B., Nedergaard, J., 2009. UCP1 ablation Richard, D., Carpentier, A., 2012. Brown adipose tissue oxidative metabolism induces obesity and abolishes diet-induced thermogenesis in mice exempt from contributes to energy expenditure during acute cold exposure in humans. J. thermal stress by living at thermoneutrality. Cell Metab. 9, 203–209. Clin. Invest. 122, 545–552. Frontini, A., Cinti, S., 2010. Distribution and development of brown adipocytes in the Parysow, O., Mollerach, A.M., Jager, V., Racioppi, S., San Roman, J., Gerbaudo, V.H., murine and human adipose organ. Cell Metab. 11, 253–256. 2007. Low-dose oral propranolol could reduce brown adipose tissue F-18 FDG Galgani, J., Ravussin, E., 2010. Effect of dihydrocapsiate on testing metabolic rate in uptake in patients undergoing PET scans. Clin. Nucl. Med. 32, 351–357. humans. Am. J. Clin. Nutr. 92, 1089–1093. Petrovic, N., Walden, T., Shabalina, I., Timmons, J., Cannon, B., Nedergaard, J., 2010. Guerra, C., Navarro, P., Valverde, A., Arribas, M., Brüning, J., Kozak, L., Kahn, C., Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) Benito, M., 2001. Brown adipose tissue-specific insulin receptor knockout activation of epididymally derived white adipocyte cultures reveals a shows diabetic phenotype without insulin resistance. J. Clin. Invest. 108, 1205– population of thermogenically competent, UCP1-containing adipocytes 1213. molecularly distinct from classic brown adipocytes. J. Biol. Chem. 285, 7153– Gunawardana, S.C., Piston, D.W., 2012. Reversal of type 1 diabetes in mice by brown 7164. adipose tissue transplant. Diabetes 61, 674–682. Saito, Y., 2010. Roles of atrial natriuretic peptide and its therapeutic use. J. Cardiol. Hadi, M., Chen, C., Whatley, M., Pacak, K., Carrasquillo, J., 2007. Brown fat imaging 56, 262–270. with (18)F-6-fluorodopamine PET/CT, (18)F-FDG PET/CT, and (123)I-MIBG Saito, M., Okamatsu-Ogura, Y., Matsushita, M., Watanabe, K., Yoneshiro, T., Nio- SPECT: a study of patients being evaluated for pheochromocytoma. J. Nucl. Kobayashi, J., Iwanaga, T., Miyagawa, M., Kameya, T., Nakada, K., Kawai, Y., Med. 48, 1077–1083. Tsujisaki, M., 2009. High incidence of metabolically active brown adipose tissue Handschin, C., Spiegelman, B., 2008. The role of exercise and PGC1alpha in in healthy adult humans: effects of cold exposure and adiposity. Diabetes 58, inflammation and chronic disease. Nature 454, 463–469. 1526–1531. Heaton, J., 1972. The distribution of brown adipose tissue in the human. J. Anat. 112, Scarpace, P., Yenice, S., Tümer, N., 1994. Influence of exercise training and age on 35–39. uncoupling protein mRNA expression in brown adipose tissue. Pharmacol. Hirata, K., 1982a. Blood flow to brown adipose tissue and norepinephrine-induced Biochem. Behav. 49, 1057–1105. calorigenesis in physically trained rats. Jpn. J. Physiol. 32, 279–291. Seebacher, F., Glanville, E., 2010. Low levels of physical activity increase metabolic Hirata, K., 1982b. Enhanced calorigenesis in brown adipose tissue in physically responsiveness to cold in a rat (Rattus fuscipes). PLoS ONE 5, e13022. trained rats. Jpn. J. Physiol. 32, 647–653. Segawa, M., Oh-Ishi, S., Kizaki, T., Ookawara, T., Sakurai, T., Izawa, T., Nagasawa, J., Huh, J., Panagiotou, G., Mougios, V., Brinkoetter, M., Vamvini, M., Schneider, B., Kawada, T., Fushiki, T., Ohno, H., 1998. Effect of running training on brown Mantzoros, C., 2012. FNDC5 and irisin in humans: I. Predictors of circulating adipose tissue activity in rats: a reevaluation. Res. Commun. Mol. Pathol. concentrations in serum and plasma and II. mRNA expression and circulating Pharmacol. 100, 77–82. concentrations in response to weight loss and exercise. Metabolism 61, 1725– Sengenès, C., Berlan, M., De Glisezinski, I., Lafontan, M., Galitzky, J., 2000. Natriuretic 1738. peptides: a new lipolytic pathway in human adipocytes. FASEB J. 14, 1345– Joshi, P., Lele, V., 2012. Unexpected visitor on FDG PET/CT-Brown Adipose Tissue 1351. (BAT) in mesentery in a case of retroperitoneal extra-adrenal Sengenès, C., Zakaroff-Girard, A., Moulin, A., Berlan, M., Bouloumié, A., Lafontan, M., pheochromocytoma: is the BAT activation secondary to catecholamine- Galitzky, J., 2002. Natriuretic peptide-dependent lipolysis in fat cells is a secreting pheochromocytoma? Clin. Nucl. Med. 37, 20. primate specificity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R257–265. Kawabata, F., Inoue, N., Masamoto, Y., Matsumura, S., Kimura, W., Kadowaki, M., Sharp, L., Shinoda, K., Ohno, H., Scheel, D., Tomoda, E., Ruiz, L., Hu, H., Wang, L., Higashi, T., Tominaga, M., Inoue, K., Fushiki, T., 2009. Non-pungent capsaicin Pavlova, Z., Gilsanz, V., Kajimura, S., 2012. Human BAT possesses molecular analogs (capsinoids) increase metabolic rate and enhance thermogenesis via signatures that resemble beige/brite cells. PLoS ONE 7, e49452. gastrointestinal TRPV1 in mice. Biosci. Biotechnol. Biochem. 73, 2690–2697. Shibata, H., Nagasaka, T., 1987. The effect of forced running on heat production in Khan, A., Cheng, S., Magnusson, M., Larson, M., Newton-Cheh, C., McCabe, E., brown adipose tissue in rats. Physiol. Behav. 39, 377–380. Coviello, A., Florez, J., Fox, C., Levy, D., Robins, S.J., Arora, P., Bhasin, S., Lam, C.S., Silva, J., 2006. Thermogenic mechanisms and their hormonal regulation. Physiol. Vasan, R.S., Melander, O., Wang, T.J., 2011. Cardiac natriuretic peptides, obesity, Rev. 86, 435–464. and insulin resistance: evidence from two community-based studies. J. Clin. Slocum, N., Durrant, J., Bailey, D., Yoon, L., Jordan, H., Barton, J., Brown, R., Clifton, L., Endocrinol. Metab. 96, 3242–3249. Milliken, T., Harrington, W., Kimbrough, C., Faber, C.A., Cariello, N., Elangbam, Kuji, I., Imabayashi, E., Minagawa, A., Matsuda, H., Miyauchi, T., 2008. Brown C.S., 2012. Responses of brown adipose tissue to diet-induced obesity, exercise, adipose tissue demonstrating intense FDG uptake in a patient with mediastinal dietary restriction and ephedrine treatment. Exp. Toxicol. Pathol. (Epub ahead pheochromocytoma. Ann. Nucl. Med. 22, 231–235. of print). Lebon, V., 2001. Effect of triiodothyronine on mitochondrial energy coupling in Soderlund, V., Larsson, S.A., Jacobsson, H., 2007. Reduction of FDG uptake in brown human skeletal muscle. J. Clin. Invest. 108. adipose tissue in clinical patients by a single dose of propranolol. Eur. J. Nucl. Lebron, L., Chou, A., Carrasquillo, J., 2010. Interesting image. Unilateral F-18 FDG Med. Mol. Imaging 34, 1018–1022. uptake in the neck, in patients with sympathetic denervation. Clin. Nucl. Med. St Peter, J., Hartley, G., Murakami, M., Apple, F., 2006. B-type natriuretic peptide 35, 899–901. (BNP) and N-terminal pro-BNP in obese patients without heart failure: Lecker, S., Zavin, A., Cao, P., Arena, R., Allsup, K., Daniels, K., Joseph, J., Schulze, P., relationship to body mass index and gastric bypass surgery. Clin. Chem. 52, Forman, D., 2012. Expression of the irisin precursor FNDC5 in skeletal muscle 680–685. correlates with aerobic exercise performance in patients with heart failure. Circ. Tikkanen, I., Fyhrquist, F., Metsärinne, K., Leidenius, R., 1985. Plasma atrial Heart. Fail. 5, 812–818. natriuretic peptide in cardiac disease and during infusion in healthy Levin, E., Gardner, D., Samson, W., 1998. Natriuretic peptides. N. Engl. J. Med. 339, volunteers. Lancet 2, 66–69. 321–328. Timmons, J.A., Baar, K., Davidsen, P.K., Atherton, P.J., 2012. Is irisin a human exercise Lowell, B.B., Spiegelman, B.M., 2000. Towards a molecular understanding of gene? Nature 480, E9–10. adaptive thermogenesis. Nature 404, 652–660. van Marken Lichtenbelt, W., Schrauwen, P., 2011. Implications of nonshivering Magnusson, M., Jujic, A., Hedblad, B., Engström, G., Persson, M., Struck, J., thermogenesis for energy balance regulation in humans. Am. J. Physiol. Regul. Morgenthaler, N., Nilsson, P., Newton-Cheh, C., Wang, T., Melander, O., 2012. Integr. Comp. Physiol. 301, 96. Low plasma level of atrial natriuretic peptide predicts development of diabetes: 50 M.J. Vosselman et al. / Molecular and Cellular Endocrinology 379 (2013) 43–50 van Marken Lichtenbelt, W.D., Vanhommerig, J.W., Smulders, N.M., Drossaerts, J.M., Wu, J., Boström, P., Sparks, L., Ye, L., Choi, J., Giang, A.-H., Khandekar, M., Virtanen, K., Kemerink, G.J., Bouvy, N.D., Schrauwen, P., Teule, G.J., 2009. Cold-activated Nuutila, P., Schaart, G., Huang, K., Tu, H., van Marken Lichtenbelt, W.D., Hoeks, J., brown adipose tissue in healthy men. N. Engl. J. Med. 360, 1500–1508. Enerbäck, S., Schrauwen, P., Spiegelman, B.M., 2012. Beige adipocytes are a Vijgen, G.H., Bouvy, N.D., Teule, G.J., Brans, B., Schrauwen, P., van Marken distinct type of thermogenic fat cell in mouse and human. Cell 150, 366–376. Lichtenbelt, W.D., 2011. Brown adipose tissue in morbidly obese subjects. Xu, X., Ying, Z., Cai, M., Xu, Z., Li, Y., Jiang, S., Tzan, K., Wang, A., Parthasarathy, S., He, PLoS ONE 6, e17247. G., Rajagopalan, S., Sun, Q., 2011. Exercise ameliorates high-fat diet-induced Vijgen, G., Bouvy, N., Teule, G.J., Brans, B., Hoeks, J., Schrauwen, P., van Marken metabolic and vascular dysfunction, and increases adipocyte progenitor cell Lichtenbelt, W., 2012. Increase in brown adipose tissue activity after weight loss population in brown adipose tissue. Am. J. Physiol. Regul. Integr. Comp. Physiol. in morbidly obese subjects. J. Clin. Endocrinol. Metab. 97, E1229–1233. 300, R1115–R1125. Virtanen, K.A., Lidell, M.E., Orava, J., Heglind, M., Westergren, R., Niemi, T., Taittonen, Yamaga, L., Thom, A., Wagner, J., Baroni, R., Hidal, J., Funari, M., 2008. The effect of M., Laine, J., Savisto, N.J., Enerback, S., Nuutila, P., 2009. Functional brown catecholamines on the glucose uptake in brown adipose tissue demonstrated by adipose tissue in healthy adults. N. Engl. J. Med. 360, 1518–1525. (18)F-FDG PET/CT in a patient with adrenal pheochromocytoma. Eur. J. Nucl. Vosselman, M.J., van der Lans, A.A., Brans, B., Wierts, R., van Baak, M.A., Schrauwen, Med. Mol. Imaging 35, 446–447. P., Lichtenbelt, W.D., 2012. Systemic b-adrenergic stimulation of thermogenesis Yoneshiro, T., Aita, S., Matsushita, M., Kameya, T., Nakada, K., Kawai, Y., Saito, M., is not accompanied by brown adipose tissue activity in humans. Diabetes 61, 2011. Brown adipose tissue, whole-body energy expenditure, and 3106–3113. thermogenesis in healthy adult men. Obesity (Silver Spring) 19, 13–16. Waldén, T., Hansen, I., Timmons, J., Cannon, B., Nedergaard, J., 2012. Recruited vs. Yoneshiro, T., Aita, S., Kawai, Y., Iwanaga, T., Saito, M., 2012. Nonpungent capsaicin nonrecruited molecular signatures of brown, ‘‘brite’’, and white adipose tissues. analogs (capsinoids) increase energy expenditure through the activation of Am. J. Physiol. Endocrinol. Metab. 302, E19–31. brown adipose tissue in humans. Am. J. Clin. Nutr. 95, 845–850. Whiting, S., Derbyshire, E., Tiwari, B., 2012. Capsaicinoids and capsinoids. A Young, P., Arch, J., Ashwell, M., 1984. Brown adipose tissue in the parametrial fat potential role for weight management? A systematic review of the evidence. pad of the mouse. FEBS Lett. 167, 10–14. Appetite 59, 341–348. Zingaretti, M.C., Crosta, F., Vitali, A., Guerrieri, M., Frontini, A., Cannon, B., Whittle, A.J., 2012. Searching for ways to switch on brown fat: are we getting Nedergaard, J., Cinti, S., 2009. The presence of UCP1 demonstrates that warmer? J. Mol. Endocrinol. 49, 79–87. metabolically active adipose tissue in the neck of adult humans truly Whittle, A.J., Vidal-Puig, A., 2012. NPs – heart hormones that regulate brown fat? J. represents brown adipose tissue. FASEB J. 23, 3113–3120. Clin. Invest. 122, 804–807. Wickler, S., Stern, J., Glick, Z., Horwitz, B., 1987. Thermogenic capacity and brown fat in rats exercise-trained by running. Metabolism 36, 76–81. Molecular and Cellular Endocrinology 379 (2013) 51–61

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review Thyroid hormones and mitochondria: With a brief look at derivatives and analogues

a,1 b,1 b, a, Federica Ciofﬁ , Rosalba Senese , Antonia Lanni ⇑, Fernando Goglia ⇑ a Dipartimento di Scienze e Tecnologie, Università degli Studi del Sannio, Via Port’Arsa 11, 82100 Benevento, Italy b Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy article info abstract

Article history: Thyroid hormones (TH) have a multiplicity of effects. Early in life, they mainly affect development and differ- Available online 13 June 2013 entiation, while later on they have particularly important influences over metabolic processes in almost all tissues. It is now quite widely accepted that thyroid hormones have two types of effects on mitochondria. The Keywords: first is a rapid stimulation of respiration, which is evident within minutes/hours after hormone treatment, Mitochondrion and it is probable that extranuclear/non-genomic mechanisms underlie this effect. The second response occurs Thyroid hormone one to several days after hormone treatment, and leads to mitochondrial biogenesis and to a change in mito- Iodothyronine chondrial mass. The hormone signal for the second response involves both T3-responsive nuclear genes and a Thyroid hormone analogue direct action of T3 at mitochondrial binding sites. T3, by binding to a specific mitochondrial receptor and affecting the transcription apparatus, may thus act in a coordinated manner with the T3 nuclear pathway to regulate mitochondrial biogenesis and turnover. Transcription factors, coactivators, corepressors, signaling pathways and, perhaps, all play roles in these mechanisms. This review article focuses chiefly on TH, but also looks briefly at some analogues and derivatives (on which the data is still somewhat patchy). We summarize data obtained recently and in the past to try to obtain an updated picture of the current research position concerning the metabolic effects of TH, with particular emphasis on those exerted via mitochondria. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Thyroid hormones and iodothyronines: the general picture ...... 52 2. Actions of thyroid hormones ...... 52 2.1. Overview of nuclear pathways ...... 52 2.2. Overview of non-nuclear pathways ...... 52 3. Mitochondria ...... 53 3.1. Mitochondrial plasticity ...... 54 4. Thyroid hormones and mitochondria ...... 55 4.1. Direct way ...... 55 4.2. Indirect ways ...... 55 5. Thyroid hormones and mitochondrial energetics...... 56 5.1. Uncoupling mechanism ...... 56 5.2. Other mechanisms ...... 56 6. Thyroid hormone derivatives and analogues ...... 57 6.1. Derivatives ...... 57 6.2. Analogues...... 57 7. Conclusions...... 58 7.1. Perspectives ...... 58 References ...... 59

Corresponding authors. Tel.: +39 0823 274542; fax: +39 0823 274545 (A. ⇑ Lanni), tel.: +39 0823 274571; fax: +39 0824 23013 (F. Goglia). E-mail addresses: [email protected] (A. Lanni), [email protected] (F. Goglia). 1 These authors contributed equally to the manuscript.

1. Thyroid hormones and iodothyronines: the general picture effects of T3 (Tata et al., 1962; Tata et al., 1963; Samuels et al., 1974; Oppenheimer et al., 1974; Bassett et al., 2003). In the ensu-

The thyroid gland produces two main iodothyronines: 3,5,30,50- ing years, efforts were made to purify the receptors, but the results tetraiodothyronine (thyroxine or T4) and 3,5,30-triiodo-L-thyronine did not allow detailed investigation of their molecular properties (T3). TH release from the thyroid occurs as part of a feedback mech- until the simultaneous cloning of the receptors by Sap et al. anism involving the pituitary–hypothalamic axis. At any given (1986) and Weinberger et al. (1986). In mammals, two genes, time, most T4 and T3 in the body is bound to transport proteins, TRalpha and TRbeta, encode several thyroid-receptor isoforms with only a small, ‘‘unbound’’ or ‘‘free’’, fraction being biologically (TRalpha1, the two splicing variants TRalpha2 and TRalpha3; active. The functions of these proteins most probably include: (a) TRbeta1, TRbeta2, and TRbeta3, respectively). All TRbeta isoforms ensuring a constant supply of TH to the cells and tissues by prevent- retain T3-binding activity, whereas only TRalpha1 of the TRalpha ing urinary loss, (b) protecting the organism against abrupt changes isoforms possesses binding activity. The existence of various iso- in thyroid-hormone production and/or degradation, and (c) ulti- forms of TRs raises the question as to whether they have distinct mately protecting against iodine deﬁciency. or redundant roles. Their tissue-dependent expressions and devel- All the circulating T4 is secreted by the thyroid gland, whereas opmentally regulated differential expression suggest that they most (about 80%) of the systemic T3 is generated by deiodination mediate speciﬁc isoform-dependent actions. In view of their of T4 within peripheral tissues. T3 is further deiodinated to yield substantial amino-acid homology with respect to steroid hormone

3,30-T2 and also, perhaps, 3,5-T2. receptors, all TR isoforms are considered to be members of the Thyroid-hormone deiodination is mediated by three iodothyro- large superfamily of nuclear receptors that also includes the recep- nine deiodinases: type I deiodinase (D1), preferentially expressed tors for retinoic acid, vitamin D and peroxisomal proliferator acti- in the liver but also present in kidney, thyroid, and pituitary; type vators. These receptors contain multiple functional domains that II deiodinase (D2), present in the central nervous system, anterior include, in particular, a DNA-binding domain (DBD) and a car- pituitary, brown adipose tissue, and placenta; type III deiodinase boxyl-terminal ligand-binding domain (LBD). The DBD domain (D3), present in the central nervous system, placenta, skin, and fe- contains about 70 amino acids forming two ‘‘zinc fingers’’. This tal tissue. For further details on deiodinases, the reader is referred region is highly conserved and interacts with the specific DNA seg- to Orozco et al., 2012; Dentice and Salvatore, 2011; Bianco, 2011. ments known as ‘‘thyroid-hormone response-elements’’ (TREs). T3 As mentioned above, T4 is synthesized entirely within the thy- receptors are transcription factors: they modulate transcription roid, while approximately 80% of T3 is formed by peripheral con- mainly by binding TREs. In the absence of T3, the TR has an intrin- version of T4. Uptake of TH into peripheral tissues is mediated by sic transcriptional repressor function. In most cases, the TRs act as specific membrane transporter proteins. Several transporter fami- heterodimers with a 9-cis retinoic acid receptor (RXR), but there lies have been identified, among which the monocarboxylate are also multiple TR complexes that bind to TREs (Farach-Carson transporter (MCT) family deserves special attention. Fourteen and Davis, 2003). In addition to RXR, many other molecules are di- members of this family have been recognized so far, but in only rectly or indirectly functionally associated with TRs (vitamin D3, 6 of them has a ligand-binding site been identified. MCT8 and peroxisome proliferator-activated receptor (PPAR), corepressors, MCT10 have been identified as specific TH transporters. However, coactivators, etc.). The transcriptional activity of TRs is regulated while MCT8 is currently known to be highly specific only for TH at multiple levels: by T3 itself; by the type of TRE located on the (Friesema et al., 2003), MCT10 also has the ability to carry different promoters of T3 target genes; by the developmental- and types of amino acid [e.g., the carrier polypeptide of various organic tissue-dependent expressions of TR isoforms; and by a host of anion transporters (OATP1C1, OATP1A2, OPTP1A4)]. Among the nuclear coregulatory factors (coactivators and corepressors) with OATPs, OATP1C1 is the most interesting for the present purposes T3-dependent activity. Deeper consideration of these mechanisms because it displays high specificity and affinity for certain iodothy- can be found in some recent reviews (Oetting and Yen, 2007; Yen ronines (especially for T4 and rT3, although not for T3). Moreover, et al., 2006; Cheng et al., 2010; Flamant and Gauthier, 2012; Tata, its preferential localization within the endothelium of brain capil- 2012). laries suggests that OATP1C1 is important for the transport of TH across the blood–brain barrier (Mayerl et al., 2012). The physiolog- 2.2. Overview of non-nuclear pathways ical roles performed by the TH transporters have been discussed in recent reviews (Kinne et al., 2011; Visser et al., 2011) and so will A number of effects mediated by iodothyronines have been de- not be described here any further. scribed for which a binding to TRs can be excluded, and it is currently assumed that these effects involve extranuclear binding sites in several compartments of the cell (including the plasma 2. Actions of thyroid hormones membrane, the cytoskeleton, the cytoplasm, and mitochondria: for review, see Cheng et al., 2010). Unlike the nuclear effects, the TH act via two distinct pathways: (1) nuclear pathways and (2) extranuclear ones: (i) are independent of thyroid hormone nuclear non-nuclear pathways. receptors; (ii) may occur within a short time (seconds to minutes); and (iii) may be mediated by signal-transducing pathways such as 2.1. Overview of nuclear pathways cAMP and protein kinases (Bassett et al., 2003; Farach-Carson and Davis, 2003; Saelim et al., 2004; Axelband et al., 2011). Some stud- At the beginning of the 1960s, Tata and coworkers were the first ies have demonstrated that plasma membrane-initiated actions to show that administration of TH to hypothyroid rats induced an begin at a binding site on integrin aVb3, a heterodimer protein that increase in their basal metabolic rate, while the simultaneous interacts both with extracellular matrix proteins and thyroid injection of an inhibitor of transcription (such as actinomycin-D) hormones (Bergh et al., 2005; Cody et al., 2007). Other molecules inhibited this effect (Tata, 1963). These data implicated the nucleus – such as stilbene, resveratrol (Lin et al., 2007, 2008), and dihydro- as the locus for the above action. In other experiments, using iso- testosterone (Lin et al., 2009a) – also bind to this integrin (Davis lated nuclei, they showed that T3 stimulated DNA-dependent et al., 2009). Lin et al. (2009b) demonstrated that the hormone- RNA-polymerase activity. Later, Samuels et al. and Oppenheimer binding domain comprises two binding sites. One site is solely et al. identified high-affinity nuclear binding sites for TH, suggest- for the binding of T3 and activates the phosphatidylinositol 3-ki- ing that thyroid hormone nuclear receptors (TR) mediated the nase (PI3K) pathway, leading to cytoplasm-to-nucleus shuttling F. Cioffi et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61 53

Table 1 Some information about nuclear and non-nuclear pathways of TH actions.(For references see text).

Nuclear pathways Non-nuclear signaling pathways Nuclear Thyroid Hormone Receptors (TR): – cAMP-activated protein kinase (AMPK) – TR-a (TR-a1, TR-a2 e TR-a3) – TR- b (TR-b1, TR-b2 e TR-b3) – TRs act as heterodimers [with i.e.: retinoid X receptor (RXR), peroxisome proliferator-activated – Akt/protein kinase B receptors (PPARs) and vitamin D3] – Corepressor [silencing mediator of TH and retinoid action (SMRT) and nuclear corepressor – Phosphatidylinositol 3-kinase (PI3K) (NCoR)] – Coactivators [p300, steroid receptor coactivator 1 (SRC-1) and Trip230] – PKC, Ras, Raf1, MEK resulting in activation of mitogen-activated protein kinase (MAPK)

of TRa1 and to transcription of the hypoxia-inducible factor-1a 3. Mitochondria gene. The second site binds both T3 and T4 and appears to trigger PKC, Ras, Raf1, and MEK, resulting in tyrosine phosphorylation, The evidence that TH affects metabolic rate dates back a long activation, and nuclear translocation of MAPK (Lin et al., 2009a; way. However, despite this and the increasing knowledge of the Lin et al., 2009b; for review, see Cheng et al., 2010). It is known that physiology and mechanism of action of TH, several aspects of their TH affect cellular calcium homeostasis, and this effect is probably effects on metabolic rate (also called calorigenic effects) remain to due to a nongenomic action. In fact, a recent study on GH3 cells be elucidated. The existing evidence and the current debate are fo- showed that both T2 and T3 exert short-term nongenomic effects cused on two possible mechanisms that might underlie the calor- on intracellular calcium by modulating plasma-membrane and igenic effects of TH: (a) a mechanism involving their interaction mitochondrial pathways (Del Viscovo et al., 2012). Those authors with nuclear receptors (TR) and (b) a mechanism involving both 2+ showed that nimodipine largely prevented the [Ca ]i increases TR and/or certain cellular sites such as mitochondria and the cell elicited by T2 and T3, suggesting that these two iodothyronines membrane. Actually, both pathways may have cellular respiration share L-type VDCC as a plasma-membrane target. Clear nonge- as their ultimate target. Mitochondria, because of their known nomic actions have been reported that involve AMP-activated physiological functions, have been and continue to be the target protein kinase (AMPK) (Irrcher et al., 2008) and Akt/protein kinase of most studies on the calorigenic effects of TH. Mitochondria, in B(Moeller et al., 2005). In skeletal muscle in vivo, T3 stimulates fact, provide about the 90% of the cellular energy supply, and they both fatty acid and glucose metabolism through rapid activations are also the headquarters for a multitude of biochemical pathways of the associated signaling pathways involving AMPK and Akt/pro- related to metabolism (for details, see Fig. 1). Indeed, besides ATP tein kinase B (de Lange et al., 2008). (For a short summary of nucle- synthesis, mitochondria are the site of other important biochemi- ar/non-nuclear pathways of TH see Table 1). cal events such as oxidation of fatty acids, production of free

Fig. 1. Schematic representation of most of the mitochondrial activities and functions. The respiratory chain transfers electrons from reduced coenzymes [coming from intra

(b-oxidation and TCA cycle) – and extra-mitochondrial (glycolysis) oxidative pathways] to O2 and, pumping out H+ from the matrix to the intermembrane space, generates an electrochemical gradient, DlH+, which provides the driving force for ATP synthesis by FoF1-ATPase. H+ can also enter the matrix by mechanisms not coupled to ATP synthesis either directly, across the lipid bilayer, or indirectly, by protein-mediated transport (mechanism not represented). Phosphate carrier (PiC), ADP/ATP carrier (ANT), and Uncoupling protein (UCP) are represented individually. Mitochondrial calcium uniporter (MCU). Anion carriers (ACs). Translocator Inner Membrane (TIM), Translocator Outer Membrane (TOM), mitochondrial transcription factor A (mtTFA), Apoptosis-inducing factor (AIF). For further details, see text. 54 F. Cioffi et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61 radical, heme synthesis, the metabolism of some amino acids, the degradation, autophagy) are now quite well elucidated, at least formation and export of Fe/S clusters, iron metabolism, and cal- in some respects. The process of mitochondrial biogenesis requires cium homeostasis. Very recently, the inner-membrane mitochon- the coordination of mitochondrial and nuclear genomes. In fact, the drial calcium uniporter has been identified as the channel mitochondrial proteome includes about 1500 proteins, most of responsible for ruthenium-red-sensitive mitochondrial Ca2+ uptake which are coded by the nuclear genome, with only 13 being coded (De Stefani et al., 2011). In addition, mitochondria contribute to by the mitochondrial genome. Accordingly, the biogenesis, abun- many processes central to both cellular function and dysfunction, dance, morphology, and physiological properties of mitochondria including calcium signaling, cell growth and differentiation, cell- are regulated primarily by the nuclear genome through a series cycle control, and cell death. of transcription factors that regulate the activity of the mitochon- Mitochondria utilize metabolic substrates to generate ATP. Such drial genome and the expressions of mitochondrial proteins (see ATP synthesis, which occurs via ATPase complex, is coupled to oxy- Fig. 2). gen consumption via the proton electrochemical gradient existing In recent decades, our knowledge regarding the dynamics of across the inner mitochondrial membrane. The inner mitochon- these organelles has greatly improved. Indeed, the old view of iso- drial membrane might be expected to be proton-proof and the lated mitochondria as static bean-shaped organelles is agonizing mechanism to be tightly coupled, but actually the coupling is not and is now replaced by the view of a dynamic and branched net- perfect, and the proton flux across the inner membrane that is work moving throughout the cell and undergoing structural transi- not coupled to ATP synthesis (the so-called proton leak) dissipates tions and changing the shape, morphology, and size. These changes part of the gradient as heat. depend on the cell-type and on the cell’s status (for review, see Lie- sa et al., 2009). In mitochondria, plasticity and function are interre- 3.1. Mitochondrial plasticity lated since plasticity may affect the activity of the organelles, while their function/dysfunction may affect their morphology and Mitochondrial shape and their positioning within cells is crucial dynamics (Kuznetsov et al., 2009). These changes are tightly regu- and is tightly regulated by processes of fission and fusion, biogen- lated by the balance between ‘‘fusion’’ and ‘‘fission’’, and determine esis and autophagy, thus ensuring a relatively stable mitochondrial the appearance of the dynamic organelles, their composition, and population (Hailey et al., 2010; Osellame et al., 2012.) In addition, finally their activities and functions (Michel et al., 2012). The prin- mitochondria are known to be involved in apoptosis, and some cipal elements participating in these events are: recent data show that they are involved in many other cellular For fusion: pathways, such as the recently highlighted ones that participate in innate immune responses (West et al., 2011). The number of – Mitofusin 1 and 2 (MFN1 and MFN2), which are located in the mitochondria varies according to the function of the cell-type outer mitochondrial membrane and form homo- and hetero- and to the physiological state of the cell/organism. The mecha- oligomeric complexes between apposing mitochondria (Koshi- nisms underlying mitochondrial turnover (i.e., biogenesis, ba et al., 2004; Meeusen et al., 2004; Chen et al., 2005; Detmer

