Subjects with Early-Onset Type 2 Diabetes Show Defective Activation of the Skeletal Muscle PGC-1␣/Mitofusin-2 Regulatory Pathway in Response to Physical Activity

Pathophysiology/Complications ORIGINAL ARTICLE

Subjects With Early-Onset Type 2 Diabetes Show Defective Activation of the Skeletal Muscle PGC-1␣/Mitofusin-2 Regulatory Pathway in Response to Physical Activity

4 MARÍA ISABEL HERNANDEZ´ -ALVAREZ, FRANCIS FINUCANE, MD tive treatments to improve insulin sensi- 1,2,3 1,2,3 MSC MARC LIESA, PHD tivity. We have been studying the effects 4 5 HOOD THABIT, MD CHIARA CHIELLINI, PHD of a variety of exercise and dietary regi- 4 1,2,3 NICOLE BURNS, MSC DEBORAH NAON, MSC mens in these very insulin-resistant pa- 4 1,2,3 SYED SHAH, MD ANTONIO ZORZANO, PHD 4 4 tients. We recently demonstrated that a IMAD BREMA, MD OHN OLAN MD 4 J J. N , 3-month, four times weekly, aerobic ex- MENSUD HATUNIC, MD ercise intervention in subjects with early- onset type 2 diabetes failed to improve OBJECTIVE — Type 2 diabetes is associated with insulin resistance and skeletal muscle VO2max and had no signiﬁcant effect on mitochondrial dysfunction. We have found that subjects with early-onset type 2 diabetes show whole-body or hepatic insulin sensitivity

incapacity to increase VO2max in response to chronic exercise. This suggests a defect in muscle (3). Equally obese nondiabetic control mitochondrial response to exercise. Here, we have explored the nature of the mechanisms subjects had a 20% increase in VO2max involved. following the same regime. This sug- gested the possibility that, in these dia- RESEARCH DESIGN AND METHODS — Muscle biopsies were collected from young betic patients, chronic exercise training type 2 diabetic subjects and obese control subjects before and after acute or chronic exercise protocols, and the expression of genes and/or proteins relevant to mitochondrial function was failed to activate a mitochondrial oxida- measured. In particular, the regulatory pathway peroxisome proliferator–activated receptor ␥ tive response. coactivator (PGC)-1␣/mitofusin-2 (Mfn2) was analyzed. Defective mitochondrial function in skeletal muscle has been reported in a va- RESULTS — At baseline, subjects with diabetes showed reduced expression (by 26%) of the ␣ riety of insulin-resistant states, including mitochondrial fusion protein Mfn2 and a 39% reduction of the -subunit of ATP synthase. Porin type 2 diabetes (4,5). Muscle mitochon- expression was unchanged, consistent with normal mitochondrial mass. Chronic exercise led to a 2.8-fold increase in Mfn2, as well as increases in porin, and the ␣-subunit of ATP synthase in dria from type 2 diabetic subjects show muscle from control subjects. However, Mfn2 was unchanged after chronic exercise in individ- reduced size and reduced activity of the uals with diabetes, whereas porin and ␣-subunit of ATP synthase were increased. Acute exercise electron transport chain (4,6). In parallel, caused a fourfold increase in PGC-1␣ expression in muscle from control subjects but not in type 2 diabetes is associated with reduced subjects with diabetes. expression of genes of oxidative metabolism as well as repression of the mito- CONCLUSIONS — Our results demonstrate alterations in the regulatory pathway that con- fusin-2 (Mfn2) gene, which encodes the trols PGC-1␣ expression and induction of Mfn2 in muscle from patients with early-onset type 2 diabetes. Patients with early-onset type 2 diabetes display abnormalities in the exercise- mitochondrial fusion protein mitofusin-2 dependent pathway that regulates the expression of PGC-1␣ and Mfn2. (7–9). Decreased expression of nuclear genes encoding proteins of oxidative Diabetes Care 33:645–651, 2010 phosphorylation has been reported in skeletal muscle of nondiabetic individuals arly-onset type 2 diabetes is increas- obesity and severe insulin resistance in with a family history of type 2 diabetes ing in prevalence, in parallel with the young people with a strong family history (8,9), along with reduced in vivo oxida- E worldwide obesity epidemic (1), and of type 2 diabetes (1,2). Weight reduction tive phosphorylation (5). These ﬁndings is typically characterized by early-onset and increased physical exercise are effec- suggest that mitochondrial abnormalities in type 2 diabetes may have a heritable ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● component. From the 1Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain; the 