Hill Et Al 2012

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 Dell’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,
Louisville, KY, USA
nerstone for establishing a framework for understanding
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
Scott Ballinger: Department of Pathology, University of Alabama at
resistance to stress. Because mitochondria are suscep-
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,
Birmingham, AL 35294, USA; and Department of Veteran Affairs
on several factors, including the integrity of mtDNA, elec-
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: Darley@uab.edu
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
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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
Stress RNS
2005; Kim et al., 2007; Youle and Narendra, 2011; Lee et RLS
al., 2012). This method of mitochondrial surveillance is ROS
↑ 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 main-
taining proper cell function. The formation of ‘new’ mito-
chondria is a tightly controlled process involving several
factors, including de novo synthesis of organelle compo-
Mitophagy
nents from cellular precursors, formation of mitochondrial
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,
nitrogen and lipid species (ROS/RNS/RLS). This oxidative stress
transcription, import, and assembly of nuclear-encoded
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.

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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
shown that the sub-populations of mitochondria destined
The cellular bioenergetic profile
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

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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
is used. Immediately upon exposure to the uncoupler,
Fundamentals of the bioenergetic 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 trans-
port typically inhibits the majority of oxygen consumption
500
in the cell, and we attribute the remaining O2 consumption
Reserve
to non-mitochondrial oxidases present in the cell. This
400
Oxygen consumption rate
Oligomycin capacity value is subtracted from other OCR measurements to deter-

mine the mitochondrial-based parameters. It should be
(pmol O2/min)
300 Maximal
oxygen
noted that for meaningful comparisons between different
consumption conditions or treatments, the basal OCR should be normal-
200 at complex
ATP- IV
ized to a factor to correct for different numbers of cells in the
Basal linked
assay or changes in cell size, e.g., cell atrophy or hypertro-
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-
ment of changes in OCR as indicated. This time course is annotated
profile
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

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A Cardiomyocytes Hepatocytes Endothelial cells
70 40 40
F A+R F A+R
F A+R

35

60 35
O
(pmol/min/μg ptn)
30
(pmol/min/μg ptn)
30
(pmol/min/μg ptn)
50
O
25 25
40 O
20 20
30
15 15
20
10 10
10 5 5
0 0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 0 10 20 30 40 50
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 con-
sumption 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 O2 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

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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
damaged d) Electron slippage
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, mito-
chondrial mass, or electron transport chain (ETC) integrity. Lastly, the effects of non-mitochondrial sources on oxygen consumption may be
examined.

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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 elimi-

nate 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 O2 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.
tial (ΔΨm) and induces a state 4-like respiratory condi-
tion (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

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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
Stateapparent = 4-
( Basal-oligo) et al., 2000) or spare respiratory (Choi et al., 2009) capacity
( FCCP-oligo) in cells under various conditions (Brand and Nicholls, 2011).
In terms of interpreting reserve capacity, the assign-
The stateapparent therefore can be modulated by changes in
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 stateapparent
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 stateapparent could involve changes in activation
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 (ΔΨM-generating processes, ATP production, and
in independent experiments.
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

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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
RCR basal =
( Basal-Anti A) of the amino acids glutamine and aspartate entering at
(Oligo-Anti A) the levels of α-ketoglutarate and oxaloacetate, respec-
tively, would influence basal metabolism. Fatty acids can
( FCCP-Anti A)
RCR max = also enter the TCA cycle through anaplerotic metabolism
(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

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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
how the glycolytic pathway responds to loss of mito-
Role for a ‘glycolytic reserve’ in the chondrial 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.

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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-
ECAR (mpH unit/min/μg protein)
0.8 tion are playing a key role (Figure 6) (Wallace, 2010, 2011;
Krzywanski et al., 2011).
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.
Basal For example, if these mutations altered mitochondrial
0 economy such that caloric utilization for ATP genera-
0 20 40 60 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
of glyceraldehyde-3-dehydrogenase) to inhibit glycolysis; alter-
better adapted to low calorie warmer climates. In the
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 avail-
ability and low energetic demand lifestyle. Figure 6 illus-
The impact of mitochondrial DNA trates this concept by showing that under conditions of
high ATP demands (e.g., adequate diet and exercise),
haplotype and damage on mito- electron flow energetics predominately manifests in ATP
chondrial quality generation with subtle differences existing in mitochon-
drial 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