Fig. 2. Schematic representation of the TH-dependent nucleus–mitochondrion cross-talk in the regulation of mitochondrial functions (biogenesis, oxygen consumption, and gene expression). Trough active transport or passive diffusion, TH move from outside the plasma membrane into the cytoplasm approaching the extra-nuclear as well as the extra-mitochondrial space. In the cytoplasm, several events can occur, among which deiodination and binding to cytosolic proteins (i.e. cytosolic TH receptors). These can activate signal transduction pathways involving MAPKs, PKC and PI3-K-AKT/PKB. Genomic action requires thyroid hormone responsive elements (TREs) for the recognition of genes for direct transcriptional regulation [a ﬁrst set of TH target genes (early expression)]. Some of these target genes serve as intermediate factors and regulate a second series of TH target genes (late expression). This group of intermediate factors encompasses transcription factors (NRF-1, NRF-2, PPARc) and transcriptional coactivators (PGC- 1a, PGC-1b). These can ultimately enter the mitochondrion and regulate a second series of T3 target genes [e.g. mitochondrial transcription factor A (mtTFA)]. For further details, see text. F. Ciofﬁ et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61 55

and Chan, 2007). They are mainly involved in the fusion of outer the c-erbA mRNA encoding the full-length TR (47 kDa) by means mitochondrial membranes (OMM) and the tethering of mito- of an internal AUG codon. Using an expression vector provided chondria to the endoplasmic reticulum (ER) (de Brito and Scorr- by these authors, Wrutniak et al. (1995) overexpressed a truncated ano, 2009). 43 kDa c-erbAK1 protein in CV1 cells, and then by cyto-immuno- – The dynamin-like GTPase protein OPA1 (optic atrophy type 1), fluorescence experiments demonstrated that this truncated TRK which is located in the intermembrane space (associated with protein is specifically imported into mitochondria. Interestingly, the inner mitochondrial membrane) and mediates the fusion the same authors have identified five sequences highly related to of the inner membranes (Cipolat et al., 2004; Chen et al., 2005). TRE within the rat mitochondrial genome, and they further showed that p43 binds to one of these sequences in the D-loop region, For fission: which contains the promoters of the mitochondrial genome. Very recently, the same group (Carazo et al., 2012) described an atypical – Recruitment of Drp1 (a GTPase belonging to the dynamin family) mechanism for the import of p43 into the mitochondrion and from the cytosol to the OMM. This step is mediated by a mito- identified the protein sequences involved in its import. Indeed, chondrial integral outer membrane protein called Fis1 (fission two alpha helices in the C-terminal part of p43 are actually mito- protein 1 homolog) (Liesa et al., 2009). Drp1 oligomerization chondrial import sequences since fusion to a cytosolic protein (regulated by post-translational modification) leads finally to induces its mitochondrial translocation. Helix 5 drives the atypical fission (for review, see Chang and Blackstone, 2010). During mitochondrial import process, whereas helices 10/11 induce a the above mitochondrial processes, there is mitochondrial classical import process. The authors further showed that despite exchange of such molecules as mtDNA, and proteins, and parts its inability to drive any mitochondrial import, the N-terminal re- of membranes (for review, see Westermann, 2010 and Otera gion of p43 also plays a permissive role since in the presence of the and Mihara, 2011). Several external stimuli may affect mito- C-terminal import sequences, different N-terminal regions deter- chondrial plasticity, including thyroid hormones; however, to mine whether the protein is imported or not imported. These re- our knowledge there are few or no data on this issue at present. sults clearly demonstrate that p43 has the ability to function as a T3-dependent mitochondrial transcription factor. The p43 mito- 4. Thyroid hormones and mitochondria chondrial T3 receptor may perform an important role in skeletal muscle since its depletion adversely affects skeletal muscle devel- Modulation of mitochondrial activity by TH may be effected in opment and activity (Pessemesse et al., 2012). one of two ways: direct or indirect. All these data raise the possibility that mitochondrial binding sites for T3 may play very important physiological roles in regulat- 4.1. Direct way ing the mitochondrial transcription apparatus, thus leading to a regulation of mitochondrial biogenesis by acting in synchrony with The direct mode requires the presence inside the organelles of the nuclear genome. This is an attractive possibility for two rea- specific binding sites for the hormone. In contrast, the indirect sons: (a) T3 influences mitochondrial biogenesis and turnover and one does not need these sites to be present but instead may be (b) the mitochondrial biogenesis or turnover needs the coordinated mediated by signaling pathways located in different parts of the participation of the nuclear and mitochondrial genetic apparatuses. cell. Concerning the first possibility, the presence of binding sites Actually, early results obtained by us and by others – showing that for T3 has been reported by several laboratories. High-affinity T3 regulates the mitochondrial population and the mitochondrial binding sites for T3 in the mitochondrial inner membrane were nucleic acid level (Gadaleta et al., 1972; Mutvei et al., 1989; Leo first reported in 1975 by Sterling and Milch (1975). The existence et al., 1976; Goglia et al., 1983) – had already suggested just such of mitochondrial binding sites for T3 were confirmed by others a possibility. Apparently confirmatory results were obtained by in 1981 (Goglia et al., 1981), but despite this and despite several re- Martino et al. (1986), who showed a direct action of T3 on mito- ports of rapid effects of T3 on mitochondria, the physiological sig- chondrial RNA-polymerase in isolated mitochondria, and subse- nificance of these sites and indeed their very existence, and the quently by Enríquez et al. (1999). The latter authors studied the physiological significance of the direct effects were controversial effect of T3 (both in vivo and in vitro) on ‘‘in organello’’ mtDNA tran- at that time. Subsequently, however, the existence of specific mito- scription and on the ‘‘in organello’’ footprinting patterns in the chondrial binding sites for T3 received additional confirmation mtDNA regions involved in the regulation of transcription. Their re- from the work of Morel et al. (1996) and Wrutniak et al. (1995). sults confirmed a direct influence of T3 on the mitochondrial tran- Morel et al. studied the kinetics of the internalization and specific scription apparatus, and in particular they showed that T3 subcellular binding of T3 in mouse liver, both in vivo and in vitro. selectively modulates the alternative H-strand transcription initia- They showed, by quantitative electron microscopic autoradiogra- tion sites without a previous activation of nuclear genes. phy, that after the injection of radiolabeled T3, specific binding was evident in five cell-compartments (including mitochondria). Surprisingly, specific binding was not evident in the cytosol, which 4.2. Indirect ways contains T3-binding proteins. Wrutniak et al., using a photoaffinity labeling technique, identified two T3-binding proteins in rat liver On the basis of the data discussed above, it seems reasonable to mitochondrial extracts. One (molecular weight 43 kDa) was lo- conclude that TH have at least three different, but probably cated in the matrix and the other (MW 28 kDa) in the inner mem- interconnected, mode of action, by which they regulate the expres- brane. These results are in partial agreement with those obtained sions of target genes contributing to mitochondrial biogenesis. The by Sterling and Milch (1975) and by us (Goglia et al., 1981). The first relies on a binding of TH to nuclear TR, and for TH to affect nu- same group (Wrutniak et al., 1995), using antibodies against the clear gene expression by binding to a TRE. The second involves TH two binding domains of c-erbA K1, identified two proteins [mito- affecting mitochondrial transcription directly by binding to a chondrial matrix T3-binding protein (p43) and inner mitochondrial mitochondrial TR. In the third, intermediate factors such as the tran- membrane T3-binding protein (p28)] whose location and molecu- scription factors NRF-1, NRF-2, and PPARc, and the coactivators lar weight were identical to the mitochondrial T3-binding proteins PGC-1alpha and PGC-1beta may be synthesized, and by entering previously described. Bigler et al. (1992) had previously demon- the nucleus, regulate other series of TH-target genes. These mecha- strated that truncated c-erbAK1 proteins are synthesized from nisms may be additionally affected by many nongenomic actions 56 F. Cioffi et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61 such as post-translational modifications, by the local bioavailability of a large number of papers, the mechanism by which TH exert or by the direct binding of TH to some cellular targets (see Fig. 2). their effects on energy metabolism is far from firmly established. The above-mentioned coactivators include the PGC-1 family. The first member of this family to be identified was PGC-1alpha 5.1. Uncoupling mechanism (peroxisome proliferator-activated receptor gamma coactivator 1alpha). PGC-1alpha is rapidly and strongly induced by TH. Indeed, One of the most intriguing hypothesis is the uncoupling PGC-1a expression levels and protein levels were increased, hypothesis. This proposes that TH stimulates metabolic rate by act- respectively, 13-fold (Weitzel and Iwen 2011) and 3-fold (Irrcher ing at the mitochondrial level to uncouple the electron transport et al., 2003; Weitzel et al., 2003; Venditti et al., 2009) 6 h after chain from ATP synthesis. This hypothesis predicts a thyroid- administration of T3 and this action is mediated by a TRE in the dependent stimulation of energy expenditure without a concomi- promoter (Wulf et al., 2008). Besides, recently, Thakran et al. have tant increase in ATP production (decreased P/O ratio). The early shown that PGC-1a partecipates in the T3 induction of CPT1a and experiments supporting such a possibility were those performed PDK4 in the liver and, for regulation of hepatic gene expression, by Lardy and Feldott (1951) and by Hess and Martius (1951), PGC-1a was deacetylated probably through the activation of the who showed that mitochondria prepared from T4-treated rats nuclear deacetylase SIRT1 (Thakran et al., 2013). It has been shown exhibited lower P/O ratios than those from untreated euthyroid that PGC-1alpha has profound influences on adaptive thermogen- controls. However, in the early 1960s its validity was questioned, esis in brown adipose tissue, on hepatic gluconeogenesis, and on principally because uncoupling was observed only with pharmaco- mitochondrial biogenesis. In addition, PGC-1alpha coactivates var- logical doses of TH. Since some effects were observable in vitro (in ious nuclear receptors and nuclear respiratory factors, including isolated mitochondria), the theory implied that TH acted directly at the thyroid hormone receptor (Puigserver et al., 1998; Sadana the mitochondrial level. In addition, the results of such in vitro et al., 2007; Attia et al., 2010). However, PGC-1alpha knock-out studies were not always reproducible, and they were widely mice and knock-down in cell culture have revealed, respectively: thought to reflect chemical artifacts. But, this hypothesis has never few alterations in mitochondrial biogenesis (Ventura-Clapier been dropped, and it continues to this day to be investigated using et al., 2008; Hock and Kralli, 2009) and few defects in TH-mediated new approaches. Indeed, it received renewed attention when the gene-expression patterns (Wulf et al., 2007). It is possible that discovery was made that uncoupling proteins are present not only other coactivators of the PGC-1 family may play roles. Recently, in brown adipose tissue (where UCP1, by the mechanism of uncou- the presence of PGC-1alpha has been demonstrated in mitochon- pling, is able to dissipate energy, so producing heat), but in almost dria. This opens interesting perspectives on the possible roles of all tissues and cells, and that their expressions are increased by T3 these coactivators, but further studies will be needed to undiscover (Lanni et al., 1997; Lanni et al., 1999; de Lange et al., 2001; for re- their functions (Aquilano et al., 2010). PGC-1beta, another member view, see also Lanni et al., 2003 and Cioffi et al., 2009). These find- of the PGC-1 family, seems to be closely related to PGC-1alpha but ings stimulated attempts to show a possible involvement of these there are some differences. PGC-1b, on the other hand, activates proteins in the calorigenic effect of T3. In particular, UCP2 (ubiqui- mithochondrial biogenesis by binding to different transcription tously expressed) and UCP3 (predominantly expressed in skeletal factors (including TR) and its expression has been shown to be rap- muscle) have attracted great interest. The realization that UCP3 idly and strongly induced by TH (Weitzel et al., 2003) suggesting a is present in skeletal muscle, a tissue that is metabolically very direct regulation via a TRE. So PGC-1a and PGC-1b are endoge- active, led to this protein being viewed as a possible candidate nously and rapidly regulated by TH in vivo via a TRE. Other activa- for the mediator of the effects of thyroid hormones on resting met- tors regulated by TH include coactivator SRC-1, which plays a role abolic rate. This hypothesis has been investigated and the authors in thermogenesis (Picard et al., 2002). SRC-1 knock-out mice dis- concluded that UCP3 does indeed have the potential to be a molec- play features of thyroid resistance (Weiss et al., 1999) highlighting ular determinant of the effects of T3 on resting metabolic rate (de a close connection between SRC-1 and TH. These important aspects Lange et al., 2001). In that study, they showed that when a single have been recently reviewed in an excellent and comprehensive injection of T3 was given to hypothyroid rats, a maximal stimula- manner by Weitzel and Iwen (2011). Recently, other studies have tion of UCP3 expression was evident at 48 h after the injection. At highlight the clinical relevance of TH. Indeed, recent observation this time-point, the resting metabolic rate also reached its maximal and animal models have shaped our understanding of signaling value and at the mitochondrial level there was a corresponding in- pathways of thyroid hormone and how this insight might be trans- crease in the proton leak. These results received support from the lated into therapeutic strategies, especially for treating hyperlipid- study by Flandin et al. (2009). In that study, to test the possibility emia and obesity but also to treat cardiac disease, cancer and that T3 might act via UCP3, a UCP3-knockout (KO) model was used. improve cognitive function (Brent, 2012). Infact, TR-b mutations This model was found to exhibit a normal phenotype except that have been identified in a broad range of cancer including hepato- upon T3 administration, the stimulation of oxygen consumption cellular carcinoma, renal cell carcinoma, erythroleukemias and was significantly weaker (by 6%) in the UCP3 KO mice than in the thyroid cancer (Rosen et al., 2011; Chan and Privalsky, 2010). In wild-type (WT) mice. These results reinforce the idea that UCP3 addition, thyroid hormone acting through TR-a regulates adult might play a role in the modulation of energy balance by TH. How- hyppocampal neurogenesis which is important in learning, mem- ever, the real uncoupling capacity of UCP3 is under debate, and a ory and moon (Desouza et al., 2005; Kapoor et al., 2010). question has been raised as to whether the uncoupling effect of UCP3 is a primary function or a secondary one (Goglia and Skula- chev, 2003; and for review, see Azzu et al., 2010).

5. Thyroid hormones and mitochondrial energetics 5.2. Other mechanisms

It is universally recognized that TH are unique in their ability to Other mechanisms have been proposed to contribute to the stimulate thermogenesis/calorigenesis (the well-known calori- uncoupling effect of T3. For instance, a recent study showed that genic effect of TH). Their main action consists in a stimulation of the mitochondrial uncoupling induced by T3 is transduced (in rats cellular respiration while at the same time reducing metabolic efﬁ- in vivo and in cultured Jurkat cells) by a gating of the mitochondrial ciency. However, despite this phenomenon being known since the permeability transition pore (PTP). This T3-induced PTP gating was end of the 19th century (Magnus-Levy, 1895) and being the subject abrogated in inositol 1,4,5-trisphosphate [IP(3)] receptor1 F. Ciofﬁ et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61 57

[IP(3)R1]( / ) cells, indicating that the endoplasmic reticulum Third, concerning 3,5-diiodothyronine: until quite recently, this À À IP(3)R1 may serve as the upstream target for the mitochondrial naturally occurring molecule was considered to be an inactive iod- activities of T3 (Yehuda-Shnaidman et al., 2010). Other mechanisms othyronine, but the discovery of metabolic effects of T2 attracted that may play a role in the calorigenic effects of TH include: (i) the the attention of several group of investigators. Early studies maintenance of transmembrane ion gradients through the action of showed that T2 was able to stimulate mitochondrial activities Na+/K+-ATPase, Ca2+-ATPase, and Ca2+ cycling in muscle and other (Lanni et al., 1992, 1993, 1994; O’Reilly and Murphy, 1992); the ef- tissues, as reviewed in a number of articles (Silva, 2006; Lanni fects of T2 seem to be mediated by a direct interaction with mito- et al., 2001; Silvestri et al., 2005) and (ii) regulation of the expres- chondria. Specific binding sites for T2 (Goglia et al., 1994b) have sions of a selected set of nuclear genes encoding mitochondrial in- been described in rat liver mitochondria, but the data concerning ner membrane proteins (Nelson et al., 1995). However, it seems mitochondrial sites need to be interpreted with some caution be- unlikely to us that a rapid regulation of mitochondrial respiration cause of the limitations inherent in such studies. Indeed, due to could be achieved by synthesizing respiratory components for the inner-ring labeling procedure the 3,5-125I-T2 used for the mea- insertion into pre-existing membranes. It seems more likely that surement of binding parameters had a low specific activity and it in the regulation of cellular respiration by TH, a double mechanism was possible to perform studies only over a narrow range of con- operates. One would be a short-term mechanism, useful for a rapid centrations. However, subsequent studies (Lanni et al., 1994; Ar- response to sudden physiological changes in energy requirements. nold et al., 1998; Goglia et al., 1994a) showed that addition of T2 The other would be a long-term mechanism, useful for responding to the COX complex isolated from bovine heart stimulated its to prolonged stimuli (days or weeks) such as a long period of cold activity, and suggested that subunit Va of the COX complex might exposure or a change in diet or developmental stage. Such a long- be the binding site for T2 (for review, see Goglia, 2005). Effects of term mechanism would ultimately produce a new mitochondrial T2 have also been observed at the level of the plasma membrane population that is more or less active (depending on the increased (Huang et al., 1999; Incerpi et al., 2002). Interestingly, we recently presence of respiratory components) and/or more or less efficient succeeded in showing that in rats that had been fed a high-fat diet, (depending on the increased presence of some specific components, administration of T2 stimulated metabolic rate, reduced the serum such as UCPs). The action of TH on calcium homeostasis may also cholesterol and triglyceride levels, and improved both glucose tol- play a role in the modulation of mitochondrial energy transduction. erance and insulin resistance, effects which involve the well known Sirtuin 1/AMP-activated protein kinase/PGC-1a pathway (Lanni et al., 2005; de Lange et al., 2011; Moreno et al., 2011,). Similar ef- 6. Thyroid hormone derivatives and analogues fects have been observed in humans (Antonelli et al., 2011). How- ever, whether or not the function of T2 is physiological remains to 6.1. Derivatives be elucidated.

Until a few years ago, it was a common assumption in the liter- 6.2. Analogues ature that T4 was a precursor, and that T3 was the only active iodothyronine. However, accumulating evidence suggest that other The metabolic effects of thyroid hormones have long been the iodothyronines – such as T4 itself, as well as some metabolites such focus of research because of the potential use of these hormones as reverse T3 (rT3), 3-iodothyronamine (T1AM), and 3,5-T2 (T2) – as drugs to stimulate body-weight loss and lipid metabolism and may be of biological relevance. This issue requires too much space to treat some disease such as obesity and diabetes (see Aguer for us to discuss it here in any detail, and since several published re- and Harper, 2012). However, the simultaneous induction of delete- views have already provided extensive analyses of the available rious side effects – such as a thyrotoxic state (tachycardia, muscle data on these molecules, we will only give a few examples. wasting, bone loss), and especially those at the cardiac level – First, concerning T4 and rT3: Farwell et al. (2006) compared the effectively stopped TH being used for these purposes. Recently, abilities of iodothyronines to initiate actin polymerization in astro- however, it has been shown that newly discovered analogues and cytes, and found that T4 and rT3 are each more potent than T3. In derivatives may have similar desirable effects without the delete- addition, they found that acute hormone replacement with either rious side effects. Indeed, since the middle of the last century much T4 or rT3 completely restored microfilament organization, while effort has been devoted to the development of analogues of thyroid acute T3 replacement failed to correct this defect (Farwell et al., hormones that might improve serum lipid profiles (i.e., plasma 2006). cholesterol, lipoprotein, etc.) without having undesirable cardiac Second, concerning T1AM: thyronamines (TAMs) are a recently effects. In the past few years, the attention of scientists has been identified class of endogenous signaling compounds. With the focused on the study of agents that are both tissue- and TRb-selec- exception that TAMs do not possess a carboxylate group, their tive (TRb-receptors are barely expressed in cardiomyocytes), with structure is identical to that of thyroid hormone and to those of the principal aim of addressing such major medical problems as deiodinated thyroid hormone derivatives. The iodothyronamines, obesity, ectopic fat accumulation, and atherosclerosis. Representa- which are probably generated by the combined action of deiodin- tive analogues endowed with these characteristics are GC-1 (sobet- ases and aromatic amino acid decarboxylase, activate a biogenic irome) and KB2115 (or eprotirome). They have the potential to amine-like G-protein-coupled receptor (GPCR): namely, trace reduce serum LDL cholesterol, lipoprotein (a), and triglyceride lev- amine receptor 1 (TAR1). T1AM, which is present both in the blood els without harmful effects on heart or muscle in humans (for re- and in peripheral tissues, seems to have actions opposite to the view, see Moreno et al., 2008; Baxter and Webb, 2009; and Cioffi classic actions of TH; indeed, when injected into mice, T1AM et al., 2010; Berkenstam et al., 2008). At cellular level, GC-1 is able induces rapid falls in body temperature and heart rate. Quite re- to stimulate mitochondrial oxidative processes (Venditti et al., cently, it has been shown that T(1)AM has significant physiological 2010). KB2115 also works additively with another cholesterol-low- effects in mammals, such as reversible, dose-dependent negative ering therapy, statins, to produce greater reductions in serum cho- inotropic and chronotropic effects on the heart and a cardioprotec- lesterol (Ladenson et al., 2010). However, very recently it has been tive effect in perfused rat hearts subjected to ischemia and reper- shown that thyroid hormone receptor b agonists prevent hepatic fusion (Frascarelli et al., 2011). A more exhaustive description of steatosis in fat-fed rats but impair insulin sensitivity (Vatner the actions and mechanisms of action of T1AM can be found in a et al., 2013. This suggest that the development of future TRb recent review by Piehl et al. (2011). agonists must consider the potential adverse effects on insulin 58 F. Cioffi et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61

Table 2 Structure of TH derivates/analogues.

Thyroid hormones Thyroid Hormones

Derivates/Analogues

sensitivity. (The structure of TH and its analogues/derivates are and thanks to the considerable efforts made by several groups of shown in Table 2). investigators around the globe, our knowledge of the influence of iodothyronines has grown and grown. 7. Conclusions 7.1. Perspectives The demands of mitochondria and their complex integration into cell biology extend far beyond the provision of ATP. This has In the future, progress in research into TH and mitochondria prompted a radical change in our perception of mitochondria, may come from investigations using methods such as proteomics. and has made these organelles a major target of investigations into Indeed, mitochondrial proteomics can be a powerful tool in the many aspects of cell biology and medicine. The identification of study of the actions of TH since its coverage can extend to mito- novel mechanisms governing mitochondrial biogenesis and chondrial proteins from all mitochondrial metabolic pathways, replication, and of the delicately poised signaling pathways coordi- including the respiratory chain. Indeed, Silvestri et al. (2010) by nating the mitochondrial and nuclear genomes, constitute funda- combining 2D-E, mass spectrometry, and blue native (BN) PAGE re- mental steps in in-depth investigations of the role of TH in the cently identified T2-induced mitochondrial proteins that may be modulation of metabolism and of the real involvement of mito- responsible for the beneficial effects of T2 on liver adiposity and chondria in these actions. TH affect many aspects of mitochondria metabolism. In addition, in the future it should be possible to make activity (bioenergetics, transcription, calcium homeostasis, etc.), progress into the possible use of TH analogues/derivatives to F. Cioffi et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61 59 improve certain worldwide diseases that result from the wide- de Brito, O.M., Scorrano, L., 2009. Mitofusin-2 regulates mitochondrial and spread adoption of an inappropriate lifestyle (involving inadequate endoplasmic reticulum morphology and tethering: the role of Ras. Mitochondrion 9 (3), 222–226. exercise and an unhealthy diet). These include obesity, cardiovas- de Lange, P., Lanni, A., Beneduce, L., Moreno, M., Lombardi, A., Silvestri, E., Goglia, F., cular disease, dyslipidemias, insulin-resistance, and type II diabe- 2001. Uncoupling protein-3 is a molecular determinant for the regulation of tes. Another future research goal may involve modulation of the resting metabolic rate by thyroid hormone. Endocrinology 142, 3414–3420. de Lange, P., Senese, R., Cioffi, F., Moreno, M., Lombardi, A., Silvestri, E., Goglia, F., network subserving signaling for TH and some analogues/deriva- Lanni, A., 2008. Rapid activation by 3,5,30-L-triiodothyronine of adenosine 50- tives as a way of improving adaptation to endocrine and other monophosphate-activated protein kinase/acetyl-coenzyme a carboxylase and environmental signals. akt/protein kinase B signaling pathways: relation to changes in fuel metabolism and myosin heavy-chain protein content in rat gastrocnemius muscle in vivo. Endocrinology 149 (12), 6462–6470. References de Lange, P., Cioffi, F., Senese, R., Moreno, M., Lombardi, A., Silvestri, E., De Matteis, R., Lionetti, L., Mollica, M.P., Goglia, F., Lanni, A., 2011. Nonthyrotoxic prevention Aguer, C., Harper, M.E., 2012. Skeletal muscle mitochondrial energetics in obesity of diet-induced insulin resistance by 3,5-diiodo-L-thyronine in rats. Diabetes 60 and type 2 diabetes mellitus: endocrine aspects. Best Pract. Res. Clin. (11), 2730–2739. Endocrinol. Metab. 26 (6), 805–819. De Stefani, D., Raffaello, A., Teardo, E., Szabo, I., Rizzuto, R., 2011. A forty-kilodalton Antonelli, A., Fallahi, P., Ferrari, S.M., Di Domenicantonio, A., Moreno, M., Lanni, A., protein of the inner membrane is the mitochondrial calcium uniporter. Nature Goglia, F., 2011. 3,5-Diiodo-L-thyronine increases resting metabolic rate and 476, 336–340. reduces body weight without undesirable side effects. J. Biol. Regul. Homeost. Del Viscovo, A., Secondo, A., Esposito, A., Goglia, F., Moreno, M., Canzoniero, L.M., Agents 25 (4), 655–660. 2012. Intracellular and plasma membrane-initiated pathways involved in the Aquilano, K., Vigilanza, P., Baldelli, S., Pagliei, B., Rotilio, G., Ciriolo, M.R., 2010. [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells. Peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC- Am. J. Physiol. Endocrinol. Metab. 302 (11), E1419–E1430. 1alpha) and sirtuin 1 (SIRT1) reside in mitochondria: possible direct function in Dentice, M., Salvatore, D., 2011. Deiodinases: the balance of thyroid hormone: local mitochondrial biogenesis. J. Biol. Chem. B 285 (28), 21590–21599. impact of thyroid hormone inactivation. J. Endocrinol. 209 (3), 273–282. Arnold, S., Goglia, F., Kadenbach, B., 1998. 3,5-Diiodothyronine binds to subunit Va Desouza, L.A., Ladiwala, U., Daniel, S.M., Agashe, S., Vaidya, R.A., Vaidya, V.A., 2005. of cytochrome-c oxidase and abolishes the allosteric inhibition of respiration by Thyroid hormone regulates hippocampal neurogenesis in the adult rat brain. ATP. Eur. J. Biochem. 252 (2), 325–330. Mol. Cell. Neurosci. 29 (3), 414–426. Attia, R.R., Connnaughton, S., Boone, L.R., Wang, F., Elam, M.B., Ness, G.C., Cook, G.A., Detmer, S.A., Chan, D.C., 2007. Complementation between mouse Mfn1 and Mfn2 Park, E.A., 2010. Regulation of pyruvate dehydrogenase kinase 4 (PDK4) by protects mitochondrial fusion defects caused by CMT2A disease mutations. J. thyroid hormone: role of the peroxisome proliferator-activated receptor Cell Biol. 176 (4), 405–414. gamma coactivator (PGC-1 alpha). J. Biol. Chem. 285, 2375–2385. Enríquez, J.A., Fernández-Silva, P., Garrido-Pérez, N., López-Pérez, M.J., Pérez- Axelband, F., Dias, J., Ferrão, F.M., Einicker-Lamas, M.J., 2011. Nongenomic signaling Martos, A., Montoya, J., 1999. Direct regulation of mitochondrial RNA synthesis pathways triggered by thyroid hormones and their metabolite 3- by thyroid hormone. Mol. Cell. Biol. 19 (1), 657–670. iodothyronamine on the cardiovascular system. J. Cell. Physiol. 226 (1), 21–28. Farach-Carson, M.C., Davis, P.J., 2003. Steroid hormone interactions with target Azzu, V., Jastroch, M., Divakaruni, A.S., Brand, M.D., 2010. The regulation and cells: cross talk between membrane and nuclear pathways. J. Pharmacol. Exp. turnover of mitochondrial uncoupling proteins. Biochim. Biophys. Acta 1797 Ther. 307 (3), 839–845. (6–7), 785–791. Farwell, A.P., Dubord-Tomasetti, S.A., Pietrzykowski, A.Z., Leonard, J.L., 2006. Bassett, J.H., Harvey, C.B., Williams, G.R., 2003. Mechanisms of thyroid hormone Dynamic nongenomic actions of thyroid hormone in the developing rat brain. receptor-specific nuclear and extra nuclear actions. Mol. Cell. Endocrinol. 213 Endocrinology 147, 2567–2574. (1), 1–11. Flamant, F., Gauthier, K., 2012. Thyroid hormone receptors: the challenge of Baxter, J.D., Webb, P., 2009. Thyroid hormone mimetics: potential applications in elucidating isotype-specific functions and cell-specific response. Biochim. atherosclerosis, obesity and type 2 diabetes. Nat. Rev. Drug Discov. 8, 308–320. Biophys. Acta June 13. Bergh, J.J., Lin, H.Y., Lansing, L., Mohamed, S.N., Davis, F.B., Mousa, S., Davis, P.J., Flandin, P., Lehr, L., Asensio, C., Giacobino, J.P., Rohner-Jeanrenaud, F., Muzzin, P., 2005. Integrin alphaVbeta3 contains a cell surface receptor site for thyroid Jimenez, M., 2009. Uncoupling protein-3 as a molecular determinant of the hormone that is linked to activation of mitogen-activated protein kinase and action of 3,5,30-triiodothyronine on energy metabolism. Endocrine 36 (2), 246– induction of angiogenesis. Endocrinology 146 (7), 2864–2871. 254. Berkenstam, A., Kristensen, J., Mellstro, M.K., Carlsson, B., Malm, J., Rehnmark, S., Frascarelli, S., Ghelardoni, S., Chiellini, G., Galli, E., Ronca, F., Scanlan, T.S., Zucchi, R., Garg, N., Andersson, C.M., Rudling, M., Sjo berg, F., Angelin, B., Baxter, J.D., 2008. 2011. Cardioprotective effect of 3-iodothyronamine in perfused rat heart The thyroid hormone mimetic compound KB2115 lowers plasma LDL subjected to ischemia and reperfusion. Cardiovasc. Drugs Ther. 25 (4), 307–313. cholesterol and stimulates bile acid synthesis without cardiac effects in Friesema, E.C., Ganguly, S., Abdalla, A., Manning Fox, J.E., Halestrap, A.P., Visser, T.J., humans. Proc. Natl. Acad. Sci. USA 105, 663–667. 2003. Identification of monocarboxylate transporter 8 as a specific thyroid Bianco, A.C., 2011. Minireview: cracking the metabolic code for thyroid hormone hormone transporter. J. Biol. Chem. 278 (41), 40128–40135. signaling. Endocrinology 152 (9), 3306–3311. Gadaleta, M.N., Barletta, A., Caldarazzo, M., De Leo, T., Saccone, C., 1972. Bigler, J., Hokanson, W., Eisenman, R.N., 1992. Thyroid hormone receptor Triiodothyronine action on RNA synthesis in rat-liver mitochondria. Eur. J. transcriptional activity is potentially autoregulated by truncated forms of the Biochem. 30 (2), 376–381. receptor. Mol. Cell. Biol. 12 (5), 2406–2417. Goglia, F., 2005. Biological effects of 3,5-diiodothyronine (T2). Biochemistry (Mosc.) Brent, G.A., 2012. Mechanisms of thyroid hormone action. J. Clin. Invest. 122 (9), 70 (2), 164–172. 3035–3043. Goglia, F., Skulachev, V.P., 2003. A function for novel uncoupling proteins: Carazo, A., Levin, J., Casas, F., Seyer, P., Grandemange, S., Busson, M., Pessemesse, L., antioxidant defense of mitochondrial matrix by translocating fatty acid Wrutniak-Cabello, C., Cabello, G., 2012. Protein sequences involved in the peroxides from the inner to the outer membrane leaflet. FASEB J. 17 (12), mitochondrial import of the 3,5,30-L-triiodothyronine receptor p43. J. Cell. 1585–1591. Physiol. 227 (12), 3768–3777. Goglia, F., Torresani, J., Bugli, P., Barletta, A., Liverini, G., 1981. In vitro binding of Chan, I.H., Privalsky, M.L., 2010. A conserved lysine in the thyroid hormone triiodothyronine to rat liver mitochondria. Pflug. Arch. 390 (2), 120–124. receptor-alpha1 DNA-binding domain, mutated in hepatocellular carcinoma, Goglia, F., Liverini, G., De Leo, T., Barletta, A., 1983. Thyroid state and mitochondrial serves as a sensor for transcriptional regulation. Mol. Cancer Res. 8 (1), 15–23. population during cold exposure. Pflug. Arch. 396 (1), 49–53. Chang, C.R., Blackstone, C., 2010. Dynamic regulation of mitochondrial fission Goglia, F., Lanni, A., Barth, J., Kadenbach, B., 1994a. Interaction of diiodothyronines through modification of the dynamin-related protein Drp1. Ann. N. Y. Acad. Sci. with isolated cytochrome c oxidase. FEBS Lett. 346 (2–3), 295–298. 1201, 34–39. Goglia, F., Lanni, A., Horst, C., Moreno, M., Thoma, R., 1994b. In vitro binding of 3,5- Chen, H., Chomyn, A., Chan, D.C., 2005. Disruption of fusion results in mitochondrial di-iodo-L-thyronine to rat liver mitochondria. J. Mol. Endocrinol. 13 (3), 275– heterogeneity and dysfunction. J. Biol. Chem. 280 (28), 26185–26192. 282. Cheng, S.Y., Leonard, J.L., Davis, P.J., 2010. Molecular aspects of thyroid hormone Hailey, D.W., Rambold, A.S., Satpute-Krishnan, P., Mitra, K., Sougrat, R., Kim, P.K., actions. Endocr. Rev. 31 (2), 139–170. Lippincott-Schwartz, J., 2010. Mitochondria supply membranes for Cioffi, F., Senese, R., de Lange, P., Goglia, F., Lanni, A., Lombardi, A., 2009. Uncoupling autophagosome biogenesis during starvation. Cell 141 (4), 656–667. proteins: a complex journey to functions discovery. BioFactors 35, 417–428. Hess, B., Martius, C., 1951. The mode of action of thyroxin. Arch. Biochem. Biophys. Cioffi, F., Lanni, A., Goglia, F., 2010. Thyroid hormones, mitochondrial bioenergetics 33 (3), 486–487. and lipid handling. Curr. Opin. Endocrinol. Diabetes Obes. 17 (5), 402–407. Hock, M.B., Kralli, A., 2009. Transcriptional control of mitochondrial biogenesis and Cipolat, S., de Brito, O.M., Dal, Zilio B., Scorrano, L., 2004. OPA1 requires mitofusin 1 function. Annu. Rev. Physiol. 71, 177–203. to promote mitochondrial fusion. Proc. Natl. Acad. Sci. USA 101 (45), 15927– Huang, C.J., Geller, H.M., Green, W.L., Craelius, W., 1999. Acute effects of thyroid 15932. hormone analogs on sodium currents in neonatal rat myocytes. J. Mol. Cell. Cody, V., Davis, P.J., Davis, F.B., 2007. Molecular modeling of the thyroid hormone Cardiol. 31 (4), 881–893. interactions with alpha v beta 3 integrin. Steroids 72 (2), 165–170. Incerpi, S., De Vito, P., Luly, P., Spagnuolo, S., Leoni, S., 2002. Short-term effects of Davis, P.J., Davis, F.B., Lin, H.Y., Mousa, S.A., Zhou, M., Luidens, M.K., 2009. thyroid hormones and 3,5-diiodothyronine on membrane transport systems in Translational implications of nongenomic actions of thyroid hormone chick embryo hepatocytes. Endocrinology 143 (5), 1660–1668. initiated at its integrin receptor. Am. J. Physiol. Endocrinol. Metab. 297 (6), Irrcher, I., Adhihetty, P.J., Sheehan, T., Joseph, A.M., Hood, D.A., 2003. PPARgamma E1238–E1246. coactivator-1alpha expression during thyroid hormone- and contractile 60 F. Cioffi et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61