2Departament de Chronic exercise induces mitochon- Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; the drial biogenesis in skeletal muscle and en- 3CIBER de Diabetes y de Enfermedades Metabo´licas Asociadas (CIBERDEM), Barcelona, Spain; the 4Met- abolic Research Unit, Department of Endocrinology, Hospital 5, St. James’ Hospital, Trinity College hances mitochondrial function (10). Dublin, Dublin, Ireland; and the 5Institute of Internal Medicine, Catholic University School of Medicine, Exercise is known to induce PGC-1␣ Rome, Italy. (11), which in turn induces the transcrip- Corresponding author: John J. Nolan, [email protected]. tion of different nuclear genes encoding Received 22 July 2009 and accepted 14 December 2009. Published ahead of print at http://care. diabetesjournals.org on 23 December 2009. DOI: 10.2337/dc09-1305. mitochondrial proteins (12,13). One ex- M.I.H.-A. and H.T. contributed equally to this work. A.Z. and J.J.N. share senior authorship. ample is Mfn2, which is induced by © 2010 by the American Diabetes Association. Readers may use this article as long as the work is properly PGC-1␣ through interaction with the cited, the use is educational and not for proﬁt, and the work is not altered. See http://creativecommons. transcription factor ERR␣ (14). This may org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby be particularly relevant, since it has been marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. reported that Mfn2 regulates not just mi-

care.diabetesjournals.org DIABETES CARE, VOLUME 33, NUMBER 3, MARCH 2010 645 PGC-1␣/mitofusin-2 pathway in early type 2 diabetes tochondrial fusion but also mitochondrial Table 1—Baseline characteristics of subjects in the chronic exercise study function through changes in mitochondrial membrane potential and the expres- Youth with type 2 sion of OXPHOS subunits (15). The Control subjects diabetes stimulatory effect of exercise on mitochondrial biogenesis and function has also been n 67 reported in muscle in insulin-resistant con- Male:female ratio 0:6 5:2 ditions such as obesity (6) and aging (16) Age (years) 22 Ϯ 127Ϯ 1* and in type 2 diabetes (17). Duration of diabetes (years) NA 3.2 Ϯ 1.5 Subjects who took part in the chronic Treatment for diabetes NA Met 7; Met/SU 1 exercise protocol (3) underwent skeletal Weight (kg) 108.7 Ϯ 10.6 108.1 Ϯ 6.7 muscle biopsies at baseline and after 3 BMI (kg/m2) 37.78 Ϯ 3.43 33.23 Ϯ 1.81 months of exercise training. To address Systolic blood pressure (mmHg) 104 Ϯ 3 122 Ϯ 5* mechanistic questions, we measured the Diastolic blood pressure (mmHg) 71 Ϯ 372Ϯ 3 expression of mitochondrial proteins Waist circumference (cm) 113.2 Ϯ 4.6 117.4 Ϯ 3.2 from this initial study. We then con- Waist-to-hip ratio 0.91 Ϯ 0.03 1.00 Ϯ 0.09 ducted an acute (short-term) exercise in- A1C (%) 5.5 Ϯ 0.2 8.2 Ϯ 0.6† tervention protocol in a similar cohort of Fasting glucose (mmol/) 5.2 Ϯ 0.2 9.1 Ϯ 0.8† patients with early-onset type 2 diabetes Fasting insulin (pmol/l) 91.1 Ϯ 9.4 77.1 Ϯ 12.3 and examined the expression in muscle Fasting C-peptide (␮g/l) 3.4 Ϯ 0.3 3.1 Ϯ 0.4 biopsies of a range of specific mitochon- Total cholesterol (mmol/l) 4.33 Ϯ 0.21 4.50 Ϯ 0.42 drial genes and proteins. We hypothe- HDL (mmol/) 1.11 Ϯ 0.08 0.88 Ϯ 0.03* sized that the lack of a whole-body LDL (mmol/l) 2.57 Ϯ 0.23 2.34 Ϯ 0.35 response to exercise training in the early- Triglycerides (mmol/l) 1.41 Ϯ 0.27 3.05 Ϯ 0.64* onset type 2 diabetic subjects may be a Free fatty acids (mmol/l) 0.635 Ϯ 0.053 0.820 Ϯ 0.104 consequence of alterations in the abun- Glucose disposal (␮mol/min/kg/mU/l) 26.25 Ϯ 4.66 16.13 Ϯ 4.16 Ϫ1 Ϫ1 Ϯ Ϯ dance or activity of relevant mitochon- Vo2max (ml kg min ) 28.61 1.94 22.67 1.57* drial proteins in skeletal muscle. Data are means Ϯ SE, unless specified otherwise. *Significantly different from control group (P Ͻ 0.05). †Significantly different from control group (P Ͻ 0.01). Met, metformin; SU, sulfonylurea. RESEARCH DESIGN AND METHODS — Subjects with early- onset type 2 diabetes (i.e., diagnosed before age 25 years and negative for GAD hypoglycemic agents were on stable Aerobic capacity (VO2peak). Maximal antibodies) were recruited from our doses throughout the duration of the oxygen consumption was measured by clinic, along with obese and otherwise study. Subjects on the combination treadmill, as previously described (3). healthy subjects who were as far as possible therapy had their insulin