Integration of Cellular Bioenergetics with Mitochondrial Quality Control and Autophagy

DOI 10.1515/hsz-2012-0198 Biol. Chem., 2012; 393(12): 1485–1512

Review

Bradford G. Hill , Gloria A. Benavides , Jack R. Lancaster Jr., Scott Ballinger , Lou Del l ’ Italia , Jianhua Zhang and Victor M. Darley-Usmar * Integration of cellular bioenergetics with mitochondrial quality control and autophagy

Abstract: Bioenergetic dysfunction is emerging as a cor- lar Biology and Physiology and Biophysics , University of Louisville, nerstone for establishing a framework for understanding Louisville, KY, USA Gloria A. Benavides: Department of Pathology, University of the pathophysiology of cardiovascular disease, diabetes, Alabama at Birmingham, Birmingham, AL 35294 , USA; and Center cancer and neurodegeneration. Recent advances in cel- for Free Radical Biology , University of Alabama at Birmingham, lular bioenergetics have shown that many cells maintain Birmingham, AL 35294 , USA a substantial bioenergetic reserve capacity, which is a Jack R. Lancaster, Jr.: Department of Anesthesiology, University of prospective index of ‘ healthy ’ mitochondrial popula- Alabama at Birmingham, Birmingham, AL 35294 , USA ; Center for Free Radical Biology , University of Alabama at Birmingham, tions. The bioenergetics of the cell are likely regulated by Birmingham, AL 35294 , USA ; Department of Physiology and energy requirements and substrate availability. Addition- Biophysics , University of Alabama at Birmingham, Birmingham, AL ally, the overall quality of the mitochondrial population 35294 , USA; and Department of Environmental Health Sciences, and the relative abundance of mitochondria in cells and Department of Medicine , University of Alabama at Birmingham, tissues also impinge on overall bioenergetic capacity and Birmingham, AL 35294 , USA resistance to stress. Because mitochondria are suscep- Scott Ballinger: Department of Pathology , University of Alabama at Birmingham, Birmingham, AL 35294 , USA; Center for Heart Failure tible to damage mediated by reactive oxygen/nitrogen Research , University of Alabama at Birmingham, Birmingham, AL and lipid species, maintaining a ‘ healthy ’ population 35294 , USA; and Center for Free Radical Biology , University of of mitochondria through quality control mechanisms Alabama at Birmingham, Birmingham, AL 35294 , USA appears to be essential for cell survival under conditions Lou Dell’Italia: Center for Free Radical Biology , University of of pathological stress. Accumulating evidence suggest Alabama at Birmingham, Birmingham, AL 35294, USA ; Center for Heart Failure Research , University of Alabama at Birmingham, that mitophagy is particularly important for preventing Birmingham, AL 35294 , USA; and Department of Veteran Affairs amplification of initial oxidative insults, which otherwise Medical Center , Birmingham, AL 35294 , USA would further impair the respiratory chain or promote Jianhua Zhang: Department of Pathology, University of mutations in mitochondrial DNA (mtDNA). The pro- Alabama at Birmingham, Birmingham, AL 35294 , USA; Center for cesses underlying the regulation of mitophagy depend Free Radical Biology , University of Alabama at Birmingham, on several factors, including the integrity of mtDNA, elec- Birmingham, AL 35294 , USA; and Department of Veteran Affairs Medical Center , Birmingham, AL 35294 , USA tron transport chain activity, and the interaction and reg- Victor M. Darley-Usmar: Center for Heart Failure Research , ulation of the autophagic machinery. The integration and University of Alabama at Birmingham, Birmingham, AL 35294 , USA ; interpretation of cellular bioenergetics in the context of Center for Free Radical Biology , University of Alabama at mitochondrial quality control and genetics is the theme Birmingham, Birmingham, AL 35294, USA; and Department of of this review. Environmental Health Sciences, Department of Medicine , University of Alabama at Birmingham, Birmingham, AL 35294 , USA Keywords: cardiovascular disease; haplotypes; mitophagy; neurodegenerative disease; spare respiratory capacity; xanthine oxidase. Introduction

*Corresponding author: Victor M. Darley-Usmar, Department of It has long been known that mitochondria show dimin- Pathology, University of Alabama at Birmingham, Birmingham, ished functional activity when isolated from a broad range AL 35294, USA, E-mail: [email protected] Bradford G. Hill: Diabetes and Obesity Center, Institute of Molecular of tissues subject to pathological stress. Well-established Cardiology, and Department of Medicine , University of Louisville, examples are alcohol-dependent hepatotoxicity, cardiac Louisville, KY, USA; and Departments of Biochemistry and Molecu- ischemia-reperfusion and neurodegenerative diseases 1486 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics

(Bailey and Cunningham , 2002 ; Jenner , 2003 ; Brookes other multiple factors coordinate the biogenesis of mito- et al. , 2004 ; Lemasters et al. , 2009 ; Carreira et al. , 2011 ; chondria (Michel et al. , 2012 ). Interesting examples of the Coskun et al., 2012; Krzywanski et al., 2011; Pilsl and pathological effects of a mismatch between energy demand Winklhofer , 2012 ). In all of these cases, a role for reac- and mitochondrial number was shown by studies where tive oxygen or nitrogen species (ROS/RNS) has been pro- expression of PGC1α was manipulated in the heart. PGC- posed in causing mitochondrial dysfunction. Typically, 1α − / − mice develop signatures of heart failure, and consti- mitochondrial damage is defined as a relative decrease in tutive, cardiac-specific overexpression of PGC-1α results in respiratory parameters, such as a change in oxygen con- excessive mitochondrial biogenesis, leading ultimately to sumption in the presence of substrates and ADP (state 3 death from heart failure (Finck and Kelly , 2007 ). It appears respiration), in the presence of substrates alone (state 4), then that mitochondrial abundance is finely tuned to the or the ratio of these parameters [i.e., respiratory control energetic needs of the cell – too few or too many mitochon- ratio (RCR)]. Other parameters include decreased activity dria can result in similar pathological outcomes. of specific enzymes, deletions in mtDNA and increases The obligation to match mitochondrial abundance in oxidative markers such as protein oxidation. The chal- and quality with energy demand is outlined in Figure 1 . lenge with these data is in integrating them into an overall Once a stable population of mitochondria is established model of cellular and tissue bioenergetic function. It is in a cell it must perform a series of integrated biological becoming apparent that mitochondria under pathological functions. These include the conversion of caloric energy stress exhibit multiple defects that overall can be viewed as a decrease in mitochondrial quality (Karbowski and Neutzner, 2012; Piantadosi and Suliman, 2012; Pilsl and Winklhofer, 2012). Importantly, it has recently been shown Biogenesis that the cell possesses a complex machinery capable of ↑ Reserve capacity ↑ Resistance to stress surveying mitochondrial quality and scheduling sections of defective organelles for demolition through a special- ized category of autophagy called mitophagy (Lemasters , ROS 2005; Kim et al., 2007; Youle and Narendra, 2011; Lee et Stress RNS RLS ROS al., 2012). This method of mitochondrial surveillance is ↑ mtDNA damage useful because it allows changes in mitochondrial param- ↑ ROS eters such as membrane potential, activity of oxidative ↑ ET damage phosphorylation, and mitochondrial damage to be placed Ca2+ ↓ Reserve capacity in the dynamic context of cell function. Fission The controlled regulation of mitochondrial abundance through biogenesis or degradation is also critical for maintaining proper cell function. The formation of ‘ new ’ mitochondria is a tightly controlled process involving several factors, including de novo synthesis of organelle components from cellular precursors, formation of mitochondrial Mitophagy membranes from pre-formed membranous structures, and Figure 1 Regulation of mitochondrial quality control and the division of pre-existing mitochondria (Michel et al., 2012). response to oxidative stress. For maintenance and modulation of energy production, An existing mitochondrial population is shown that was subjected the cell then must coordinate the biogenic process: the to a pathological stress with the formation of reactive oxygen, transcription, import, and assembly of nuclear-encoded nitrogen and lipid species (ROS/RNS/RLS). This oxidative stress damages mtDNA, impairing the ability of the organelle to replace genes must match with the replication and transcription damaged electron transport proteins and decreasing bioenergetic of the mitochondrial genome and with biogenesis of mito- reserve capacity; the resulting increased mitochondrial ROS then chondrial membranes. External stimuli have been shown oxidatively damages previously unmodified mitochondria. The to initiate this dynamic process. For example, caloric damaged mitochondria are turned over by a mitophagic mecha- restriction, exercise, hypothermic conditions, and energy nism that then suppresses this vicious cycle. The mitochondrial population is now renewed through mitochondrial biogenesis. The deprivation result in the activation of signaling pathways bioenergetic reserve capacity is essential for resistance to oxidative responsible for increasing mitochondrial abundance (Cao stress and supplying ATP demand. Once the bioenergetic reserve is et al., 2001; Wu et al., 2002 ; Nisoli et al., 2003; Bordicchia depleted, bioenergetic failure occurs and the cell is programmed for et al. , 2012 ). The PGC1 family, especially PGC1α , along with cell death. B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1487 into metabolic intermediates for cell differentiation, of mitochondria (mitophagy) is essential for mitochon- molecular energy (ATP), thermal energy (heat), and oxi- drial quality control (Kim et al. , 2007 ). It therefore follows dants. The generation of ROS from mitochondria appears that damaged mitochondria arise during normal metabo- to serve a cell signaling function when controlled at low lism and are potentially hazardous because of their ability levels but can contribute to pathology if it leads to damage to amplify ROS-dependent damage (Figure 1). Under to electron transport proteins and ‘ electron leak’ to oxygen pathological stressors several interrelated mechanisms at a non-physiological locations (Gutierrez et al. , 2006 ; can contribute to decreased mitochondrial quality. These Murphy, 2009). Under normal conditions mitochondrial include mitochondrial calcium overload, increased levels populations in cells appear highly stable. For example, it of ROS/RNS, endoplasmic reticulum stress and accumu- has been shown that mitochondria have a half-life of ∼ 9 lation of aggregated proteins. Where extra-mitochondrial days in the liver, ∼10 days in the kidney, ∼16 days in the sources of ROS/RNS are present, the quality of the mito- heart, and ∼26 days in the brain (Menzies and Gold, 1971). chondrial population declines, as evidenced by increased Quality control of individual mitochondrial proteins mtDNA damage, decreased membrane potential, and a is mediated by intra-mitochondrial proteases (Bota and diminished bioenergetic reserve capacity (Hill et al. , 2009 ; Davies , 2001 ; Ugarte et al. , 2010 ). The ATP-stimulated Dranka et al. , 2010 ; Zelickson et al. , 2011 ; Higdon et al. , mitochondrial Lon protease plays an important role in the 2012 ). Overwhelming the mitophagy pathway exacerbates degradation of oxidized proteins (Bayot et al., 2008). This the problem as the low quality mitochondria now impact process is important in the maintenance of proteins that on the remaining healthy mitochondrial population are highly susceptible to damage and therefore need to be through release of ROS and calcium from the organelle. turned over more frequently to sustain normal mitochon- Thus, it is clear that the dynamics, turnover and biogen- drial function. For example, oxidatively damaged acon- esis collectively maintain quality control. These processes itase has been shown to be selectively degraded by Lon are regulated by intracellular and extracellular signals protease (Bota and Davies , 2002 ). In response to hypoxia, that coordinate the biogenesis transcriptional program, Lon protease degrades cytochrome c oxidase subunit IV cellular bioenergetics, redox signaling and mitophagy. In isoform 1 to allow isoform 2 to replace its position (Fukuda this review we will discuss emerging concepts in assess- et al., 2007). The mitochondrial AAA-proteases have been ing cellular bioenergetic function, and how mitochondrial shown to be important for preventing mtDNA escape from quality is maintained in a cellular setting. In particular, mitochondria to the nucleus, possibly through ensuring we address the following questions: appropriate fission and fusion, as well as rapid proteolysis 1. How can bioenergetics be measured and placed in of non-assembled inner membrane proteins (Thorsness context with disease ? and Fox, 1990; Thorsness et al., 1993; Hori et al., 2002). 2. What is the role of the mitochondrial genetic land- In response to more extensive mitochondrial damage, scape in diseases based in bioenergetic dysfunction ? increased mitochondrial fragmentation and decreased 3. How does mitophagy contribute to mitochondrial fusion can separate damaged mitochondrial proteins, quality control ? lipids and DNA from functional components, and the entire damaged organelle is processed by mitophagy (Kim et al., 2007; Lee et al., 2012). It has recently been The cellular bioenergetic profile shown that the sub-populations of mitochondria destined for turnover are segregated through a dynamic process and its interpretation involving fission of areas of low functioning mitochondria and fusion of healthy populations (Twig et al. , 2008b ; Kim Typically, experiments with isolated mitochondria have and Lemasters , 2011 ). This turnover of mitochondria by been performed in the context of established protocols lysosomes is executed in a process now called autophagy performed with oxygen electrodes. The outputs from such of mitochondria, or mitophagy (Lemasters, 2005; Kim experiments are the rates of state 3 and 4 respiration and et al. , 2007 ; Green et al. , 2011 ; Youle and Narendra, 2011 ). the ratio of these two rates – the respiratory control ratio Because of their importance in providing cellu- (RCR) (Chance and Baltscheffsky, 1958a,b). Mitochondrial lar energy, modulating ROS and controlling apoptosis, dysfunction has often been defined as a deviation of these mitochondrial quality control is critical for normal cell parameters in a treatment or disease group relative to function. Mitochondrial morphology and function are control. Classic experimentation with isolated mitochon- perturbed in genetically engineered autophagy-deficient dria cannot effectively address this issue because cellular animals, further supporting the hypothesis that autophagy regulation of mitochondrial function is removed during 1488 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics isolation. To bridge this gap, methods have been devel- Dranka et al., 2011). In the first series of measurements, the oped to determine the behavior of mitochondria in a cel- basal oxygen consumption of the cells is established. The lular setting using extracellular flux analysis (Wu et al. , basal OCR represents the net sum of all processes in the cell