activity-induced mitochondrial adaptations. Am. J. Physiol. Cell Physiol. 284 (6), thyronine prevents high-fat-diet-induced insulin resistance in rat skeletal C1669–C1677. muscle through metabolic and structural adaptations. FASEB J. 25 (10), 3312– Irrcher, I., Walkinshaw, D.R., Sheehan, T.E., Hood, D.A., 2008. Thyroid hormone 3324. (T3)rapidly activates p38 and AMPK in skeletal muscle in vivo. J. Appl. Physiol. Mutvei, A., Kuzela, S., Nelson, B.D., 1989. Control of mitochondrial transcription by 104, 178–185. thyroid hormone. Eur. J. Biochem. 180 (1), 235–240. Kapoor, R., van Hogerlinden, M., Wallis, K., Ghosh, H., Nordstrom, K., Vennstrom, B., Nelson, B.D., Luciakova, K., Li, R., Betina, S., 1995. The role of thyroid hormone and Vaidya, V.A., 2010. Unliganded thyroid hormone receptor alpha1 impairs adult promoter diversity in the regulation of nuclear encoded mitochondrial proteins. hippocampal neurogenesis. FASEB J. 24 (12), 4793–4805. Biochim. Biophys. Acta 271 (1), 85–91. Kinne, A., Schülein, R., Krause, G., 2011. Primary and secondary thyroid hormone Oetting, A., Yen, P.M., 2007. New insights into thyroid hormone action. Best Pract. transporters Thyroid Research 4 (Suppl. 1), S7. Res. Clin. Endocrinol. Metab. 21 (2), 193–208. Koshiba, T., Detmer, S.A., Kaiser, J.T., Chen, H., McCaffery, J.M., Chan, D.C., 2004. Oppenheimer, J.H., Schwartz, H.L., Surks, M.I., 1974. Tissue differences in the Structural basis of mitochondrial tethering by mitofusin complexes. Science concentration of triiodothyronine nuclear binding sites in the rat: liver, kidney, 305 (5685), 858–862. pituitary, heart, brain, spleen and testis. Endocrinology 95, 897–903. Kuznetsov, A.V., Hermann, M., Saks, V., Hengster, P., Margreiter, R., 2009. The cell- O’Reilly, I., Murphy, M.P., 1992. Studies on the rapid stimulation of mitochondrial type specificity of mitochondrial dynamics. Int. J. Biochem. Cell Biol. 41 (10), respiration by thyroid hormones. Acta Endocrinol. (Copenh.) 127 (6), 542–546. 1928–1939. Orozco, A., Valverde, R.C., Olvera, A., García, G.C., 2012. Iodothyronine deiodinases: a Ladenson, P.W., Kristensen, J.D., Ridgway, E.C., Olsson, A.G., Carlsson, B., Klein, I., functional and evolutionary perspective. J. Endocrinol. 215 (2), 207–219. Baxter, J.D., Angelin, B., 2010. Use of the thyroid hormone analogue eprotirome Osellame, L.D., Blacker, T.S., Duchen, M.R., 2012. Cellular and molecular in statin-treated dyslipidemia. N. Engl. J. Med. 362 (10), 906–916. mechanisms of mitochondrial function. Best Pract. Res. Clin. Endocrinol. Lanni, A., Moreno, M., Cioffi, M., Goglia, F., 1992. Effect of 3,30-diiodothyronine and Metab. 26 (6), 711–723. 3,5-diiodothyronine on rat liver oxidative capacity. Mol. Cell. Endocrinol. 86, Otera, H., Mihara, K., 2011. Molecular mechanisms and physiologic functions of 143–148. mitochondrial dynamics. J. Biochem. 149 (3), 241–251. Lanni, A., Moreno, M., Cioffi, M., Goglia, F., 1993. Effect of 3,30-di-iodothyronine and Pessemesse, L., Schlernitzauer, A., Sar, C., Levin, J., Grandemange, S., Seyer, P., Favier, 3,5-di-iodothyronine on rat liver mitochondria. J. Endocrinol. 136, 59–64. F.B., Kaminski, S., Cabello, G., Wrutniak-Cabello, C., Casas, F., 2012. Depletion of Lanni, A., Moreno, M., Lombardi, A., Goglia, F., 1994. Rapid stimulation in vitro of rat the p43 mitochondrial T3 receptor in mice affects skeletal muscle development liver cytochrome oxidase activity by 3,5-diiodo-L-thyronine and by 3,30-diiodo- and activity. FASEB J. 26 (2), 748–756. L-thyronine. Mol. Cell. Endocrinol. 99 (1), 89–94. Picard, F., Géhin, M., Annicotte, J., Rocchi, S., Champy, M.F., O’Malley, B.W., Lanni, A., De Felice, M., Lombardi, A., Moreno, M., Fleury, C., Ricquier, D., Goglia, F., Chambon, P., Auwerx, J., 2002. SRC-1 and TIF2 control energy balance 1997. Induction of UCP2 mRNA by thyroid hormones in rat heart. FEBS Lett. 418, between white and brown adipose tissues. Cell 111 (7), 931–941. 171–174. Piehl, S., Hoefig, C.S., Scanlan, T.S., Köhrle, J., 2011. Thyronamines past, present, and Lanni, A., Beneduce, L., Lombardi, A., Moreno, M., Boss, O., Muzzin, P., Giacobino, J.P., future. Endocr. Rev. 32 (1), 64–80. Goglia, F., 1999. Expression of uncoupling protein-3 and mitochondrial activity Puigserver, P., Wu, Z., Park, C.W., Graves, R., Wright, M., Spiegelman, B.M., 1998. A in the transition from hypothyroid to hyperthyroid state in rat skeletal muscle. cold-inducible coactivator of nuclear receptors linked to adaptive FEBS Lett. 444, 250–254. thermogenesis. Cell 92, 829–839. Lanni, A., Moreno, M., Lombardi, A., de Lange, P., Goglia, F., 2001. Control of energy Rosen, M.D., Chan, I.H., Privalsky, M.L., 2011. Mutant thyroid hormone receptors metabolism by iodothyronines. J. Endocrinol. Invest. 24 (11), 897–913. (TRs) isolated from distinct cancer types display distinct target gene Lanni, A., Moreno, M., Lombardi, A., Goglia, F., 2003. Thyroid hormone and specificities: a unique regulatory repertoire associated with two renal clear uncoupling proteins. FEBS Lett. 543, 5–10. cell carcinomas. Mol. Endocrinol. 25 (8), 1311–1325. Lanni, A., Moreno, M., Lombardi, A., de Lange, P., Silvestri, E., Ragni, M., Farina, P., Sadana, P., Zhang, Y., Song, S., Cook, G.A., Elam, M.B., Park, E.A., 2007. Regulation of Baccari, G.C., Fallahi, P., Antonelli, A., Goglia, F., 2005. 3,5-Diiodo-L-thyronine carnitine palmitoyltransferase I (CPT-I alpha) gene expression by the powerfully reduces adiposity in rats by increasing the burning of fats. FASEB J. peroxisome proliferator activated receptor gamma coactivator (PGC-1) 19 (11), 1552–1554. isoforms. Mol. Cell. Endocrinol. 267, 6–16. Lardy, H.A., Feldott, G., 1951. Metabolic effects of thyroxine in vitro. Ann. N. Y. Acad. Saelim, N., John, L.M., Wu, J., Park, J.S., Bai, Y., Camacho, P., Lechleiter, J.D., 2004. Sci. 54 (4), 636–648. Nontranscriptional modulation of intracellular Ca2+ signaling by ligand Leo, T., Meo, S., Barletta, A., Martino, G., Goglia, F., 1976. Modification of nucleic acid stimulated thyroid hormone receptor. J. Cell Biol. 167 (5), 915–924. levels per mitochondrion induced by thyroidectomy or triiodothyronine Samuels, H.H., Tsai, J.S., Casanova, J., 1974. Thyroid hormone action: in vitro administration. Pflug. Arch. 366 (1), 73–77. demonstration of putative receptors in isolated nuclei and soluble nuclear Liesa, M., Palacín, M., Zorzano, A., 2009. Mitochondrial dynamics in mammalian extracts. Science 184 (4142), 1188–1191. health and disease. Physiol. Rev. 89 (3), 799–845. Sap, J., Muñoz, A., Damm, K., Goldberg, Y., Ghysdael, J., Leutz, A., Beug, H., Lin, H.Y., Tang, H.Y., Shih, A., Keating, T., Cao, G., Davis, P.J., Davis, F.B., 2007. Thyroid Vennström, B., 1986. The c-erb-A protein is a high-affinity receptor for thyroid hormone is a MAPK-dependent growth factor for thyroid cancer cells and is hormone. Nature 324 (6098), 635–640. anti-apoptotic. Steroids 72 (2), 180–187. Silva, J.E., 2006. Thermogenic mechanisms and their hormonal regulation. Physiol. Lin, H.Y., Tang, H.Y., Keating, T., Wu, Y.H., Shih, A., Hammond, D., Sun, M., Hercbergs, Rev. 86 (2), 435–464. A., Davis, F.B., Davis, P.J., 2008. Resveratrol is pro-apoptotic and thyroid Silvestri, E., Schiavo, L., Lombardi, A., Goglia, F., 2005. Thyroid hormones as hormone is anti-apoptotic in glioma cells: both actions are integrin and ERK molecular determinants of thermogenesis. Acta Physiol. Scand. 184, 265–283. mediated. Carcinogenesis 29, 62–69. Silvestri, E., Cioffi, F., Glinni, D., Ceccarelli, M., Lombardi, A., de Lange, P., Chambery, Lin, H.Y., Sun, M., Lin, C., Tang, H.Y., London, D., Shih, A., Davis, F.B., Davis, P.J., 2009a. A., Severino, V., Lanni, A., Goglia, F., Moreno, M., 2010. Pathways affected by 3,5- Androgen induced human breast cancer cell proliferation is mediated by diiodo-L-thyronine in liver of high fat-fed rats: evidence from two-dimensional discrete mechanisms in estrogen receptor-alpha-positive and -negative breast electrophoresis, blue-native PAGE, and mass spectrometry. Mol. BioSyst. 6 (11), cancer cells. J. Steroid Biochem. Mol. Biol. 113, 182–188. 2256–2271. Lin, H.Y., Sun, M., Tang, H.Y., Lin, C., Luidens, M.K., Mousa, S.A., Incerpi, S., DrusanoG, Sterling, K., Milch, P.O., 1975. Thyroid hormone binding by a component of L., Davis, F.B., Davis, P.J., 2009b. L-Thyroxine vs. 3,5,3-triiodo-L-thyronine and mitochondrial membrane. Proc. Natl. Acad. Sci. USA 72 (8), 3225–3229. cell proliferation: activation of mitogen-activated protein kinase and Tata, J.R., 1963. Inhibition of the biological action of thyroid hormones by phosphatidylinositol 3-kinase. Am. J. Physiol. Cell Physiol. 296, C980–C991. actinomycin D and puromycin. Nature 197, 1167–1168. Magnus-Levy, A., 1895. Ueber den respiratorishen Gaswechsel unter Einfluss der Tata, J.R., 2012. The road to nuclear receptors of thyroid hormone. Biochim. Biophys. Thyroidea sowie unter verschieden pathologische Zustand. Berliner Klinische Acta March 17. Wochenshrift 32, 650–652. Tata, J.R., Ernster, L., Lindberg, O., 1962. Control of basal metabolic rate by thyroid Martino, G., Covello, C., De Giovanni, R., Filippelli, R., Pitrelli, G., 1986. Direct in vitro hormones and cellular function. Nature 193, 1058–1060. action of thyroid hormones on mitochondrial RNA-polymerase. Mol. Biol. Rep. Tata, J.R., Ernster, L., Lindberg, O., Arrhenius, E., Pedersen, S., Hedman, R., 1963. The 11 (4), 205–211. action of thyroid hormones at the cell level. Biochem. J. 86 (3), Mayerl, S., Visser, T.J., Darras, V.M., Horn, S., Heuer, H., 2012. Endocrinology 153 (3), 408–428. 1528–1537. Thakran, S., Sharma, P., Attia, R.R., Hori, R.T., Deng, X., Elam, M.B., Park, E.A., 2013. Meeusen, S., McCaffery, J.M., Nunnari, J., 2004. Mitochondrial fusion intermediates Role of sirtuin 1 in the regulation of hepatic gene expression by thyroid revealed in vitro. Science 305 (5691), 1747–1752. hormone. J. Biol. Chem. 288 (2), 807–818. Michel, S., Wanet, A., De Pauw, A., Rommelaere, G., Arnould, T., Renard, P., 2012. Vatner, D.F., Weismann, D., Beddow, S.A., Kumashiro, N., Erion, D.M., Liao, X-H., Crosstalk between mitochondrial (dys)function and mitochondrial abundance. Grover, G.J., Webb, P., Phillips, K.J., Weiss, R.E., Bogan, J.S., Baxter, J., Shulman, J. Cell. Physiol. 227 (6), 2297–2310. G.I., Varman, T., Samuel, V.T., 2013. Thyroid hormone receptor b agonists Moeller, L.C., Dumitrescu, A.M., Refetoff, S., 2005. Cytosolic action of thyroid prevent hepatic steatosis in fat-fed rats but impair insulin sensitivity via hormone leads to induction of hypoxia-inducible factor-1_ and glycolytic genes. discrete pathways. Am. J. Physiol. Endocrinol. Metab. May 7. http://dx.doi.org/ Mol. Endocrinol. 19, 2955–2963. 10.1152/ajpendo.00573.20. Morel, G., Ricard-Blum, S., Ardail, D., 1996. Kinetics of internalization and Venditti, P., Bari, A., Di Stefano, L., Cardone, A., Della, Ragione.F., D’Esposito, M., Di subcellular binding sites for T3 in mouse liver. Biol. Cell 86 (2–3), 167–174. Meo, S., 2009. Involvement of PGC-1, NRF-1, and NRF-2 in metabolic response Moreno, M., de Lange, P., Lombardi, A., Silvestri, E., Lanni, A., Goglia, F., 2008. by rat liver to hormonal and environmental signals. Mol. Cell. Endocrinol. 305 Metabolic effects of thyroid hormone derivatives. Thyroid 18 (2), 239–253. (1–2), 22–29. Moreno, M., Silvestri, E., De Matteis, R., de Lange, P., Lombardi, A., Glinni, D., Senese, Venditti, P., Chiellini, G., Di Stefano, L., Napolitano, G., Zucchi, R., Columbano, A., R., Cioffi, F., Salzano, A.M., Scaloni, A., Lanni, A., Goglia, F., 2011. 3,5-Diiodo-L- Scanlan, T.S., Di Meo, S., 2010. The TRbeta-selective agonist, GC-1, stimulates F. Cioffi et al. / Molecular and Cellular Endocrinology 379 (2013) 51–61 61

mitochondrial oxidative processes to a lesser extent than triiodothyronine. J. Westermann, B., 2010. Mitochondrial fusion and ﬁssion in cell life and death. Nat. Endocrinol. 2010 (205), 279–289. Rev. Mol. Cell Biol. 11 (12), 872–884. Ventura-Clapier, R., Garnier, A., Veksler, V., 2008. Transcriptional control of Wrutniak, C., Cassar-Malek, I., Marchal, S., Rascle, A., Heusser, S., Keller, J.M., mitochondrial biogenesis: the central role of PGC-1alpha. Cardiovasc. Res. 79 Fléchon, J., Dauça, M., Samarut, J., Ghysdael, J., Cabello, G., 1995. A 43-kDa (2), 208–217. protein related to c-Erb A alpha 1 is located in the mitochondrial matrix of rat Visser, W.E., Friesema, E.C., Visser, T.J., 2011. Minireview: thyroid hormone liver. J. Biol. Chem. 270 (27), 16347–16354. transporters: the knowns and the unknowns. Mol. Endocrinol. 25 (1), 1–14. Wulf, A., Harneit, A., Weitzel, J.M., 2007. T3-mediated gene expression is Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, D.J., Evans, R.M., 1986. The independent of PGC-1alpha. Mol. Cell. Endocrinol. 270 (1–2), 57–63. c-erb-A gene encodes a thyroid hormone receptor. Nature 324 (6098), 641–646. Wulf, A., Harneit, A., Kröger, M., Kebenko, M., Wetzel, M.G., Weitzel, J.M., 2008. T3- Weiss, R.E., Xu, J., Ning, G., Pohlenz, J., O’Malley, B.W., Refetoff, S., 1999. Mice mediated expression of PGC-1alpha via a far upstream located thyroid hormone deﬁcient in the steroid receptor co-activator 1 (SRC-1) are resistant to thyroid response element. Mol. Cell. Endocrinol. 287 (1–2), 90–95. hormone. EMBO J. 18 (7), 1900–1904. Yehuda-Shnaidman, E., Kalderon, B., Azazmeh, N., Bar-Tana, J., 2010. Gating of the Weitzel, J.M., Iwen, K.A., 2011. Coordination of mitochondrial biogenesis by thyroid mitochondrial permeability transition pore by thyroid hormone. FASEB J. 24, hormone. Mol. Cell. Endocrinol. 342 (1–2), 1–7. 93–104. Weitzel, J.M., Iwen, K.A., Seitz, H.J., 2003. Regulation of mitochondrial biogenesis by Yen, P.M., Ando, S., Feng, X., Liu, Y., Maruvada, P., Xia, X., 2006. Thyroid hormone thyroid hormone. Exp. Physiol. 88 (1), 121–128. action at the cellular, genomic and target gene levels. Mol. Cell. Endocrinol. 246 West, A.P., Shadel, G.S., Ghosh, S., 2011. Mitochondria in innate immune responses. (1–2), 121–127. Nat. Rev. Immunol. 11 (6), 389–402. Molecular and Cellular Endocrinology 379 (2013) 62–73

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Steroid hormone synthesis in mitochondria

Walter L. Miller ⇑

Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143-1346, USA Division of Endocrinology, University of California San Francisco, San Francisco, CA 94143-1346, USA article info abstract

Article history: Mitochondria are essential sites for steroid hormone biosynthesis. Mitochondria in the steroidogenic cells Available online 28 April 2013 of the adrenal, gonad, placenta and brain contain the cholesterol side-chain cleavage enzyme, P450scc, and its two electron-transfer partners, ferredoxin reductase and ferredoxin. This enzyme system converts Keywords: cholesterol to pregnenolone and determines net steroidogenic capacity, so that it serves as the chronic Cholesterol transport regulator of steroidogenesis. Several other steroidogenic enzymes, including 3b-hydroxysteroid dehydro- Cholesterol side chain cleavage genase, 11b-hydroxylase and aldosterone synthase also reside in mitochondria. Similarly, the mitochon- Outer mitochondrial membrane dria of renal tubular cells contain two key enzymes participating in the activation and degradation of Steroidogenesis vitamin D. The access of cholesterol to the mitochondria is regulated by the steroidogenic acute regula- Steroidogenic acute regulatory protein Vitamin D tory protein, StAR, serving as the acute regulator of steroidogenesis. StAR action requires a complex multi-component molecular machine on the outer mitochondrial membrane (OMM). Components of this machine include the 18 kDa translocator protein (TSPO), the voltage-dependent anion chanel (VDAC-1), TSPO-associated protein 7 (PAP7, ACBD3), and protein kinase A regulatory subunit 1a (PKAR1A). The precise fashion in which these proteins interact and move cholesterol from the OMM to P450scc, and the means by which cholesterol is loaded into the OMM, remain unclear. Human deﬁciency diseases have been described for StAR and for all the mitochondrial steroidogenic enzymes, but not for the electron transfer proteins or for the components of the cholesterol import machine. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction thetic pathways that are initiated by specialized, tissue-specific enzymes found in mitochondria. These hormones include Six classes of steroid hormones, all of which are indispensable glucocorticoids (cortisol, corticosterone) and mineralocorticoids for mammalian life, are made from cholesterol via complex biosyn- (aldosterone) produced in the adrenal cortex; estrogens (estradiol), progestins (progesterone) and androgens (testosterone, dihydrotestosterone) produced in the gonads; and calciferols (1,25-dihy- Abbreviations: 1,25(OH)2D, 1,25 dihydroxy vitamin D (calcitriol); 3bHSD, 3b- hydroxysteroid dehydrogenase; ACAT, acyl transferase; ACTH, adrenocorticotropic droxy vitamin D [1,25OH2D]) produced in the kidney. The hormone; ANT, adenine nucleotide transporter; ER, endoplasmic reticulum; CRAC, biosynthesis of the steroid hormones (Miller and Auchus, 2011) cholesterol recognition amino acid consensus domain; FAD, flavin adenine dinu- and of 1,25OH2D (a sterol) (Feldman et al., 2013) from cholesterol cleotide; HDL, high density lipoproteis; HSL, hormone-sensitive neutral lipase; have been reviewed recently. There are two specialized aspects to HMGCoA, 3-hydroxy-3-methylglutaryl co-enzyme A; IMM, inner mitochondrial membrane; IMS, intramembranous space; Km, Michaelis constant; LAL, lysosomal the mitochondria of these steroidogenic tissues – the specialized acid lipase; LDL, low-density lipoproteins; LH, luteinizing hormone; MENTAL, mechanisms by which cholesterol is delivered to the mitochondria MLN64 N-terminal; MENTHO, MLN64 N-terminal domain homologue; MLN64, and the specialized intra-mitochondrial enzymes that paricipate in metastatic lymph node clone 64; NADPH, nicotinamide adenine dinucleotide the synthesis of hormonal steroids. phosphate; NPC, Niemann Pick type C; OMM, outer mitochondrial membrane; PAP7, TSPO-associated protein 7 (ACBD3); PBR, peripheral benzodiazepine receptor; PCP, phosphate carrier protein; PKA, protein kinase A; PKAR1A, protein kinase A 2. Delivery of cholesterol to mitochondria regulatory subunit 1a; PRAX1, TSPO-associated protein 1; PTH, parathyroid hormone; P450scc, mitochondrial cytochrome P450 specific for cholesterol side- chain cleavage; SF1, steroidogenic factor 1; SOAT, sterol O-acetyltransferase; SR-B1, 2.1. Sources of cholesterol scavenger receptor B1; StAR, steroidogenic acute regulatory protein; START, StAR- related lipid transfer domain; SREBPs, sterol regulatory element binding proteins; The intracellular transport and distribution of cholesterol prior TSPO, 18 kDa translocator protein; VDAC1, voltage-dependent anion channel. Address: Department of Pediatrics, University of California San Francisco, San to its delivery to the mitochondria has been reviewed recently ⇑ Francisco, CA 94143-1346, USA. (Miller and Bose, 2011). Cholesterol may be produced de novo from E-mail address: [email protected] acetate via a complex pathway primarily found in the endoplasmic

0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.04.014 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 63 reticulum (ER) (Porter and Herman, 2011), but most steroidogenic (ACAT) also known as sterol-O-acetyltransferase (SOAT1). Simi- cholesterol is derived from circulating lipoproteins. High density larly, HDL cholesteryl esters that enter the cell via SR-B1 are acted lipoproteins (HDL) may be taken up via scavenger receptor B1 on by hormone-sensitive neutral lipase (HSL), following which the (SR-B1) and low-density lipoproteins (LDL) are taken up by recep- free cholesterol may also be used or re-esterified for storage. ACTH tor-mediated endocytosis via LDL receptors. LDL can suppress the and luteinizing hormone (LH) respectively increase intracellular rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-meth- levels of cAMP in the adrenal and gonad, stimulating HSL and ylglutaryl co-enzyme A (HMGCoA) reductase. Rodents preferenti- inhibiting ACAT, thus increasing the availability of free cholesterol aly use the HDL/SR-B1 pathway to obtain steroidogenic for steroid hormone synthesis. ACTH, LH and other factors that in- cholesterol, but the principal human source is receptor-mediated crease cAMP stimulate the activity of HMGCoA reductase and the endocytosis of LDL. Nevertheless, patients with congenital abeta- uptake of LDL cholesterol. When intracellular cholesterol concen- lipoproteinemia have low LDL cholesterol but have normal basal trations are high, the genes for the LDL receptor, HMGCoA reduc- cortisol concentrations, and only mildly impaired cortisol re- tase and LAL are repressed while ACAT is induced, thereby sponses to adrenocorticotropic hormone (ACTH) (Illingworth decreasing cholesterol uptake, synthesis and de-esterification. et al., 1982), and those treated with high doses of statins have no Conversely, when intracellular cholesterol concentrations are impairment of cortisol secretion (Dobs et al., 2000). Thus endoge- low, this process is reversed. nously produced cholesterol is sufficient in most situations, and Mutations in the LIPA gene encoding LAL cause Wolman disease, the HDL/SR-B1 system plays a relatively minor role in human ste- characterized by visceral accumulation of cholesteryl esters and roidogenesis. The regulation of cholesterol uptake, intracellular triglycerides, with secondary adrenal insufficiency; cholesterol es- transport, and utilization is coordinated by a family of basic he- ter storage disease is a milder, adult variant (Lohse et al., 1999). Af- lix-loop-helix transcription factors called the sterol regulatory ele- fected infants quickly develop hepatosplenomegaly, malabsorptive ment binding proteins (SREBPs). The cell biology of intracellular malnutrition and developmental delay; the diagnosis may be sug- cholesterol trafficking is summarized in Fig. 1. gested by calcification that outlines the adrenal glands, and is established by finding deficient lysosomal acid lipase activity in 2.2. Lipases and the processing of cholesterol esters leukocytes, fibroblasts or prenatal amniocytes. Bone marrow transplantation may ameliorate the disease, but the mechanism is un- After circulating LDL is internalized by receptor-mediated endo- clear. In contrast to LAL, no known human disease is associated cytosis, the resulting endocytic vesicles fuse with lysosomes, with HSL deficiency. where the LDL proteins are degraded by proteolysis, liberating the cholesteryl esters, which are then hydrolyzed to ‘free’ choles- 2.3. Endosomal processing of cholesterol terol by lysosomal acid lipase (LAL). However, cholesterol is never truly free, as its solubility is only about 20 lmol per liter, so that The entry and exit of cholesterol from lipid droplets involves the term ‘free cholesterol’ refers to cholesterol that is bound to pro- the NPC proteins, so named because their mutation causes Nie- teins or membranes, but lacks a covalently linked group. Free cho- mann Pick type C (NPC) disease, which is characterized by endo- lesterol may be used by the cell or stored in lipid droplets following somal accumulation of LDL-cholesterol and glycosphingolipids. re-esterification by acyl-coenzyme-A-cholesterol-acyl-transferase Patients are normal in infancy but develop ataxia, dementia, loss