doses reduced Muscle biopsy. Muscle biopsies were ϳ matched for age and BMI, but with normal by 20% during the course of the taken either after an overnight fast or im- glucose tolerance and without family his- study. mediately after the most recent session of tory of diabetes. All subjects were sedentary. exercise, as described below. All gave written informed consent for the Baseline studies Biopsies were obtained under local study, which had been approved by the lo- Baseline studies were identical for both anesthesia from the vastus lateralis mus- cal research ethics committee. the chronic and acute exercise studies (3) cle. The muscle samples were immedi- Clinical and metabolic characteristics and included the following. ately frozen in liquid nitrogen and stored of both groups of subjects, in each study, Screening. Each subject was screened for protein extraction for samples from are summarized in Tables 1 and 2. with a medical history and physical exam- pre- and 12 weeks post-exercise program ination and routine blood and urine bio- (chronic exercise study) and subse- Concurrent medications chemistries. Waist-to-hip ratio, weight, quently for both RNA and protein extrac- None of the obese control subjects was height, and BMI were measured. Blood tion (acute exercise study: for samples receiving medications during the course pressure was measured using the left arm pre- and 1-h and 7 days post-exercise). of these studies. after the subject had been sitting comfort- Hyperinsulinemic-euglycemic clamp. A clamp study (with insulin infusion 40 Chronic exercise study. All subjects ably for 5 min, using an oscillometric de- Ϫ Ϫ were receiving metformin. One subject vice (Omron 705 CP). Three readings mU m 2 min 1) was performed at was also receiving gliclazide. Doses of oral were taken and the lowest one recorded. baseline and after exercise training in the hypoglycemic agents were unchanged Body composition was assessed using an chronic exercise study (3). Subjects tak- during the course of the study. electrical impedance device (Tanita Body ing insulin omitted the basal dose on the Acute exercise study. Three subjects Composition Analyzer). An exercise night before the clamp as well as the dose on were on dietary management alone. Seven stress test with electrocardiogram and ox- the morning of the clamp study. The glu- of the 12 subjects were receiving oral hy- ygen uptake was performed. Subjects cose disposal rate (between 80 and 120 min poglycemic agents alone. Two subjects with any abnormal stress response were after commencement of the insulin infu- were on oral and insulin combination excluded, as were those with clinically sion) was calculated after correction for re- therapy (metformin plus basal bolus significant abnormalities on routine lab sidual hepatic glucose production using the 2 insulin treatment). All subjects on oral testing. [6,6- H2] glucose tracer.

646 DIABETES CARE, VOLUME 33, NUMBER 3, MARCH 2010 care.diabetesjournals.org Herna´ndez-Alvarez and Associates

␮ Table 2—Baseline characteristics of subjects in the acute exercise study mm MgCl2, pH adjusted to 6.8, 2 m leupeptin, 2 ␮m pepstatin, 0.5 mm phenyl- Youth with type 2 methylsulphonylfluoride) and then Control subjects diabetes disrupted with a motor-driven Teflon/glass homogenizer. The entire procedure was n 712performed at 0–4°C. The protein concen- Male:female ratio 2:5 10:2 tration was determined using a Micro BCA Age (years) 26 Ϯ 227Ϯ 1 protein assay (Pierce, Rockford, IL). West- Duration of diabetes (years) NA 2.5 Ϯ 0.7 ern blot assays were performed as reported Treatment for diabetes NA Met 9; Met/Ins 2; Diet 3 (15) (see online appendix). Weight (kg) 113.9 Ϯ 11.2 114.5 Ϯ 7.3 BMI (kg/m2) 39.13 Ϯ 2.54 36.13 Ϯ 1.78 Statistical analysis Systolic blood pressure (mmHg) 117 Ϯ 3 127 Ϯ 4 Unpaired t tests were performed to compare Diastolic blood pressure (mmHg) 75 Ϯ 481Ϯ 3 muscle protein expression between control Waist circumference (cm) 120.2 Ϯ 6.1 111.4 Ϯ 4.8 subjects and subjects with diabetes at base- Waist-to-hip ratio 0.98 Ϯ 0.03 0.99 Ϯ 0.02 line (Fig. 1A). Paired t tests were performed A1C (%) 5.6 Ϯ 0.1 7.5 Ϯ 0.5† to compare the effects of chronic exercise Fasting glucose (mmol/l) 5.2 Ϯ 0.1 8.0 Ϯ 0.7† (Fig. 1B) and acute exercise (Fig. 1C)onthe Fasting insulin (pmol/l) 163.0 Ϯ 65.3 169.6 Ϯ 61.7 expression of a range of proteins and RNAs. Fasting C-peptide (␮g/l) 5.3 Ϯ 1.2 3.4 Ϯ 0.4* In all