2007 ; Ferrick et al. , 2008 ; Gerencser et al. , 2009 ; Dranka capable of consuming O2 , including mitochondria and other et al., 2010; Nicholls et al., 2010; Brand and Nicholls, oxidases. Next, oligomycin, an inhibitor of mitochondrial 2011). Recent advances in this technology have allowed for ATP synthase, is injected. In support of the assumption that real-time measurements of O2 consumption and acidifica- the basal respiration represents the demand on the proton tion in cell culture with the capacity to inject respiratory motive force (for making either ATP or moving other ions inhibitors. This has quickly allowed for the establishment across the inner membrane), the pharmacological inhibi- of methodical assays to interrogate bioenergetic function tion of energy-requiring processes results in a decrease in in cells under a broad range of conditions (Ferrick et al. basal respiration. The decrease in OCR in response to oli- 2008 ; Wu et al. 2010 ; Dranka et al. , 2011 ). How the rates gomycin is then related to the proportion of mitochondrial of O2 consumption (OCR) and extracellular acidification activity used to generate ATP. (ECAR), which are global properties of the cell prepara- In contrast to experiments with isolated mitochon- tions, can be ascribed to specific biological processes will dria, respiration is sustained in these conditions by res- be discussed next (Sridharan et al., 2008 ; Hill et al., 2009). piratory substrates provided by cellular metabolism. To estimate the maximal potential respiration sustainable by the cells, a proton ionophore (uncoupler) such as FCCP Fundamentals of the bioenergetic is used. Immediately upon exposure to the uncoupler, oxygen consumption increases as the mitochondrial inner profile assay membrane becomes permeable to protons, and electron transfer is no longer controlled by the proton gradient. Information on how mitochondria function in intact cells An important feature emerging from such assays is the has largely been derived from assays similar to that shown in mitochondrial reserve capacity, which is calculated by Figure 2 . Here, intracellular mitochondrial function can be subtracting the FCCP-stimulated rate from the basal OCR. determined by sequentially adding pharmacological inhibi- To determine the extent of non-mitochondrial oxygen- tors of oxidative phosphorylation (Brand and Nicholls , 2011 ; consuming processes, the respiratory chain is inhibited with antimycin A either alone or in combination with rote- FCCP Antimycin-A none. Such pharmacological inhibition of electron transport typically inhibits the majority of oxygen consumption 500 in the cell, and we attribute the remaining O2 consumption to non-mitochondrial oxidases present in the cell. This 400 Reserve Oligomycin capacity value is subtracted from other OCR measurements to determine the mitochondrial-based parameters. It should be 300

/min) Maximal 2 oxygen noted that for meaningful comparisons between different consumption conditions or treatments, the basal OCR should be normal- 200 at complex (pmol O ATP- ized to a factor to correct for different numbers of cells in the Basal IV linked assay or changes in cell size, e.g., cell atrophy or hypertro- Oxygen consumption rate 100 Proton leak phy. Insights into the specific activity of the mitochondria Non-mitochondrial can be obtained by normalizing to a mitochondrial protein 0 Time (min) measured in the cell lysates after the assay. Taken together, these data have interesting implications for understanding Figure 2 The cellular bioenergetic profile. mitochondrial function in a cellular context. This mitochondrial stress test can be used to measure several indices of mitochondrial function in intact cells in real time and allows for the identification of critical respiratory defects. A typical experiment is shown, in which basal oxygen consumption rate (OCR) is allowed to stabilize before the sequential addition of Interpretation of the bioenergetic oligomycin, FCCP, and antimycin A and/or rotenone with a measure- profile ment of changes in OCR as indicated. This time course is annotated to show the relative contribution of non-respiratory chain oxygen consumption, ATP-linked oxygen consumption, the maximal OCR In Figure 3 we have selected examples with diverse energy after the addition of FCCP, and the reserve capacity of the cells. requirements in vivo to illustrate how the bioenergetic B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1489

A Cardiomyocytes Hepatocytes Endothelial cells

70 40 40 F A+R F A+R F A+R 60 35 35 O 50 30 30 O g ptn) g ptn) g ptn) 25 25 μ μ 40 μ O 20 20 30 15 15 20 (pmol/min/

(pmol/min/ 10

(pmol/min/ 10 10

Oxygen consumption rate 5 5 Oxygen consumption rate Oxygen consumption rate 0 0 0 0 102030405060 0 1020304050 0 1020304050 Time (min) Time (min) Time (min)

B 0%

17%

39% 38% 40% 17%

60% 66%

23%

C 0%

11% 10% 9% 6% 26% 36%

56% 18% 46% 73% 9%

ATP-linked Proton leak Reserve capacity Non-mitochondrial

RCRbasal =0.95±0.05 RCRbasal =2.59±0.2 RCRbasal =5.0±0.3

RCRmaximal =3.20±0.32 RCRmaximal =18.59±1.1 RCRmaximal =10.0±1.0

Stateapparent =4.00±0.00 Stateapparent =3.90±0.01 Stateapparent =3.5±0.0

Figure 3 Cellular bioenergetics in different cell types. Extracellular flux analysis using the mitochondrial stress test assay: Panel A: Adult rat cardiomyocytes, primary hepatocytes or bovine aortic endothelial cells were subject to a metabolic stress test as shown, and the OCR rates were normalized to protein. Panel B: in this analysis basal OCR was established as 100% OCR and the proportion of ATP-linked OCR, proton leak and non-mitochondrial oxygen consumption is shown. Panel C: in this analysis maximal OCR is established as 100% and the oxygen consumption is apportioned between

ATP-linked OCR, proton leak, reserve capacity and non-mitochondrial O 2 consumption. The RCR for basal and maximal OCR is reported together with the stateapparent for each cell type. profile differs between cell type. Aortic endothelial cells cardiomyocytes, which leads to a high reserve capacity are typically regarded as having low energetic demand, in both cells. At first glance the profiles look remarkably primary hepatocytes a demand and cardiomyocytes similar but on closer examination differences are appar- highly energetic. Differences in ATP-linked oxygen con- ent. For example, the lack of sensitivity of cardiomyocytes sumption and proton leak are clearly evident between the to oligomycin is likely due to the fact that under these cell types, with endothelial cells having a much higher conditions the cells are not contracting. This means that proportion of the basal OCR ascribed to ATP turnover. the activity of ATP synthase required to supply the ATP Both hepatocytes and cardiomyocytes show little change demand is likely very low and therefore the effect of oli- when treated with oligomycin. The FCCP-stimulated gomycin is minimal. The pie chart in panel B represents rate is highest in the more energetic hepatocytes and basal OCR distributed between ATP-linked OCR, proton 1490 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics leak and non-mitochondrial functions. It is interesting genetics and maintenance of organelle quality, as will be to note that proton leak (or ion movements requiring discussed below. proton motive force) and non-mitochondrial consump- To assist the investigator, Figure 4 shows a typical tion of oxygen is quite different between the different basis for interpretation of the most common changes in cell types. Data in Panel C are expressed as a percent- the bioenergetic profile observed in routine experiments. age of the maximal respiration, and these data reinforce Below we describe in detail these interpretations of cel- the conclusion that the contribution of mitochondria to lular bioenergetic profiles and some of the principles that oxygen consumption in cells is both diverse and distinct. underlie changes in the key parameters of basal respira- This finding assumes an additional importance when tion, ATP-linked oxygen consumption, proton leak and we consider the potential role and extent of ROS genera- reserve capacity (sometimes also called spare respiratory tion in mitochondria both in relation to mitochondrial capacity).