Fig. 1. Intracellular cholesterol trafficing. Human steroidogenic cells take up circulating low-density lipoproteins (LDLs) by receptor-mediated endocytosis, directing the cholesterol to endosomes; rodent cells utilize cholesterol from high-density lipoproteins (HDLs) via scavenger receptor B1 (SRB1). Cholesterol may also be synthesized from acetate in the ER. Cholesteryl esters are cleaved by lysosomal acid lipase (LAL); free cholesterol is then bound by NPC1, transferred to NPC2, and exported. The MLN64/ MENTHO system resides in the same endosomes as the NPC system, but its role in cholesterol trafficking remains uncertain. Cholesterol may be re-esterified by acyl-CoA: cholesterol transferase (ACAT) and stored in lipid droplets as cholesteryl esters. Free cholesterol may be produced by hormone-sensitive lipase (HSL). Cholesterol can reach the outer mitochondrial membrane (OMM) by non-vesicular means by utilizing START-domain proteins or other cholesterol transport proteins. Movement of cholesterol from the OMM to the inner mitochondrial membrane (IMM) requires a multi-protein complex on the OMM. In the adrenals and gonads, the steroidogenic acute regulatory protein, StAR, is responsible for the rapid movement of cholesterol from the OMM to the IMM, where it can be converted to pregnenolone by P450scc. (ÓWL Miller). 64 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 of speech and spasticity at 2–4 years, and usually die during their terminal signal sequences, suggesting they are cytosolic proteins. second decade (Vanier and Millat, 2003). Cholesterol and other lip- StarD1 (StAR), StarD3 (MLN64), StarD4 and StarD5 bind cholesterol ids accumulate in Purkinje cells and other neurons, and there is ro- with high affinity and specificity, facilitate cholesterol transport bust glial infiltration. The diagnosis is typically made by finding through an aqueous environment, and appear to play important characteristic foamy Niemann-Pick cells and ‘sea-blue’ histiocytes roles in cellular cholesterol homeostasis (Rodriguez-Agudo et al., in the bone marrow. The NPC1 or NPC2 proteins participate in 2008). StAR and StarD6 stimulate the movement of cholesterol endosomal/lysosomal cholesterol transport. NPC1 is a 1278 AA gly- from the OMM to the inner mitochondrial membrane (IMM), but coprotein containing 13 transmembrane domains that span the StarD4 and StarD5 do not (Bose et al., 2008a). StarD4 and/or StarD5 endo-lysosomal membrane (Kwon et al., 2009), and NPC2 is a sol- may bring cholesterol to the OMM; however, StarD4 knockout uble 151 AA glycoprotein found in the lysosomal lumen (Xu et al., mice have no changes in steroidogenesis and minimal changes in 2007). NPC2 binds cholesteryl esters with the cholesterol side- weight and serum lipids, hence an essential function of StarD4 is chain buried is in a hydrophobic pocket and the polar 3bOH group not established (Riegelhaupt et al., 2010). While all the mecha- exposed, allowing LAL to cleave cholesteryl esters while they are nisms that move cholesterol to the mitochondria have not been bound to NPC2. The free cholesterol is then transferred to the N- determined, the current view is that the principal mechanism is terminal domain of NPC1 in the lysosomal lumen, which binds by non-vesicular transport via StarD proteins, but that StAR itself cholesterol the 3bOH group buried in the protein and the side plays a minor role in this step. The OMM of adrenal mitochondria chain partially exposed. Thus, the NPC2 and NPC1 proteins act to- contains abundant cholesterol, while the IMM contains little cho- gether to insert cholesterol into the lysosomal membrane with the lesterol. Whether or not all OMM cholesterol is potentially avail- hydrophobic side-chain going in first. able for steroidogenesis has been unclear. Early studies identified Two proteins named MLN64 (metastatic lymph node clone 64) a distinct pool of ‘‘steroidogenic’’ OMM cholesterol that was dis- and MENTHO (MLN64 N-terminal domain homologue) may also tinct from the structural membrane cholesterol and could be mobi- participate in endosomal cholesterol trafficking. MLN64 is a cho- lized by cAMP (Stevens et al., 1992). Movement of cholesterol from lesterol-binding protein that co-localizes with NPC1 in late endo- the OMM to the IMM is central to the initiation of steroidogenesis, somes (Zhang et al., 2002; Alpy and Tomasetto, 2006). The N- because the cholesterol side-chain cleavage enzyme (P450scc) sys- terminal ‘MENTAL’ domain (for MLN64 N-TerminAL) is structurally tem is localized to the IMM. Whether or nor there are two kineti- related to the late-endosomal protein, MENTHO (Alpy et al., 2002), cally distinct pools of cholesterol in the OMM, and how cholesterol contains 4 transmembrane domains, and targets MLN64 to late is transferred to the IMM remain under investigation. endosomal membranes. The C-terminal domain of MLN64 is called the START (StAR-related lipid transfer) domain because it is similar 3. Entry of cholesterol into steroidogenic mitochondria: action to the lipid-biding domain of the steroidogenic acute regulatory of the steroidogenic acute regulatory protein, StAR protein (StAR) (Alpy et al., 2001; Clark, 2012) (see Section 3). The MENTAL domains of MENTHO and MLN64 can interact to form 3.1. Acute and chronic regulation of steroidogenesis homo- and heterodimers and to bind cholesterol, suggesting a role in endosomal cholesterol transport. MLN64 lacking the MENTAL Unlike cells that produce polypeptide hormones, which store domain (N-234 MLN64) has 50–60% of StAR-like activity to stimu- large amounts of mature hormone available for rapid release, cells late mitochondrial uptake of cholesterol (Bose et al., 2000). The that produce steroid homones store very little steroid, so that ste- START domain of MLN64 may interact with cytoplasmic HSP60 roid secretion requires induction of steroid synthesis. There are to stimulate steroidogenesis in placental mitochondria (Olvera- several distinct mechanisms regulating steroidogenesis. In the Sanchez et al., 2011). The function of MLN64 remains unclear: adrenal, ACTH promotes steroidogenesis at three levels. First, over knockout of the START domain of MLN64 yielded viable, neurolog- the course of months ACTH promotes adrenal growth via fibroblast ically intact, fertile mice with normal plasma and hepatic lipids growth factor, epidermal growth factor, insulin-like growth factor (Kishida et al., 2004), and no human disorders of MLN64 or MEN- 2, and possibly other factors. Second, over the course of days, THO have been described. Accumulation of cholesterol in NPC1- ACTH, acting via cAMP in the adrenal zona fasciculata and angio- deficient cells increased MLN64-mediated cholesterol transport tensin II acting via calcium/calmodulin in the zona reticularis pro- to mitochondria and accumulation of cholesterol in the outer mote transcription of genes for steroidogenic enzymes, especially mitochondiral membrane (OMM), suggesting that cholesterol the gene encoding P450scc (CYP11A1) thus increasing the amounts transport from endosomes to mitochondria may involve MLN64 of steroidogenic enzymes. Third, acting on a 15-60 min time scale, (Charman et al., 2010). ACTH rapidly stimulates both the activation of pre-existing StAR and the synthesis of new StAR. ACTH/cAMP stimulates phosphory- 2.4. Non-vesicular intracytoplasmic cholesterol transport lation of StAR at Ser195 almost immediately, doubling its activity (Arakane et al., 1997), and induces StAR transcription within min- Intracellular cholesterol transport may may be ‘vesicular’ (med- utes (Jo et al., 2005; Manna et al., 2009). StAR then interacts with a iated by membrane fusion) or ‘non-vesicular’ (bound to proteins). complex macromolecular machine on the OMM to increase the Because membranes participating in vesicular transport are fluid, flow of cholesterol from the OMM to the IMM where it becomes the lipid compositions of the mitochondrial membranes may vary. the substrate for P450scc. The first two modes of steroid regulation Protein-protein interactions between lipid droplets, mitochondria, comprise the chronic steroidogenic response and the action of StAR and other organelles may facilitate vesicular cholesterol transport. comprises the acute steroidogenic response. Both vesicular and non-vesicular cholesterol transport occur in steroidogenic cells, but non-vesicular transport involving high-affinity cholesterol-binding START-domain proteins appears to be the 3.2. The steroidogenic acute regulatory protein, StAR principal means of cholesterol transport from lipid droplets to mitochondria (Miller and Bose, 2011). START-domain proteins StAR was discovered in the search for the factor that triggers the are found in fungi, plants and animals; the mammalian START-do- acute steroidogenic response. Autoradiographic spots were noted main proteins are termed StarD1-15 (Clark, 2012). The START pro- on 2-dimentional gels that appeared within 30 min of stimulating teins most closely related to StAR (StarD4, D5 and D6), bind steroidogenic tissues (Pon and Orme-Johnson, 1986; Pon et al., cholesterol and are induced by SREBP. StarD4, D5, and D6 lack N- 1986; Epstein and Orme-Johnson, 1991; Stocco and Sodeman W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 65

1991); the cDNA was then cloned (Clark et al., 1994; Sugawara OMM (see below) and requires pH-induced conformational et al., 1995), and the factor was named the steroidogenic acute reg- changes in StAR that are needed for StAR to accept and discharge ulatory protein, StAR (reviewed in Stocco and Clark, 1996). StAR is cholesterol (Bose et al., 1999; Baker et al., 2005). StAR can transfer a 37-kDa protein with an N-terminal mitochondrial targeting se- cholesterol between synthetic, protein-free membranes in vitro, quence that is cleaved off during mitochondrial import to yield but with non-physiologic stoichiometry (Tuckey et al., 2002), and 30-kDa intramitochondrial StAR. Co-expression of StAR and a biologically inactive StAR mutant (R182L) can also transfer cho- P450scc in nonsteroidogenic cells rapidly increases conversion of lesterol to membranes in vitro (Baker et al., 2007). Thus choles- cholesterol to pregnenolone, suggesting that StAR triggers the terol-binding is necessary but not sufficient for StAR activity. acute steroidogenic response (Stocco and Clark, 1996). The indis- Proteolysis of StAR bound to synthetic membranes that model pensible role of StAR was demonstrated by finding that StAR muta- OMM lipid composition showed that only the exterior surface of tions cause congenital lipoid adrenal hyperplasia, in which very the C-terminal a-helix and small segments of the adjacent X-loops little steroid is made (Lin et al., 1995; Bose et al., 1996). However, were protected, suggesting that only these domains interact with some steroidogenesis can take place in the absence of StAR. In vivo, the OMM (Yaworsky et al., 2005). Molecular dynamics studies the human placenta synthesizes steroids via mitochondrial show that cholesterol is blocked from reaching the cholesterol- P450scc but does not express StAR (Bose et al, 2000). In vitro, cells binding pocket of StAR by a set of hydrogen bonds that are dis- expressing the P450scc enzyme system but not StAR can convert rupted when the surface residues of StAR are protonated (as hap- cholesterol to pregnenolone (Harikrishna et al., 1993; Black et al., pens when StAR interacts with charged phospholipids on the 1994) at 14% of the maximal StAR-induced rate (Lin et al., OMM), thus eliciting a conformational change that permits choles- 1995; Bose et al., 1996). StAR-independent steroidogenesis might terol access (Bose et al., 1999; Baker et al., 2005). When the move- occur without a triggering protein, possibly using intracellular ment of the C-helix is prohibited by mutagenesis that creates hydroxysterols that bypass StAR action; alternatively, another pro- disulfide bonds, activity is lost, but reducing these bonds restores tein, such as the 30 kDa proteolytic cleavage product of MLN64, activity, showing that the immobilized C-helix, and not the muta- may exert StAR-like activity to promote mitochondrial cholesterol genesis, is responsible for the lost activity (Baker et al., 2005). Thus import, but the precise mechanism(s) are unclear. the activity of StAR on the OMM requires an acid-induced disruption of hydrogen bonds and a consequent conformational change 3.3. StAR acts on the outer mitochondrial membrane in StAR to permit it to bind and release cholesterol. The phosphorylation of StAR on Ser 195 approximately doubles The presence of a typical mitochondrial ‘leader’ sequence ini- StAR’s activity (Arakane et al., 1997). It appars that the protein ki- tially suggested that 37 kDa cytoplasmic StAR is a ‘precursor. and nase A (PKA) anchor protein AKAP121 recruits the type II PKA reg- that the 30 kDa intramitochondrial protein is the ‘mature form’, ulatory subunit a (PKAR2A) to the OMM, which phosphorylates but these terms do not describe the biology of StAR (Miller, 2007). StAR, whereas the type I kinase drives StAR transcription (Dyson The crystallographic structure of the START domain of MLN64 (N- et al., 2009). A physical interaction between the 37 kDa cytoplas- 216 MLN64), which shares 37% amino acid sequence identity with mic form of StAR and HSL has also been reported (Shen et al., StAR, suggested that 30-kDa StAR acts in the intramembranous 2003). StAR is recycled, with each molecule moving hundreds of space (IMS) to shuttle cholesterol from the OMM to the IMM (Tsuj- molecules of cholesterol into the mitochondria before StAR is im- ishita and Hurley, 2000). This structure shows a globular protein ported into the mitochondria and is inactivated (Artemenko with an a/b helix-grip fold and an elongated hydrophobic pocket et al., 2001). approximately 26 Å deep and 10 Å across at its widest diameter. The structure of StAR was then modeled based on MLN64 (Mathieu et al., 2002; Yaworsky et al., 2005) and these models have been con- 4. StAR interacts with an OMM protein complex firmed by crystallography at 3.4 Å resolution (Thorsell et al., 2011). The sterol-binding pocket of StAR accommodates a single molecule 4.1. The peripheral benzodiazepine receptor or mitochondrial of cholesterol with its 3b-OH group coordinated by the two polar transporter protein residues. These structures and the structure of StarD4 (Romanowski et al., 2002) are characterized by long N- and C-terminal a-helixes, StAR acts on the OMM via a complex that consists of several two short a-helixes, and 9 antiparallel b sheets that form a helix- proteins, most of which are now identified, even though it remains grip fold. However, deletion of StAR’s mitochondrial leader has no unclear what role each plays in the mitochondrial importation of effect on its activity (Arakane et al., 1996), and both the 37 kDa cholesterol (Bose et al., 2008b; Rone et al., 2012; Papadopoulos and 30 kDa forms of StAR are equally active in vivo and in vitro (Bose and Miller, 2012). The first of these proteins to be identified is et al., 2002). When StAR was fused to the cytoplasmic C-terminus of the peripheral benzodiazepine receptor (PBR) (Lacapere and the OMM protein Tom-20, thus immobilizing StAR an OMM, it was Papadopoulos, 2003), now also called the mitochondrial trans- constituitively active, but when StAR was immobilized in the IMS or porter protein (TSPO) (Papadopoulos et al., 2006). It was initially on the matrix side of the IMM, it was wholly inactive. Furthermore, proposed that PBR/TSPO was the ‘acute trigger’ of steroidogenesis, manipulating the speed of StAR’s mitochondrial entry by manipu- but it is now clear that StAR plays that role, and that PBR/TSPO is lating the leader sequence showed that faster import decreased part of the molecular machine on the OMM through which StAR activity whereas slower import increased activity (Bose et al., acts (Papadopoulos and Miller, 2012). PBR/TSPO is a ubiquitously 2002). Thus, StAR acts exclusively on the OMM and its activity is expressed 18 kDa protein that comprises up to 2% of OMM protein. proportional to how long it remains on the OMM. Thus it is the PBR/TSPO ligands can stimulate cholesterol movement from the OMM localization of StAR, and not its cleavage from the 37 kDa form OMM to the IMM and stimulate steroidogenesis (Lacapere and to the 30 kDa form, that determines its activity. Papadopoulos, 2003). Knockdown of the gene encoding PBR/TSPO in Leydig cells disrupts cholesterol transport and steroidogenesis, 3.4. StAR’s action requires conformational changes and serine and transfection of the PBR/TSPO-disrupted cells with the wild- phosphorylation type cDNA rescues steroidogenesis (Papadopoulos et al., 1997). However, knocking out the PBR/TSPO gene in mice caused early The mechanism by which StAR triggers cholesterol flux from embryonic lethality, indicating its indispensible role during devel- the OMM to the IMM involves a multi-protein complex on the opment (Lacapere and Papadopoulos, 2003). 66 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73

As is typical of proteins associated with the OMM, PBR/TSPO et al., 1995; Bose et al., 1996), so that lipoid CAH represents the lacks a cannonical mitochondrial targeting sequence; instead, the StAR knockout experiment of nature (Miller, 1997). C-terminal half targets PBR/TSPO to the OMM (Rone et al., 2009). The pathophysiology of lipoid CAH is explained by the two-hit PBR/TSPO appears to have five transmembrane a-helices spanning model (Bose et al., 1996). The first hit is lost StAR activity, causing the OMM (Joseph-Liauzun et al., 1998; Korkhov et al., 2010), sug- diminished steroidogenesis and a compensatory rise in ACTH and gesting that TSPO functions as a cholesterol channel, consistent gonadotropins. These increased tropic hormones stimulate intra- with data indicating that PBR/TSPO acts downstream from StAR cellular cAMP production, increasing LDL receptors, LDL choles- (Hauet et al., 2005; Liu et al., 2006). PBR/TSPO and has a cytoplas- terol uptake, and de novo synthesis of cholesterol. Intracellular mic domain containing a ‘‘cholesterol recognition amino acid con- cholesterol then accumulates, causing the second hit: the loss of sensus’’ (CRAC) domain (Li et al., 2001). PBR/TSPO binds cholesterol remaining steroidogenic capacity due to mitochondrial damage at the CRAC domain, suggesting that this domain participates in from the accumulated cholesterol, cholesteryl esters, and their transferring cholesterol from the OMM to the IMM (Jamin et al., auto-oxidation products (Bose et al., 1996)(Fig. 2). The absence 2005). Mutagenesis of the CRAC domain interferes with cholesterol of StAR activity affects fetal testicular Leydig cells early in gesta- binding and transfer of cholesterol to the IMM, and blocking the tion, preventing synthesis of the testosterone needed for the devel- binding of cholesterol to the CRAC domain prevents steroidogene- opment of male external genitalia, hence affected 46,XY fetuses are sis (Midzak et al., 2011). Targeted deletion of the TSPO gene in an born with female external genitalia. However, the testicular Sertoli immortalized Leydig cell line blocked cholesterol transport into the cells function normally and produce anti-Müllerian hormone, so mitochondria and reduced steroid production; reintroduction of the phenotypically female 46,XY fetus lacks female internal repro- TSPO into the deficient cell line restored steroidogenic capacity ductive organs. The fetal adrenal typically produces very little (Li et al., 2001). aldosterone, so that the zona glomerulosa is not tropicly stimulated and hence usually remains undamaged until after birth. Hence, newborns with lipoid CAH may not have a salt-wasting cri- 4.2. Other components of the OMM protein complex sis until several months of life, when chronic stimulation then leads to cellular damage. PBR/TSPO is a component of a 140–200 kDa multi-protein com- The two-hit model has been confirmed in clinical studies, plex consisting of PBR/TSPO, the 34-kDa voltage-dependent anion explaining why affected 46,XX patients feminize at the age of pub- channel (VDAC1), the 30-kDa the adenine nucleotide transporter erty (Bose et al., 1997; Fujieda et al., 1997), and also in knockout (ANT), the 10-kDa diazepam-binding inhibitor (acyl-CoA-binding mice (Hasegawa et al., 2000). The fetal ovary makes essentially domain 1, ACBD1), the TSPO-associated protein-1 (PRAX-1), and no steroids and remains unstimulated and hence undamaged until the PKA regulatory subunit RIa-associated protein 7 (PAP7) (Rone adolescence, when it is stimulated by pubertal gonadotropins, et al., 2012). PAP7, also known as acyl-CoA binding domain-con- yielding small amounts of estrogen produced by StAR-independent taining protein 3 (ACBD3), binds both TSPO and the regulatory sub- steroidogenesis. However, this low level of steroidogenesis is insuf- unit RIa of PKA (Fan et al., 2010). PBR/TSPO interacts with VDAC on ficient to generate progesterone in response to the mid-cycle LH the OMM, helping to anchor the multi-protein complex to the surge; continued stimulation results in cholesterol accumulation OMM and assisting with the binding and import of StAR (Liu and cellular damage, impairing the later biosynthesis of progester- et al., 2006); VDAC1 also interacts with ANT on the IMM (McEnery one, resulting in anovulatory cycles. Gonadotropin stimulation et al., 1992). While a functional interaction between StAR and PBR/ does not promote global ovarian steroidogenesis, but only recruits TSPO is clear (Hauet et al., 2005), a physical interaction has not individual follicles, hence most follicles remain undamaged and been demonstrated; protein cross-linking experiments indicate available for future cycles. A new follicle is recruited with each suc- that StAR directly interacts with VDAC1 and with phosphate carrier cessive cycle, producing estradiol by StAR-independent steroido- protein (PCP), but not with PBR/TSPO (Bose et al., 2008b). VDAC1 genesis, resulting in breast development, so that the patient also appears to interact with PBR/TSPO (Liu et al., 2006; Rone experiences general feminization, monthly estrogen withdrawal et al., 2009). It is not clear how VDAC-1 participates in cholesterol and cyclic vaginal bleeding. import, as VDAC-1 has a ring-like structure with a hydrophilic inte- Lipoid CAH is relatively common in Japan and Korea, where the rior that is well-suited to anion transport, but is unlikely to form a carrier frequency is approximately one in 300, and most affected channel for hydrophobic cholesterol (Bayrhuber et al., 2008; Hiller individuals carry the mutation Q258X (Bose et al., 1996; Nakae et al., 2008). VDAC is found at contact sites between the OMM and et al., 1997; Kim et al., 2011). Mutations retaining about 10–25% the IMM (Mannella et al., 1992) where it may complex with hexo- of normal StAR activity, especially R188C, cause a milder disease kinase, ANT, creatine kinase and proteins of the Bcl-2 family called ‘‘non-classic lipoid CAH’’, characterized by adrenal insuffi- (Brdiczka et al., 2006). ciency several years after infancy, nearly normal masculinization of 46,XY individuals, and minimally affected mineralocorticoid 5. Mutations in StAR – congenital lipoid adrenal hyperplasia secretion (Baker et al., 2006). (Lipoid CAH)

Lipoid CAH is characterized by absent or very low serum con- 6. The cholesterol side chain cleavage enzyme – the prototypical centrations of all steroids, high basal ACTH and plasma renin activ- mitochondrial P450 ity, and grossly enlarged adrenals ﬁlled with cholesterol and cholesteryl esters (Miller, 1997). Lipoid CAH was initially thought 6.1. Cytochrome P450 enzymes to be an enzymatic disorder and was mis-termed ‘20,22-desmolase deﬁciency’, but the CYP11A1 gene for P450scc is not mutated in Once cholesterol reaches the IMM, it may be converted to preg- these patients (Lin et al., 1991), and the placenta (a fetal tissue) nenolone to initiate steroidogenesis. Most steroidogenic enzymes continues to produce progesterone in lipoid CAH (Saenger et al., are cytochrome P450 enzymes, all of which have approximately 1995), indicating a lesion upstream from P450scc, such as in a fac- 500 residues, contain a single heme group and absorb light at tor involved in mitochondrial cholesterol transport (Lin et al., 450 nm when reduced with carbon monoxide. The human genome 1991). Mutations in the gene for PBR/TSPO were sought and ex- contains 57 CYP genes encoding cytochrome P450 enzymes; the cluded (Lin et al., 1993), but mutations were found in StAR (Lin corresponding proteins may be given the same name without the W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 67

Fig. 2. Two-hit model of lipoid CAH. (A) In normal adrenal cells, cholesterol is primarily derived from low-density lipoproteins, and the rate-limiting step in steroidogenesis is movement of cholesterol from the OMM to the IMM. (B) Early in lipoid CAH, StAR-independent steroidogenesis moves small amounts of cholesterol into mitochondria, yielding sub-normal steroidogenesis; ACTH secretion increases, stimulating further accumulation of cholesteryl esters in lipid droplets. (C) As lipids accumulate, they damage the cell through physical engorgement and by the action of cholesterol auto-oxidation products; steroidogenic capacity is destroyed, but tropic stimulation continues. Ovarian follicular cells remain unstimulated and undamaged until puberty, when small amounts of estradiol are produced, as in B, causing phenotypic feminization, with infertility and hypergonadotropic hypogonadism. Modified from (Bose et al., 1996), with permission. use of italics (thus CYP11A1 encodes the cholesterol side chain cleavage enzyme, P450scc; which may also be termed CYP11A1, without italics). Seven human cytochrome P450 enzymes are targeted to the mitochondria, the other 50 are targeted to the ER; the roles of the human P450 enzymes have been reviewed recently (Nebert et al., 2013). P450 enzymes activate molecular oxygen via their heme iron using electrons donated by nicotinamide adenine dinucleotide phosphate (NADPH). Mitochondrial (type 1) P450 enzymes receive electrons from NADPH via an electron transfer chain consisting of a flavoprotein termed ferredoxin reductase and a small iron-sulfur protein termed ferredoxin (Miller, 2005) (Fig. 3). The type 2 P450 enzymes in the ER receive electrons via a single 2-flavin protein termed P450 oxidoreductase (Miller, 2005). All P450 enzymes can catalyze multiple chemical reactions, Fig. 3. Organization of mitochondrial P450 enzyme systems. NADPH first donates often with very different substrates. electrons to the FAD moiety of ferredoxin reductase (FeRed); ferredoxin reductase then interacts with ferredoxin (Fedx) by charge-charge attraction, permitting

electron transfer of the Fedx to the Fe2S2 center (ball and stick diagram). Ferredoxin then dissociates from ferredoxin reductase and diffuses through the mitochrondrial 6.2. Steps in steroidogenesis matrix. The same surface of ferredoxin that received the electrons from ferredoxin reductase then interacts with the redox-partner binding-site of a mitochondrial Six P450 enzymes participate in steroidogenesis, and at least P450, such as P450scc, and the electrons then travel to the heme ring of the P450. three more participate in the processing of vitamin D; ﬁve of these The heme iron then mediates catalysis with substrate bound to the P450. ÓWL Miller. are found in mitochondria. Steroidogenesis is initiated by mitochondrial P450scc. P450scc cleaves the 20,22 bond of insoluble cholesterol to produce soluble pregnenolone, and is the hormon- pregnenolone may exit the mitochondrion and become the sub- ally regulated, rate-limiting step in steroidogenesis. It is the strate for P450c17 in the ER, which catalyzes both 17a-hydroxy- expression of the CYP11A1 gene that renders a cell ‘steroidogenic’. lase and 17,20 lyase activities. Pregnenolone appears to exit the Pregnenolone may then be converted to progesterone by 3b- mitochondrion unaided; no transport protein has been found, hydroxysteroid dehydrogenase (3bHSD), which may be found both and physiologic evidence does not suggest the presence of such a in the mitochondria and in the ER (see Section 9). Alternatively, transporter. Following the activities of 3bHSD and P450c17, 68 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73

P450c21 catalyzes the 21-hydroxylation of both glucocorticoids 1993). Genetic disorders of human ferredoxin reductase and ferre- and mineralocorticoids. The final steps in the synthesis of both glu- doxin have not been described, and mouse knockouts have not cocorticoids and mineralocorticoids again takes place in the mito- been reported. Mutation of the Drosophila ferredoxin reductase chondria, where two proteins that share 93% sequence identity, homologue dare causes developmental arrest and degeneration of 11b-hydroxylase (P450c11b, CYP11B1), and aldosterone synthase the adult nervous system secondary to disrupted ecdysone produc- (P450c11AS, CYP11B2) reside. P450c11b catalyzes the 11b-hydrox- tion (Freeman et al., 1999). ylation of 11-deoxycortisol to cortisol, and P450c11AS catalyzes The human FDXR gene produces two alternatively spliced ferre- the 11b-hydroxylation, 18-hydroxylation, and 18-methyl oxidation doxin reductase mRNAs differing by 18 bp (Solish et al., 1988), but to convert deoxycorticosterone to aldosterone. Histologic and elec- only the protein encoded by the more abundant, shorter mRNA is tron microscopic examination of steroidogenic cells suggests that active (Brandt and Vickery, 1992). Ferredoxin reductase mRNA is domains of the ER containing the steroidogenic P450 enzymes widely expressed, but is far more abundant in steroidogenic tissues come close to the OMM during hormonally-induced steroidogene- (Brentano et al, 1992). Ferredoxin reductase is a 54.5 kDa flavopro- sis, forming a steroidogenic complex, so that the movement of ste- tein affixed to the IMM that consists of two domains, each com- roidal intermediates from the mitochondrion to the ER involves prising a b-sheet core surrounded by a-helices (Ziegler et al., very small distances. 1999). The NADP(H)-binding domain is compact, whereas the domain that binds flavin adenine dinucleotide (FAD) is more open; this domain binds the dinucleotide portion of FAD across a Ross- 6.3. Chemistry and transcription of P450scc man fold with the redox-active flavin isoalloxazine ring abutting the NADP(H) domain. Electron transfer occurs in the cleft formed P450scc catalyzes three reactions: 22-hydroxylation of choles- by these two domains. This cleft is characterized by basic residues terol, 20-hydroxylation of 22(R)-hydroxycholesterol, and oxidative that interact with acidic residues on ferredoxin. scission of the C20-22 bond of 20(R),22(R)-dihydroxycholesterol, yielding pregnenolone and isocaproaldehyde. The binding of cholesterol and 22-hydroxylation are rate-limiting, as the efficiencies 7.2. Ferredoxin