cases, significance level for the t tests Total cholesterol (mmol/l) 4.13 Ϯ 0.32 4.29 Ϯ 0.26 was set at P Ͻ 0.05. HDL (mmol/l) 1.30 Ϯ 0.17 0.95 Ϯ 0.05* LDL (mmol/l) 2.21 Ϯ 0.29 2.63 Ϯ 0.28 RESULTS Triglycerides (mmol/l) 1.35 Ϯ 0.20 1.76 Ϯ 0.26 Free fatty acids (mmol/l) 0.67 Ϯ 0.16 0.77 Ϯ 0.22 Clinical and metabolic Ϫ1 Ϫ1 Ϯ Ϯ Vo2max (ml kg min ) 22.85 2.71 23.79 1.79 measurements Data are means Ϯ SE, unless specified otherwise. *Significantly different from control group (P Ͻ 0.05). Chronic exercise study. Baseline char- †Significantly different from control group (P Ͻ 0.01). Ins, insulin; Met, metformin. acteristics (Table 1) of the chronic exercise participants have been described previously (3). The nondiabetic control Exercise training after the last exercise session at the end of subjects were matched for BMI with the For more information on exercise train- the study (3) (supplementary Fig. 1). diabetic group, but the diabetic subjects ing, see supplementary Figs. 1 and 2 in Acute exercise. After completion of all were slightly older. The 3-month exercise the online appendix at http://care. baseline measurements and a baseline training program led to no significant diabetesjournals.org/cgi/content/full/dc09- muscle biopsy, subjects exercised for a change in whole-body or hepatic insulin 1305/DC1. The subjects maintained a sta- single 1-h session at 70% of VO2max.A sensitivity in either the control subjects or ble diet and treatment for diabetes during second muscle biopsy was taken immedi- the subjects with diabetes (3). VO2max in- both exercise programs. Subjects exer- ately after the first session of exercise. The creased by 20% in the control group cised either on a treadmill or a stationary subjects then exercised for 1 h daily for 7 (from 28.61 Ϯ 1.94 to 35.15 Ϯ 2.95 ml Ϫ Ϫ bicycle ergometer. Each session lasted for a days, followed by a final muscle biopsy kg 1 min 1, P Ͻ 0.01), but was un- total of 70 min (5-min warm-up, 60-min immediately after the final exercise ses- changed in the group with diabetes Ϫ exercise, 5-min cool down) and was fully sion at the end of this study (supplemen- (22.67 Ϯ 1.57 vs. 24.40 Ϯ 1.50 ml kg 1 Ϫ supervised by an exercise physiologist or tary Fig. 2). min 1, P ϭ NS) after exercise. Fasting physician. Each exercise session was con- RNA extraction and real-time quantita- plasma free fatty acid concentrations tended ducted at the same intensity, i.e., at 70% of tive PCR (acute exercise study). For to be lower at baseline, although not the subject’s VO2max. This intensity of exer- more information on the RNA extraction reaching significance, in the control cise has previously been shown by us and and real-time quantitative PCR, see the group (0.635 vs. 0.820 mmol/l, P ϭ NS) others to improve insulin sensitivity in online appendix. and were significantly lower (0.561 vs. obese middle-aged subjects with type 2 di- Homogenate extracts from muscle bi- 0.826 mmol/l, P ϭ 0.003) in the control abetes. Blood glucose was checked before opsies and Western blot assays (acute subjects after the 3-month exercise in- exercise, and heart rate and blood pressure and chronic exercise studies). Protein tervention (3). No correlation was found ϳ were monitored throughout exercise. Com- homogenate was extracted from 25 mg between either age or sex and VO2max (data pliance with the exercise regimens was skeletal muscle. The muscle was im- not shown). greater than 90%. mersed in ice-cold medium buffer A (0.1 Acute exercise study. The acute exer- Chronic exercise. After completion of all mM KCL, 5 mm MgCl2, 5 mm EGTA, 5 cise participants (Table 2) had a similar baseline measurements and a baseline mus- mm sodium pyrophosphate, pH adjusted phenotype to individuals we have re- cle biopsy, subjects exercised for 4 1-h ses- to 7.4, 2 ␮m leupeptin, 2 ␮m pepstatin, ported from previous studies (2,3). The sions each week. Each exercise session was 0.5 mm phenylmethylsulphonylfluoride control subjects were matched for age ϫ conducted at 70% of the subject’s VO2max. for 2 10 min). After this, the muscle and BMI and had similar VO2max at The study continued for a total of 12 weeks. was finely minced in 1/10 (wt/vol) buffer baseline to individuals with diabetes Baseline measurements were repeated and a B (0.25 mM sacharose, 50 mm KCl, 5 mm (22.85 Ϯ 2.71 vs. 23.79 Ϯ 1.79 ml Ϫ Ϫ final muscle biopsy was taken immediately EDTA, 1 mm sodium pyrophosphate, 5 kg 1 min 1, respectively; P ϭ NS).