High: a) ATP turnover in cell has increased b) Proton leak has increased c) Non-mitochondrial (N.M.) ROS has increased

Low: a) ATP demand has Basal decreased b) Proton leak has decreased Test: Oligo c) ETC or ATP synthase inhibited d) Substrate supply High: High ATP decreased demand High: a) UCP activity ATP Leak increased b) Inner membrane Low: a) Low ATP damaged demand c) ETC complexes b) ETC damaged d) Electron slippage damaged Test: FCCP c) Low substrate Low: a) UCP activity availability decreased Maximal capacity b) Good membrane and ETC integrity

High: a) Substrate Test: A.A./Rot. availability increased b) Mitochondrial N.M. mass increased c) Good ETC integrity

Low: a) Substrate availability High: Cytosolic Low: Low decreased ROS increased extramitochondrial b) Mitochondrial ROS mass decreased c) Poor ETC integrity

Figure 4 Interpretation tree for the mitochondrial bioenergetic profile. In cells, differences in basal oxygen consumption rate (OCR) could be because of several factors, including ATP turnover reactions, proton leak, non-mitochondrial (NM) oxygen consumption, or damage to the electron transport chain. This can be tested by adding oligomycin, and some potential outcomes are shown. Next, the changes in the maximal capacity (and the reserve capacity) can be identified using the FCCP-stimulated rate. An increase or a decrease in this rate compared with controls could be due to changes in substrate availability, mitochondrial mass, or electron transport chain (ETC) integrity. Lastly, the effects of non-mitochondrial sources on oxygen consumption may be examined. B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1491

Basal oxygen consumption rate Maximal OCR

Taken in isolation, a change in the value of basal OCR may Experimentally, the use of FCCP to estimate the maximal be due to either a physiological adaptation or response respiratory rate has several limitations. In each cell type to stress. An increase in basal OCR could be due to an the FCCP concentration has to be titrated, because at high increase in ATP turnover, an increase in proton leak, and/ concentrations it can be inhibitory (Dranka et al., 2011). or an increase in non-mitochondrial oxygen consump- A lower maximal capacity could indicate decreased sub- tion (e.g., ROS formation). Oligomycin treatment gives an strate availability or that mitochondrial mass or integrity insight into the former two possibilities (Figure 4). is compromised. A high FCCP-stimulated rate compared with the basal rate suggests that the cell has a substantial mitochondrial reserve capacity, which could be important ATP-linked OCR and proton leak for responding to acute insults.

The addition of oligomycin is assumed to completely eliminate control of respiration by oxidative phosphorylation, Non-mitochondrial OCR and the remaining rate of mitochondrial respiration is then taken to represent the cumulative proton leak that Very few measurements have been performed in cells to was present prior to addition of oligomycin. An increase determine the contribution of non-mitochondrial sources in the ATP-linked rate of O 2 consumption would indicate of oxygen consumption. Using isolated mitochondria an increase in ATP demand, and a decrease would indicate and respiratory chain inhibitors, it was calculated in the either low ATP demand, a lack of substrate availability, or early 1970s that mitochondrial superoxide production was severe damage to the ETC, which would impede the flow of 1– 2 % of oxygen consumption (Boveris and Chance, 1973; electrons and result in a lower OCR (Figure 4); of note, this Turrens , 2003 ). Using cell fractionation and the measure- would also decrease the maximal capacity (see below). ment of hydrogen peroxide generation in hepatocytes, it An increase in apparent proton leak could be due to a was calculated that hydrogen peroxide production was number of factors: uncoupling protein (UCP) activity could 10% of the total oxygen consumption by the cell (Boveris be increased (Echtay et al., 2003; Divakaruni and Brand, et al., 1972). In Figure 3B we show the distribution of 2011); the inner mitochondrial membrane and/or ETC oxygen consumption in the cells calculated, assuming complexes could be damaged, allowing H + to leak into the basal respiration is 100% ; in Figure 3C this is calculated matrix; or there could be electron slippage, which would assuming maximal respiration is 100% . In the hepatocytes result in oxygen consumption in the absence of proton and cardiomyocytes, non-mitochondrial oxygen consump- translocation (Brown, 1992). Treatment of cardiomyocytes tion constitutes approximately 40% of basal OCR, and with 4-hydroxynonenal (HNE), for example, results in an in endothelial cells approximately 15% . A more detailed increase in both ATP-linked oxygen consumption and investigation of the potential sources of these oxygen con- proton leak (Hill et al., 2009 ; Sansbury et al., 2011a,b ). suming pathways needs to be performed as not all of these In such a result, it is important to consider that the pathways will generate hydrogen peroxide. However, it is pharmacological interrogation of the metabolic profile intriguing that this value is different between cell types. can bias data output and interpretation. For example, The widely held view that mitochondria consume over 95% oligomycin increases the mitochondrial membrane poten- of the oxygen in the body should be reevaluated. ΔΨ tial ( m ) and induces a state 4-like respiratory condition (Brown et al. , 1990 ), which will also increase proton leak through the mitochondrial membrane (Brand , 1990 ; Brown, 1992). The value for ATP-linked respiration is Cellular respiratory stateapparent then modestly under-estimated and proton leak over- estimated. It is important to consider also that the rate of It is becoming clear that in most cells mitochondria func- apparent proton leak could represent the passage of any tion at an intermediate respiratory state between state 3 ion, e.g., calcium, across the inner membrane, which can and state 4 and have RCR values far in excess of those dissipate the proton gradient. Technologies that allow the found in isolated mitochondria (Dranka et al. , 2010 ; simultaneous measurement of mitochondrial membrane Brand and Nicholls , 2011 ; Dranka et al. , 2011 ; Sansbury potential in conjunction with cellular respiration would et al. , 2011a ). The fact that cells have measurable respira- need to be developed to test this experimentally. tory state values that vary with insult or injury suggests 1492 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics that the redox status of individual components of the and quantitative framework for examining mitochondrial respiratory chain populate a dynamic range of intermedi- respiratory control in cells. The overall approach involves ate metabolic conformations. When comparing the state determining the relative extent to which one individual 4 condition (oligomycin) to the state 3/uncoupled condi- step in a pathway, such as oxidative phosphorylation, tion (FCCP-stimulated), cytochrome c oxidase appears to exerts control of overall flux through the entire pathway. exhibit a potential range of activity of approximately 5 – 10 The parameter that quantitates this control is the control fold. This is interesting because experiments with the iso- coefficient and can assume a value between 0 and 1. Impor- lated enzyme have shown the existence of intermediate tantly, if the overall flux is at steady-state the sum of control active forms of cytochrome c oxidase that depend on elec- coefficients for all steps of a pathway is equal to 1. Func- tron flux, and these forms of the enzyme are differentially tionally, the closer the value of a control coefficient for an reactive with respiratory modulators such as CO (carbon individual step is to 1 the more control this single step has − monoxide), NO (nitric oxide) and NO2 (nitrite) (Sarti et al. , over the flux through the entire pathway. Metabolic control 2003 ; Cooper and Giulivi , 2007 ). analysis has been applied to both isolated mitochondria