(kcat/Km) are much higher for the subsequent reactions, and the high K of 3000 nM drives the dissociation of pregnenolone from The human FDX1 gene encodes ferredoxin (Fdx1), a 14 kDa, sol- D P450scc (Miller and Auchus, 2011). Alternatively, soluble hydrox- uble, iron/sulfur (Fe2S2) protein that resides either free in the mito- ysterols such as 22(R)-hydroxycholesterol can enter the mitochon- chondrial matrix or is loosely bound to the inner mitochondrial drion readily, without the action of StAR and its associated membrane (Miller, 2005). There are two human ferredoxins, Fdx1 machinery. Catalysis by P450scc is slow, with a net turnover of and Fdx2, but only Fdx1 supports steroidogenic mitochondrial approximately 6–20 molecules of cholesterol per molecule of P450 enzymes; Fdx2 participates in the synthesis of heme and P450scc per second. The crystal structures of bovine (Mast et al., Fe/S cluster proteins (Sheftel et al., 2010). Ferredoxin has a core re- 2011) and human (Strushkevich et al., 2011) P450scc, the latter gion containing four cysteine residues that tether the Fe2S2 cluster, in complex with ferredoxin, show that the single active site of and an interaction domain containing a helix with several charged P450scc is in contact with the IMM. residues, producing a negatively charged surface above the Fe2S2 Transcription of the CYP11A1 gene determines cellular steroido- cluster that interacts with the mitochondrial P450 (Muller et al., genic capacity. This transcription is regulated by hormonally- 1998). responsive factors that differ in different types of steroidogenic cells; both the PKA and PKC second messenger systems can induce 7.3. Mitochondrial P450 fusion proteins CYP11A1 transcription via different promoter elements. Adrenal and gonadal transcription of P450scc and other steroidogenic en- The same surface of ferredoxin interacts with both ferredoxin zymes requires the action of steroidogenic factor 1 (SF1) (Schim- reductase and the P450; nevertheless, creation of three-component mer and White, 2010). By contrast, placental expression of fusion proteins of the general scheme H2N-P450-FerredoxinReduc- P450scc is constitutive, independent of SF1 and requires members tase-Ferredoxin-COOH (termed F2) increases Vmax. This design of the CP2 (graineyhead) family of transcription factors (also known was first tested with P450scc (Harikrishna et al., 1993) and has as LBP proteins) (Huang and Miller, 2000; Henderson et al., 2007) been confirmed with P450c27 (Dilworth et al., 1996) and and TreP-132 (Gizard et al., 2002). Thus, long-term cellular stimu- P450c11b (Cao et al., 2000). In such fusions, the ferredoxin moiety lation over the course of days will increase the content of P450scc, is at the C-terminus, tethered by a hydrophilic linker that permits and the level of basal steroid produced, as well as the capacity of rotational freedom, so that the same surface of the ferredoxin can the cell to mount a steroidogenic response. access both the P450 and the ferredoxin reductase. The requirement for ferredoxin reductase and ferredoxin is not absolute, at least in vitro. When P450scc is fused to an alternative electron do- 7. Electron transfer to P450scc: ferredoxin reductase and nor, microsomal P450 oxidoreductase and targeted of to the mito- ferredoxin chondria, it remains active; by contrast when P450scc, its fusion protein, or other P450scc constructs are targeted to the ER, they 7.1. Ferredoxin reductase are inactive even when supplied with the 22(R)-hydroxycholesterol substrate that bypasses the StAR system (Black et al., 1994). Catalysis by P450scc and other mitochondrial P450 enzymes re- Thus the mitochondrial localiztion is essential for the enzymatic quires two electron-transfer intermediates, ferredoxin reductase activity of P450scc. and ferredoxin (Miller, 2005). Ferredoxin reductase receives electrons from NADPH then forms a 1:1 complex with ferredoxin, which then dissociates and forms an analogous 1:1 complex with 8. P450scc deficiency syndromes a mitochondrial P450 such as P450scc, thus functioning as an indiscriminate, diffusible electron shuttle for all mitochondrial Three models of defective P450scc function, a spontaneously forms of P450 (Fig. 3). The relative abundances of ferredoxin reduc- occurring CYP11A1 deletion in the rabbit (Yang et al., 1993), knock- tase and ferredoxin, as well as the inherent properties of the mito- out of the gene in the mouse (Hu et al., 2002), and rare patients chondrial P450, determine catalytic activity (Harikrishna et al., with P450scc mutations confirm that P450scc is the only enzyme W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 69 that converts cholesterol to pregnenolone. Because progesterone is are 93.5% identical with nearly indistinguishable enzymology, but needed to suppress uterine contractility and thus prevent sponta- with distinct distributions of expression. The type 1 enzyme cata- neous abortion, it would appear that P450scc mutations would be lyzes 3bHSD activity in placenta, breast, liver, brain and some other incompatible with term gestation; the mouse and rabbit models tissues, whereas the type 2 enzyme is expressd in the adrenals and are explained by the persistence of the maternal corpus luteum gonads. Deficiency of 3bHSD2 causes a rare form of congenital in these species, providing an alternative source of progesterone. adrenal hyperplasia (3bHSD deficiency); mutations have not been Nevertheless, beginning in 2001 (Tajima et al., 2001) 19 patients found in 3bHSD1, possibly reflecting the impact of such a mutation have been described with mutations in CYP11A1 that affect on plcental progesterone synthesis. P450scc activity (Tee et al., 2013). Most of these patients have mutations that ablate all P450scc activity; they probably reached 10. Other steroidogenic mitochondrial P450 enzymes term gestation because of the maternal corpus luteum remained functional beyond the second trimester, when it normally invo- Adrenocortical mitochondria contain two additional P450 en- lutes. These patients may be clinically indistinguishable from those zymes: P450c11b (11b-hydroxylase) is found in zona fasciculata with lipoid CAH. The 46,XY genetic males fail to produce testoster- cells where it catalyzes the conversion of 11-deoxycortisol to cor- one during fetal life, and are born with female external genitalia, tisol; P450c11AS (aldosterone synthase) is found in zona glomerul- although their internal reproductive structures are male, as their osa cells where it catalyzes the three distinct reactions needed to testes produced anti-Müllerian hormone. Following birth, these convert deoxycorticosterone to aldosterone (White et al., 1994; patients require steroid hormone replacement therapy and may Fardella and Miller, 1996; Miller and Auchus, 2011). These two have long-term survival. As with non-classical lipoid CAH, a milder proteins share 93% amino acid sequence identity and are encoded ‘‘non-classical’’ form of P450scc deficiency has been described by duplicated genes termed CYP11B1 (producing P450c11b) and caused by missense mutations that retain 10–20% of normal activ- CYP11B2 (producing P450c11AS). Like P450scc, both proteins have ity (Rubtsov et al., 2009; Sahakitrungruang et al., 2011). No hor- typical mitochondrial targeting sequence and are associated with monal test distinguishes lipoid CAH from P450scc deficiency, but the IMM. These enzymes must compete with P450scc for reducing the adrenals are typically grossly enlarged in lipoid CAH but nor- equivalents provided via ferredoxin reductase and ferredoxin. mal-sized in P450scc deficiency, sometimes permitting radiologic Mutations in CYP11B1 cause 11b-hydroxylase deficiency, a rare distinction, but the only definitive test to distinguish these disor- form of virilizing congenital adrenal hyperplasia in which the over- ders is DNA sequencing (Gucev et al., 2013). production of deoxycorticosterone may lead to mineralocorticoid hypertension. Mutations in CYP11B2 cause aldosterone synthase deficiency. An unusual recombination between the CYP11B1 and 9. 3b-hydroxysteroid dehydrogenase CYP11B2 genes can place the CYP11B1 promoter upstream from the CYP11B2 gene, thus producing aldosterone synthase in re- The 42 kDa 3b-hydroxysteroid dehydrogenase (3bHSD) is a sponse to ACTH, causing glucocorticoid suppressible member of the short-chain dehydrogenase/reductase (SDR) family hyperaldosteronism. of enzymes, which are are b-a-b proteins having up to seven parallel b-strands that fan across the center of the molecule, forming the so-called ‘‘Rossman fold’’, which is characteristic of oxida- 11. Mitochondrial P450 enzymes in vitamin D synthesis tion/reduction enzymes that use nicotinamide cofactors (Agarwal and Auchus, 2005; Penning, 1997). 3bHSD converts D5 steroids Vitamin D and its metabolites are not steroids in the strict (pregnenolone, 17OH-pregneneolone, DHEA), having a double chemical sense, as the B ring of cholesterol is opened (Fig. 4). Nev- bond in the B ring to D4 steroids, having a double bond in the A ertheless, these sterols are derived from cholesterol, assume ring (Miller and Auchus, 2011). Members of this family of enzymes shapes that are very similar to steroids, and bind to a similar, lack mitochondrial leader peptides and are generally found in the zinc-finger receptor that regulates gene transcription (Feldman cytosol. However, 3bHSD was first isolated from mitochondria et al., 2013). The final step in the biosynthesis of cholesterol is con- (Thomas et al., 1989). This unexpected cellular localization was version of 7-dehydrocholesterol to cholesterol. In human skin, confirmed by immunogold electron microscopy showing that ultraviolet radiation at 270–290 nm directly cleaves the 9–10 car- 3bHSD immunoreactivity is found in mitochondria and endoplas- bon–carbon bond of the cholesterol B ring, converting 7-dehydro- mic reticulum as well as in the cytoplasm of bovine adrenal zona cholesterol to cholecalciferol (vitamin D3)(Norman, 1998). Plants glomerulosa cells (Cherradi et al., 1997; Pelletier et al., 2001). It produce ergocalciferol (vitamin D2), which has essentially the same is not clear if this is also true for human 3bHSD, or if this subcellu- properties as cholecalciferol. Both calciferols are biologically inac- lar distribution differs in various types of steroidogenic cells, but tive pro-hormones that are then activated, and subsequently inac- this property could be a novel mechanism for regulating the direc- tivated, by mitochondrial P450 enzymes. tion of steroidogenesis. 3bHSD appears to be associated with The initial step in the activation of vitamin D is its hepatic 25- P450scc on the IMM, and possibly at OMM-IMM contact sites hydroxylation to 25(OH)D, which may be catalyzed by several en- (Cherradi et al., 1995). 3bHSD and 17a-hydroxylase (P450c17) zymes. The principal 25-hydroxylase is CYP2R1 (Cheng et al., compete for the pregnenolone produced by P450scc. The Michaelis 2003), and deficiency of this enzyme causes the very rare 25- constant (Km) for P450c17 in the ER is about 0.8 lM(Auchus et al., hydroxylase deficiency syndrome (Cheng et al., 2004; Dong and 1998), whereas the Km of 3bHSD is about 5.2–5.5 lM(Lee et al., Miller, 2004). A hepatic mitochondrial P450 (variously termed 1999), so that an intramitochondrial location of 3bHSD will facili- P450c25 and P450c27) encoded by the CYP27A1 gene also has vita- tate the formation of progesterone rather than 17OH-pregneno- min D 25-hydroxylase activity, but its mutation causes cerebroten- lone. During its mitochondrial entry, 3bHSD associates with dinous xanthomatosis without a disorder in calcium metabolism several mitochondrial translocase proteins to reach the IMM (Paw- (Cali et al., 1991; Leitersdorf et al., 1993), hence this mitochondrial lak et al., 2011) and undergoes a pH-dependent conformational enzyme is of marginal importance to vitamin D metabolism. change in the intramembranous space that facilitates its two enzy- The active, hormonal form of vitamin D, 1,25(OH)2D, is pro- matic activities (Prasad et al., 2012). duced by the 1a-hydroxylation of 25(OH)D by the mitochondrial There are two 3bHSD genes and several pseudogenes in a gene 1a-hydroxylase, P450c1a, encoded by the CYP27B1 gene (Fu cluster on chromosome 1p13.1. The two encoded 3bHSD enzymes et al., 1997a,b). 1,25(OH)2D in the circulation derives primarily 70 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73

Fig. 4. Biosynthesis of vitamin D3. Ultraviolet light at 290–320 nm acts on the skin to cleave the B ring of 7-dehydrocholesterol to yield cholecalciferol (vitamin D3). Vitamin D, is converted to 25OHD by several enzymes in the liver, principally CYP2R1. 25OHD is activated by 1a-hydroxylation in the kidney, yielding the active hormone 1,25(OH)2D, which acts by binding to the vitamin D receptor (VDR). Both 25OHD and 1,25(OH)2D may be inactivated by P450c24 to yield 24,25(OH)2D or 1,24,25(OH)3D, respectively. from the kidney, but 1a-hydroxylase activity is also found in kerat- implications for the mechanism of StAR action. Proc. Natl. Acad. Sci. USA 93, inocytes, macrophages, osteoblasts and placenta. 1 -hydroxylation 13731–13736. a Arakane, F., King, S.R., Du, Y., Kallen, C.B., Walsh, L.P., Watari, H., Stocco, D.M., is the rate-limiting step in the activation of vitamin D, and renal Strauss 3rd, J.F., 1997. Phosphorylation of steroidogenic acute regulatory enzyme activity is tightly regulated by parathyroid hormone protein (StAR) modulates its steroidogenic activity. J. Biol. Chem. 272, 32656– 32662. (PTH), calcium, phosphorus, and 1,25(OH)2D itself. The first human Artemenko, I.P., Zhao, D., Hales, D.B., Hales, K.H., Jefcoate, C.R., 2001. Mitochondrial clone was obtained from human keratinocytes, but multiple stud- processing of newly synthesized steroidogenic acute regulatory protein ies, including finding mutations in a patient affected with renal 1a- (StAR), but not total StAR, mediates cholesterol transfer to cytochrome hydroxylase deficiency, proved that the same gene was expressed P450 side chain cleavage enzyme in adrenal cells. J. Biol. Chem. 276, 46583– 46596. in kidney (Fu et al., 1997a). Mutations in this gene cause the disor- Auchus, R.J., Lee, T.C., Miller, W.L., 1998. Cytochrome b5 augments the 17,20 lyase der variously termed ‘vitamin D-dependent rickets, Type 1’, ‘pseu- activity of human P450c17 without direct electron transfer. J. Biol. Chem. 273, do vitamin D-deficient rickets’ and ‘vitamin D 1a-hydroxylase 3158–3165. Baker, B.Y., Yaworsky, D.C., Miller, W.L., 2005. A pH-dependent molten globule deficiency’ (Wang et al., 1998; Kim et al., 2007). The single CYP27B1 transition is required for activity of the steroidogenic acute regulatory protein, gene for 1a-hydroxylase is only 5 kb long and has an intron/exon StAR. J. Biol. Chem. 280, 41753–41760. organization that is very similar to that of other mitochondrial Baker, B.Y., Lin, L., Kim, C.J., Raza, J., Smith, C.P., Miller, W.L., Achermann, J.C., 2006. P450 enzymes, especially the mitochondrial cholesterol side-chain Nonclassic congenital lipoid adrenal hyperplasia: a new disorder of the steroidogenic acute regulatory protein with very late presentation and cleavage enzyme, P450scc (Fu et al., 1997b). Thus, even though the normal male genitalia. J. Clin. Endocrinol. Metab. 91, 4781–4785. mitochondrial P450 enzymes retain only 30–40% amino acid se- Baker, B.Y., Epand, R.F., Epand, R.M., Miller, W.L., 2007. Cholesterol binding does not quence identity with each other, they all belong to a single evolu- predict activity of the steroidogenic acute regulatory protein, StAR. J. Biol. Chem. 282, 10223–10232. tionary lineage. More than 100 patients with CYP27B1 mutations Bayrhuber, M., Meins, T., Habeck, M., Becker, S., Giller, K., Villinger, S., Vonrhein, C., have been described (Edouard et al 2011). Griesinger, C., Zweckstetter, M., Zeth, K., 2008. Structure of the human voltage- dependent anion channel. Proc. Natl. Acad. Sci. USA 105, 15370–15375. 1,25(OH)2D may be inactivated by the principal hepatic drug- Black, S.M., Harikrishna, J.A., Szklarz, G.D., Miller, W.L., 1994. The mitochondrial metabolizing enzyme, microsomal CYP3A4, or by its 24-hydroxyl- environment is required for activity of the cholesterol side-chain cleavage ation by vitamin D 24-hydroxylase (P450c24), encoded by the enzyme, cytochrome P450scc. Proc. Natl. Acad. Sci. USA 91, 7247–7251. CYP24A1 gene (Feldman et al., 2013). This mitochondrial enzyme Bose, H.S., Sugawara, T., Strauss 3rd, J.F., Miller, W.L., 1996. The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. N. Engl. J. Med. 335, can catalyze the 24-hydroxylation of 25(OH)D to 24,25(OH)2D 1870–1878. and of 1,25(OH)2D to 1,24,25(OH)3D, primarily in the kidney and Bose, H.S., Pescovitz, O.H., Miller, W.L., 1997. Spontaneous feminization in a 46XX intestine, thus inactivating vitamin D (Ohyama et al., 1991; Chen female patient with congenital lipoid adrenal hyperplasia caused by a homozygous frame-shift mutation in the steroidogenic acute regulatory et al., 1993). The expression of P450c24 is induced by protein. J. Clin. Endocrinol. Metab. 82, 1511–1515. 1,25(OH)2D, providing a direct feedback mechanism to autoregu- Bose, H.S., Whittal, R.M., Baldwin, M.A., Miller, W.L., 1999. The active form of the steroidogenic acute regulatory protein, StAR, appears to be a molten globule. late circulating levels of 1,25(OH)2D(Xie et al., 2002). The crystal structure of rat P450c24 has been determined, showing a classic Proc. Natl. Acad. Sci. USA 96, 7250–7255. Bose, H.S., Whittal, R.M., Huang, M.C., Baldwin, M.A., Miller, W.L., 2000. N-218 cytochrome P450 fold with an open conformation which would MLN64, a protein with StAR-like steroidogenic activity, is folded and cleaved suggest that this enzyme might bind multiple different substrates, similarly to StAR. Biochemistry 39, 11722–11731. nevertheless, like all other mitochondrial P450’s, P450c24 exhibits Bose, H.S., Lingappa, V.R., Miller, W.L., 2002. Rapid regulation of steroidogenesis by mitochondrial protein import. Nature 417, 87–91. a narrow substrate specificity (Annalora et al., 2010). Rare muta- Bose, H.S., Whittal, R.M., Ran, Y., Bose, M., Baker, B.Y., Miller, W.L., 2008a. StAR-like tions in P450c24 result in elevated circulating concentrations of activity and molten globule behavior of StARD6, a male germ-line protein. Biochemistry 47, 2277–2288. 1,25(OH)2D, causing severe neonatal hypercalcemia (Schlingmann Bose, M., Whittal, R.M., Miller, W.L., Bose, H.S., 2008b. Steroidogenic activity of StAR et al., 2011; Dauber et al., 2012). requires contact with mitochondrial VDAC1 and phosphate carrier protein. J. Biol. Chem. 283, 8837–8845. Brandt, M.E., Vickery, L.E., 1992. Expression and characterization of human References mitochondrial ferredoxin reductase in Escherichia coli. Arch. Biochem. Biophys. 294, 735–740. Agarwal, A.K., Auchus, R.J., 2005. Cellular redox state regulates hydroxysteroid Brdiczka, D.G., Zorov, D.B., Sheu, S.S., 2006. Mitochondrial contact sites: their role in dehydrogenase activity and intracellular hormone potency. Endocrinology 146, energy metabolism and apoptosis. Biochim. Biophys. Acta 1762, 148–163. 2531–2538. Brentano, S.T., Black, S.M., Lin, D., Miller, W.L., 1992. CAMP post-transcriptionally Alpy, F., Tomasetto, C., 2006. MLN64 and MENTHO, two mediators of endosomal diminishes the abundance of adrenodoxin reductase mRNA. Proc. Natl. Acad. cholesterol transport. Biochem. Soc. Trans. 34, 343–345. Sci. USA 89, 4099–4103. Alpy, F., Stoeckel, M.E., Dierich, A., Escola, J.M., Wendling, C., Chenard, M.P., Vanier, Cali, J.J., Hsieh, C.L., Francke, U., Russell, D.W., 1991. Mutations in the bile acid M.T., Gruenberg, J., Tomasetto, C., Rio, M.C., 2001. The steroidogenic acute biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous regulatory protein homolog MLN64, a late endosomal cholesterol-binding xanthomatosis. J. Biol. Chem. 266, 7779–7783. protein. J. Biol. Chem. 276, 4261–4269. Cao, P., Bulow, H., Dumas, B., Bernhardt, R., 2000. Construction and characterization Alpy, F., Wendling, C., Rio, M.C., Tomasetto, C., 2002. MENTHO, a MLN64 homologue of a catalytic fusion protein system: P-45011-adrenodoxin reductase- devoid of the START domain. J. Biol. Chem. 277, 50780–50787. adrenodoxin. Biochim. Biophys. Acta 1476, 253–264. Annalora, A.J., Goodin, D.B., Hong, W.X., Zhang, Q., Johnson, E.F., Stout, C.D., 2010. Charman, M., Kennedy, B.E., Osborne, N., Karten, B., 2010. MLN64 mediates egress of Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in cholesterol from endosomes to mitochondria in the absence of functional vitamin D metabolism. J. Mol. Biol. 396, 441–451. Niemann-Pick Type C1 protein. J. Lipid Res. 51, 1023–1034. Arakane, F., Sugawara, T., Nishino, H., Liu, Z., Holt, J.A., Pain, D., Stocco, D.M., Miller, Chen, K.S., Prahl, J.M., DeLuca, H.F., 1993. Isolation and expression of human 1,25- W.L., Strauss 3rd., J.F., 1996. Steroidogenic acute regulatory protein (StAR) dihydroxyvitamin D3 24-hydroxylase cDNA. Proc. Natl. Acad. Sci. USA 90, retains activity in the absence of its mitochondrial import sequence: 4543–4547. W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 71

Cheng, J.B., Motola, D.L., Mangelsdorf, D.J., Russell, D.W., 2003. De-orphanization of Henderson, Y.C., Frederick, M.J., Jayakumar, A., Choi, Y., Wang, M.T., Kang, Y., Evans, cytochrome P450 2R1: a microsomal vitamin D 25-hydroxilase. J. Biol. Chem. R., Spring, P.M., Uesugi, M., Clayman, G.L., 2007. Human LBP-32/MGR is a 278, 38084–38093. repressor of the P450scc in human choriocarcinoma cell line JEG-3. Placenta 28, Cheng, J.B., Levine, M.A., Bell, N.H., Mangelsdorf, D.J., Russell, D.W., 2004. Genetic 152–160. evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Hiller, S., Garces, R.G., Malia, T.J., Orekhov, V.Y., Colombini, M., Wagner, G., 2008. Proc. Natl. Acad. Sci. USA 101, 7711–7715. Solution structure of the integral human membrane protein VDAC-1 in Cherradi, N., Chambaz, E.M., Defaye, G., 1995. Organization of 3b-hydroxysteroid detergent micelles. Science 321, 1206–1210. dehydrogenase/isomerase and cytochrome P450scc into a catalytically active Hu, M.C., Hsu, N.C., El Hadj, N.B., Pai, C.I., Chu, H.P., Wang, C.K.L., Chung, B.C., 2002. molecular complex in bovine adrenocortical mitochondria. J. Steroid Biochem. Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1. Mol. Mol. Biol. 55, 507–514. Endocrinol. 16, 1943–1950. Cherradi, N., Rossier, M.F., Vallotton, M.B., Timberg, R., Friedberg, I., Orly, J., Wang, Huang, N., Miller, W.L., 2000. Cloning of factors related to HIV-inducible LBP X.J., Stocco, D.M., Capponi, A.M., 1997. Submitochondrial distribution of three proteins that regulate steroidogenic factor-1-independent human placental key steroidogenic proteins (steroidogenic acute regulatory protein and transcription of the cholesterol side-chain cleavage enzyme, P450scc. J. Biol. cytochrome P450scc and 3-hydroxysteroid dehydrogenase isomerase Chem. 275, 2852–2858. enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa Illingworth, D., Kenney, T.A., Orwoll, E.S., 1982. Adrenal function in heterozygous cells. J. Biol. Chem. 272, 7899–7907. and homozygous hypobetalipoproteinemia. J. Clin. Endocrinol. Metab. 54, 27– Clark, B.J., 2012. The mammalian START domain protein family in lipid transport in 33. health and disease. J. Endocrinol. 212, 257–275. Jamin, N., Neumann, J.M., Ostuni, M.A., Vu, T.K., Yao, Z.X., Murail, S., Robert, J.C., Clark, B.J., Wells, J., King, S.R., Stocco, D.M., 1994. The purification, cloning and Giatzakis, C., Papadopoulos, V., Lacapere, J.J., 2005. Characterization of the expression of a novel luteinizing hormone-induced mitochondrial protein in cholesterol recognition amino acid consensus sequence of the peripheral-type MA-10 cells mouse Leydig tumor cells. Characterization of the steroidogenic benzodiazepine receptor. Mol. Endocrinol. 19, 588–594. acute regulatory protein (StAR). J. Biol. Chem. 269, 28314–28322. Jo, Y., King, S.R., Khan, S.A., Stocco, D.M., 2005. Involvement of protein kinase C and Dauber, A., Nguyen, T.T., Sochett, E., Cole, D.E., Horst, R., Abrams, S.A., Carpenter, cyclic adenosine 30,50-monophosphate-dependent kinase in steroidogenic acute T.O., Hirschhorn, J.N., 2012. Genetic defect in CYP24A1, the vitamin D 24- regulatory protein expression and steroid biosynthesis in Leydig cells. Biol. hydroxylase gene, in a patient with severe infantile hypercalcemia. J. Clin. Reprod. 73, 244–255. Endocrinol. Metab. 97, E268–E274. Joseph-Liauzun, E., Delmas, P., Shire, D., Ferrara, P., 1998. Topological analysis of the Dilworth, F.J., Black, S.M., Guo, Y.D., Miller, W.L., Jones, G., 1996. Construction of a peripheral benzodiazepine receptor in yeast mitochondrial membranes P450c27 fusion enzyme – a useful tool for analysis of vitamin D3–25- supports a five-transmembrane structure. J. Biol. Chem. 273, 2146–2152. hydroxylase activity. Biochem. J. 320, 267–271. Kim, C.J., Kaplan, L.E., Perwad, F., Huang, N., Sharma, A., Choi, Y., Miller, W.L., Portale, Dobs, A.S., Schrott, H., Davidson, M.H., Bays, H., Stein, E.A., Kush, D., Wu, M., Mitchel, A.A., 2007. Mutations in the gene for 1-hydroxylase, CYP27B1, in patients with Y., Illingworth, R.D., 2000. Effects of high-dose simvastatin on adrenal and vitamin D 1-hydroxylase deficiency. J. Clin. Endocrinol. Metab. 92, 3177–3182. gonadal steroidogenesis in men with hypercholesterolemia. Metabolism 49, Kim, J.M., Choi, J.H., Lee, J.H., Kim, G.H., Lee, B.H., Kim, H.S., Shin, J.H., Shin, C.H., Kim, 1234–1238. C.J., Yu, J., Lee, D.Y., Cho, W.K., Suh, B.K., Lee, J.E., Chung, H.R., Yoo, H.W., 2011. Dong, Q., Miller, W.L., 2004. Vitamin D 25-hydroxylase deficiency. Mol. Genet. High allele frequency of the p.Q258X mutation and identification of a novel Metab. 83, 197–198. mis-splicing mutation in the STAR gene in Korean patients with congenital Dyson, M.T., Kowalewski, M.P., Manna, P.R., Stocco, D.M., 2009. The differential lipoid adrenal hyperplasia. Eur. J. Endocrinol. 165, 771–778. regulation of steroidogenic acute regulatory protein-mediated steroidogenesis Kishida, T., Kostetskii, I., Zhang, Z., Martinez, F., Liu, P., Walkley, S.U., Dwyer, N.K., by type I and type II PKA in MA-10 cells. Mol. Cell. Endocrinol. 300, 94–103. Blanchette-Mackie, E.J., Radice, G.L., Strauss 3rd., J.F., 2004. Targeted mutation Edouard, T., Alos, N., Chabot, G., Roughley, P., Glorieux, F.H., Rauch, F., 2011. Short- of the MLN64 START domain causes only modest alterations in cellular sterol and long-term outcome of patients with pseudo-vitamin D deficiency rickets metabolism. J. Biol. Chem. 279, 19276–19285. treated with calcitriol. J. Clin. Endocrinol. Metab. 96, 82–89. Korkhov, V.M., Sachse, C., Short, J.M., Tate, C.G., 2010. Three-dimensional structure Epstein, L.F., Orme-Johnson, N.R., 1991. Regulation of steroid hormone biosynthesis. of TspO by electron cryomicroscopy of helical crystals. Structure 18, 677–687. Identification of precursors of a phosphoprotein targeted to the mitochondrion Kwon, H.J., Abi-Mosleh, L., Wang, M.L., Deisenhofer, J., Goldstein, J.L., Brown, M.S., in stimulated rat adrenal cortex cells. J. Biol. Chem. 266, 19739–19745. Infante, R.E., 2009. Structure of N-terminal domain of NPC1 reveals distinct Fan, J., Liu, J., Culty, M., Papadopoulos, V., 2010. Acyl-coenzyme A binding domain subdomains for binding and transfer of cholesterol. Cell 137, 1213–1224. containing 3 (ACBD3; PAP7; GCP60): an emerging signaling molecule. Prog. Lacapere, J.J., Papadopoulos, V., 2003. Peripheral-type benzodiazepine receptor: Lipid Res. 49, 218–234. structure and function of a cholesterol-binding protein in steroid and bile acid Fardella, C.E., Miller, W.L., 1996. Molecular biology of mineralocorticoid biosynthesis. Steroids 68, 569–585. metabolism. Annu. Rev. Nutr. 16, 443–470. Lee, T.C., Miller, W.L., Auchus, R.J., 1999. Medroxyprogesterone acetate and Feldman, D., Malloy, P.J., Miller, W.L., 2013. Genetic disorders of vitamin D synthesis dexamethasone are competitive inhibitors of different human steroidogenic and action. In: Thakker, R.V., Whyte, M.P., Eisman, J.A., Igarashi, T. (Eds.), enzymes. J. Clin. Endocrinol. Metab. 84, 2104–2110. Genetics of Bone Biology and Skeletal. Academic Press (Elsevier), London, UK, Leitersdorf, E., Reshef, A., Meiner, V., Levitzki, R., Schwartz, S.P., Dann, E.J., Berkman, pp. 537–552. N., Cali, J.J., Klapholz, L., Berginer, V.M., 1993. Frameshift and splice-junction Freeman, M.R., Dobritsa, A., Gaines, P., Segraves, W.A., Carlson, J.R., 1999. The dare mutations in the sterol 27-hydroxylase gene cause cerebrotendinous gene: steroid hormone production, olfactory behavior, and neural degeneration xanthomatosis in Jews or Moroccan origin. J. Clin. Invest. 91, 2488–2496. in Drosophila. Development 126, 4591–4602. Li, H., Yao, Z., Degenhardt, B., Teper, G., Papadopoulos, V., 2001. Cholesterol binding Fu, G.K., Lin, D., Zhang, M.Y.H., Bikle, D.D., Shackleton, C.H.L., Miller, W.L., Portale, at the cholesterol recognition/interaction amino acid consensus (CRAC) of the A.A., 1997a. Cloning of human 25-hydroxyvitamin D 1-hydroxylase and peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by mutations causing vitamin D-dependent rickets type I. Mol. Endocrinol. 11, an HIV TAT-CRAC peptide. Proc. Natl. Acad. Sci. USA 98, 1267–1272. 1961–1970. Lin, D., Gitelman, S.E., Saenger, P., Miller, W.L., 1991. Normal genes for the Fu, G.K., Portale, A.A., Miller, W.L., 1997b. Complete structure of the human gene for cholesterol side chain cleavage enzyme, P450scc, in congenital lipoid adrenal the vitamin D 1hydroxylase, P450clDNA. Cell Biol. 16, 1499–1507. hyperplasia. J. Clin. Invest. 88, 1955–1962. Fujieda, K., Tajima, T., Nakae, J., Sageshima, S., Tachibana, K., Suwa, S., Sugawara, T., Lin, D., Chang, Y.J., Strauss III, J.F., Miller, W.L., 1993. The human peripheral Strauss, J.F.I.I.I., 1997. Spontaneous puberty in 46, XX subjects with congenital benzodiazepine receptor gene: cloning and characterization of alternative lipoid adrenal hyperplasia. Ovarian steroidogenesis is spared to some extent splicing in normal tissues and in a patient with congenital lipoid adrenal despite inactivating mutations in the steroidogenic acute regulatory protein hyperplasia. Genomics 18, 643–650. (StAR) gene. J. Clin. Invest. 99, 1265–1271. Lin, D., Sugawara, T., Strauss 3rd, J.F., Clark, B.J., Stocco, D.M., Saenger, P., Rogol, A., Gizard, F., Lavallee, B., DeWitte, F., Teissier, E., Staels, B., Hum, D.W., 2002. The Miller, W.L., 1995. Role of steroidogenic acute regulatory protein in adrenal and transcriptional regulating protein of 132 kDa (TReP-132) enhances P450scc gonadal steroidogenesis. Science 267, 1828–1831. gene transcription through interaction with steroidogenic factor-1 in human Liu, J., Rone, M.B., Papadopoulos, V., 2006. Protein-protein interactions mediate adrenal cells. J. Biol. Chem. 277, 39144–39155. mitochondrial cholesterol transport and steroid biosynthesis. J. Biol. Chem. 281, Gucev, Z., Tee, M.K., Chitayat, D., Wherrett, D., Miller, W.L., 2013. Distinguishing 38879–38893. deficiencies of StAR and P450scc causing neonatal adrenal failure. J. Pediatr. Lohse, P., Maas, S., Sewell, A.C., van Diggelen, O.P., Seidel, D., 1999. Molecular defects 162, 819–822. underlying Wolman disease appear to be more heterogeneous than those Harikrishna, J.A., Black, S.M., Szklarz, G.D., Miller, W.L., 1993. Construction and resulting in cholesteryl ester storage disease. J. Lipid Res. 40, 221–228. function of fusion enzymes of the human cytochrome P450scc system. DNA Cell Manna, P.R., Dyson, M.T., Stocco, D.M., 2009. Regulation of the steroidogenic acute Biol. 12, 371–379. regulatory protein gene expression: present and future perspectives. Mol. Hum. Hasegawa, T., Zhao, L.P., Caron, K.M., Majdic, G., Suzuki, T., Shizawa, S., Sasano, H., Reprod. 15, 321–333. Parker, K.L., 2000. Developmental roles of the steroidogenic acute regulatory Mannella, C.A., Forte, M., Colombini, M., 1992. Toward the molecular structure of protein (StAR) as revealed by StAR knockout mice. Mol. Endocrinol. 14, 1462– the mitochondrial channel, VDAC. J. Bioenergy Biomembr. 24, 7–19. 1471. Mast, N., Annalora, A.J., Lodowski, D.T., Palczewski, K., Stout, C.D., Pikuleva, I.A., Hauet, T., Yao, Z.X., Bose, H.S., Wall, C.T., Han, Z., Li, W., Hales, D.B., Miller, W.L., 2011. Structural basis for three-step sequential catalysis by the cholesterol side Culty, M., Papadopoulos, V., 2005. Peripheral-type benzodiazepine receptor- chain cleavage enzyme CYP11A1. J. Biol. Chem. 286, 5607–5613. mediated action of steroidogenic acute regulatory protein on cholesterol entry Mathieu, A.P., Fleury, A., Ducharme, L., Lavigne, P., LeHoux, J.G., 2002. Insights into into Leydig cell mitochondria. Mol. Endocrinol. 19, 540–554. steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in 72 W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73