care.diabetesjournals.org DIABETES CARE, VOLUME 33, NUMBER 3, MARCH 2010 647 PGC-1␣/mitofusin-2 pathway in early type 2 diabetes

Figure 1—A: Baseline: Young type 2 diabetic subjects show an impaired expression of mitochondrial proteins in skeletal muscle. Western blot assays were performed in extracts obtained from skeletal muscle biopsies from 11 normal glucose tolerant (control) and 16 young type 2 diabetic patients (young T2D). Data are means Ϯ SE. *Statistically significant difference compared with the control group at P Ͻ 0.05. Representative autoradiograms are also shown. B: Chronic exercise. Chronic exercise causes a deficient induction of muscle mitochondrial proteins in young type 2 diabetic subjects. Western blot assays were performed in extracts obtained from skeletal muscle biopsies from normal glucose tolerant and young type 2 diabetic patients before and after a protocol of chronic exercise. Representative autoradiograms are shown in the lower right corner. Data are means Ϯ SE. *Statistical significant difference compared with basal values at P Ͻ 0.05. C: Acute exercise. Acute exercise induces skeletal muscle PGC-1␣ gene expression in control but not in young type 2 diabetic subjects. Real-time PCR was performed in skeletal muscle biopsies from nine normal glucose tolerant and six young type 2 diabetic patients before and after an acute session of exercise. Data are means Ϯ SE. *Statistical significant difference compared with basal values at P Ͻ 0.05.

Neither VO2max nor insulin sensitivity The young type 2 diabetic group porin (1.6-fold increase), and p37 sub- were re-measured at the end of this pro- showed a reduced expression of the mi- unit of Complex I (Ndufa9) (1.7-fold tocol, since neither of these parameters tochondrial fusion protein, Mfn2 (26% ncrease) in skeletal muscle from control were expected to change after only 1 week reduction in those with diabetes) and subjects (Fig. 1B). In the young type 2 of exercise training. the alpha subunit of ATP synthase diabetic group, chronic exercise caused a (ATP5a1) (39% reduction in those with different pattern of muscle changes. The Expression of skeletal muscle diabetes) (Fig. 1A). Under these condi- expression of Mfn2 was unchanged, while mitochondrial proteins at baseline tions, the abundance of porin (a marker there was induction in both Ndufa9 (2.0- (combined baseline data from the of mitochondrial mass) and of the p37 fold increase) and porin (1.6-fold increase chronic and acute studies) subunit of Complex I of the respiratory but not reaching statistical signiﬁcance) Muscle biopsies were collected from chain (Ndufa9) was unaltered indicat- (Fig. 1B). young type 2 diabetes or control sub- ing that the changes of Mfn2 and ATP jects, homogenates were obtained and synthase were not secondary to changes Mitochondrial gene expression in the expression of mitochondrial pro- in mitochondrial mass (Fig. 1A). skeletal muscle in response to acute teins was studied. The yield of total pro- exercise: acute exercise study teins was similar in homogenates from Expression of skeletal muscle Muscle samples taken after 7 days of ex- control and young type 2 diabetic sub- mitochondrial proteins after 3 months ercise showed no signiﬁcant changes in jects (54.1 Ϯ 2.6 and 54.8 Ϯ 3.1 mg exercise: chronic exercise study gene expression (data not shown). How- protein/g of tissue in control and young iChronic exercise was associated with the ever, acute exercise after 1 h caused a sub- type 2 diabetic groups, respectively). induction of Mfn2 (2.8-fold increase), stantial induction in PGC-1␣ gene