We have proposed that the cellular stateapparent can be and also to mitochondrial respiration in intact cells and has calculated as follows: revealed information that can be used for the quantitative and mechanistic interpretation of the reserve (Pan -Zhou ()Basal-oligo State= 4- et al., 2000 ) or spare respiratory (Choi et al. , 2009 ) capacity apparent () FCCP-oligo in cells under various conditions (Brand and Nicholls , 2011 ). In terms of interpreting reserve capacity, the assign- The state therefore can be modulated by changes in apparent ment of each component of OCR to the biological activities the basal OCR, the coupling status, and/or the maximal depicted in Figure 2 makes specific assumptions regard- respiratory rate. ing the control coefficients for the various components It is important to note that the calculation of state apparent under the different experimental conditions. Specifically, simplifies the underlying and highly interactive phenom- the control coefficient for the proton leak component must ena that act together to determine overall flux through the dominate respiration after addition of oligomycin and the respiratory chain. As the factors that determine respira- coefficient for electron transfer (implicating the integrity of tion are multiparametric then it is clear that several dif- respiratory complexes and/or substrate conditions) must ferent conditions could lead to similar values for these dominate after addition of uncoupler. Previous applica- parameters. The underlying molecular mechanisms for tion of MCA to studies with hepatocytes has demonstrated changes in state could involve changes in activation apparent that under baseline conditions (similar to the basal OCR) or levels of expression of the metabolic proteins control- control of oxidation is distributed among all three com- ling these processes, which then must then be determined ponents (Δ Ψ -generating processes, ATP production, and in independent experiments. M proton leak) (Brown et al., 1990). Comparison with Figure 3 indeed supports the concept that under basal conditions control of respiration is distributed among these compo- Cellular RCR nents but this differs between different cell types. Biological regulation of the RCR is also complex, and The respiratory control ratio was originally defined for it is now known that the RCR varies with both the respira- isolated rat heart muscle sarcosomes and referred to the tory substrate used and the activation of uncoupling pro- extent to which respiration is slowed after added ADP is cesses in the inner mitochondrial membrane. These can phosphorylated completely (state 3 to state 4 transition) be specifically regulated by channels known as uncou- (Chance and Baltscheffsky , 1958b ). This ratio, in its various pling proteins, proton slip in respiratory chain complexes, forms, is generally regarded as the ‘ gold standard’ for or non-specific damage to the mitochondrial membrane measuring mitochondrial coupling, and the concept has such as would occur with lipid peroxidation. been extended to cells. In the original paradigm, respira- The bioenergetic profiles shown in Figures 2 and 3 tory control can be viewed as a result of the buildup of a can be used to calculate a respiratory control ratio (RCR) high-energy intermediate that links respiration to ATP syn- for oxidative phosphorylation in a cellular setting. To do thesis, which occurs because all ADP is phosphorylated. this, we assume that the OCR after the addition of oligo- In reality, the phenomena that are responsible for res- mycin represents state 4 respiration and that after FCCP piratory control are much more complex. Metabolic control is equivalent to state 3. The cellular RCR without (basal analysis (MCA) has undoubtedly been the most useful RCR) and with (maximal RCR) the addition of FCCP is then B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1493 calculated as the state 3 rate divided by the state 4 rate. including isocitrate dehydrogenase, α -ketoglutarate The non-cytochrome c oxidase OCR (i.e., in the presence dehydrogenase, and malate dehydrogenase (Williamson of antimycin A; Anti A) is subtracted from all rates. and Cooper, 1980). This is even more important because anaplerotic metabolism, for instance through metabolism ()Basal-Anti A RCR = of the amino acids glutamine and aspartate entering at basal () Oligo-Anti A the levels of α -ketoglutarate and oxaloacetate, respec- tively, would influence basal metabolism. Fatty acids can (FCCP-Anti A) RCR = also enter the TCA cycle through anaplerotic metabolism max () Oligo-Anti A to succinyl CoA (Brunengraber and Roe , 2006 ). These selected examples illustrate how basal metabolism could The RCR calculations for the three cell types are be altered by different substrates. The use of alternative shown in Figure 3. The high RCR values calculated using pathways may also differentially modulate the control these data are clearly much above that reported for iso- coefficients for mitochondrial respiration. lated mitochondria. There could be several reasons for Substrate utilization is cell- and context-specific. Experi- this difference. mentally, this has been validated by simply providing differ- 1. The isolation process damages mitochondria and ent concentrations, types, or combinations of substrate(s) in detaches them from the cellular matrix, which the extracellular medium and examining their effects on res- results in higher proton leak. piration. For example, hepatocyte respiration is increased by 2. The mitochondria respond dynamically to substrate fatty acids, pyruvate and lactate (Korzeniewski et al. , 1995 ; demand in a cellular setting through metabolic regu- Nobes et al. , 1989, 1990 ), while insulinoma basal respira- lation which is lost in the isolated mitochondria. tion depends primarily on glucose concentration (Affourtit and Brand, 2008). In synaptosomes, inhibition of pyruvate Both factors likely contribute. In any event, it is clear that uptake leads to a decrease in the reserve capacity by inhibit- mitochondria in many cells are normally functioning at a ing the maximal respiratory rate (Kauppinen and Nicholls, sub-maximal capacity under basal conditions. 1986 ; Brand and Nicholls , 2011 ). Such a result would indicate that glycolysis, in some cells, can exert considerable control over respiration rate. In cardiomyocytes, it appears that gly- Bioenergetic reserve capacity colysis can negatively regulate mitochondrial respiration and that pyruvate is the substrate of choice (Sansbury et al., In addition to the RCR, calculation of the mitochondrial 2011a ). Interestingly, neonatal myocytes in the presence of % reserve capacity (FCCPrate - Basalrate ) is a useful qualitative pyruvate alone have a 20 higher reserve capacity compared indicator of mitochondrial energetic status in cells. The with cells having both pyruvate and glucose as substrate, factors controlling the mitochondrial reserve capacity and this difference is due to a suppression of the maximal are complex. Substrate supply, coupling, ATP demand respiratory rate when glucose is present. The reasons for and allosteric regulation of key metabolic enzymes, and such inhibition are unclear but may be due to allosteric regu- the integrity or level of expression of respiratory chain lation of the electron transport chain by glycolytic interme- components are critical features that regulate oxygen diates (Diaz -Ruiz et al., 2008 ). Recent data suggest that the consumption. bioenergetic reserve capacity may be particularly important Substrate supply appears to be extremely impor- in the cell cycle and related to mitochondrial fission/fusion tant in regulating the bioenergetic reserve in the cellular (Mitra et al. , 2009 ; Mitra and Lippincott -Schwartz, 2010 ). In context. In contrast to experiments using isolated mito- addition, it has recently been shown that the regulation of chondria, substrate delivery in intact cells is subject to glycolysis is central to controlling the cell cycle suggesting more complex regulation. Allosterism of key metabolic a complex interaction between mitochondrial dynamics and enzymes and the cadence of metabolic cell signaling are metabolism (Garedew et al. , 2012 ). Collectively, these results two fundamental ways in which respiration is controlled. exemplify the importance of substrate availability in regulat- For example, pyruvate dehydrogenase (PDH), which is ing the reserve capacity, which, as discussed below, translate the rate limiting step in glucose oxidation, is regulated to the cellular response to differentiation, division and stress. allosterically by NAD + /NADH, ATP and AMP, CoA/Acetyl The bioenergetic reserve capacity is also influenced CoA, and calcium as well as by both PDH phosphatase by overall mitochondrial mass, the activity of individual and kinase (Patel and Korotchkina, 2006). Furthermore, mitochondrial complexes and the mitochondrial network. the TCA cycle is regulated allosterically at several points It is important to note that an underlying bioenergetic 1494 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics dysfunction can counter-intuitively increase the appar- 3. The synthesis of amino sugars (hexosamine biosyn- ent reserve capacity. For example, conditions that act to thetic pathway); inhibit the ATP synthase (or any of its associated func- 4. The synthesis of glycerophospholipids (dihydroxyac- tions) would decrease the basal OCR, and, assuming etone phosphate pathway). no change in the maximally uncoupled rate, this would produce a ‘ false-positive ’ increase in the reserve capacity. Metabolism of glucose to pyruvate results in two primary However, this effect would in fact compromise the pro- possibilities: the metabolism of pyruvate to lactate or duction of ATP and actually result in a decreased bioen- the oxidative decarboxylation of pyruvate to acetyl CoA ergetic capacity. This can be identified and tested for, as for entry into the TCA cycle. It is the former of these two described in Figure 4. The ability of a cell to meet a sus- possibilities that can be measured as an index of glyco- tained but high ATP demand may be a less vulnerable lytic flux. While lactate assays can give a snapshot view metabolic state than a transient but severe ATP demand in of whether glycolytic flux is increased or decreased over which reserve capacity is exceeded (Brand and Nicholls , a period of time, XF ECAR analysis gives a time-resolved 2011 ). It is then important to consider not just the magni- view of changes in glycolysis (Ferrick et al., 2008). This tude of the reserve capacity but the physiological context has allowed for new concepts to develop concerning how in which the mitochondria are functioning. glycolysis could regulate cell function. The challenge as we move forward is to develop Interestingly, in hypertrophied neonatal myocytes, methods to compare different conditions quantitatively it appears that the glycolytic reserve rather than the and draw mechanistic conclusions. A limitation in the mitochondrial reserve capacity helps the cell survive analysis is the assumption that addition of inhibitors/ HNE-induced injury and cell death. Cellular hypertro- uncouplers can be used to selectively eliminate certain phy driven by adrenergic stimuli resulted in a decrease ‘ blocks or metabolic units ’ , leaving intact and unchanged in the mitochondrial reserve capacity concomitant with the functions of the other ‘ components ’ . This leads to some an increase in the glycolytic reserve, and this reserve error in assignment of metabolic functions such as ATP- appeared to help the cell through the oxidative insult linked respiration and proton leak as discussed above. (Sansbury et al., 2011b). Extant evidence suggests that the Similarly, the presence of respiratory substrates capable hypertrophy of the heart in vivo also leads to an increase of increasing NADH may increase the control coefficient in glycolytic capacity. For example, in a rodent model of for chain control of respiration, perhaps increasing the pressure-overload left ventricular hypertrophy (LVH), respiration rate. This effect could decrease the apparent insulin-independent glucose uptake was increased 3-fold reserve capacity when actually proton flux (and thus ATP and activators of phosphofructokinase were increased synthesis) increases, resulting in a true increase in bio- up to 10-fold, leading to a 2-fold increase in glycolysis energetic capacity. Lastly, it has been shown that under (Nascimben et al. , 2004 ). conditions of increased bioenergetic stress (ATP con- In extracellular flux assays, the glycolytic reserve sumption) a substantial amount of ATP production is via capacity can be examined through an assay analogous glycolysis in addition to mitochondrial phosphorylation, to the mitochondrial function assay where oligomycin and this can be estimated by measurement of ECAR under and the GAPDH inhibitor koningic acid (KA) are used basal or stressed conditions (Dranka et al. , 2011 ). to examine glycolysis and non-glycolytic ECAR. An example of how this assay could be used is shown in Figure 5 . Oligomycin is used in this assay to examine Role for a ‘ glycolytic reserve’ in the how the glycolytic pathway responds to loss of mitochondrial ATP production. To further inhibit mitochon- response to stress drial electron flux and remove proton leak, which could act a small sink for reducing equivalents derived from It is well known that glucose metabolism plays a fun- glycolysis (e.g., pyruvate), antimycin-A and rotenone damental role in maintaining metabolic homeostasis. can be added. This can give a maximal rate of glycoly- Branching off of the primary glycolysis conduit are path- sis that could possibly occur if all mitochondrial elec- ways regulating multiple cellular processes and functions, tron flux is inhibited. Inhibitors of glycolysis such as KA including: or 2-deoxyglucose (2-DG) can then be used to measure 1. The biosynthesis of nucleotides and NADPH (pentose non-glycolytic ECAR, which is comparable to the use of phosphate pathway); antimycin-A and rotenone in the mitochondrial function 2. Cellular osmolarity (polyol pathway); assay. B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1495

Oligo Anti-A/R KA or 2-DG 1.0 mitochondrial quality and the susceptibility to diseases in which the interface between bioenergetics and inflamma- 0.8 tion are playing a key role (Figure 6 ) (Wallace , 2010 , 2011; Krzywanski et al. , 2011 ). g protein) μ 0.6 As the ancestors for contemporary humans migrated Glycolytic out of sub-Saharan Africa, they accumulated missense reserve Maximal 0.4 capacity mutations in their mtDNAs. It is now becoming clear that Non-glycolytic some of these mutations may have had a selective advan- ECAR 0.2 tage through their impact on mitochondrial economy. ECAR (mpH unit/min/ Basal For example, if these mutations altered mitochondrial 0 economy such that caloric utilization for ATP genera- 0204060 80 100 tion was decreased while that used for generating heat Time (min) increased, then this would have been a metabolic advan- Figure 5 Glycolysis stress test. tage for our ancestors as they migrated into northern cli- This extracellular flux assay demonstrates how basal glycolytic mates (Ruiz-Pesini et al., 2004; Wallace, 2005). Although rate, maximal glycolytic rate, and the glycolytic reserve capacity these mutations redistributed the economics of electron can be determined. After measurement of the basal extracellular transfer with a decrease in efficiency in ATP generation, acidification rate (ECAR), oligomycin can be added, which gener- these changes could be accommodated in our migra- ally increases glycolytic rate in response to loss of mitochondrial ATP production. The addition of antimycin A/rotenone, depending tory ancestors due to changes in their diet (increased on experimental conditions and cell type, may further increase the caloric intake associated with animal fats) (Cordain ECAR, giving an index of lactate production occurring when all mito- et al., 2000). In contrast, the mtDNA haplotypes associ- chondrial electron transport is inhibited. Non-glycolytic extracel- ated with an economic distribution of electrons to ATP lular acidification (i.e., acidification not due to lactate production) synthesis (e.g., those having sub-Saharan origins) are can then be measured by introducing koningic acid (KA; an inhibitor better adapted to low calorie warmer climates. In the of glyceraldehyde-3-dehydrogenase) to inhibit glycolysis; alter- natively, 2-deoxyglucose (2-DG) may be used. This assay may be modern era with massive and rapid migration of popu- prefaced by addition of oxidants or other stressors. lations over decades, the mitochondrial haplotype can frequently be maladapted for an excessive caloric availability and low energetic demand lifestyle. Figure 6 illus- The impact of mitochondrial DNA trates this concept by showing that under conditions of haplotype and damage on mito- high ATP demands (e.g., adequate diet and exercise), electron flow energetics predominately manifests in ATP chondrial quality generation with subtle differences existing in mitochondrial oxidant and heat generation between individuals A unique feature of the molecular machinery control- harboring mtDNA haplotypes originally associated with ling cellular bioenergetics is that the proteins necessary northern or sub-Saharan latitudes. In contrast, under for electron transport and oxidative phosphorylation are conditions of low ATP demand (e.g., excess calorie intake encoded by both nuclear and mitochondrial genomes. and physical inactivity) mitochondrial oxidant produc- Each cell contains hundreds to thousands of mitochon- tion will be differentially increased between individuals dria and each mitochondrion contains 5 – 10 copies of with distinct patterns of electron distribution between maternally inherited mitochondrial DNA (mtDNA). The ATP/ROS and heat (associated with mtDNA haplotype). mammalian mtDNA encodes for the 13 key structural Specifically, under conditions of excess caloric intake subunits required for the catalytic activity of four of the and sedentary lifestyle individuals with sub-Saharan five OXPHOS enzyme complexes (I, III, IV and V). Because maternal origins will have greater mitochondrial oxidant of the pivotal role of these proteins in bioenergetics it has production relative to those having origins associated been proposed that mutations in the mtDNA-encoded with more northern latitudes because excess electron subunits modulate the economics of oxidative phospho- flow in those individuals (sub-Saharan origins) are less rylation in controlling the relative distribution of elec- likely to be utilized for heat generation and will therefore trons to ATP generation, heat and the formation of ROS. be diverted to ROS formation. We and others are now exploring the hypothesis that While cybrid cell culture studies have provided con- these features of mitochondrial economy, which have flicting data regarding the role of mtDNA haplotype on in evolved over thousands of years, can ultimately affect vitro bioenergetics, other studies have shown that mtDNA 1496 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics

e- flow ATP Heat Oxidants

High ATP demand Oxidants

mtDNA associated with Calories ATP northern latitudes

Heat

Oxidants

mtDNA Calories associated with ATP sub-Saharan latitudes

Heat

Low energy demand Oxidants

mtDNA Calories* associated with ATP northern latitudes

Heat

Oxidants

mtDNA Calories* associated with ATP sub-Saharan latitudes

Heat

Figure 6 Hypothetical presentation of electronic energy utilization between mitochondria having mtDNA haplotypes representing Northern or Sub-Saharan origins in terms of latitude. Under conditions of high energy (ATP) demand, differences in energy utilization exist between mitochondria having different mtDNAs that manifest in differences in electrons used to generate ATP, oxidants and heat as illustrated by the arrows and pie charts. Under conditions of low energy (ATP) demand and excessive caloric intake (*), these profiles shift to decreased use of electronic energy for ATP generation and greater production of oxidants, with those mitochondria having sub-Saharan latitude mtDNAs generating more oxidants compared with those with Northern latitude mtDNAs as indicated by the arrows and pie charts. haplotype can influence tumor growth, age-related deaf- (De Benedictis et al. , 1999 ; Rose et al. , 2001 ; Petros et al. , ness, cognition, behavior, reproductive behavior, and 2005 ; Wallace , 2005 ; Bhopal and Rafnsson, 2009 ). susceptibility to autoimmune disease in mice (Moreno- Loshuertos et al., 2006 ; Amo and Brand , 2007 ; Amo et al. , 2008 ; Gomez -Duran et al., 2010 ). Similarly, molecular Mitochondrial quality, mtDNA epidemiologic studies have shown that human longevity correlates with mtDNA haplotypes having temperate damage and disease and arctic origins (yet have increased risk for illnesses related with energetic insufficiency) whereas those of From the previous discussion it is clear that as mitochon- sub-Saharan origins appear to be more frequently associ- drial function declines with the accumulation of mito- ated with certain types of cancer and diseases thought to chondrial damage, then assessment of overall mitochon- be due to increased oxidative stress and/or mutagenesis drial quality can serve as a prognostic indicator for disease B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1497 risk. For example, studies on primary vascular endothe- 1962). During development, mitophagy plays a key role in lial and smooth muscle cells have shown preferential and eliminating the paternal mitochondria after fertilization sustained mtDNA damage, altered mitochondrial tran- in C. elegans (Sato and Sato , 2011 ). As maternal inherit- script levels, and decreased mitochondrial protein syn- ance of mtDNA is shared by many organisms, including thesis in response to oxidant treatments (Ballinger et al. , humans, mitophagy may play a critical role in the genet- 2000 ). High levels of nitric oxide have also been shown to ics and diversity of mtDNA haplotype and, as discussed decrease the levels of mitochondrial respiratory proteins previously, disease susceptibility. in endothelial cells and increase the susceptibility to cell death (Ramachandran et al., 2004). In vivo studies have shown increased mtDNA damage in atherosclerotic and Mitophagy and mitochondrial quality aged tissues from both humans and animals, and moreover, that vascular mtDNA damage occurs prior to or coin- In addition to the developmental process, tissues that cidental to pathologic changes (atherogenesis) in mice are highly dependent on mitochondrial quality, includ- and non-human primates (Ballinger et al. , 2002 ; West- ing liver, heart and brain, maintain active mitophagy brook et al., 2010). It is also well established that numer- that appears to be protective against toxins, hypoxia, and ous environmental oxidants, cardiotoxicants and disease age-dependent diseases (Osiewacz, 2011; Karbowski and risk factors (e.g., hypercholesterolemia, hyperglycemia) Neutzner, 2012; Lee et al., 2012; Okamoto and Kondo- cause significant mtDNA damage, and it has been sug- Okamoto, 2012). Oxidative stress may be a key mechanism gested that developmental exposure to these factors can through which mitochondria are damaged and targeted significantly increase the risk for adult disease develop- for mitophagy (Apostolova et al., 2011). We have recently ment by causing early damage to the mtDNA (Gutierrez et shown in heme-dependent toxicity in endothelial cells al. , 2006 ; Cakir et al. , 2007 ; Yang et al. , 2007 ; Chuang et that one of the earliest events is bioenergetic dysfunction al. , 2009 ). In addition to the obvious impact of damaged and the engagement of mitophagy (Higdon et al., 2012). mtDNA on organelle function, oxidant production, and In a number of human diseases with mitochondrial defi- energy generation, studies are now suggesting that the cits, mitochondrial respiratory chain activities and CoQ10 damaged mtDNA can serve as a D amage A ssociated levels are decreased, and oxidative stress and mitochon- M olecular P attern (DAMP) which contributes to a pro- drial membrane permeabilization are increased (Cotan inflammatory cellular environment (Krysko et al. , 2011 ). et al., 2011). Mitophagic turnover in these cells is important Finally, certain mtDNA haplotypes may be more prone to for preventing accumulation of additional damage. For mtDNA damage by virtue of mitochondrial oxidant gener- example, in MELAS fibroblasts prevention of autophagy ation associated with conditions of chronic excess energy resulted in apoptotic cell death, supporting the idea that balance (e.g., mtDNA haplotypes associated with greater mitophagy plays a protective role in cell survival (James oxidant production will sustain more mtDNA damage et al. , 1996 ). compared with those that generate less oxidants). Con- Mitophagy is important for control of mitochondrial sequently, the mitochondrial bioenergetics and organelle quality and ROS. Key factors in the mitophagy pathway quality are likely dependent upon organelle economy that include Atg1/Atg13/FIP200 activation, an ubiquitin-like may be influenced by mtDNA haplotype, which in turn conjugation pathway involving Atg 3, 5, 7 and 22 leading may influence the accumulation of mtDNA damage. to lipidation of LC3I and its conversion to LC3II, and LC3II insertion into autophagosomal membranes (Figure 7 ). Autophagosomal expansion is also stimulated by Beclin/ Regulation of mitochondrial quality VPS34 complex activities. Some of the key molecular mediators are shown in Figure 7 and are important for control and the role of mitophagy mitochondrial quality control. Deformed mitochondria have been observed in Atg3 − /− (Jia and He, 2011), Atg5 − /− , Mitochondrial turnover is stimulated by a broad range Atg7 −/− (Pua et al. , 2009 ), and Beclin (Atg6 ) + / − cells. These of conditions, including nutrient deprivation. Electron effects also impact on pathology becuase livers of VPS34 microscopy studies in the early 1960s showed that lys- knockout mice exhibit significant increases in mitochon- osomes are increased in number in glucagon-treated rat drial mass and steatosis (Jaber et al. , 2012 ). Significantly liver and many of the lysosomal structures contained increased levels of ROS have been found in various cells mitochondria or remnants that appeared to be in various in FIP200 , Atg3 , Atg5 , and Atg7 knockout mice (Jia and stages of breakdown or hydrolysis (Ashford and Porter , He , 2011 ). LC3B knockout macrophages generate more 1498 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics

FIP200 Beclin/VPS 34 complex LC3I

Atg 3, 5, 7 p-ERK, p-AMPA LC3II

Drp1 Fis1

LC3II Nix Opa1 BNIP Mfn1/2

LC3II LC3II LC3II p62

Cathepsins D, LC3II B, C and L

LC3II Autophagosome Lysosome

Figure 7 General pathway for activation of autophagy. Autophagy is mediated by the interaction or activation of several components including Atg1/Atg13/FIP200 and a ubiquitin-like conjugation pathway involving Atg 3, 5, 7 and 22. This leads to the lipidation of LC3I, converting it to LC3II. LC3II is inserted into autophagosomal membranes and is essential for autophagosomal formation. Autophagosomal expansion is also stimulated by Beclin/VPS34 complex activities. Autophagosomes then fuse with lysosomes and the content of autophagosomes are degraded in the acidic environment of the lysosomes. Nix and BNIP can target to mitochondria and bind to LC3II via a LIR consensus sequence and are thus important for mitophagy. ERK and AMPA activities have been shown to stimulate mitophagy. Mitochondrial fission and fusion proteins such as Drp1, Fis1, Opa1, and Mfn1/2 are also critical in regulating mitophagy. superoxide and in response to LPS these macrophages processes that detect nutrient conditions and energy displayed more swollen mitochondria (Xu et al. , 2011 ). status (Figure 7). Importantly, there exists a module of It is important to note that inhibiting mitophagy has not key proteins that regulate the interaction of mitochon- always been shown to be detrimental. For instance, a dria with autophagosomes, and considerable progress mouse model with a targeted deletion of Atg7 in adipose has been made in identifying these proteins and their tissue had increased amounts of brown-like adipose mechanisms of mitochondrial recognition. BNIP3 and Nix tissue containing higher levels of mitochondria and (BNIP3L), both BH3-domain containing Bcl-2 family of pro- increased fatty acid oxidation. The mice were resistant to teins, have been shown to play critical roles in mitophagy diet-induced obesity and insulin resistance (Singh et al., (Band et al., 2009 ; Zhang and Ney, 2009; Ding et al., 2009; Zhang et al., 2009). These results illustrate that, in 2010 ). These proteins localize to mitochondria and are some organs and under certain pathological conditions, a important in targeting mitochondria for degradation. The dysfunctional mitochondrial quality control system may significance of the involvement of Nix in mitochondrial have unexpected advantages. clearance is supported by deficient reticulocyte matura- tion in Nix knockout mice (Zhang and Ney, 2009). Another important regulator of mitochondria-autophagosome tar- Molecular control of mitophagy geting is FUNDC1, which is an outer mitochondrial membrane protein that binds to LC3, and may be important in Because mitophagy impacts mitochondrial homeostasis, mitochondrial quality control in response to hypoxia (Liu it is not surprising that it is a tightly regulated process. et al. , 2012 ). Depending on the cell type, genetic makeup and envi- The rapid pace of investigations concerning mitophagy ronmental stimuli, mitophagic regulatory mechanisms has led to a detailed understanding of two particularly include: mitochondrial membrane depolarization fol- important components of the mitophagic machinery – lowed by recruitment of Parkin (Elmore et al. , 2001 ; Jin PTEN-induced putative kinase 1 (PINK1) and Parkin. It and Youle, 2012), fission and fusion activities, phosphoryl- is clear that PINK1 is critical in identifying mitochondria ated ERK targeting to the mitochondria, and cell signaling destined for autophagy. In ‘ healthy ’ mitochondria, PINK1 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1499 is rapidly turned over by proteolysis; however, mitochon- colocalization of mitochondria with autophagosomes dria that become damaged appear to allow mitochondrial (Twig et al., 2008a). It is important to draw attention to accumulation of PINK, which leads to the recruitment of the fact that all mitophagic events appear to be exquisitely Parkin (Matsuda et al., 2010; Narendra et al., 2010b). How sensitive to changes in mitochondrial function. In most membrane potential and bioenergetic function regulate cases, mitochondrial membrane depolarization appears this PINK-Parkin mechanism is unknown; nevertheless, to serve as a mechanism to recruit signaling molecules it is well established that the machinery is acutely sensi- that target damaged mitochondria to be degraded. This tive to changes in mitochondrial function. Within 1 h after may then help prevent injured mitochondria from releas- administration of uncoupling agents, Parkin translocates ing pro-apoptotic agents and producing undesirable ROS to mitochondria; this translocation can also be induced (Figure 8 ). Additional evidence suggests that the bioener- by mitochondrial poisons such as azide (Narendra et al., getic reserve capacity may be sensed by the mitophagic 2008; Suen et al., 2010). The induction of mitophagy fol- machinery (Higdon et al. , 2012 ). An attractive hypothesis lowing this recruitment is thought to involve the ubiquitin that integrates all of these findings is that loss of the ability ligase activity of parkin, which ubiquitinates substrates to proteolyze initial recruitment proteins such as PINK such as mitofusin 1 (Mfn1), mitofusin 2 (Mfn 2) (Gegg occurs once the bioenergetic reserve is breached. Such a et al. , 2010 ; Tanaka et al. , 2010 ), and voltage-dependent hypothesis integrates the bioenergetic status of each mito- anion channel (VDAC) (Geisler et al. , 2010 ). The ubiquitin- chondrion tightly with targeted mitophagic processes. binding adaptor p62 may then be involved in recruiting the mitochondrial cargo into autophagosomes (Pankiv et al., 2007 ; Ding et al., 2010; Geisler et al., 2010), although Regulation of mitochondrial quality contradictory reports exist concerning whether p62 is indeed essential for mitophagy (Narendra et al. , 2010a ; in disease Okatsu et al. , 2010 ). Parkin also interacts with Ambra1; this is important for Ambra1 to activate class III PI3K The concept that mitochondrial quality control is a critical which is needed for subsequent mitochondrial clearance element in the role the organelle plays in the pathogenesis (Van Humbeeck et al. , 2011 ). of disease is gaining support. Some selected examples are discussed below.

Role of fission and fusion in mitophagy The reserve capacity and its regulation The shape and functional attributes of mitochondria of cell (patho)physiology appear to be important for determining whether they are targeted for mitophagy (Gomes et al. , 2011 ). In hepatocytes, An emerging concept in the energetics field is that the fission is associated with mitophagy (Kim and Lemasters , reserve capacity can be used as an index of mitochondrial 2011 ). The blocking of mitochondrial fusion results in loss health (Hill et al. , 2009 ; Dranka et al. , 2010 ; Brand and of membrane potential in distinct mitochondrial subpop- Nicholls , 2011 ; Zelickson et al. , 2011 ; Higdon et al. , 2012 ). ulations and recruitment of parkin (Youle and Narendra, By indicating how close a cell is to operating at its bio- 2011 ). Parkin, once localized to mitochondria, develops energetic limit, predictions can be made regarding the an increase in its E3 ubiquitin ligase activity (Matsuda cellular response to stress or increased energy demand. et al. , 2010 ), and ubiquitinylates mitofusins (Gegg et al. , This has been demonstrated in the response of cells to 2010 ; Tanaka et al. , 2010 ). In turn, this appears to mediate oxidative stress. In intact myocytes, stressors that are both the proteolytic degradation of mitofusin and to promote causes and consequences of oxidative stress can impact mitochondrial fragmentation. Hence, Parkin may inter- the reserve capacity. For example, the lipid peroxidation act dynamically with the fission/fusion machinery and product, HNE, increases ATP demand and proton leak facilitate the removal of only damaged mitochondria by to a point where the reserve capacity is depleted (Hill autophagy. et al., 2009). It appears that a higher bioenergetic reserve Other proteins affecting fission and fusion, includ- capacity results in a greater ability to withstand oxida- ing Fis1, Drp1, and Opa1, have been shown to also be tive stress. In cells given only glucose to respire on, the important (Chan, 2006). For example, in INS1 β -cells, Fis1 reserve capacity was only 30% of that of cells provided knockdown, overexpression of Opa1 or dominant nega- with both glucose and pyruvate, and the cells given only tive Drp1 resulted in decreased mitophagy as assayed by glucose showed a much earlier HNE-induced bioenergetic 1500 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics

Normal mito Damaged mito irreversible oxidative protein modification to metabolic Pink 1 PARL and respiratory chain components or non-repairable ROS damage to proteins or organelles that regulate ion fluxes. RLS The role of the bioenergetic reserve has been extended Δψ Δψ beyond its relationship simply with cell death. In par- VDAC ticular, emerging evidence implicates the mitochondrial