mitochondria: evidence from molecular modeling and structure-based Rone, M.B., Midzak, A.S., Issop, L., Rammouz, G., Jagannathan, S., Fan, J., Ye, X., thermodynamics supporting the existence of partially unfolded states of StAR. Blonder, J., Veenstra, T., Papadopoulos, V., 2012. Identification of a dynamic J. Mol. Endocrinol. 29, 327–345. mitochondrial protein complex driving cholesterol import, trafficking, and McEnery, M.W., Snowman, A.M., Trifiletti, R.R., Snyder, S.H., 1992. Isolation of the metabolism to steroid hormones. Mol. Endocrinol. 26, 1868–1882. mitochondrial benzodiazepine receptor: association with the voltagedependent Rubtsov, P., Karmanov, M., Sverdlova, P., Spirin, P., Tiulpakov, A., 2009. A novel anion channel and the adenine nucleotide carrier. Proc. Natl. Acad. Sci. USA 89, homozygous mutation in CYP11A1 gene is associated with late onset adrenal 3170–3174. insufficiency and hypospadias in a 46 XY patient. J. Clin. Endocrinol. Metab. 94, Midzak, A., Akula, N., Lecanu, L., Papadopoulos, V., 2011. Novel androstenetriol 936–939. interacts with the mitochondrial translocator protein and controls Saenger, P., Klonari, Z., Black, S.M., Compagnone, N., Mellon, S.H., Fleischer, A., steroidogenesis. J. Biol. Chem. 286, 9875–9887. Abrams, C.A.L., Shackleton, C.H.L., Miller, W.L., 1995. Prenatal diagnosis of Miller, W.L., 1997. Congenital lipoid adrenal hyperplasia: the human gene knockout congenital lipoid adrenal hyperplasia. J. Clin. Endocrinol. Metab. 80, 200–205. of the steroidogenic acute regulatory protein. J. Mol. Endocrinol. 19, 227–240. Sahakitrungruang, T., Tee, M.K., Blackett, P.R., Miller, W.L., 2011. Partial defect in the Miller, W.L., 2005. Minireview: regulation of steroidogenesis by electron transfer. cholesterol side-chain cleavage enzyme P450scc (CYP11A1) resembling Endocrinology 146, 2544–2550. nonclassic congenital lipoid adrenal hyperplasia. J. Clin. Endocrinol. Metab. Miller, W.L., 2007. StAR search–what we know about how the steroidogenic acute 96, 792–798. regulatory protein mediates mitochondrial cholesterol import. Mol. Endocrinol. Schimmer, B.P., White, P.C., 2010. Steroidogenic factor 1: its roles in differentiation, 21, 589–601. development and disease. Mol. Endocrinol. 24, 1322–1337. Miller, W.L., Auchus, R.J., 2011. The molecular biology, biochemistry, and physiology Schlingmann, K.P., Kaufmann, M., Weber, S., Irwin, A., Goos, C., John, U., Misselwitz, of human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151. J., Klaus, G., Kuwertz-Broking, E., Fehrenbach, H., Wingen, A.M., Guran, T., Miller, W.L., Bose, H.S., 2011. Early steps in steroidogenesis: intracellular cholesterol Hoenderop, J.G., Bindels, R.J., Prosser, D.E., Jones, G., Konrad, M., 2011. Mutations trafficking. J. Lipid Res. 52, 2111–2135. in CYP24A1 and idiopathic infantile hypercalcemia. N. Engl. J. Med. 365, 410– Muller, A., Muller, J.J., Muller, Y.A., Uhlmann, H., Bernhardt, R., Heinemann, U., 1998. 421. New aspects of electron transfer revealed by the crystal structure of a truncated Sheftel, A.D., Stehling, O., Pierik, A.J., Elsasser, H.P., Muhlenhoff, U., Webert, H., bovine adrenodoxin, Adx(4-108). Structure 6, 269–280. Hobler, A., Hannemann, F., Bernhardt, R., Lill, R., 2010. Humans possess two Nakae, J., Tajima, T., Sugawara, T., Arakane, F., Hanaki, K., Hotsubo, T., Igarashi, N., mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in Igarashi, Y., Ishii, T., Koda, N., Kondo, T., Kohno, H., Nakagawa, Y., Tachibana, K., steroidogenesis, heme, and Fe/S cluster biosynthesis. Proc. Natl. Acad. Sci. Takeshima, Y., Tsubouchi, K., Strauss III, J.F., Fujieda, K., 1997. Analysis of the USA 107, 11775–11780. steroidogenic acute regulatory protein (StAR) gene in Japanese patients with Shen, W.J., Patel, S., Natu, V., Hong, R., Wang, J., Azhar, S., Kraemer, F.B., 2003. congential lipoid adrenal hyperplasia. Hum. Mol. Genet. 6, 571–576. Interaction of hormone-sensitive lipase with steroidogenic acute regulatory Nebert, D.W., Wikvall, K., Miller, W.L., 2013. Human cytochromes P450 in health protein. J. Biol. Chem. 278, 43870–43876. and disease. Phil. Trans. R. Soc. B 368, 20120431, . Miller, W.L., 1988. Human adrenodoxin reductase: two mRNAs encoded by a Norman, A.W., 1998. Sunlight, season, skin pigmentation, vitamin D, and 25- single gene of chromosome 17cen?25 are expressed in steroidogenic tissues. hydroxyvitamin D: integral components of the vitamin D endocrine system. Proc. Natl. Acad. Sci. USA 85, 7104–7108. Am. J. Clin. Nutr. 67, 1108–1110. Stevens, V.L., Xu, T., Lambeth, J.D., 1992. Cholesterol pools in rat adrenal Ohyama, Y., Noshiro, M., Okuda, K., 1991. Cloning and expression of cDNA encoding mitochondria: use of cholesterol oxidase to infer a complex pool structure. 25-hydroxyvitamin D3 24-hydroxylase. FEBS Lett. 278, 195–198. Endocrinology 130, 1557–1563. Olvera-Sanchez, S., Espinosa-Garcia, M.T., Monreal, J., Flores-Herrera, O., Martinez, Stocco, D.M., Clark, B.J., 1996. Regulation of the acute production of steroids in F., 2011. Mitochondrial heat shock protein participates in placental steroidogenic cells. Endocr. Rev. 17, 221–244. steroidogenesis. Placenta 32, 222–229. Stocco, D.M., Sodeman, T.C., 1991. The 30 kDa mitochondrial proteins induced by Papadopoulos, V., Miller, W.L., 2012. Role of mitochondria in steroidogenesis. Best hormone stimulation in MA-10 mouse Leydig tumor cells are processed from practice & research. Clin. Endocrinol. Metab. 26, 771–790. larger precursors. J. Biol. Chem. 266, 19731–19738. Papadopoulos, V., Amri, H., Li, H., Boujrad, N., Vidic, B., Garnier, M., 1997. Targeted Strushkevich, N., MacKenzie, F., Cherkesova, T., Grabovec, I., Usanov, S., Park, H.W., disruption of the peripheral-type benzodiazepine receptor gene inhibits 2011. Structural basis for pregnenolone biosynthesis by the mitochondrial steroidogenesis in the R2C Leydig tumor cell line. J. Biol. Chem. 272, 32129– monooxygenase system. Proc. Natl. Acad. Sci. USA 108, 10139–10143. 32135. Sugawara, T., Holt, J.A., Driscoll, D., Strauss III, J.F., Lin, D., Miller, W.L., Patterson, D., Papadopoulos, V., Baraldi, M., Guilarte, T.R., Knudsen, T.B., Lacapere, J.J., Lindemann, Clancy, K.P., Hart, I.M., Clark, B.J., Stocco, D.M., 1995. Human steroidogenic acute P., Norenberg, M.D., Nutt, D., Weizman, A., Zhang, M.R., Gavish, M., 2006. regulatory protein (StAR): functional activity in COS-1 cells, tissue-specific Receptor based on its structure and molecular function. Trends Pharmacol. Sci. expression, and mapping of the structural gene to 8p11.2 and an expressed 27, 402–409. pseudogene to chromosome 13. Proc. Natl. Acad. Sci. USA 92, 4778–4782. Pawlak, K.J., Prasad, M., Thomas, J.L., Whittal, R.M., Bose, H.S., 2011. Inner Tajima, T., Fujieda, K., Kouda, N., Nakae, J., Miller, W.L., 2001. Heterozygous mitochondrial translocase Tim50 interacts with 3b-hydroxysteroid mutation in the cholesterol side chain cleavage enzyme (P450scc) gene in a dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis. J. Biol. patient with 46, XY sex reversal and adrenal insufficiency. J. Clin. Endocrinol. Chem. 286, 39130–39140. Metab. 86, 3820–3825. Pelletier, G., Li, S., Luu-The, V., Tremblay, Y., Belanger, A., Labrie, F., 2001. Tee, M.K., Abramsohn, M., Loewenthal, N., Harris, M., Siwach, S., Kaplinsky, A., Immunoelectron microscopic localization of three key steroidogenic enzymes Markus, B., Birk, O., Sheffield, V.C., Pavari, R., Hershkovitz, E., Miller, W.L., 2013. (cytochrome P450(scc), 3b-hydroxysteroid dehydrogenase and cytochrome Varied clinical presentations with mutations in CYP11A1 encoding the P450(c17)) in rat adrenal cortex and gonads. J. Endocrinol. 171, 373–383. cholesterol side chain cleavage enzyme, P450scc. J. Clin. Endocrinol. Metab. Penning, T.M., 1997. Molecular endocrinology of hydroxysteroid dehydrogenases. 98, 713–720. Endocr. Rev. 18, 281–305. Thomas, J.L., Myers, R.P., Strickler, R.C., 1989. Human placental 3-hydroxy-5-ene- Pon, L.A., Orme-Johnson, N.R., 1986. Acute stimulation of steroidogenesis in steroid dehydrogenase and steroid 5 D 4-ene-isomerase: purification from corpus luteum and adrenal cortex by peptide hormones. J. Biol. Chem. 261, mitochondria and kinetic profiles, biophysical characterization of the purified 6594–6599. mitochondrial and microsomal enzymes. J. Steroid Biochem. 33, 209–217. Pon, L.A., Hartigan, J.A., Orme-Johnson, N.R., 1986. Acute ACTH regulation of adrenal Thorsell, A.G., Lee, W.H., Persson, C., Siponen, M.I., Nilsson, M., Busam, R.D., corticosteroid biosynthesis: rapid accumulation of a phosphoprotein. J. Biol. Kotenyova, T., Schuler, H., Lethio, L., 2011. Comparative structural analysis of Chem. 261, 13309–13316. lipid binding START domains. PLoS ONE 6, e19521–e19532. Porter, F.D., Herman, G.E., 2011. Malformation syndromes caused by disorders of Tsujishita, Y., Hurley, J.H., 2000. Structure and lipid transport mechanism of a StAR- cholesterol synthesis. J. Lipid Res. 52, 6–34. related domain. Nat. Struct. Biol. 7, 408–414. Prasad, M., Thomas, J.L., Whittal, R.M., Bose, H.S., 2012. Mitochondrial 3- Tuckey, R.C., Headlam, M.J., Bose, H.S., Miller, W.L., 2002. Transfer of cholesterol hydroxysteroid dehydrogenase enzyme activity requires reversible pH- between phospholipid vesicles mediated by the steroidogenic acute regulatory dependent conformational changes at the intermembrane space. J. Biol. Chem. protein (StAR). J. Biol. Chem. 277, 47123–47128. 287, 9534–9546. Vanier, M.T., Millat, G., 2003. Niemann-Pick disease type C. Clin. Genet. 64, 269– Riegelhaupt, J.J., Waase, M.P., Garbarino, J., Cruz, D.E., Breslow, J.L., 2010. Targeted 281. disruption of steroidogenic acute regulatory protein D4 leads to modest weight Wang, J.T., Lin, C.J., Burridge, S.M., Fu, G.K., Labuda, M., Portale, A.A., Miller, W.L., reduction and minor alterations in lipid metabolism. J. Lipid Res. 51, 1134– 1998. Genetics of vitamin D 1a-hydroxylase deficiency in 17 families. Am. J. 1143. Hum. Genet. 63, 1694–1702. Rodriguez-Agudo, D., Ren, S., Wong, E., Marques, D., Redford, K., Gil, G., Hylemon, White, P.C., Curnow, K.M., Pascoe, L., 1994. Disorders of steroid 11-hydroxylase P.B., Pandak, W.M., 2008. Intracellular cholesterol transporter StarD4 binds free isozymes. Endocr. Rev. 15, 421–438. cholesterol and increases cholesteryl ester formation. J. Lipid Res. 49, 1409– Xie, Z.J., Munson, S.J., Huang, N., Portale, A.A., Miller, W.L., Bikle, D.D., 2002. The 1419. mechanism of 1,25-dihydroxyvitamin D3 auto-regulation in keratinocytes. J. Romanowski, M.J., Soccio, R.E., Breslow, J.L., Burley, S.K., 2002. Crystal structure of Biol. Chem. 277, 36987–36990. the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a Xu, S., Benoff, B., Liou, H.L., Lobel, P., Stock, A.M., 2007. Structural basis of sterol StAR-related lipid transfer domain. Proc. Natl. Acad. Sci. USA 99, 6949–6954. binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 Rone, M.B., Liu, J., Blonder, J., Ye, X., Veenstra, T.D., Young, J.C., Papadopoulos, V., disease. J. Biol. Chem. 282, 23525–23531. 2009. Targeting and insertion of the cholesterol-binding translocator protein Yang, X., Iwamoto, K., Wang, M., Artwohl, J., Mason, J.I., Pang, S., 1993. Inherited into the outer mitochondrial membrane. Biochemistry 48, 6909–6920. congenital adrenal hyperplasia in the rabbit is caused by a deletion in the gene W.L. Miller / Molecular and Cellular Endocrinology 379 (2013) 62–73 73

encoding cytochrome P450 cholesterol side-chain cleavage enzyme. mediates mobilization of lysosomal cholesterol to steroidogenic Endocrinology 132, 1977–1982. mitochondria. J. Biol. Chem. 277, 33300–33310. Yaworsky, D.C., Baker, B.Y., Bose, H.S., Best, K.B., Jensen, L.B., Bell, J.D., Baldwin, M.A., Ziegler, G.A., Vonrhein, C., Hanukoglu, I., Schulz, G.E., 1999. The structure of Miller, W.L., 2005. PH-dependent interactions of the carboxyl-terminal helix of adrenodoxin reductase of mitochondrial P450 systems: electron transfer for steroidogenic acute regulatory protein with synthetic membranes. J. Biol. Chem. steroid biosynthesis. J. Mol. Biol. 289, 981–990. 280, 2045–2054. Zhang, M., Liu, P., Dwyer, N.K., Christenson, L.K., Fujimoto, T., Martinez, F., Comly, M., Hanover, J.A., Blanchette-Mackie, E.J., Strauss 3rd, J.F., 2002. MLN64 Molecular and Cellular Endocrinology 379 (2013) 74–84

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review Mitochondria and mammalian reproduction

a,b, a João Ramalho-Santos ⇑, Sandra Amaral a CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Portugal b Department of Life Sciences, University of Coimbra, Portugal article info abstract

Article history: Mitochondria are cellular organelles with crucial roles in ATP synthesis, metabolic integration, reactive Available online 13 June 2013 oxygen species (ROS) synthesis and management, the regulation of apoptosis (namely via the intrinsic pathway), among many others. Additionally, mitochondria in different organs or cell types may have dis- Keywords: tinct properties that can decisively influence functional analysis. In terms of the importance of mitochon- Reproduction dria in mammalian reproduction, and although there are species-specific differences, these aspects Mitochondria involve both energetic considerations for gametogenesis and fertilization, control of apoptosis to ensure Gametogenesis the proper production of viable gametes, and ROS signaling, as well as other emerging aspects. Crucially, Fertilization mitochondria are the starting point for steroid hormone biosynthesis, given that the conversion of Steroidogenesis cholesterol to pregnenolone (a common precursor for all steroid hormones) takes place via the activity of the cytochrome P450 side-chain cleavage enzyme (P450scc) on the inner mitochondrial membrane. Furthermore, mitochondrial activity in reproduction has to be considered in accordance with the very distinct strategies for gamete production in the male and female. These include distinct gonad morpho-physiologies, different types of steroids that are more prevalent (testosterone, estrogens, progesterone), and, importantly, the very particular timings of gametogenesis. While spermatogenesis is complete and continuous since puberty, producing a seemingly inexhaustible pool of gametes in a fixed environment; oogenesis involves the episodic production of very few gametes in an environment that changes cyclically. These aspects have always to be taken into account when considering the roles of any common element in mammalian reproduction. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 75 2. Mitochondria in gametogenesis and early embryo development ...... 76 2.1. Primordial germ cells and gonad specification ...... 76 2.2. Mitochondria in spermatogenesis ...... 76 2.3. Mitochondria in sperm ...... 79 2.4. Mitochondria in oogenesis ...... 79 2.5. Mitochondria in early embryo development ...... 79 3. The endocrine role of mitochondria in reproduction ...... 80 3.1. The mitochondrial step in steroid biosynthesis...... 80 3.2. Sex-specific steroidogenesis ...... 80 4. Conclusions and future perspectives...... 80 Acknowledgements ...... 81 References ...... 81

Corresponding author. Address: Department of Life Sciences, University of Coimbra, PO Box 3046, 3001-401 Coimbra, Portugal. Tel.: +351 (239) 855 760; fax: +351 (239) ⇑ 855 789. E-mail address: [email protected] (J. Ramalho-Santos).

0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.06.005 J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84 75

1. Introduction Rowland and Voeltz, 2012). Indeed, studies of mitochondrial (dys)function related to aging, degenerative and metabolic disor- Mitochondria are usually mentioned primarily in terms of cellu- ders or cancer encompass several of these aspects, from abnormal lar ATP production by oxidative phosphorylation (OXPHOS) via the OXPHOS activity and ROS production, to defective apoptosis and electron transport chain (ETC) located in the inner mitochondrial mitophagy/autophagy, to changes in mtDNA and mitochondrial membrane. ETC activity generates a transmembrane proton gradi- structure (Amaral et al., 2008b; Amaral and Ramalho-Santos, ent (Fig. 1), of which the mitochondrial membrane potential 2009; Cereghetti and Scorrano, 2011; Correia et al., 2012; Dorn (MMP) is the main component, driving the ATP synthase (Kakkar and Scorrano, 2010; Martinou and Youle, 2011; Nunnari and and Singh, 2007; Newmeyer and Ferguson-Miller, 2003; Scheffler, Suomalainen, 2012; Oettinghaus et al., 2012; Palmeira and Rama- 2001). A few components of this machinery are encoded by resident lho-Santos, 2011; Ramalho-Santos and Rodrigues, 2013; Ramalho- mitochondrial DNA (mtDNA) a prokaryotic-like genome that is Santos et al., 2009; St John et al., 2010). In short, mitochondria are inherited maternally (Jansen and de Boer, 1998; St John et al., 2010). involved in many other duties while (also) making ATP. However, recent mitochondrial research focuses on other top- In this review we will focus specifically on the role of mitochon- ics, such as the production of reactive oxygen species (ROS) by dria in gametogenesis, fertilization and early embryo development. the ETC and their role(s) in both physiological cell signaling and It should noted that mitochondrial function is most often studied pathological processes (related to oxidative stress); the regulation in terms of dysfunction induced by pathological conditions or toxic of the intrinsic apoptosis pathway and intracellular calcium levels; substances (pharmacological agents, environmental contaminants, the production of steroid hormones; quality control of cellular distinct pathologies, etc.), and how these dysfunctions may ulti- mitochondria via autophagy/mitophagy pathways, or the central mately affect the reproductive system (Aly and Khafagy, 2011; position of mitochondria in integrating several metabolic and sig- Amaral et al., 2008a, 2009; Banu et al., 2011; Miyamoto et al., naling pathways, epigenetics and the cell cycle (Folmes et al., 2010; Mota et al., 2011; Svechnikov et al., 2009; Wang et al., 2012; Kakkar and Singh, 2007; Nichols and Ferguson, 2002; Nun- 2009, 2010). Using different aspects of mitochondrial function as nari and Suomalainen, 2012). damage indicators in several disease models and, conversely, as Moreover, although previously mitochondria were thought to diagnostic tools in Assisted Reproductive Technologies (ART), has have a fixed and individual morphology, it is now known that increased in recent years, in terms of functional sperm analysis changes in shape (both in terms of cristae structure and matrix (Aitken et al., 2012; Dorn and Scorrano, 2010; Gallon et al., 2006; texture), size (regulated by the fission/fusion machinery) and Marchetti et al., 2002; Marchetti et al., 2012; Nakada et al., 2006; relationships with other cellular features (the cytoskeleton, Ruiz-Pesini et al., 1998; Sanchez-Partida et al., 2008; Sousa et al., the endoplasmic reticulum) can have important functional 2011), and oocyte quality assessment (Van Blerkom, 2011; Wang consequences (Bereiter-Hahn and Voth, 1994; Collins et al., 2002; and Sun, 2007).

Fig. 1. Possible roles of mitochondria in reproduction. Mitochondria are double membrane organelles with their own genome (mtDNA). Mitochondrial substrates derived from glycolysis, beta-oxidation of fatty acids and the Krebs cycle (Tricarboxylic acid cycle- TCA) provide energy for ATP production through oxidative phosphorylation (OXPHOS) by the activity of the electron transfer chain (ETC) on the inner mitochondrial membrane, composed of four inner membrane (IMM)-associated enzyme complexes (I–IV), plus cytochrome c (Cytc) and the mobile electron carrier ubiquinone (Q). This electron transfer generates a proton gradient across the inner membrane that drives ATP synthase (often known as complex V). However, at several sites of the electron transport chain (mainly complexes I and III) electrons can react with oxygen forming ROS. The energy dissipation mechanism promoted by UCPs (uncoupling proteins) can reduce ROS formation. Both beta-oxidation of fatty acids and amino acid catabolism provide TCA intermediates. The initial step of steroidogenesis also takes place in mitochondria. The ﬁrst step involves cholesterol (Chol) transport into the mitochondria facilitated by StAR protein via its interaction with Translocator protein (TSPO) and voltage dependent anion channel (VDAC) that constitute the transduceosome, located on the outer mitochondrial membrane (OMM). Once in the mitochondria, cholesterol will be converted to pregnenolone through the action of side chain cleavage cytochrome P450 (P450scc) that depends on the Adrenoxin reductase (AdxRed)–adrenoxin (Adx) system to receive electrons from NADPH. Pregnenolone then diffuses to the smooth endoplasmatic reticulum (SER) where it is further metabolized. See text for discussion. 76 J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84

2. Mitochondria in gametogenesis and early embryo controlled proton leak characteristics that distinguish them from development mitochondria from other organs, consuming less oxygen in order to generate approximately the same maximum electric potential 2.1. Primordial germ cells and gonad specification (Amaral et al., 2008a, 2009; Mota et al., 2009; Rodrigues et al., 2010). This suggests that, unlike what is usually the case, testicular Mammalian gametogenesis is commonly defined by important mitochondria should be considered as the primary mitochondrial sex-specific differences, although the starting point is identical. models to test the effect of distinct substances on male gametogen- Gonadal tissue derives from the mesoderm, into which primordial esis, and not be substituted by other in vitro models, such as com- germ cells (PGCs) migrate from outside the developing embryo and monly used liver mitochondria (Mota et al., 2011; Tavares et al., are subjected to distinct sex determination signals. PGCs divide 2009). several times, and establish functional relationships with somatic Although descriptive studies, or those that consider cells out- cells that will have supportive, protective, nutritional, and endocr- side of their biological context (isolated from tissue architecture, inal roles in gamete formation. In the testis these include Sertoli grown in nutrient-rich media, under normoxia), must be inter- and Leydig cells, while their homologous equivalents in the ovary preted with caution, it is well known that different testicular cells are granulosa and theca cells, respectively (Gilbert, 2010; Soder, have morphologically different mitochondria. These differences 2007). may be due to the mitochondrial fusion/fission machinery (Aihara While PGCs that colonize the fetal testes ultimately differenti- et al., 2009), and could have functional consequences, as is the case ate into spermatogonial stem cells, which remain mostly quies- in other systems (Bereiter-Hahn and Voth, 1994; Campello and cent, retinoic acid exposure causes ovarian oogonia to commit to Scorrano, 2010; Collins et al., 2002; De Martino et al., 1979; Hom meiosis in utero. Therefore, at puberty the testis still contains stem and Sheu, 2009; Mannella, 2006, 2008). In fact both somatic cells which can both self-renew and enter meiosis to form sperm, (Sertoli, Leydig) and germline (spermatogonia, spermatocytes, allowing for the continuous production of a large number of male spermatids, sperm) cells have distinct metabolic preferences and gametes. On the other hand, the ovary contains only a finite activities, which are translated into distinct mitochondrial contri- amount of committed primary oocytes, which will mature cycli- butions (Bajpai et al., 1998; De Martino et al., 1979; Grootegoed cally until the gamete pool is exhausted, ultimately resulting in et al., 1984; Meinhardt et al., 1999; Nakamura et al., 1984; menopause (Gassei and Schlatt, 2007; Pereda et al., 2006; Soder, Robinson and Fritz, 1981)(Table 1). Interestingly, putative 2007). This is one of the main reasons why more information is substrate availability does not fully explain the differences encoun- available on male gametogenesis. Anatomical differences are also tered in the testis, as spermatogonia on the basal membrane evident between gonads: the testis is comprised of an extensive remain mostly glycolytic although they are closer to blood vessels duct system formed by seminiferous tubules onto which millions (and therefore oxygen sources), while spermatocytes in the semi- of small mature sperm are constantly released (ultimately matur- niferous tubules seem to rely more on OXPHOS, despite being far- ing in the epidydimis); while the ovary consists of a series of follic- ther away from the oxygen supply. This seems to be a peculiarity ular structures embedded in the ovarian stroma, each containing spermatogonia share with other stem cells (Ramalho-Santos and one large female gamete which will be released upon ovulation, Rodrigues, 2013; Ramalho-Santos et al., 2009). with the oocyte and follicle developing in unison (Gilbert, 2010; The importance of ATP formed via OXPHOS for spermatogenesis Holstein et al., 2003; McLaughlin and McIver, 2009). In terms of is exemplified by the meiotic arrest found in mice that do not ex- mitochondrial characteristics and metabolic activity there are also press a testis-specific adenine nucleotide translocase (ANT4), several sex- and stage-specific differences (Table 1). essential for the translocation of ADP and ATP across the inner mitochondrial membrane (Brower et al., 2009). The regulation of apoptosis is also another aspect of mitochondrial function in the 2.2. Mitochondria in spermatogenesis testis, both to ensure a manageable number of germ cells that can be supported by existing Sertoli cells (Ramalho-Santos et al., Besides providing support and assisting in sperm formation and 2009), or as result of different environmental stimuli (Jia et al., transport Sertoli cells form the blood-testis barrier, creating a sep- 2010; Reyes et al., 2012; Shaha et al., 2010). The former aspect is arate and immuneprivileged site (Meinhardt and Hedger, 2011; highlighted by several experiments involving genetically modified Smith and Braun, 2012). Testosterone-secreting Leydig cells are mice that lack different components of the intrinsic apoptosis found in the intertubular tissue surrounding the capillaries and pathway. For example, the deletion of pro-apoptotic BCL-2 family have a prominent role in spermatogenesis maintenance, the differ- proteins BAX, BAK, as well as the simultaneous deletion of BIM entiation of male sexual organs and secondary sex characteristics and BIK (possibly due to redundant functions), results in an excess (Ge et al., 2008). Testis-specific morphogenetic events in early go- of germ cells, increased mutagenesis and testicular tumorigenesis nad differentiation suggest that male gonads have a higher energy (Coultas et al., 2005; Katz et al., 2012; Knudson et al., 1995; Russell requirement than ovaries, and that these distinct metabolic fea- et al., 2002; Xu et al., 2010), a process somewhat mirrored follow- tures, focused on mitochondrial activity, might even have a role ing overexpression of the pro-survival BCL-W in the testis (Yan in sex determination itself (Matoba et al., 2008; Mittwoch, 2004). et al., 2003). On the other hand deletion of this protein also results Spermatogenesis takes place in the seminiferous tubules and is in male infertility (Ross et al., 2001; Russell et al., 2001), and the a highly dynamic and metabolically active biological process dur- same is true for BCL-2 (Yamamoto et al., 2001), although in the for- ing which haploid spermatozoa are produced through a gradual mer this seems due to BAX-induced death of Sertoli cells, while in transformation of an interdependent population of germ cells. the latter germ cells were more affected. Mice devoid of apoptotic These cells sequentially migrate from the basal compartment to- protease-activating factor-1 (Apaf-1), which is usually activated by wards the luminal regions of the tubules, passing the blood-testis cytochrome c, are also infertile, in this case due to degenerated barrier (Holstein et al., 2003). The existence of numerous mito- spermatogonia leading to an almost absence of viable sperm in chondria in male germ cells (Meinhardt et al., 1999), as well as the seminiferous tubules (Honarpour et al., 2000). Additionally, the presence of several testis-specific mitochondrial protein iso- mice lacking the testis-specific form of cytochrome c have im- forms (Hess et al., 1993; Huttemann et al., 2003) highlights their paired sperm function (Narisawa et al., 2002). importance in testicular metabolism. As a whole testis mitochon- Pathophysiological processes such as mitochondrial ROS dria have been shown to possess specific bioenergetical and production may also have an (usually detrimental) effect on Table 1 Mitochondrial characteristics and energy metabolism throughout mammalian gametogenesis and early embryo development.