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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
epidemiologic studies have shown that human longev-
Mitochondrial quality, mtDNA
ity 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

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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 more-
over, 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 Damage Associated levels are decreased, and oxidative stress and mitochon-
Molecular Pattern (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/
VPS34 complex activities. Some of the key molecular
Regulation of mitochondrial quality 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

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FIP200 Beclin/VPS
34 complex
LC3I
Atg 3, 5, 7 p-ERK,
p-AMPA
LC3II
Drp1
Fis1
LC3II
Nix Opa1
BNIP Mfn1/2
LC LC
3II 3II
LC
3II
p6
LC3II2 Cathepsins D,
B, C and L
I
3I
LC
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 mem-
branes 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 mem-
brane 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

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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
contradictory reports exist concerning whether p62 is
Regulation of mitochondrial quality
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

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Normal mito irreversible oxidative protein modification to metabolic

Damaged mito
Pink 1 and respiratory chain components or non-repairable
PARL
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
respiratory capacity in immunity. For example, it was
LC3
shown that CD8+ memory T-cells possessed a substan-

LC
tial reserve capacity that was responsive to interleukin-15

3
(IL-15), which regulated the stability of memory T-cells

LC3 Up
tak
e Parkin, also
after infection (van der Windt et al., 2012). A high reserve
capacity may also be linked to proliferation of certain cell
LC3
activated UPS
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, mito-
chondrial 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

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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
the pH and ΔΨM 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
ΔΨM 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
these normal conditions mitochondrial ΔΨM 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 stateapparent
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

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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
increased levels of ADP and AMP. ADP and AMP are then degraded
the ability to move toward a more energetic condition
to hypoxanthine (HX) via purine catabolism. XO reacts with HX,
using the available reserve cellular bioenergetic capac- forming superoxide and hydrogen peroxide (H2O2) 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 31P-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 H2O2.
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 31P magnetic reso-
ity to Ca2+-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.

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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

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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
accumulate less lipid when stimulated to develop into
Summary
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
References
Affourtit, C. and Brand, M.D. (2008). Uncoupling protein-2 Amo, T., Yadava, N., Oh, R., Nicholls, D.G., and Brand, M.D. (2008).
contributes significantly to high mitochondrial proton leak in Experimental assessment of bioenergetic differences caused
INS-1E insulinoma cells and attenuates glucose-stimulated by the common European mitochondrial DNA haplogroups H
insulin secretion. Biochem. J. 409, 199–204. and T. Gene 411, 69–76.
Ahmed, M.I., Gladden, J.D., Litovsky, S.H., Lloyd, S.G., Gupta, Apostolova, N., Blas-Garcia, A., and Esplugues, J.V. (2011).
H., Inusah, S., Denney, T., Jr., Powell, P., McGiffin, D.C., and Mitochondria sentencing about cellular life and death: a matter
Dell’Italia, L.J. (2010). Increased oxidative stress and cardio- of oxidative stress. Curr. Pharm. Des. 17, 4047–4060.
myocyte myofibrillar degeneration in patients with chronic Arteel, G., Marsano, L., Mendez, C., Bentley, F., and McClain, C.J.
isolated mitral regurgitation and ejection fraction > 60%. J. Am. (2003). Advances in alcoholic liver disease. Best. Pract. Res.
Coll. Cardiol. 55, 671–679. Clin. Gastroenterol. 17, 625–647.
Amo, T. and Brand, M.D. (2007). Were inefficient mitochondrial Ashford, T.P. and Porter, K.R. (1962). Cytoplasmic components in
haplogroups selected during migrations of modern hepatic cell lysosomes. J. Cell. Biol. 12, 198–202.
humans? A test using modular kinetic analysis of coupling in Bailey, S.M. (2003). A review of the role of reactive oxygen and
mitochondria from cybrid cell lines. Biochem. J. 404, nitrogen species in alcohol-induced mitochondrial dysfunction.
345–351. Free Radic. Res. 37, 585–596.