648 DIABETES CARE, VOLUME 33, NUMBER 3, MARCH 2010 care.diabetesjournals.org Hernańdez-Alvarez and Associates expression in skeletal muscle from con- whole-body findings in these subjects. PGC-1␣ expression was detected in mus- trol subjects (fourfold induction) (Fig. Thus, in the obese nondiabetic subjects, cle from obese subjects in response to ex- 1C). These changes in PGC-1␣ had re- acute exercise intervention was associated ercise (22). Under these conditions, the verted to baseline after 7 days of exercise with increased muscle expression of expression of genes downstream of (data not shown). Control subjects also genes encoding for PGC-1␣ and ERR␣ PGC-1␣ such as NRF-1 or cytochrome c showed an increased ERR␣ gene expres- under conditions in which Mfn2 or porin oxidase subunit VIc also showed a re- sion (2.2-fold increase), although this did was unchanged. These data are consistent duced response in obese subjects after ex- not reach statistical significance and no with prior observations indicating that ercise (22). changes in the gene expression of PGC- AMPK activity is stimulated by acute ex- Interestingly, we have also noted a 1␤, porin, Mfn2, or the mitochondrial ercise in skeletal muscle in humans (18) markedly different response to bariatric gene COXIII (Fig. 1C). In contrast, acute and that PGC-1␣ gene expression and surgery in morbidly obese diabetic sub- exercise led to no change in PGC-1␣ gene promoter activity are stimulated by jects compared with a matched nondia- expression in skeletal muscle in the young AMPK (19). Moreover, and based on the betic cohort (23). Despite similar weight type 2 group, and the expression of ERR␣ observations that Mfn2 gene transcription loss (ϳ60 kg) and a marked improvement (23% reduction), Mfn2 (30% reduction), is induced by ERR␣, and coactivated by in insulin sensitivity in both groups, the or porin (23% reduction) was signifi- PGC-1␣ (14), we propose that enhanced patients with diabetes showed a blunted cantly reduced (Fig. 1C). No changes in PGC-1␣ activity will promote an increase response in terms of glucose oxidation PGC-1␤ or COXIII gene expression were in Mfn2 gene transcription. In contrast and no significant changes in the expres- detected in young type 2 diabetic subjects with this profile, acute exercise caused a sion of Mfn2, porin, and citrate synthase (Fig. 1C). We analyzed whether AMPK reduction in the expression of genes en- (23). In all, these data together with data was stimulated under these conditions; coding Mfn2 and porin in muscle from from our current study suggest a link be- however, we detected no phosphoryla- the subjects with diabetes under condi- tween insulin resistance and a defective tion of AMPK in either the control or type tions in which PGC-1␣ remained un- regulation of PGC-1␣ and downstream 2 diabetic subjects (data not shown). No changed. These data can be explained by mitochondrial proteins in response to ex- correlation was found between VO2max the incapacity of acute exercise to stimu- ercise, so that under conditions of severe and the expression of mitochondrial late AMPK activity in patients with diabe- insulin resistance, the induction of genes or proteins (data not shown). tes, which cancels the induction of PGC-1␣ in response to exercise is abol- PGC-1␣ gene expression. We also pro- ished. Further studies should investigate CONCLUSIONS — We have previ- pose that the reduced expression of the whether the defective PGC-1␣ expression ously reported that young subjects with Mfn2 gene (and perhaps the porin gene, is a primary defect or whether it is second- type 2 diabetes are severely insulin resis- which also encodes for a mitochondrial ary to insulin resistance or reversible tant, achieving a maximal glucose dis- protein) may be due to reduced transcrip- lipotoxicity. Ϫ posal rate of only 2.15 Ϯ 0.42 mg kg 1 tional activities of either PGC-1␣ or PGC- There are further possible explana- Ϫ min 1, compared with 4.09 Ϯ 0.58 mg 1␤, key regulators of Mfn2 transcription tions to link environmental effects such as Ϫ Ϫ kg 1 min 1 in very obese control sub- (14,20). In the obese nondiabetic sub- diet and physical activity with abnormal- jects (3). We undertook the current stud- jects, chronic exercise training led to an ities of mitochondrial oxidative proteins ies to directly analyze a range of increase in both Mfn2 (2.8-fold) and in patients with type 2 diabetes. In a re- parameters of muscle mitochondrial porin (1.6-fold), consistent with the in- cent muscle biopsy study in human sub- function in these subjects at baseline and crease in VO2max observed after 3 months. jects including control subjects, subjects after different durations of exercise train- In subjects with diabetes, however, we with impaired glucose