LC3 respiratory capacity in immunity. For example, it was + LC3 shown that CD8 memory T-cells possessed a substantial reserve capacity that was responsive to interleukin-15 (IL-15), which regulated the stability of memory T-cells LC3 Uptake Parkin, also after infection (van der Windt et al., 2012). A high reserve activated UPS capacity may also be linked to proliferation of certain cell LC3 Ubiquitination of types, which could result in pathology. For example, it VDAC, Drp1, and Mfn by Parkin was shown that cancer cells proliferate more rapidly in ubiquitination of the presence of exogenous pyruvate when compared with other mitochondrial lactate and that pyruvate supplementation increased the proteins by UPS and recruitment reserve respiratory capacity. Inhibition of cellular uptake of p62 of pyruvate led to decreased mitochondrial respiration p62 and cell growth (Diers et al. , 2012 ). Hence, cells that trans- form to a phenotype with high proliferative potential Figure 8 Regulation of mitophagy in response to mitochondrial could use the mitochondrial reserve capacity to increase membrane depolarization. energy for biosynthesis reactions that drive cell division. Mitochondria with normal membrane potential and active presenilin- associated rhomboid-like protein (PARL) prevent mitochondrial PINK1 accumulation. When mitochondria become damaged, mitochondrial membrane potential decreases, PARL is inactivated, and Neurodegenerative diseases mitochondrial PINK1 is stabilized and accumulates in the affected mitochondria. This recruits Parkin, which promotes the ubiquitina- Some of the most interesting mechanisms implicating tion of Drp1 and Mfn, as well as a large number of other mitochon- mitochondrial quality control in pathologies are associ- drial proteins. Ubiquitinated proteins can be recognized by p62 ated with bioenergetic dysfunction characteristic of many which binds LC3 and may help traffic damaged mitochondria to autophagosomes for degradation. neurodegenerative diseases. Mutations in Parkin, Pink1 and DJ-1 have been shown to be responsible for a small subset of autosomal-recessive collapse compared with cells having extracellular pyru- Parkinson’ s disease cases. The recent finding that these vate available (Sansbury et al. , 2011a ). Another example proteins also play an important role for mitochondrial was shown in neurons, where glutamate excitotoxicity quality control and mitophagy in a variety of cell types similarly depleted the reserve respiratory capacity, result- have stimulated new research regarding neurodegenera- ing in respiratory failure (Johnson -Cadwell et al., 2007 ; tion pathogenesis in Parkinson ’ s disease (Jin and Youle , Yadava and Nicholls, 2007). In the development of resist- 2012 ). As discussed above, studies in HEK294, HeLa cells, ance to chemotherapy, changes to the mitochondrial res- cardiomyocytes and primary cortical neurons have shown piratory chain (predominantly at cytochrome c oxidase) that Parkin accumulates in depolarized mitochondria were associated with an increase in reserve capacity (Oliva (Narendra et al. , 2008 ). PINK1-Parkin interactions lead et al., 2010). In endothelial cells, exposure to nontoxic to ubiquitination of VDAC and involve changes in the concentrations of NO or low levels of hydrogen perox- activity of fission and fusion proteins such as Drp1 and ide reversibly decreased mitochondrial reserve capacity. Mfn (Chu, 2010; Geisler et al., 2010; Rakovic et al., 2011). However, combined NO and superoxide and hydrogen per- In addition, Parkin activates the ubiquitin-proteasome oxide treatment resulted in an irreversible loss of reserve system (UPS) which in turn ubiquitinates a large number capacity and was associated with cell death (Dranka of mitochondrial proteins (Chan et al. , 2011 ), which may et al., 2010). These findings suggest that the reversibility serve as signals for mitophagy. DJ-1, has been shown to of deleterious changes in the reserve capacity may also act in a parallel pathway to Parkin and PINK1 to regulate be critical; the mechanisms controlling the recoverability mitochondrial fragmentation and is critical in Parkinson ’ s of the reserve capacity are unclear, but likely relate with disease (Schapira and Gegg , 2011 ). Knockout of DJ-1 in flies B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1501 decreased the respiratory control ratio (RCR) and mito- et al., 2003; Bailey, 2003; Scaglioni et al., 2011). The chondrial DNA/nuclear DNA ratio without changing mem- reserve capacity of the mitochondria after chronic alcohol brane potential or complex I subunit NDUFS3 level (Hao exposure is decreased and this is exacerbated by the com- et al. , 2010 ). DJ-1− /− mice exhibit decreased skeletal muscle bined exposure of the cells to hypoxia and nitric oxide. ATP (Hao et al. , 2010 ), associated with down regulation of Nitric oxide (NO) in combination with reactive oxygen uncoupling protein 4 and 5 (and decreased mitochondrial species (ROS) and the associated formation of potent length and fusion rate. DJ-1− /− MEFs exhibit a profound prooxidants such as peroxynitrite contributes to steato- decrease in mitochondrial quality. This is evident from sis and decreased mitochondrial quality (Jaeschke et al., impaired mitochondrial respiration, increased mitochon- 2002 ; Shiva et al. , 2005 ; Pacher et al. , 2007 ). For example, drial ROS, decreased mitochondrial membrane potential alcohol consumption is associated with increased mtDNA and characteristic alterations of mitochondrial shape, and damage, enhanced sensitivity to the mitochondrial per- depression of autophagy (Krebiehl et al. , 2010 ). meability transition, increased sensitivity to the NO- The concepts of reserve capacity and cellular RCR can dependent control of respiration, extensive modification be applied to mitochondrial quality control via fusion/ of the mitochondrial proteome, protein nitration and lipid fission in the context of these Parkinson’ s disease asso- peroxidation (Venkatraman et al., 2004a,b,c; Zelickson ciated proteins. The sensing of mitochondrial depolariza- et al. , 2011 ). Consistent with these findings, a mitochon- tion during mitophagy occurs via PINK1 whereby depo- drially targeted antioxidant decreases oxidative stress larization prevents the engagement of the N-terminus and steatosis (Chacko et al., 2011). It is now becoming mitochondrial targeting sequence (MTS) with the Tim23 clear that repair of damaged mitochondria is a complex pathway and prevents localization to the mitochon- process, mediated by the autophagy-lysosomal system, drial matrix side of the inner mitochondrial membrane which is defective in EtOH toxicity (Lemasters, 2007; (Narendra and Youle , 2011 ). This interruption in locali- Donohue , 2009 ; Ding et al. , 2011 ). Interestingly, enhanc- zation inhibits the normally active proteolytic cleavage ing autophagy with the therapeutic agent rapamycin of PINK1 and it accumulates at the outer mitochondrial decreased acute EtOH dependent hepatotoxicity (Ding membrane where it recruits and activates Parkin, resulting et al. , 2011 ). In this context, autophagy is protective in polyubiquitination of multiple mitochondrial protein because inhibition of the autophagic process resulted targets and subsequent targeting for mitophagy. It is note- in increased mitochondrial-dependent apoptosis. These worthy that either uncoupler (which will collapse both data also suggest that inhibition of the autophagic process ΔΨ the pH and M component of the proton motive force) occurs and is associated with increased steatosis and or valinomycin plus potassium (which collapses only the marked deterioration of mitochondrial quality probably ΔΨ M component) (Narendra et al., 2010b; Youle and Nar- through a Cyp2E1 mediated mechanism (Wu et al., 2010). endra , 2011 ) will prevent processing of PINK1, suggesting This is consistent with the observation that damaged that it is the transmembrane electrical field component of mitochondria that are not removed by mitophagy are the proton motive force that is responsible. Studies with susceptible to uncoupling and may promote cell death. isolated rat hepatocytes (Hafner et al. , 1990 ) indicate that The formation of Mallory-Denk bodies is characteristic under normal conditions there is only modest control of of alcoholic steatohepatitis in human patients and are the proton motive force exerted by the respiratory chain, comprised of protein aggregates containing cytokeratins, phosphorylating system, or proton leaks, illustrating the ubiquitin and p62 (Zatloukal et al., 2007; Rautou et al., high degree of homeostasis of this component of mito- 2010). These findings suggest that therapeutic strategies chondrial quality. This result supports the idea that under that link mitochondrial oxidative damage and autophagy ΔΨ these normal conditions mitochondrial M homeosta- may be beneficial in alcoholic liver disease. sis is maintained and mitophagy is avoided, even under changing metabolic conditions (e.g., ‘ state 3– 4 ’ -like transitions). Cardiovascular disease

Given the high energy demands of the cardiovascular Alcohol-dependent hepatotoxicity system it is not surprising that defects in bioenergetics are associated with the pathology of ischemia-reperfu-

In the liver, an early event in response to ethanol, endo- sion and heart failure. An example of how the state apparent toxin and cytokines are released, this depresses mito- changes based on cell phenotype can be drawn from our chondrial function, leading to hepatotoxicity (Arteel previous work, where cardiomyocytes were exposed to 1502 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics phenylephrine (Sansbury et al., 2011b). This resulted in hypertrophy of the cells and changes in cellular bioener- Increased ATP demand of volume overload getics. Notably, the hypertrophied cardiomyocytes dem- Increased catabolism onstrated a small movement toward state 3 compared of ATP/ADP with non-hypertrophied cells. In this instance, the dif- ATP ference in respiratory state was driven by a single factor, MitoQ i.e., a higher basal OCR due to increased ATP demand. HX XDH A similar response was shown in vivo in hypertrophied H2O2 and failing hearts, where failure was shown to deplete XO bioenergetic capacity almost completely (Gong et al., H2O2 Allopurinol 2003 ), which would be thought to push the stateapparent to near 3.0. These are several examples illustrating that an Figure 9 Mitochondrial quality control and xanthine oxidase in the intermediate respiratory state exists under physiological heart. conditions and that intracellular mitochondria possess The increase in VO leads to increased usage of ATP, resulting in the ability to move toward a more energetic condition increased levels of ADP and AMP. ADP and AMP are then degraded to hypoxanthine (HX) via purine catabolism. XO reacts with HX, using the available reserve cellular bioenergetic capac- forming superoxide and hydrogen peroxide (H2 O2 ) which damage ity. Evidence for a reserve capacity in functioning organs mitochondria, leading to bioenergetic dysfunction and increased and tissues has also been presented using 31 P-NMR tech- ATP catabolism. Collectively, this causes increased electron leak niques in tissues under metabolic stress (Sako et al., to form more ROS and decreased ATP production. The increase 1988 ). in mitochondrial ROS leads to increased conversion of xanthine dehydrogenase (XDH) to xanthine oxidase. The enhanced ROS During heart failure, the heart decompensates when generation from mitochondria and XO cause further damage to there is an imbalance between energy production and use, the mitochondria and left ventricular dysfunction. Mitochondrially leading to a decreased energy reserve in the heart (Liao targeted ubiquinone (Mito Q) inhibits the conversion of XDH to XO, et al. , 1996 ; Ingwall , 2009 ). For example, with volume which may depend on mitochondrial H2 O2 . overload (VO), there is a 2-fold increase in myocardial oxygen consumption and increased ATP consumption (Strauer , 1987 ; Yun et al. , 1992 ). As shown in Figure 9 , the shown that cyclical stretch of adult rat cardiomyocytes increased ATP demand in VO is also associated with the causes increased oxidative stress, mitochondrial swell- activation of the ROS-generating enzyme xanthine oxidase ing, and cytoskeletal disruption – all of which are pre- (XO) (Gladden et al., 2011a,b). This finding was translated vented by an XO inhibitor or the mitochondrial targeted to the clinic in a recent study which showed that left ven- drug MitoQ (Gladden et al., 2011b). More importantly, tricular biopsies taken from patients with isolated mitral MitoQ prevented XO activation, suggesting that the regurgitation had significant cardiomyocyte myofibrillar mitochondria themselves represent a more direct target degeneration along with increased protein nitration and in preventing oxidative stress, XO activation, and cel- lipofuscin accumulation, consistent with increased forma- lular remodeling in cardiomyocyte stretch. In a similar tion of ROS/RNS (Ahmed et al. , 2010 ). These oxidants are fashion, 24 h of VO in vivo also results in increased car- generated from a number of sources including XO. In addi- diomyocyte XO activity with decreased state 3 mitochon- tion to being a major source of reactive oxygen species, XO drial respiration and decreased LV contractility, which is linked to bioenergetic dysfunction since its substrates are significantly improved with allopurinol (Gladden derive from ATP catabolism (Figure 9). Correspondingly, et al. , 2011b ; Ulasova et al. , 2011 ). Most recently, ROS there was also evidence of aggregates of small mitochon- production from XO has been linked to myocardial dria in cardiomyocytes, which is generally considered high energy phosphates and ATP flux through creatine a response to bioenergetic deficit in cells. This increase kinase in the failing human heart (Hirsch et al., 2012). pro-oxidant environment could decrease mitochondrial Treatment of heart failure patients with an XO inhibi- quality and underlie the finding that mitochondria iso- tor increased phosphocreatine to ATP ratio and creatine lated from rats with chronic VO have a greater sensitiv- kinase flux (measured using in vivo 31 P magnetic reso- ity to Ca 2 +-induced PTP opening, and these volume-over- nance spectroscopy), which is consistent with the high loaded hearts have increased ischemia-reperfusion injury susceptibility of creatine kinase to oxidative modifica- (Marcil et al. , 2006 ). tions resulting in decreased activity and efficiency of To gain further insight into changes from oxida- high energy phosphate transfer from mitochondria to tive stress and bioenergetic dysfunction in VO, we have the contractile apparatus. B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics 1503