Cell type Mitochondria Mitochondria Energy source Metabolic particularities Morphology Cellular localization Male Spermatogonia Ovoid shaped, lamellar cristae, Scattered through the Glycolysis – Existence of the blood-testis barrier and Oxygen gradient in the seminiferous electron translucent matrix – cytoplasm tubules Orthodox

Primary spermatocyte Orthodox (Leptotene) ? Condensed Around the nucleus Glycolysis – Associations between germ cell mitochondrial morphology and metabolic (Pachytene, Diplotene) (round (Zygotene, early Pachytene). status have been suggested in which condensed mitochondria are more efﬁcient shaped, dense matrix, expansion of Small cytoplasmic clusters the intracristal spaces) with the nuage (intermitochondrial

cement; late Pachytene) 74–84 (2013) 379 Endocrinology Cellular and Molecular / Amaral S. Ramalho-Santos, J. OXPHOS – Germ cells have some pentose phosphate pathway activity, mainly spermatocytes Secondary spermatocyte Condensed No cluster arrangement OXPHOS – There are several testis-speciﬁc mitochondrial protein isoforms

Spermatid Condensed (early No cluster arrangement. OXPHOS Spermatid) ? intermediate (late Start to localize close to Spermatid) (elongated, crescent plasma membrane shaped cristae, matrix less condensed) In late spermatids localize close to the ﬂagellum. Sperm Intermediate Arranged in the midpiece Glycolysis OXPHOS b-oxidation Female/embryo Oogonia Spherical-ovoid shape. Tubulo- Typically clustered in close Glycolysis – Mitochondrial number increases throughout oocyte maturation vesicular cristae ? lamellar cristae association with the nuage (intermitochondrial cement) Pale matrix – Despite their primitive state, mitochondria are active in OXPHOS and are the primary source of ATP in the human oocyte and early embryo – The oocyte contains two populations of mitochondria; the more abundant mitochondria have low MMP and the smaller population is highly polarized. Mitochondrial MMP increases as the oocyte progress through meiotic maturation – Changes in mitochondrial distribution during oocyte growth may be a response to different energy demands. – Mammalian oocytes have limited ability in using glucose and therefore rely on cumulus cells. These cells convert glucose into readily utilizable substrates that enter the oocyte and are further metabolized via TCA followed by OXPHOS. The origin of these substrates may also be external (i.e. female reproductive tract) – While growing oocytes preferentially metabolize pyruvate over glucose, the somatic compartment of ovarian follicles is more gycolitic – The pentose phosphate pathway is important for oocyte development. -Triglycerides provide an additional rich energy supply for oocyte maturation through beta-oxidation – Mammalian oocytes may also utilize amino acids mainly via cumulus cells. Aminoacids serve as substrates for the synthesis of proteins, nucleotides, GSH, signaling molecules and provide substrates for the TCA cycle – Bioenergetic deﬁciencies have been associated with failure of oocyte maturation and fertilization and embryo demise during pre-implantation stages

(continued on next page) 77 78 Table 1 (continued)

Cell type Mitochondria Mitochondria Energy source Metabolic particularities Morphology Cellular localization 1ry Oocyte Spherical. Cristae with lamellar Random cytoplasmic (L) OXPHOS pattern (L,Z,P) ? arch like pattern or disposed parallel to the outer membrane (D) Z,P-high dense matrix perinuclear(Z) D-lighter matrix form a crescent shape mass near nucleus (D), in association with other organelles(balbiani’s vitelline body) Growing Oocyte Spherical, cristae pattern change and Dispersed in cytoplasm OXPHOS

increasingly dense matrix 74–84 (2013) 379 Endocrinology Cellular and Molecular / Amaral S. Ramalho-Santos, J. Beta- oxidation Preovulatory Oocyte Round with arched cristae, dense In the ooplasm. Form OXPHOS (mature) matrix voluminous aggregates with smooth endoplasmic reticulum tubules and vesicles.

Zygote Round or oval with few cristae Concentrated around OXPHOS Although early embryos have poorly differentiated mitochondria, they are active parallel to the outer mitochondrial pronuclei and the main source of ATP. A more complex form is gradually achieved, membrane. Some dumb-bell shaped. matching increasing development energetic requirements. Electrodense matrix. – A subset of high-polarized mitochondria is observed in zygotes and early embryos, and this population increases with cleavage state. – A transient increase in the ratio of high to low MMP was observed in 2-cell stage mouse embryos, synchronized with embryonic genome activation (maternal-embryonic transition) 2 cell Round shape with few small Uniformly dispersed in the OXPHOS – In human 8-cell embryos an increased ratio of mitochondria with high- to low- peripheral cristae. Dense matrix blastomeres with a MMP correlates with embryo fragmentation tendency towards perinuclear arrangement – It has been hypothesized that up regulation of beta-oxidation might result in increased availability of carbohydrates such as glucose for use in other pathways. This situation may also aid metabolic regulation and rapid cell proliferation via the Warburg effect 4 cell More elongated with numerous Dispersed in blastomeres OXPHOS transverse cristae. Lighter matrix

6-8 cell Most with elongated shape Associated with nuage Glycolysis (intermitochondrial cement) Blastocyst –Mitochondriainthetrophoblastaremorenumerousandhyperpolarized.

Trophoblast Orthodox-like OXPHOS Mitochondrial cristae transversely Glycolysis oriented. ICM Quiescent Matrix less dense

Information collected from the following sources: Amaral et al. (2013), Amaral et al. (2009), Bajpai et al. (1998), Bentov et al. (2011), Boussouar and Benahmed (2004), Collado-Fernandez et al. (2012) , De Martino et al. (1979), Dumollard et al. (2009), Dunning et al. (2010), Hess et al. (1993), Meinhardt et al. (1999), Mota et al. (2009), Motta et al. (2000), Ramalho-Santos et a l. (2009), Songsasen et al. (2012), Van Blerkom (2008), Van Blerkom (2009), Van Blerkom (2011), Wilding et al. (2001) . J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84 79 spermatogenesis and sperm quality (Agarwal et al., 2003; Tremel- ovary (BCL-X, BOK) were shown to have no apparent role (Ke len, 2008). For example, mice with a mutation in the inner mito- et al., 2012; Riedlinger et al., 2002). chondrial membrane peptidase 2-like (Immp2l) gene show Mitochondria are the most abundant and prominent organelle impairment in processing of signal peptide sequences from mito- in the oocyte and early embryo (Motta et al., 2000; Sathananthan chondrial cytochrome c and glycerol phosphate dehydrogenase 2, and Trounson, 2000)(Table 1). Depending on the species, a mam- and this causes testicular damage and subfertility, possibly due malian oocyte contains around 105 to 108 mitochondria (Chen to excessive ROS production (George et al., 2012). et al., 1995; Jansen and de Boer, 1998), descending from a restricted founder population in PGCs. Interestingly, female mice 2.3. Mitochondria in sperm seem to select against mutated mtDNA that cause extensive damage and mitochondrial dysfunction, not including these mutations In the final step of sperm differentiation (spermiogenesis) most in ovulated oocytes (Fan et al., 2008). As noted previously, mito- of the cytoplasm (including most mitochondria) is lost in the so- chondria are transmitted exclusively from the maternal gamete called residual bodies. The remaining 22–75 mitochondria rear- (Cummins, 2001; St John et al., 2010). Contradicting a common no- range end to end in the midpiece (Ho and Wey, 2007; Olson and tion, in mammals the entire sperm enters the oocyte at fertilization Winfrey, 1990; Otani et al., 1988). The fact that some mitochondria however sperm mitochondria are diluted or destroyed inside the are evolutionarily retained in a very specialized sperm region sug- embryo (Ankel-Simons and Cummins, 1996; Ramalho-Santos, gests that these organelles have a role in sperm function. Indeed 2011). In rare cases where paternal mitochondria are not destroyed the tight arrangement of mitochondria around the sperm midpiece a mixture of mtDNA types in the embryo (mtDNA heteroplasmy) often is used to exemplify a strategy to concentrate ATP production might result, and could impair development (St John et al., 2010). for a specific function, in this case sperm movement. In fact, mito- During oocyte maturation mitochondria are relocated to different chondrial parameters (MMP, ETC complex activity) correlate posi- regions, in response to localized energy demands (Bavister and tively with sperm function (Gallon et al., 2006; Marchetti et al., Squirrell, 2000; Van Blerkom, 2011), and bursts on ATP production 2002, 2012; Nakada et al., 2006; Ruiz-Pesini et al., 1998; Sousa are correlated with mitochondrial redistribution and oocyte matu- et al., 2011), mitochondrial inhibition impairs sperm activity ration (Yu et al., 2010). (Ruiz-Pesini et al., 2000; St John et al., 2005), and the introduction Mitochondrial function may determine mammalian oocyte of a mutant mtDNA with a pathogenic 4696-bp deletion in mice re- quality and mitochondrial activity, mtDNA copy number and sulted in male infertility (Nakada et al., 2006), with comparable mtDNA mutations, have been associated with fertilization rates, data being reported in human patients (St John et al., 2005). How- embryo development and maternal age, and proposed as bioindi- ever, this is probably not due to ATP production specifically direc- cators for oocyte competence (Wang and Sun, 2007). Additionally, ted to fuel movement, as other pathways (such as glycolysis) seem mitochondria-related factors such as ATP, pyruvate dehydrogenase more prevalent in mammalian sperm for this specific purpose complex and ROS are necessary for correct spindle assembly and (Amaral et al., 2011; Nascimento et al., 2008). The available evi- chromosome alignment in female meiosis (Choi et al., 2007; John- dence seems to demonstrate that in the few days it can spend in son et al., 2007; Van Blerkom, 2011; Zhang et al., 2006). On the the female reproductive tract mammalian sperm might be able other hand the mitochondrial Immp2l mutation mentioned earlier to utilize both glycolysis and OXPHOS to produce ATP for different in the context of spermatogenesis causes female infertility, by purposes. The balance between these (and other) metabolic path- affecting MMP and ROS production (Lu et al., 2008). Finally, oocyte ways may vary between species, according to the substrates avail- mitochondria also contribute in regulating calcium waves, essen- able during in the female reproductive tract and the specific tial for zygote activation (Dumollard et al., 2003; Dumollard function to be carried out (Amaral et al., 2013). Finally, the ability et al., 2004). of sperm mitochondria to accumulate calcium has also been suggested to have a role in sperm signaling pathways (Publicover 2.5. Mitochondria in early embryo development et al., 2008; Publicover et al., 2007). Similarly to what has been described for spermatogenesis, 2.4. Mitochondria in oogenesis mitochondrial structure and metabolic activity seem to vary in distinct stages of oocyte and embryo development (Biggers et al., Essentially the same roles are postulated for mitochondria in fe- 1967; Gott et al., 1990; Harris et al., 2009; Houghton, 2006; Leese, male gametogenesis, adapted to the circumstances related to cyclic 1995; Van Blerkom, 2009; Van Blerkom, 2011; Wycherley et al., oogenesis/folliculogenesis. Oogenesis involves the production of 2005)(Table 1). Nevertheless, OXPHOS is clearly important at cer- very few gametes with high developmental competence, rather tain stages of follicular development/meiotic maturation, during than millions of gametes with reduced (individual) potential, fertilization, and in the first stages on embryo development. and, as in the testis, intrinsic apoptotic pathways involving mito- In the final stage of pre-implantation development (i.e. the blas- chondria also seem to play a role in follicle survival and selection tocyst stage) there is a clear division of cellular lineages, with a (Hunzicker-Dunn and Mayo, 2006). Indeed, recent mouse data sug- small cluster of Inner Cell Mass (ICM)/pluriblast cells, surrounded gests that the mitochondrial-dependent intrinsic apoptotic path- by a thin layer of trophoblast (called trophectoderm after implan- way is constitutively active in oocytes, and might help eliminate tation) cells. Interestingly, while ICM cells have low MMP and are female gametes with meiotic defects (Ene et al., 2013). Interest- almost quiescent in terms of mitochondrial activity, trophoblast ingly there also seem to be sex-specific differences, as noted in cells are highly polarized and very active, producing more ATP mice devoid of BCL-2: while males show decreased spermatogen- and consuming more oxygen, and both aspects seem to be impor- esis (as discussed above), folliculogenesis was increased and folli- tant for implantation (Houghton, 2006; Leese, 2012; Van Blerkom, cle apoptosis inhibited (Yamamoto et al., 2001). Female mice 2009; Van Blerkom, 2011)(Table 1). Pluripotent embryonic stem without BAX also had an increased number of ovarian follicles cells (ESCs) isolated from the ICM maintain this characteristic, and extended fertility (Greenfeld et al., 2007; Perez et al., 2007), and favor aerobic glycolysis over OXPHOS in terms of ATP produc- although this could be due to an indirect effect on PGC migration tion (Ramalho-Santos et al., 2009; Van Blerkom, 2008; Varum et al., (Greenfeld et al., 2007). At any rate targeted expression of BCL-2 2009). More importantly, somatic cell reprogramming to pluripo- seemed to provide equivalent results (Morita et al., 1999). Using tency to generate induced pluripotent stem cells (iPSCs) also in- similar strategies other BCL-2 family proteins expressed in the volves a glycolytic shift away from OXPHOS, and concomitant 80 J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84 changes in mitochondrial function and morphology (Armstrong that then takes place seems to include these different cell types, et al., 2010; Folmes et al., 2011; Prigione et al., 2010; Varum resulting in the formation of the corpus luteum, a transient endo- et al., 2011). As noted above some of these features are also found crine gland crucial for the establishment of a viable pregnancy, and in spermatogonial stem cells, suggesting that they may be common that produces mainly progesterone (Niswender, 2002; Stouffer, to all cells with differentiation potential and roles in the transmis- 2006). The continuous production of the same steroid hormones sion of information. The reasons for this remain unknown, by a defined cell type in the male (following the continuous nature although it has been suggested (Ramalho-Santos et al., 2009) that of spermatogenesis) is thus contrasted by the cyclic production of low mitochondrial activity would also prevent ROS-induced different steroid hormones by changing cell types in the female. cell damage, which might be detrimental both to embryo Several signaling pathways can regulate steroid production by development (ICM cells), and genetic transmission via the germ- activating/inactivating distinct factors, or changing their expres- line (spermatogonia). sion levels. This may take place under physiological circumstances (puberty in either sex, different stages of the ovarian cycle), or as a result of a pathological event. Molecular tools devised to specifi- 3. The endocrine role of mitochondria in reproduction cally target the gonads have provided information focusing mostly downstream of initial mitochondrial intervention. Thus, ovarian 3.1. The mitochondrial step in steroid biosynthesis StAR expression is upregulated during the periovulatory period in parallel with steroid biosynthesis. It is mainly present in the the- The initial enzymatic reaction in the biosynthesis of all steroids ca interna at the beginning of the ovulatory process, increasing in takes place in mitochondria, and involves the conversion of choles- the granulosa layer when ovulatory follicles begin producing sub- terol to pregnenolone (Manna et al., 2009; Stouffer, 2006). This stantial amounts of progesterone, and continues to be prevalent in reaction is dependent on cytochrome P450 side-chain cleavage the corpus luteum (Richards and Pangas, 2010a, 2010b). Con- (P450scc; or CYP11A1), located on the matrix-facing side of the inversely in Leydig cells StAR and P450scc expression is reduced as ner mitochondrial membrane (Fig. 1). In turn, P450cc activity is a function of aging, and this might therefore compromise the early dependent on electron transfer from NADPH mediated by the steps of steroidogenesis (Luo et al., 2001). Furthermore, the impor- adrenoxin-adrenoxin reductase system (Miller, 2005). Pregneno- tance of StAR in this process was confirmed with KO mice, which lone is exported from mitochondria (although it can also be further showed undescended testicles, problems with sperm maturation, processed there in some cases) and can be converted to other com- and premature ovarian failure (Hasegawa et al., 2000). Similar ap- pounds (progesterone, testosterone) by enzymes in the endoplas- proaches had been employed to study the role of other participants mic reticulum/microsomal system (Stocco and McPhaul, 2006). in this process, such as the importance of VDAC and of the phos- Other crucial factors for the mitochondrial step in steroid biosyn- phate transporter in StAR function (Bose et al., 2008). thesis are the steroidogenic acute regulatory protein (StAR; (Sto- Additionally, different in vitro models have been used to study cco, 2001) and the transducesome complex, which includes the role of mitochondria in steroidogenesis. For example, in Leydig components such as a 18 kDa translocator protein (TPSO), the volt- cell models it has been convincingly shown that synthesis of preg- age dependent anion channel (VDAC-1), TPSO-associated protein 7 nenolone from cholesterol via P450scc requires ETC activity, high and protein kinase A subunit 1a (Hauet et al., 2005; Li et al., 2001; MMP and the ability to produce ATP (Allen et al., 2006; Hales Papadopoulos et al., 2007; Papadopoulos and Miller, 2012). Preg- et al., 2005; Levine et al., 2007; Midzak et al., 2011; Stocco and nenolone synthesis requires the processing of cholesterol by an in- McPhaul, 2006). However, these requirements may well vary with ner mitochondrial membrane cytochrome, i.e., it takes place in a the system used (e.g. primary cells isolated from the testis, versus membrane devoid of cholesterol (Tuckey et al., 2002), possibly to immortal Leydig cell lines), an important point that has been high- avoid changes in membrane fluidity/functionality that might occur lighted in a recent study (Midzak et al., 2011). Similar studies have elsewhere. Although cholesterol sources for steroid biosynthesis also been developed in ovarian cells, mainly regarding the effects may vary, a limiting step is the transport of this lipid from the out- of different substances on mitochondrial function and associated er to the inner mitochondrial membrane, a process catalyzed by steroidogenesis, including putative therapeutic agents (Ortega StAR, and in which transduceosome also participates, although de- et al., 2012) and toxicants (Svechnikova et al., 2007). It has also tails regarding the interaction of StAR with this complex need to be been shown that P450cc induction takes place before steroidogen- further clarified (Manna et al., 2009). esis (Hanukoglu et al., 1990). Such models should provide novel insights into the role of mito- 3.2. Sex-specific steroidogenesis chondria in reproduction, although they must always accurately specify, and report back to, the particularities of gametogenesis Male steroidogenesis involves the final production of testoster- in either sex. It should also be noted that gonad steroidogenesis one (or of the more potent testosterone-derived androgen dihydro- may link back to other mitochondrial attributes, for example par- testosterone), and also of some estrogens. As noted previously this ticipating in the regulation apoptosis in both Sertoli cells and ovar- takes place mostly in Leydig cells (Ge et al., 2008). Although in the ian follicles (Simoes et al., 2013; Yacobi et al., 2007). ovary theca cells are homologous to Leydig cells, steroidogenesis (notably the production of estrogens and progesterone) also occurs in granulosa cells, from androgens initially produced in theca cells, 4. Conclusions and future perspectives and varies (both in quantity and in quality) in conjunction with the folliculogenesis/ovulation cycle (Bjersing, 1968; Gelety and Magof- Although some reproductive processes are hard to model fin, 1997). In fact, follicle growth is related to granulosa cell divi- in vitro, or monitor in vivo, many studies have highlighted the sev- sion, maturation and increased steroidogenic activity, which also eral roles played by mitochondria in mammalian reproduction, as influences/is influenced by oocyte growth and maturation within stressed by the fact that mitochondrial dysfunction has been linked the follicle, due to gap junctions established between the gamete to subfertility and infertility at distinct levels, including poor OX- and its supporting cells (Albertini et al., 2001; Gilchrist et al., PHOS activity, changes in mtDNA, excessive ROS production, the 2008). Following ovulation the ruptured follicle contains theca abnormal triggering of apoptosis, or defects in steroidogenesis. cells and granulosa cells that did not accompany the oocyte (sur- These studies are extremely relevant, both in terms of fertility rounding it as cumulus cells). The extensive cellular remodeling management and for reproductive toxicology. However, there are J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84 81 a few other emerging topics where the study of mitochondrial Aly, H.A., Khafagy, R.M., 2011. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced function may also prove useful. cytotoxicity accompanied by oxidative stress in rat Sertoli cells: Possible role of mitochondrial fractions of Sertoli cells. Toxicol. Appl. Pharmacol. 252, 273–280. Although the mechanisms involved remain obscure, recent data Amaral, S., Ramalho-Santos, J., 2009. Aging, mitochondria and male reproductive showing a putative transgenerational (and sex-specific) influence function. Curr. Aging Sci. 2, 165–173. of certain conditions (high fat or low protein diets), or even the Amaral, S., Mota, P., Rodrigues, A.S., Martins, L., Oliveira, P.J., Ramalho-Santos, J., 2008a. Testicular aging involves mitochondrial dysfunction as well as an use of modified ART, on offspring (Calle et al., 2012; Carone increase in UCP2 levels and proton leak. FEBS Lett. 582, 4191–4196. et al., 2010; Ng et al., 2010) is particularly interesting, and may also Amaral, S., Oliveira, P.J., Ramalho-Santos, J., 2008b. Diabetes and the impairment of involve changes in mitochondrial function. Indeed, mitochondrial reproductive function: possible role of mitochondria and reactive oxygen species. Curr. Diabetes Rev. 4, 46–54. dysfunction in oocytes and cumulus cells cultured under diabetic Amaral, S., Mota, P.C., Lacerda, B., Alves, M., Pereira Mde, L., Oliveira, P.J., Ramalho- or insulin-resistance conditions has been recently related to poor Santos, J., 2009. Testicular mitochondrial alterations in untreated fertility, (Ou et al., 2012; Wang et al., 2009, 2010) and infertility streptozotocin-induced diabetic rats. Mitochondrion 9, 41–50. Amaral, A., Paiva, C., Baptista, M., Sousa, A.P., Ramalho-Santos, J., 2011. Exogenous in obese Leptin-deficient (ob/ob) mice has been linked to ovarian glucose improves long-standing human sperm motility, viability, and dysfunction, and notably to higher levels of apoptosis and de- mitochondrial function. Fertil. Steril. 96, 848–850. creased steroidogenesis (Serke et al., 2012). Amaral, A., Castillo, J., Estanyol, J.M., Ballesca, J.L., Ramalho-Santos, J., Oliva, R., 2013. The integration of mitochondrial functions (especially ETC and Human sperm tail proteome suggests new endogenous metabolic pathways. Mol. Cell. Proteomics 12, 330–342. TCA) in the wider context of cell homeostasis has also been sug- Ankel-Simons, F., Cummins, J.M., 1996. Misconceptions about mitochondria and gested (Folmes et al., 2012; Hitchler and Domann, 2009), as it per- mammalian fertilization: implications for theories on human evolution. Proc. tains to signaling and epigenetic status (for example, with Natl. Acad. Sci. USA 93, 13859–13863. Armstrong, L., Tilgner, K., Saretzki, G., Atkinson, S.P., Stojkovic, M., Moreno, R., mitochondria providing intermediates for epigenetic post-transla- Przyborski, S., Lako, M., 2010. Human induced pluripotent stem cell lines show tional modifications). This may provide novel insights into repro- stress defense mechanisms and mitochondrial regulation similar to those of ductive function, where both erasure of imprints in PGCs and the human embryonic stem cells. Stem Cells 28, 661–673. Bajpai, M., Gupta, G., Setty, B.S., 1998. Changes in carbohydrate metabolism of re-placing of sex-specific marks upon gonad colonization are well testicular germ cells during meiosis in the rat. Eur. J. Endocrinol. 138, 322–327. known phenomena (Abramowitz and Bartolomei, 2012). Banu, S.K., Stanley, J.A., Lee, J., Stephen, S.D., Arosh, J.A., Hoyer, P.B., Burghardt, R.C., Finally, mitochondrial function in gonads may also be unex- 2011. Hexavalent chromium-induced apoptosis of granulosa cells involves selective sub-cellular translocation of Bcl-2 members, ERK1/2 and p53. Toxicol. pectedly related to regulatory RNA processing. Recently male Appl. Pharmacol. 251, 253–266. (but not female) KO mice for the mitochondria-specific phospholi- Bavister, B.D., Squirrell, J.M., 2000. Mitochondrial distribution and function in pase D, were shown by two independent groups to be infertile due oocytes and early embryos. Hum. Reprod. 15 (Suppl. 2), 189–198. Bentov, Y., Yavorska, T., Esfandiari, N., Jurisicova, A., Casper, R.F., 2011. The to meiotic arrest, and this was correlated with fission-fusion de- contribution of mitochondrial function to reproductive aging. J. Assist. Reprod. fects and, interestingly, also with impaired production of piRNAs Genet. 28, 773–783. that are crucial for proper spermatogenesis (Huang et al., 2011; Bereiter-Hahn, J., Voth, M., 1994. Dynamics of mitochondria in living cells: shape Watanabe et al., 2011). changes, dislocations, fusion, and fission of mitochondria. Microsc. Res. Tech. 27, 198–219. Biggers, J.D., Whittingham, D.G., Donahue, R.P., 1967. The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci USA 58, 560–567. Acknowledgements Bjersing, L., 1968. On the morphology and endocrine function of granulosa cells in ovarian follicles and corpora lutea. Biochemical, histochemical, and Given the extent of available literature, and the specific multi- ultrastructural studies on the porcine ovary with special reference to steroid hormone synthesis (Copenh). Acta Endocrinol. (Suppl. 12), 121–123. disciplinary nature of this paper, readers have been often referred Bose, M., Whittal, R.M., Miller, W.L., Bose, H.S., 2008. Steroidogenic activity of StAR to review articles. Apologies are due to all authors whose work was requires contact with mitochondrial VDAC1 and phosphate carrier protein. J. not directly cited. Alexandra Amaral is thanked for critical reading Biol. Chem. 283, 8837–8845. Boussouar, F., Benahmed, M., 2004. Lactate and energy metabolism in male germ of the Manuscript and J. Saints is gratefully acknowledged for lin- cells. Trends Endocrinol. Metab. 15, 345–350. guistic suggestions. All lab members are thanked for helpful dis- Brower, J.V., Lim, C.H., Jorgensen, M., Oh, S.P., Terada, N., 2009. Adenine nucleotide cussions, especially Paula Mota, Ana Sofia Rodrigues and Ana translocase 4 deficiency leads to early meiotic arrest of murine male germ cells. Reproduction 138, 463–470. Paula Sousa. Part of the work in the Authors lab was funded by Calle, A., Miranda, A., Fernandez-Gonzalez, R., Pericuesta, E., Laguna, R., Gutierrez- FEDER and COMPETE, via FCT (Fundação para a Ciência e Tecnolo- Adan, A., 2012. Male mice produced by in vitro culture have reduced fertility gia), Portugal in grants PTDC/EBB-EBI/101114/2008, PTDC/EBB- and transmit organomegaly and glucose intolerance to their male offspring. Biol. Reprod. 87, 34. EBI/120634/2010 and PTDC/QUI-BIQ/120652/2010. Sandra Amaral Campello, S., Scorrano, L., 2010. Mitochondrial shape changes: orchestrating cell is the recipient of a FCT fellowship (SFRH/BPD/63190/2009) and pathophysiology. EMBO Rep. 11, 678–684. the Center for Neuroscience and Cell Biology (CNC) funding is also Carone, B.R., Fauquier, L., Habib, N., Shea, J.M., Hart, C.E., Li, R., Bock, C., Li, C., Gu, H., Zamore, P.D., Meissner, A., Weng, Z., Hofmann, H.A., Friedman, N., Rando, O.J., supported by FCT (PEst-C/SAU/LA0001/2011). 2010. Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell 143, 1084–1096. Cereghetti, G.M., Scorrano, L., 2011. Phagocytosis: coupling of mitochondrial References uncoupling and engulfment. Curr. Biol. 21, R852–854. Chen, X., Prosser, R., Simonetti, S., Sadlock, J., Jagiello, G., Schon, E.A., 1995. Abramowitz, L.K., Bartolomei, M.S., 2012. Genomic imprinting: recognition and Rearranged mitochondrial genomes are present in human oocytes. Am. J. Hum. marking of imprinted loci. Curr. Opin. Genet. Dev. 22, 72–78. Genet. 57, 239–247. Agarwal, A., Saleh, R.A., Bedaiwy, M.A., 2003. Role of reactive oxygen species in the Choi, W.J., Banerjee, J., Falcone, T., Bena, J., Agarwal, A., Sharma, R.K., 2007. Oxidative pathophysiology of human reproduction. Fertil. Steril. 79, 829–843. stress and tumor necrosis factor-alpha-induced alterations in metaphase II Aihara, T., Nakamura, N., Honda, S., Hirose, S., 2009. A novel potential role for mouse oocyte spindle structure. Fertil. Steril. 88, 1220–1231. gametogenetin-binding protein 1 (GGNBP1) in mitochondrial morphogenesis Collado-Fernandez, E., Picton, H.M., Dumollard, R., 2012. Metabolism throughout during spermatogenesis in mice. Biol. Reprod. 80, 762–770. follicle and oocyte development in mammals. Int. J. Dev. Biol. 56, 799–808. Aitken, R.J., Gibb, Z., Mitchell, L.A., Lambourne, S.R., Connaughton, H.S., De Iuliis, Collins, T.J., Berridge, M.J., Lipp, P., Bootman, M.D., 2002. Mitochondria are G.N., 2012. Sperm motility is lost in vitro as a consequence of mitochondrial free morphologically and functionally heterogeneous within cells. EMBO J. 21, radical production and the generation of electrophilic aldehydes but can be 1616–1627. significantly rescued by the presence of nucleophilic thiols. Biol. Reprod. 87, Correia, S.C., Santos, R.X., Perry, G., Zhu, X., Moreira, P.I., Smith, M.A., 2012. 110. Mitochondrial importance in Alzheimer’s, Huntington’s and Parkinson’s Albertini, D.F., Combelles, C.M., Benecchi, E., Carabatsos, M.J., 2001. Cellular basis for diseases. Adv. Exp. Med. Biol. 724, 205–221. paracrine regulation of ovarian follicle development. Reproduction 121, 647– Coultas, L., Bouillet, P., Loveland, K.L., Meachem, S., Perlman, H., Adams, J.M., 653. Strasser, A., 2005. Concomitant loss of proapoptotic BH3-only Bcl-2 antagonists Allen, J.A., Shankara, T., Janus, P., Buck, S., Diemer, T., Hales, K.H., Hales, D.B., 2006. Bik and Bim arrests spermatogenesis. EMBO J. 24, 3963–3973. Energized, polarized, and actively respiring mitochondria are required for acute Cummins, J.M., 2001. Mitochondria: potential roles in embryogenesis and Leydig cell steroidogenesis. Endocrinology 147, 3924–3935. nucleocytoplasmic transfer. Hum. Reprod. Update 7, 217–228. 82 J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84