tolerance, and sub- ing. The mitochondrial parameters reveal found no increase in Mfn2, consistent jects with type 2 diabetes, it was shown interesting new abnormalities in the sub- with the lack of stimulation of whole- that type 2 diabetes is associated with hy- jects with diabetes both at baseline and body oxygen uptake. In spite of the lack permethylation of PGC-1␣, concomitant after exercise. At baseline, subjects with of induction of PGC-1␣ or Mfn2, with reduced mitochondrial content (24). diabetes display modestly (ϳ25%) re- chronic exercise induced porin or Epigenetic effects may also modulate the duced expression of Mfn2 and some Ndufa9 expression in the type 2 dia- effect of diet and activity on the pathogen- OXPHOS subunits. Thus, in the baseline betic subjects. The induction of porin or esis of diabetes and could explain at least state, without any intervention, subjects Ndufa9 in the absence of PGC-1␣ sug- some of the abnormalities observed in the with diabetes showed a defective pattern gests the existence of mechanisms of current study. of mitochondrial protein expression in mitochondrial biogenesis, alternative to We acknowledge some limitations muscle compared with equally obese PGC-1␣ gene induction, that are trig- in the current studies. The overall num- young people without diabetes. The pa- gered by physical exercise. ber of subjects included is small, and it tients with type 2 diabetes, in comparison Several studies have analyzed the ef- was not possible with these numbers to with matched obese control subjects, fect of dietary and/or exercise interven- match groups for sex. Early-onset type 2 were markedly dyslipidemic. Lipotoxicity tions aimed to promote weight loss in diabetes remains uncommon, and it is in the patients with diabetes at baseline obese or type 2 diabetic subjects. These challenging to recruit young subjects of could either cause or result from the mi- studies have detected that weight loss in- working age for these relatively com- tochondrial abnormalities that we have duced by diet/exercise stimulate mito- plex protocols. In the initial (chronic demonstrated in the current study. chondrial activity in skeletal muscle both exercise) study, the skeletal muscle in- After exercise, the mitochondrial in obese subjects as well as in type 2 dia- vestigations were limited by the biopsy changes we have observed are much more betic patients (17,21,22). In a recent amount, and it was not possible to mea- pronounced and are consistent with the study, a delayed and reduced response in sure PGC-1␣ or other gene expression. care.diabetesjournals.org DIABETES CARE, VOLUME 33, NUMBER 3, MARCH 2010 649 PGC-1␣/mitofusin-2 pathway in early type 2 diabetes

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In addition, subjects with early- sulin secretion, severe insulin resistance specify PGC-1alpha-dependent oxidative onset type 2 diabetes are characterized by and a lack of response to aerobic exercise phosphorylation gene expression that is a deficient capacity to induce PGC-1␣ or training. Diabetologia 2007;50:1500– altered in diabetic muscle. Proc Natl Acad Mfn2 in response to aerobic exercise 1508 SciUSA2004;101:6570–6575 training. These alterations in Mfn2 ex- 4. Kelley DE, He J, Menshikova EV, Ritov 13. Wu Z, Puigserver P, Andersson U, Zhang pression and the failure to stimulate VB. Dysfunction of mitochondria in hu- C, Adelmant G, Mootha V, Troy A, Cinti PGC-1␣ may be relevant to the ob- man skeletal muscle in type 2 diabetes. S, Lowell B, Scarpulla RC, Spiegelman served incapacity in these patients to Diabetes 2002;51:2944–2950 BM. Mechanisms controlling mitochon- 5. Petersen KF, Dufour S, Befroy D, Garcia drial biogenesis and respiration through enhance whole-body VO in re- 2max R, Shulman GI. Impaired mitochondrial the thermogenic coactivator PGC-1. Cell sponse to exercise training. Further activity in the insulin-resistant offspring 1999;98:115–124 mechanistic studies of these pathways of patients with type 2 diabetes. N Engl 14. Soriano FX, Liesa M, Bach D, Chan DC, in this patient group are clearly indi- J Med 2004;350:664–671 Palacín M, Zorzano A. Evidence for a mi- cated. A more complete