Obesity and diabetes overexpression of UCP1 in adipose tissue (Kopecky et al. , 1996 ) and skeletal muscle (Li et al. , 2000 ) prevented An excess consumption of high calorie foods appears diet-induced obesity in mice, suggesting that uncoupling to be one of the key factors in the epidemic of obesity of oxidative phosphorylation in these two organs is bene- (Nielsen and Popkin, 2003; Briefel and Johnson, 2004; ficial. Interestingly, oxidative phosphorylation is more Popkin et al., 2006; Duffey and Popkin, 2007; Kant and uncoupled in endurance athletes compared with sed- Graubard , 2004 ; Wang et al. , 2008 ), which increases the entary subjects (Befroy et al., 2008), and this appears to risk for developing diseases with a bioenergetic com- enhance fatty acid oxidation and diminish oxidative stress. ponent (Haslam and James , 2005 ). As described above, Moreover, gene programs regulating fat oxidation capacity these include cardiovascular disease (Roger et al. , 2012 ), in skeletal muscle are improved in atheletes compared hepatotoxicity (Zakhari and Li, 2007), and neurodegen- with sedentary subjects (Schrauwen -Hinderling et al., erative diseases (Bruce-Keller et al., 2009). While a high 2006 ), which display an increased gene profile favoring fat caloric intake can be offset by exercise (for example, the storage (Schrauwen-Hinderling et al., 2005). However, it is gold medalist Michael Phelps remains lean despite con- important to note that a higher metabolic capacity does not suming up to ∼ 12 000 calories per day), increasing evi- always equate with a decreased risk for metabolic disease. dence suggests that physical activity, on average, is declin- For example, American subjects of Asian Indian ancestry ing (Williamson et al., 1993; Smith et al., 1994; Robinson, are predisposed to obesity and type 2 diabetes and have 1999; Hu et al., 2003; Roger et al., 2012). This has led to higher mitochondrial genomic content and oxidative heightened efforts to promote increased energy utilization phosphorylation capacity compared with Americans of and to better understand how altered intermediary metab- European descent (Nair et al. , 2008 ). The South Asian pop- olism, perturbations in cellular bioenergetics, and genetic ulation does appear to have more coupled mitochondria, differences contribute to the risk for becoming obese and which has led to a ‘ mitochondrial efficiency hypothesis’ insulin resistant. that could explain the propensity for obesity in some pop- Mitochondria lie at the heart of systemic metabolic ulations (Bhopal et al., 2009). Other hypotheses relating to regulation. They act as end-point regulators of metabolic genetic predispositions such as the ‘ thrifty gene ’ hypoth- rate and affect thermogenesis primarily by increasing esis and the ‘ predation release ’ hypothesis have also been proton leak. In brown fat, the relatively high expression proposed (Speakman , 2008 ). The fact that mtDNA variants of uncoupling protein 1 (UCP1) allows re-entry of protons relate with the risk of type 2 diabetes does support a role for into the mitochondrial matrix without generating ATP. genetic factors relating to energetics and metabolism (Sun This uncoupling therefore generates an increase in sub- et al., 2003; Irwin et al., 2009; Wallace, 2011). However, the strate utilization and electron transport chain turnover sudden increase in obesity in areas that have genetically as well as energy in the form of heat (Tseng et al., 2010). dissimilar populations (such as the south-eastern United Despite the low amounts of brown fat in humans, as little States) does not appear to support any of these hypotheses as 50 g of brown fat has been estimated to be capable of exclusively. It appears that a stronger hypothesis integrat- utilizing up to 20% of basal caloric needs (Rothwell and ing physiological, genetic, and environmental frameworks Stock , 1983 ). This suggests that decreasing mitochondrial is required to explain the relatively abrupt increase in the efficiency is an attractive therapeutic option for obesity. prevalence of obesity and diabetes. Indeed, synthetic uncouplers such as 2,4-dinitrophenol The regulation of autophagy also appears to be impor- were used in the 1930s as a weight loss drug, and it was tant in the context of metabolic diseases such as obesity. capable of increasing metabolic rate by up to 40% (Cutting Interestingly, autophagy appears to be upregulated in and Tainter, 1933 ; Harper et al. , 2008 ). However, DNP has adipose tissues of obese humans (Kovsan et al. , 2011 ), a narrow therapeutic window (similar to the narrow dose- suggesting that it may be involved in adipocyte hypertro- response curves observed in XF experiments), and side phy, and inhibiting autophagy appears to have beneficial effects led to removal from the market (Harper et al., 2008). effects in the context of obesity. Conditional knock-out Nevertheless, it is clear that decreasing mitochondrial effi- of atg7 in adipocytes was shown to prevent diet-induced ciency may be a robust treatment option for obesity. obesity and to increase insulin sensitivity in mice (Singh The obvious drawback for such an approach is its sys- et al., 2009; Zhang et al., 2009). Interestingly, deletion of temic effects. Mitochondria in organs with high energetic atg7 was associated with significantly increased levels of needs (e.g., the heart) should likely maintain a high level mitochondria in white adipocytes, supporting a critical of mitochondrial coupling, while other organs such as role of autophagy in regulating mitochondrial number in adipose tissue could afford to be less economical. Indeed, adipose tissue. These changes were also associated with 1504 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics an increase in adipocyte fatty acid oxidation as well as complex and regulate much more than autophagy and changes in whole body metabolism (Singh et al., 2009; mitophagy. Zhang et al., 2009). Inactivation of other autophagy genes has resulted in similar results in vitro . For example, mouse − − embryonic fibroblasts (MEFs) isolated from atg5 / mice Summary accumulate less lipid when stimulated to develop into adipocytes, and atg7 −/− MEFs similarly cannot undergo Collectively, these studies illustrate how differences in adipogenesis to the same extent as their wild-type coun- mitochondrial economy, bioenergetic reserve capacity, terpart cells. This suggests that inhibiting autophagy may and autophagy regulate health and disease. The control be a therapeutic option for treating or preventing obesity of mitochondrial quality in which biogenesis is balanced or type 2 diabetes although the impact on mitochondrial with mitophagy is likely to serve as a productive frame- quality is unclear. In line with this view, a prospective, work for the design of mitochondrial therapeutics. As observational multicenter clinical study showed that expected, many of the factors controlling metabolism and hydroxychloroquine, an inhibitor of autophagy, is associ- susceptibility to disease appear to be driven by genetics ated with a reduced risk of diabetes (Wasko et al. , 2007). and the environment. It is now also becoming clear that While the above-mentioned studies suggest that physiological processes such as cell differentiation are inhibiting autophagy is beneficial in the context of associated with profound changes in cellular bioener- obesity, the role of autophagy and components critical to getics including reserve capacity (Schneider et al., 2011). the autophagic machinery remain unclear in the context The challenge for us is to continue to strive to more fully of insulin resistance. For example, an interesting recent understand how bioenergetics regulates cell and tissue study demonstrated that autophagy deficits undermine function. Certainly, this would best position us to develop the benefit of exercise in protection against high-fat-diet- better and more targeted therapies. induced glucose intolerance (He et al., 2012). Moreover, systemic knock-out of the parkin gene, which as described Acknowledgements: The authors acknowledge funding in the previous sections is involved in mitophagy, has also from the following sources: B.G.H. was supported by a been shown to prevent diet-induced obesity, steatohepa- grant from the NIH-NCRR (P20 RR024489). J.L. is sup- titis, and insulin resistance. However, the effect of parkin ported by NIH CA131653; S.B. is supported by NIH HL94518, deletion did not appear to be due to regulation of mito- HL103859. L.D.-I. was supported by NIH HL097176. J.Z. was chondria or intermediary metabolism. A lack of parkin supported by NIHR01-NS064090 and a VA merit award. expression prevented high fat diet-induced upregula- V.D.U. was supported by NIH Grants (HL109785, ES10167, tion of lipid transport proteins such as CD36, Sr-B1, and AA 13395 and DK 75865). FABP and the uptake of fat from the diet (Kim et al. , 2011 ). Hence, it appears that the metabolic effects of parkin are Received May 13, 2012; accepted June 22, 2012

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Bradford G. Hill received his PhD in Biochemistry from the Scott Ballinger received his undergraduate degrees from Texas University of Louisville. He completed postdoctoral training in A&M University and his PhD in Biochemistry from Emory University. mitochondrial biology in the laboratory of Victor Darley-Usmar at He completed postdoctoral training at the University of Vermont’s the University of Alabama-Birmingham before moving to U of L as Genetics and Toxicology Laboratory, as an Environmental Pathology an Assistant Professor of Medicine in the Center for Diabetes and Fellow and as a Department of Energy Alexander Hollaender Obesity Research. Dr. Hill’s research focuses on understanding Distinguished Fellow. He is currently a Professor of Pathology in the role of intermediary metabolism in cardiovascular health and the Division of Molecular and Cellular Pathology, Department of disease. The long-term objective of these studies is to identify Pathology and a senior scientist in the Center for Free Radical Biology derangements in metabolism that underlie the development of at the University of Alabama at Birmingham. His laboratory studies obesity and cardiovascular disease and to target these metabolic the role of the mitochondrion and its genetics upon influencing processes therapeutically. disease susceptibility with focus upon diseases influenced by the environment, particularly the cardiometabolic diseases.

Louis Dell’Italia received his MD and subsequent Internal Medicine Gloria A. Benavides holds a BSc in Clinical Chemist, and a MSc and training at Georgetown University and his Cardiology Fellowship a PhD in Biomedical Chemistry from the Autonomous University of Training at the University of Texas Health Science Center at San Nuevo Leon, Mexico. She was a Visiting Scholar in the Chemistry Antonio (UTSA). He served as Cardiology Faculty at UTSA and moved Department of Louisiana State University (1998–1999) and the to the University of Alabama-Birmingham (UAB) where he has been Pharmacognosy Department in The University of Mississippi (2001); since 1988 serving as a staff physician at the Birmingham VA Medical a Postdoctoral Researcher in the Departments of Environmental Center and Faculty at the UAB School of Medicine in the Department Health Sciences (2004–2007) and Pathology (2007–2011), and of Medicine and Division of Cardiovascular Disease. Dr. Dell’Italia’s is currently a Research Associate in the Pathology Department at research focuses on understanding the molecular and cellular University of Alabama at Birmingham (2011–current). mechanisms in the pathophysiology of a pure volume overload of the heart, in particular in patients with mitral regurgitation.

Jack R. Lancaster received his PhD in Biochemistry from the Jianhua Zhang was trained at University of Texas Medical Center University of Tennessee Health Sciences Center, and pursued at Dallas for her PhD. She then completed her postdoctoral postdoctoral work at Cornell and Duke universities. He assumed studies at the Whitehead Institute for Biomedical Research. faculty positions in the Department of Chemistry and Biochemistry, She was appointed to her first faculty position at University of Utah State University; Department of Surgery, University of Cincinnati Medical Center. In 2005 she joined the University of Pittsburgh; Department of Physiology, LSU Health Sciences Center Alabama at Birmingham where she is now an Associate Professor New Orleans; and the Department of Anesthesiology, University of in the Department of Pathology. She leads a research program Alabama at Birmingham. His research focus is on the chemical and to understand the mechanisms of apoptosis and autophagy in physical foundations of the biological actions of nitric oxide. neurodegeneration. 1512 B.G. Hill et al.: Mitochondrial quality control and cellular bioenergetics

Victor M. Darley-Usmar is presently Professor of Pathology and Director of the UAB Center for Free Radical Biology at UAB. In this role, he is able to combine major research interests in mitochondriology and reactive oxygen and nitrogen species with a pursuit of an understanding of the mechanisms of cell death in cardiovascular disease. He was trained as biochemist at the University of Essex in England and then moved to the University of Oregon to pursue his interests in the structure and function of mitochondrial proteins in human disease. After a period as a lecturer in Japan and a Research Scientist at the Wellcome Research Foundation in London he joined UAB to establish his own research group in the Department of Pathology.