De Martino, C., Floridi, A., Marcante, M.L., Malorni, W., Scorza Barcellona, P., Bellocci, Honarpour, N., Du, C., Richardson, J.A., Hammer, R.E., Wang, X., Herz, J., M., Silvestrini, B., 1979. Morphological, histochemical and biochemical studies 2000. Adult Apaf-1-deficient mice exhibit male infertility. Dev. Biol. 218, on germ cell mitochondria of normal rats. Cell Tissue Res. 196, 1–22. 248–258. Dorn 2nd, G.W., Scorrano, L., 2010. Two close, too close: sarcoplasmic reticulum- Houghton, F.D., 2006. Energy metabolism of the inner cell mass and trophectoderm mitochondrial crosstalk and cardiomyocyte fate. Circ. Res. 107, 689–699. of the mouse blastocyst. Differentiation 74, 11–18. Dumollard, R., Hammar, K., Porterfield, M., Smith, P.J., Cibert, C., Rouviere, C., Sardet, Huang, H., Gao, Q., Peng, X., Choi, S.Y., Sarma, K., Ren, H., Morris, A.J., Frohman, M.A., C., 2003. Mitochondrial respiration and Ca2+ waves are linked during 2011. PiRNA-associated germline nuage formation and spermatogenesis require fertilization and meiosis completion. Development 130, 683–692. MitoPLD profusogenic mitochondrial-surface lipid signaling. Dev. Cell 20, 376– Dumollard, R., Marangos, P., Fitzharris, G., Swann, K., Duchen, M., Carroll, J., 2004. 387. Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have Hunzicker-Dunn, M., Mayo, K., 2006. Gonadotropin signaling in the ovary. In: Neill, an absolute requirement for mitochondrial ATP production. Development 131, J.e. (Ed.), Knobil and Neill’s Physiology of Reproduction, third ed. Academic 3057–3067. Press/Elsevier, St. Louis, MO, USA, pp. 547–592. Dumollard, R., Carroll, J., Duchen, M.R., Campbell, K., Swann, K., 2009. Mitochondrial Huttemann, M., Jaradat, S., Grossman, L.I., 2003. Cytochrome c oxidase of mammals function and redox state in mammalian embryos. Semin. Cell Dev. Biol. 20, 346– contains a testes-specific isoform of subunit VIb – the counterpart to testes- 353. specific cytochrome c? Mol. Reprod. Dev. 66, 8–16. Dunning, K.R., Cashman, K., Russell, D.L., Thompson, J.G., Norman, R.J., Robker, R.L., Jansen, R.P., de Boer, K., 1998. The bottleneck: mitochondrial imperatives in 2010. Beta-oxidation is essential for mouse oocyte developmental competence oogenesis and ovarian follicular fate. Mol. Cell. Endocrinol. 145, 81–88. and early embryo development. Biol. Reprod. 83, 909–918. Jia, Y., Lee, K.W., Swerdloff, R., Hwang, D., Cobb, L.J., Sinha Hikim, A., Lue, Y.H., Cohen, Ene, A.C., Park, S., Edelmann, W., Taketo, T., 2013. Caspase 9 is constitutively P., Wang, C., 2010. Interaction of insulin-like growth factor-binding protein-3 activated in mouse oocytes and plays a key role in oocyte elimination during and BAX in mitochondria promotes male germ cell apoptosis. J. Biol. Chem. 285, meiotic prophase progression. Dev. Biol.. 1726–1732. Fan, W., Waymire, K.G., Narula, N., Li, P., Rocher, C., Coskun, P.E., Vannan, M.A., Johnson, M.T., Freeman, E.A., Gardner, D.K., Hunt, P.A., 2007. Oxidative metabolism Narula, J., Macgregor, G.R., Wallace, D.C., 2008. A mouse model of mitochondrial of pyruvate is required for meiotic maturation of murine oocytes in vivo. Biol. disease reveals germline selection against severe mtDNA mutations. Science Reprod. 77, 2–8. 319, 958–962. Kakkar, P., Singh, B.K., 2007. Mitochondria: a hub of redox activities and cellular Folmes, C.D., Nelson, T.J., Martinez-Fernandez, A., Arrell, D.K., Lindor, J.Z., Dzeja, P.P., distress control. Mol. Cell. Biochem. 305, 235–253. Ikeda, Y., Perez-Terzic, C., Terzic, A., 2011. Somatic oxidative bioenergetics Katz, S.G., Fisher, J.K., Correll, M., Bronson, R.T., Ligon, K.L., Walensky, L.D., 2012. transitions into pluripotency-dependent glycolysis to facilitate nuclear Brain and testicular tumors in mice with progenitor cells lacking BAX and BAK. reprogramming. Cell Metab. 14, 264–271. Oncogene. Folmes, C.D., Dzeja, P.P., Nelson, T.J., Terzic, A., 2012. Metabolic plasticity in stem Ke, F., Voss, A., Kerr, J.B., O’Reilly, L.A., Tai, L., Echeverry, N., Bouillet, P., Strasser, A., cell homeostasis and differentiation. Cell Stem Cell 11, 596–606. Kaufmann, T., 2012. BCL-2 family member BOK is widely expressed but its loss Gallon, F., Marchetti, C., Jouy, N., Marchetti, P., 2006. The functionality of has only minimal impact in mice. Cell Death Differ. 19, 915–925. mitochondria differentiates human spermatozoa with high and low fertilizing Knudson, C.M., Tung, K.S., Tourtellotte, W.G., Brown, G.A., Korsmeyer, S.J., 1995. capability. Fertil. Steril. 86, 1526–1530. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Gassei, K., Schlatt, S., 2007. Testicular morphogenesis: comparison of in vivo and Science 270, 96–99. in vitro models to study male gonadal development. Ann. N. Y. Acad. Sci. 1120, Leese, H.J., 1995. Metabolic control during preimplantation mammalian 152–167. development. Hum. Reprod. Update 1, 63–72. Ge, R., Chen, G., Hardy, M.P., 2008. The role of the Leydig cell in spermatogenic Leese, H.J., 2012. Metabolism of the preimplantation embryo: 40 years on. function. Adv. Exp. Med. Biol. 636, 255–269. Reproduction 143, 417–427. Gelety, T.J., Magoffin, D.A., 1997. Ontogeny of steroidogenic enzyme gene Levine, S.L., Han, Z., Liu, J., Farmer, D.R., Papadopoulos, V., 2007. Disrupting expression in ovarian theca-interstitial cells in the rat: regulation by a mitochondrial function with surfactants inhibits MA-10 Leydig cell paracrine theca-differentiating factor prior to achieving luteinizing hormone steroidogenesis. Cell Biol. Toxicol. 23, 385–400. responsiveness. Biol. Reprod. 56, 938–945. Li, H., Degenhardt, B., Tobin, D., Yao, Z.X., Tasken, K., Papadopoulos, V., 2001. George, S.K., Jiao, Y., Bishop, C.E., Lu, B., 2012. Oxidative stress is involved in age- Identification, localization, and function in steroidogenesis of PAP7: a dependent spermatogenic damage of Immp2l mutant mice. Free Radic. Biol. peripheral-type benzodiazepine receptor- and PKA (RIalpha)-associated Med. 52, 2223–2233. protein. Mol. Endocrinol. 15, 2211–2228. Gilbert, S., 2010. Developmental Biology, ninth ed.. Sinauer, New York. Lu, B., Poirier, C., Gaspar, T., Gratzke, C., Harrison, W., Busija, D., Matzuk, M.M., Gilchrist, R.B., Lane, M., Thompson, J.G., 2008. Oocyte-secreted factors: regulators of Andersson, K.E., Overbeek, P.A., Bishop, C.E., 2008. A mutation in the inner cumulus cell function and oocyte quality. Hum. Reprod. Update 14, 159–177. mitochondrial membrane peptidase 2-like gene (Immp2l) affects mitochondrial Gott, A.L., Hardy, K., Winston, R.M., Leese, H.J., 1990. Non-invasive measurement of function and impairs fertility in mice. Biol. Reprod. 78, 601–610. pyruvate and glucose uptake and lactate production by single human Luo, L., Chen, H., Zirkin, B.R., 2001. Leydig cell aging: steroidogenic acute preimplantation embryos. Hum. Reprod. 5, 104–108. regulatory protein (StAR) and cholesterol side-chain cleavage enzyme. J. Greenfeld, C.R., Pepling, M.E., Babus, J.K., Furth, P.A., Flaws, J.A., 2007. BAX regulates Androl. 22, 149–156. follicular endowment in mice. Reproduction 133, 865–876. Manna, P.R., Dyson, M.T., Stocco, D.M., 2009. Regulation of the steroidogenic acute Grootegoed, J.A., Jansen, R., Van der Molen, H.J., 1984. The role of glucose, pyruvate regulatory protein gene expression: present and future perspectives. Mol. Hum. and lactate in ATP production by rat spermatocytes and spermatids. Biochim. Reprod. 15, 321–333. Biophys. Acta 767, 248–256. Mannella, C.A., 2006. The relevance of mitochondrial membrane topology to Hales, D.B., Allen, J.A., Shankara, T., Janus, P., Buck, S., Diemer, T., Hales, K.H., 2005. mitochondrial function. Biochim. Biophys. Acta 1762, 140–147. Mitochondrial function in Leydig cell steroidogenesis. Ann. N. Y. Acad. Sci. 1061, Mannella, C.A., 2008. Structural diversity of mitochondria: functional implications. 120–134. Ann. N. Y. Acad. Sci. 1147, 171–179. Hanukoglu, I., Suh, B.S., Himmelhoch, S., Amsterdam, A., 1990. Induction and Marchetti, C., Obert, G., Deffosez, A., Formstecher, P., Marchetti, P., 2002. Study of mitochondrial localization of cytochrome P450scc system enzymes in normal mitochondrial membrane potential, reactive oxygen species, DNA and transformed ovarian granulosa cells. J. Cell Biol. 111, 1373–1381. fragmentation and cell viability by flow cytometry in human sperm. Hum. Harris, S.E., Leese, H.J., Gosden, R.G., Picton, H.M., 2009. Pyruvate and oxygen Reprod. 17, 1257–1265. consumption throughout the growth and development of murine oocytes. Mol. Marchetti, P., Ballot, C., Jouy, N., Thomas, P., Marchetti, C., 2012. Influence of Reprod. Dev. 76, 231–238. mitochondrial membrane potential of spermatozoa on in vitro fertilisation Hasegawa, T., Zhao, L., Caron, K.M., Majdic, G., Suzuki, T., Shizawa, S., Sasano, H., outcome. Andrologia 44, 136–141. Parker, K.L., 2000. Developmental roles of the steroidogenic acute regulatory Martinou, J.C., Youle, R.J., 2011. Mitochondria in apoptosis: Bcl-2 family members protein (StAR) as revealed by StAR knockout mice. Mol. Endocrinol. 14, 1462– and mitochondrial dynamics. Dev. Cell 21, 92–101. 1471. Matoba, S., Hiramatsu, R., Kanai-Azuma, M., Tsunekawa, N., Harikae, K., Kawakami, Hauet, T., Yao, Z.X., Bose, H.S., Wall, C.T., Han, Z., Li, W., Hales, D.B., Miller, W.L., H., Kurohmaru, M., Kanai, Y., 2008. Establishment of testis-specific SOX9 Culty, M., Papadopoulos, V., 2005. Peripheral-type benzodiazepine receptor- activation requires high-glucose metabolism in mouse sex differentiation. Dev. mediated action of steroidogenic acute regulatory protein on cholesterol entry Biol. 324, 76–87. into leydig cell mitochondria. Mol. Endocrinol. 19, 540–554. McLaughlin, E.A., McIver, S.C., 2009. Awakening the oocyte: controlling primordial Hess, R.A., Miller, L.A., Kirby, J.D., Margoliash, E., Goldberg, E., 1993. follicle development. Reproduction 137, 1–11. Immunoelectron microscopic localization of testicular and somatic Meinhardt, A., Hedger, M.P., 2011. Immunological, paracrine and endocrine aspects cytochromes c in the seminiferous epithelium of the rat. Biol. Reprod. 48, of testicular immune privilege. Mol. Cell. Endocrinol. 335, 60–68. 1299–1308. Meinhardt, A., Wilhelm, B., Seitz, J., 1999. Expression of mitochondrial marker Hitchler, M.J., Domann, F.E., 2009. Metabolic defects provide a spark for the proteins during spermatogenesis. Hum. Reprod. Update 5, 108–119. epigenetic switch in cancer. Free Radic. Biol. Med. 47, 115–127. Midzak, A.S., Chen, H., Aon, M.A., Papadopoulos, V., Zirkin, B.R., 2011. ATP synthesis, Ho, H.C., Wey, S., 2007. Three dimensional rendering of the mitochondrial sheath mitochondrial function, and steroid biosynthesis in rodent primary and tumor morphogenesis during mouse spermiogenesis. Microsc. Res. Tech. 70, 719–723. Leydig cells. Biol. Reprod. 84, 976–985. Holstein, A.F., Schulze, W., Davidoff, M., 2003. Understanding spermatogenesis is a Miller, W.L., 2005. Minireview: regulation of steroidogenesis by electron transfer. prerequisite for treatment. Reprod. Biol. Endocrinol. 1, 107. Endocrinology 146, 2544–2550. Hom, J., Sheu, S.S., 2009. Morphological dynamics of mitochondria–a special Mittwoch, U., 2004. The elusive action of sex-determining genes: mitochondria to emphasis on cardiac muscle cells. J. Mol. Cell. Cardiol. 46, 811–820. the rescue? J. Theor. Biol. 228, 359–365. J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84 83

Miyamoto, K., Sato, E.F., Kasahara, E., Jikumaru, M., Hiramoto, K., Tabata, H., Ramalho-Santos, J., Varum, S., Amaral, S., Mota, P.C., Sousa, A.P., Amaral, A., 2009. Katsuragi, M., Odo, S., Utsumi, K., Inoue, M., 2010. Effect of oxidative stress Mitochondrial functionality in reproduction: from gonads and gametes to during repeated ovulation on the structure and functions of the ovary, oocytes, embryos and embryonic stem cells. Hum. Reprod. Update 15, 553–572. and their mitochondria. Free Radic. Biol. Med. 49, 674–681. Reyes, J.G., Farias, J.G., Henriquez-Olavarrieta, S., Madrid, E., Parraga, M., Zepeda, Morita, Y., Perez, G.I., Maravei, D.V., Tilly, K.I., Tilly, J.L., 1999. Targeted expression of A.B., Moreno, R.D., 2012. The hypoxic testicle: physiology and pathophysiology. Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents Oxid. Med. Cell Longev. 2012, 929285. spontaneous and chemotherapy-induced oocyte apoptosis in vitro. Mol. Richards, J.S., Pangas, S.A., 2010a. New insights into ovarian function. Handb. Exp. Endocrinol. 13, 841–850. Pharmacol., 3–27. Mota, P., Amaral, S., Martins, L., de Lourdes Pereira, M., Oliveira, P.J., Ramalho- Richards, J.S., Pangas, S.A., 2010b. The ovary: basic biology and clinical implications. Santos, J., 2009. Mitochondrial bioenergetics of testicular cells from the J. Clin. Invest. 120, 963–972. domestic cat (Felis catus)-a model for endangered species. Reprod. Toxicol. Riedlinger, G., Okagaki, R., Wagner, K.U., Rucker 3rd, E.B., Oka, T., Miyoshi, K., 27, 111–116. Flaws, J.A., Hennighausen, L., 2002. Bcl-x is not required for maintenance of Mota, P.C., Cordeiro, M., Pereira, S.P., Oliveira, P.J., Moreno, A.J., Ramalho-Santos, J., follicles and corpus luteum in the postnatal mouse ovary. Biol. Reprod. 66, 2011. Differential effects of p, p’-DDE on testis and liver mitochondria: 438–444. implications for reproductive toxicology. Reprod. Toxicol. 31, 80–85. Robinson, R., Fritz, I.B., 1981. Metabolism of glucose by Sertoli cells in culture. Biol. Motta, P.M., Nottola, S.A., Makabe, S., Heyn, R., 2000. Mitochondrial morphology in Reprod. 24, 1032–1041. human fetal and adult female germ cells. Hum. Reprod. 15 (Suppl. 2), 129–147. Rodrigues, A.S., Lacerda, B., Moreno, A.J., Ramalho-Santos, J., 2010. Proton leak Nakada, K., Sato, A., Yoshida, K., Morita, T., Tanaka, H., Inoue, S., Yonekawa, H., modulation in testicular mitochondria affects reactive oxygen species Hayashi, J., 2006. Mitochondria-related male infertility. Proc Natl Acad Sci USA production and lipid peroxidation. Cell Biochem. Funct. 28, 224–231. 103, 15148–15153. Ross, A.J., Amy, S.P., Mahar, P.L., Lindsten, T., Knudson, C.M., Thompson, C.B., Nakamura, M., Okinaga, S., Arai, K., 1984. Metabolism of pachytene primary Korsmeyer, S.J., MacGregor, G.R., 2001. BCLW mediates survival of postmitotic spermatocytes from rat testes: pyruvate maintenance of adenosine Sertoli cells by regulating BAX activity. Dev. Biol. 239, 295–308. triphosphate level. Biol. Reprod. 30, 1187–1197. Rowland, A.A., Voeltz, G.K., 2012. Endoplasmic reticulum-mitochondria contacts: Narisawa, S., Hecht, N.B., Goldberg, E., Boatright, K.M., Reed, J.C., Millan, J.L., 2002. function of the junction. Nat. Rev. Mol. Cell Biol. 13, 607–625. Testis-specific cytochrome c-null mice produce functional sperm but undergo Ruiz-Pesini, E., Diez, C., Lapena, A.C., Perez-Martos, A., Montoya, J., Alvarez, E., early testicular atrophy. Mol. Cell. Biol. 22, 5554–5562. Arenas, J., Lopez-Perez, M.J., 1998. Correlation of sperm motility with Nascimento, J.M., Shi, L.Z., Tam, J., Chandsawangbhuwana, C., Durrant, B., Botvinick, mitochondrial enzymatic activities. Clin. Chem. 44, 1616–1620. E.L., Berns, M.W., 2008. Comparison of glycolysis and oxidative phosphorylation Ruiz-Pesini, E., Lapena, A.C., Diez-Sanchez, C., Perez-Martos, A., Montoya, J., Alvarez, as energy sources for mammalian sperm motility, using the combination of E., Diaz, M., Urries, A., Montoro, L., Lopez-Perez, M.J., Enriquez, J.A., 2000. Human fluorescence imaging, laser tweezers, and real-time automated tracking and mtDNA haplogroups associated with high or reduced spermatozoa motility. trapping. J. Cell. Physiol. 217, 745–751. Am. J. Hum. Genet. 67, 682–696. Newmeyer, D.D., Ferguson-Miller, S., 2003. Mitochondria: releasing power for life Russell, L.D., Warren, J., Debeljuk, L., Richardson, L.L., Mahar, P.L., Waymire, K.G., and unleashing the machineries of death. Cell 112, 481–490. Amy, S.P., Ross, A.J., MacGregor, G.R., 2001. Spermatogenesis in Bclw-deficient Ng, S.F., Lin, R.C., Laybutt, D.R., Barres, R., Owens, J.A., Morris, M.J., 2010. Chronic mice. Biol. Reprod. 65, 318–332. high-fat diet in fathers programs beta-cell dysfunction in female rat offspring. Russell, L.D., Chiarini-Garcia, H., Korsmeyer, S.J., Knudson, C.M., 2002. Bax- Nature 467, 963–966. dependent spermatogonia apoptosis is required for testicular development Nichols, D.G., Ferguson, S.J., 2002. Bioenergetics, third ed. Academic Press, and spermatogenesis. Biol. Reprod. 66, 950–958. Amsterdam. Sanchez-Partida, L.G., Simerly, C.R., Ramalho-Santos, J., 2008. Freeze-dried primate Niswender, G.D., 2002. Molecular control of luteal secretion of progesterone. sperm retains early reproductive potential after intracytoplasmic sperm Reproduction 123, 333–339. injection. Fertil. Steril. 89, 742–745. Nunnari, J., Suomalainen, A., 2012. Mitochondria: in sickness and in health. Cell 148, Sathananthan, A.H., Trounson, A.O., 2000. Mitochondrial morphology during 1145–1159. preimplantational human embryogenesis. Hum. Reprod. 15 (Suppl. 2), 148– Oettinghaus, B., Licci, M., Scorrano, L., Frank, S., 2012. Less than perfect divorces: 159. dysregulated mitochondrial fission and neurodegeneration. Acta Neuropathol. Scheffler, I.E., 2001. A century of mitochondrial research: achievements and 123, 189–203. perspectives. Mitochondrion 1, 3–31. Olson, G.E., Winfrey, V.P., 1990. Mitochondria-cytoskeleton interactions in the Serke, H., Nowicki, M., Kosacka, J., Schroder, T., Kloting, N., Bluher, M., Kallendrusch, sperm midpiece. J. Struct. Biol. 103, 13–22. S., Spanel-Borowski, K., 2012. Leptin-deficient (ob/ob) mouse ovaries show fatty Ortega, I., Cress, A.B., Wong, D.H., Villanueva, J.A., Sokalska, A., Moeller, B.C., Stanley, degeneration, enhanced apoptosis and decreased expression of steroidogenic S.D., Duleba, A.J., 2012. Simvastatin reduces steroidogenesis by inhibiting acute regulatory enzyme. Int. J. Obes. (Lond.) 36, 1047–1053. Cyp17a1 gene expression in rat ovarian theca-interstitial cells. Biol. Reprod. 86, Shaha, C., Tripathi, R., Mishra, D.P., 2010. Male germ cell apoptosis: regulation and 1–9. biology. Philos. Trans. Roy. Soc. Lond. B Biol. Sci. 365, 1501–1515. Otani, H., Tanaka, O., Kasai, K., Yoshioka, T., 1988. Development of mitochondrial Simoes, V.L., Alves, M.G., Martins, A.D., Dias, T.R., Rato, L., Socorro, S., Oliveira, P.F., helical sheath in the middle piece of the mouse spermatid tail: regular 2013. Regulation of apoptotic signaling pathways by 5alpha- dispositions and synchronized changes. Anat. Rec. 222, 26–33. dihydrotestosterone and 17beta-estradiol in immature rat Sertoli cells. J. Ou, X.H., Li, S., Wang, Z.B., Li, M., Quan, S., Xing, F., Guo, L., Chao, S.B., Chen, Z., Liang, Steroid Biochem. Mol. Biol. 135, 15–23. X.W., Hou, Y., Schatten, H., Sun, Q.Y., 2012. Maternal insulin resistance causes Smith, B.E., Braun, R.E., 2012. Germ cell migration across Sertoli cell tight junctions. oxidative stress and mitochondrial dysfunction in mouse oocytes. Hum. Reprod. Science 338, 798–802. 27, 2130–2145. Soder, O., 2007. Sexual dimorphism of gonadal development. Best Pract. Res. Clin. Palmeira, C.M., Ramalho-Santos, J., 2011. Mitochondrial dysfunction in reproductive Endocrinol. Metab. 21, 381–391. and developmental toxicity. In: Gupta, C. (Ed.), Reproductive and Songsasen, N., Wesselowski, S., Carpenter, J.W., Wildt, D.E., 2012. The ability to Developmental Toxicology R. Elsevier, New York, pp. 815–824. achieve meiotic maturation in the dog oocyte is linked to glycolysis and Papadopoulos, V., Miller, W.L., 2012. Role of mitochondria in steroidogenesis. Best glutamine oxidation. Mol. Reprod. Dev. 79, 186–196. Pract. Res. Clin. Endocrinol Metab. 26, 771–790. Sousa, A.P., Amaral, A., Baptista, M., Tavares, R., Caballero Campo, P., Caballero Papadopoulos, V., Liu, J., Culty, M., 2007. Is there a mitochondrial signaling complex Peregrin, P., Freitas, A., Paiva, A., Almeida-Santos, T., Ramalho-Santos, J., 2011. facilitating cholesterol import? Mol. Cell. Endocrinol. 265–266, 59–64. Not all sperm are equal: functional mitochondria characterize a subpopulation Pereda, J., Zorn, T., Soto-Suazo, M., 2006. Migration of human and mouse primordial of human sperm with better fertilization potential. PLoS ONE 6, e18112. germ cells and colonization of the developing ovary: an ultrastructural and St John, J.C., Jokhi, R.P., Barratt, C.L., 2005. The impact of mitochondrial genetics on cytochemical study. Microsc. Res. Tech. 69, 386–395. male infertility. Int. J. Androl. 28, 65–73. Perez, G.I., Jurisicova, A., Wise, L., Lipina, T., Kanisek, M., Bechard, A., Takai, Y., Hunt, St John, J.C., Facucho-Oliveira, J., Jiang, Y., Kelly, R., Salah, R., 2010. Mitochondrial P., Roder, J., Grynpas, M., Tilly, J.L., 2007. Absence of the proapoptotic Bax DNA transmission, replication and inheritance: a journey from the gamete protein extends fertility and alleviates age-related health complications in through the embryo and into offspring and embryonic stem cells. Hum. Reprod. female mice. Proc. Natl. Acad. Sci. USA 104, 5229–5234. Update 16, 488–509. Prigione, A., Fauler, B., Lurz, R., Lehrach, H., Adjaye, J., 2010. The senescence-related Stocco, D.M., 2001. StAR protein and the regulation of steroid hormone mitochondrial/oxidative stress pathway is repressed in human induced biosynthesis. Annu. Rev. Physiol. 63, 193–213. pluripotent stem cells. Stem Cells 28, 721–733. Stocco, D.M., McPhaul, M.J., 2006. Physiology of testicular steroidogenesis. In: Neill, Publicover, S.J., Harper, C.V., Barratt, C., 2007. [Ca2+]i signalling in sperm–making J.e. (Ed.), Knobil and Neill’s Physiology Of Reproduction, third ed. Academic the most of what you’ve got. Nat. Cell Biol. 9, 235–242. Press/Elsevier, St. Louis, MO, USA, pp. 977–1016. Publicover, S.J., Giojalas, L.C., Teves, M.E., de Oliveira, G.S., Garcia, A.A., Barratt, C.L., Stouffer, R., 2006. Structure, function, and regulation of the corpus luteum. In: Neill, Harper, C.V., 2008. Ca2+ signalling in the control of motility and guidance in J.e. (Ed.), Knobil and Neill’s Physiology of Reproduction, third ed. Academic mammalian sperm. Front. Biosci. 13, 5623–5637. Press/Elsevier, St. Louis, MO, USA, pp. 475–526. Ramalho-Santos, J., 2011. A sperm’s tail: the importance of getting it right. Hum. Svechnikov, K., Spatafora, C., Svechnikova, I., Tringali, C., Soder, O., 2009. Effects of Reprod. 26, 2590–2591. resveratrol analogs on steroidogenesis and mitochondrial function in rat Leydig Ramalho-Santos, J., Rodrigues., A.S., 2013. From oocytes and pluripotent stem cells cells in vitro. J. Appl. Toxicol. 29, 673–680. to fully differentiated fates: (Also) a mitochondrial odyssey. In: St. John, J.C. Svechnikova, I., Svechnikov, K., Soder, O., 2007. The influence of di-(2-ethylhexyl) (Ed.), Mitochondrial DNA Mitochondria Disease and Stem Cells. Humana Press/ phthalate on steroidogenesis by the ovarian granulosa cells of immature female Springer-Verlag, New York, pp. 69–86. rats. J. Endocrinol. 194, 603–609. 84 J. Ramalho-Santos, S. Amaral / Molecular and Cellular Endocrinology 379 (2013) 74–84

Tavares, R.S., Martins, F.C., Oliveira, P.J., Ramalho-Santos, J., Peixoto, F.P., 2009. Lin, Sasaki, H., 2011. MITOPLD is a mitochondrial protein essential for nuage Parabens in male infertility-is there a mitochondrial connection? Reprod. formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375. Toxicol. 27, 1–7. Wilding, M., Carotenuto, R., Infante, V., Dale, B., Marino, M., Di Matteo, L., Tremellen, K., 2008. Oxidative stress and male infertility–a clinical perspective. Campanella, C., 2001. Confocal microscopy analysis of the activity of Hum. Reprod. Update 14, 243–258. mitochondria contained within the ‘mitochondrial cloud’ during oogenesis in Tuckey, R.C., Headlam, M.J., Bose, H.S., Miller, W.L., 2002. Transfer of cholesterol Xenopus laevis. Zygote 9, 347–352. between phospholipid vesicles mediated by the steroidogenic acute regulatory Wycherley, G., Kane, M.T., Hynes, A.C., 2005. Oxidative phosphorylation and the protein (StAR). J. Biol. Chem. 277, 47123–47128. tricarboxylic acid cycle are essential for normal development of mouse ovarian Van Blerkom, J., 2008. Mitochondria as regulatory forces in oocytes, follicles. Hum. Reprod. 20, 2757–2763. preimplantation embryos and stem cells. Reprod. Biomed. Online 16, 553–569. Xu, G., Vogel, K.S., McMahan, C.A., Herbert, D.C., Walter, C.A., 2010. BAX and tumor Van Blerkom, J., 2009. Mitochondria in early mammalian development. Semin. Cell suppressor TRP53 are important in regulating mutagenesis in spermatogenic Dev. Biol. 20, 354–364. cells in mice. Biol. Reprod. 83, 979–987. Van Blerkom, J., 2011. Mitochondrial function in the human oocyte and embryo and Yacobi, K., Tsafriri, A., Gross, A., 2007. Luteinizing hormone-induced caspase their role in developmental competence. Mitochondrion 11, 797–813. activation in rat preovulatory follicles is coupled to mitochondrial Varum, S., Momcilovic, O., Castro, C., Ben-Yehudah, A., Ramalho-Santos, J., Navara, steroidogenesis. Endocrinology 148, 1717–1726. C.S., 2009. Enhancement of human embryonic stem cell pluripotency through Yamamoto, C.M., Hikim, A.P., Lue, Y., Portugal, A.M., Guo, T.B., Hsu, S.Y., Salameh, inhibition of the mitochondrial respiratory chain. Stem Cell Res. 3, 142–156. W.A., Wang, C., Hsueh, A.J., Swerdloff, R.S., 2001. Impairment of Varum, S., Rodrigues, A.S., Moura, M.B., Momcilovic, O., Easley, C.A.t., Ramalho- spermatogenesis in transgenic mice with selective overexpression of Bcl-2 in Santos, J., Van Houten, B., Schatten, G., 2011. Energy metabolism in human the somatic cells of the testis. J. Androl. 22, 981–991. pluripotent stem cells and their differentiated counterparts. PLoS ONE 6, Yan, W., Huang, J.X., Lax, A.S., Pelliniemi, L., Salminen, E., Poutanen, M., Toppari, J., e20914. 2003. Overexpression of Bcl-W in the testis disrupts spermatogenesis: Wang, Q., Sun, Q.Y., 2007. Evaluation of oocyte quality: morphological, cellular and revelation of a role of BCL-W in male germ cell cycle control. Mol. Endocrinol. molecular predictors. Reprod. Fertil. Dev. 19, 1–12. 17, 1868–1879. Wang, Q., Ratchford, A.M., Chi, M.M., Schoeller, E., Frolova, A., Schedl, T., Moley, K.H., Yu, Y., Dumollard, R., Rossbach, A., Lai, F.A., Swann, K., 2010. Redistribution of 2009. Maternal diabetes causes mitochondrial dysfunction and meiotic defects mitochondria leads to bursts of ATP production during spontaneous mouse in murine oocytes. Mol. Endocrinol. 23, 1603–1612. oocyte maturation. J. Cell. Physiol. 224, 672–680. Wang, Q., Frolova, A.I., Purcell, S., Adastra, K., Schoeller, E., Chi, M.M., Schedl, T., Zhang, X., Wu, X.Q., Lu, S., Guo, Y.L., Ma, X., 2006. Deﬁcit of mitochondria-derived Moley, K.H., 2010. Mitochondrial dysfunction and apoptosis in cumulus cells of ATP during oxidative stress impairs mouse MII oocyte spindles. Cell Res. 16, type I diabetic mice. PLoS ONE 5, e15901. 841–850. Watanabe, T., Chuma, S., Yamamoto, Y., Kuramochi-Miyagawa, S., Totoki, Y., Toyoda, A., Hoki, Y., Fujiyama, A., Shibata, T., Sado, T., Noce, T., Nakano, T., Nakatsuji, N.,