understanding 6. Toledo FG, Watkins S, Kelley DE. tochondrial regulatory pathway defined of these mechanisms will be crucial to Changes induced by physical activity and by peroxisome proliferator-activated re- the design of lifestyle interventions to weight loss in the morphology of inter- ceptor-gamma coactivator-1 alpha, estro- prevent and treat type 2 diabetes in ad- myofibrillar mitochondria in obese men gen-related receptor-alpha, and mitofusin olescents and young adults. and women. J Clin Endocrinol Metab 2. Diabetes 2006;55:1783–1791 2006;91:3224–3227 15. Pich S, Bach D, Briones P, Liesa M, Camps 7. Bach D, Naon D, Pich S, Soriano FX, Vega M, Testar X, Palacín M, Zorzano A. The Acknowledgments— This study was funded N, Rieusset J, Laville M, Guillet C, Boirie Charcot-Marie-Tooth type 2A gene prod- in part by grant support from the European Y, Wallberg-Henriksson H, Manco M, uct, Mfn2, up-regulates fuel oxidation Foundation for the Study of Diabetes/Novo Calvani M, Castagneto M, Palacín M, Min- through expression of OXPHOS system. Nordisk (to J.J.N.) and by a grant from the EU grone G, Zierath JR, Vidal H, Zorzano A. Hum Mol Genet 2005;14:1405–1415 Commission (LHSM-CT-2003-503041) (to Expression of Mfn2, the Charcot-Marie- 16. Menshikova EV, Ritov VB, Fairfull L, Fer- J.J.N.). This study was also supported by re- Tooth neuropathy type 2A gene, in hu- rell RE, Kelley DE, Goodpaster BH. Effects search grants from the “Ministerio de Educa- man skeletal muscle: effects of type 2 of exercise on mitochondrial content and cioń y Cultura” (SAF 2005-00445 and SAF diabetes, obesity, weight loss, and the reg- function in aging human skeletal muscle. 2008-03803) and grant 2005SGR00947 from ulatory role of tumor necrosis factor alpha J Gerontol A Biol Sci Med Sci 2006;61: the “Generalitat de Catalunya.” CIBER de Di- and interleukin-6. Diabetes 2005;54: 534–540 abetes y Enfermedades Metabo´licas Asociadas 2685–2693 17. Toledo FG, Menshikova EV, Ritov VB, is an Instituto de Salud Carlos III project. M.H. 8. Mootha VK, Lindgren CM, Eriksson KF, Azuma K, Radikova Z, DeLany J, Kelley is the recipient of a predoctoral fellowship Subramanian A, Sihag S, Lehar J, Puig- DE. Effects of physical activity and from Consejo Nacional de Ciencia y Tecnolo- server P, Carlsson E, Ridderstråle M, Lau- weight loss on skeletal muscle mito- gia, Mexico. M.L. was the recipient of a pre- rila E, Houstis N, Daly MJ, Patterson N, chondria and relationship with glucose doctoral fellowship from the Ministerio de Mesirov JP, Golub TR, Tamayo P, controlintype2diabetes.Diabetes2007; Educacioń y Cultura, Spain. A.Z. was the re- Spiegelman B, Lander ES, Hirschhorn JN, 56:2142–2147 cipient of a Science Intensification Award from Altshuler D, Groop LC. PGC-1alpha-re- 18. Musi N, Fujii N, Hirshman MF, Ekberg I, the University of Barcelona. sponsive genes involved in oxidative Fro¨berg S, Ljungqvist O, Thorell A, Good- No other potential conflicts of interest rele- phosphorylation are coordinately down- year LJ. AMP-activated protein kinase vant to this article were reported. regulated in human diabetes. Nat Genet (AMPK) is activated in muscle of subjects The authors wish to thank the study pa- 2003;34:267–273 with type 2 diabetes during exercise. Di- tients and volunteers and their families, as 9. 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22. De Filippis E, Alvarez G, Berria R, Cusi K, 23. Herna´ndez-Alvarez MI, Chiellini C, diabetics. Diabetologia 2009;52:1618– Everman S, Meyer C, Mandarino LJ. Insu- Manco M, Naon D, Liesa M, Palacín M, 1627 lin-resistant muscle is exercise resistant: Mingrone G, Zorzano A. Genes involved 24. Barres R, Osler M, Yan J, Rune A, Fritz T, evidence for reduced response of nuclear- in mitochondrial biogenesis/function are Caidahl K, Krook A, Zierath J. Non-CpG encoded mitochondrial genes to exercise. induced in response to bilio-pancreatic methylation of the PGC-1␣ promoter Am J Physiol Endocrinol Metab 2008; diversion in morbidly obese subjects with through DNMT3B controls mitochondrial 294:E607–E614 normal glucose tolerance but not in type 2 density. Cell Metab 2009;1:189–198

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