Authenticated
Bailey, S.M. and Cunningham, C.C. (2002). Contribution of Brown, G.C., Lakin-Thomas, P.L., and Brand, M.D. (1990). Control of
mitochondria to oxidative stress associated with alcoholic liver respiration and oxidative phosphorylation in isolated rat liver
disease. Free Radic. Biol. Med. 32, 11–16. cells. FEBS 192, 355–362.
Ballinger, S.W., Patterson, C., Yan, C.N., Doan, R., Burow, D.L., Bruce-Keller, A.J., Keller, J.N., and Morrison, C.D. (2009). Obesity
Young, C.G., Yakes, F.M., Van Houten, B., Ballinger, C.A., and vulnerability of the CNS. Biochim. Biophys. Acta 1792,
Freeman, B.A., et al. (2000). Hydrogen peroxide- and 395–400.
peroxynitrite-induced mitochondrial DNA damage and Brunengraber, H. and Roe, C.R. (2006). Anaplerotic molecules:
dysfunction in vascular endothelial and smooth muscle cells. current and future. J. Inherit. Metab. Dis. 29, 327–331.
Circ. Res. 86, 960–966. Cakir, Y., Yang, Z., Knight, C.A., Pompilius, M., Westbrook, D.,
Ballinger, S.W., Patterson, C., Knight-Lozano, C.A., Burow, D.L., Bailey, S.M., Pinkerton, K.E., and Ballinger, S.W. (2007). Effect
Conklin, C.A., Hu, Z., Reuf, J., Horaist, C., Lebovitz, R., Hunter, of alcohol and tobacco smoke on mtDNA damage and athero-
G.C., et al. (2002). Mitochondrial integrity and function in genesis. Free Radic. Biol. Med. 43, 1279–1288.
atherogenesis. Circulation 106, 544–549. Cao, W., Medvedev, A.V., Daniel, K.W., and Collins, S. (2001).
Band, M., Joel, A., Hernandez, A., and Avivi, A. (2009). Hypoxia- beta-Adrenergic activation of p38 MAP kinase in adipocytes:
induced BNIP3 expression and mitophagy: in vivo comparison cAMP induction of the uncoupling protein 1 (UCP1) gene
of the rat and the hypoxia-tolerant mole rat, Spalax ehrenbergi. requires p38 MAP kinase. J. Biol. Chem. 276, 27077–27082.
FASEB J. 23, 2327–2335. Carreira, R.S., Lee, P., and Gottlieb, R.A. (2011). Mitochondrial
Bayot, A., Basse, N., Lee, I., Gareil, M., Pirotte, B., Bulteau, A.L., therapeutics for cardioprotection. Curr. Pharm. Des. 17,
Friguet, B., and Reboud-Ravaux, M. (2008). Towards the 2017–2035.
control of intracellular protein turnover: mitochondrial Lon Chacko, B.K., Srivastava, A., Johnson, M.S., Benavides, G.A., Chang,
protease inhibitors versus proteasome inhibitors. Biochimie M.J., Ye, Y., Jhala, N., Murphy, M.P., Kalyanaraman, B., and
90, 260–269. Darley-Usmar, V.M. (2011). Mitochondria-targeted ubiquinone
Befroy, D.E., Petersen, K.F., Dufour, S., Mason, G.F., Rothman, (MitoQ) decreases ethanol-dependent micro and macro
D.L., and Shulman, G.I. (2008). Increased substrate hepatosteatosis. Hepatology 54, 153–163.
oxidation and mitochondrial uncoupling in skeletal muscle of Chan, D.C. (2006). Mitochondrial fusion and fission in mammals.
endurance-trained individuals. Proc. Natl. Acad. Sci. USA 105, Annu. Rev. Cell. Dev. Biol. 22, 79–99.
16701–16706. Chan, N.C., Salazar, A.M., Pham, A.H., Sweredoski, M.J., Kolawa,
Bhopal, R.S. and Rafnsson, S.B. (2009). Could mitochondrial N.J., Graham, R.L., Hess, S., and Chan, D.C. (2011). Broad
efficiency explain the susceptibility to adiposity, metabolic activation of the ubiquitin-proteasome system by Parkin is
syndrome, diabetes and cardiovascular diseases in South critical for mitophagy. Hum. Mol. Genet. 20, 1726–1737.
Asian populations? Int. J. Epidemiol. 38, 1072–1081. Chance, B. and Baltscheffsky, H. (1958a). Respiratory enzymes in
Bordicchia, M., Liu, D., Amri, E.Z., Ailhaud, G., Dessi-Fulgheri, P., oxidative phosphorylation. VII. Binding of intramitochondrial
Zhang, C., Takahashi, N., Sarzani, R., and Collins, S. (2012). reduced pyridine nucleotide. J. Biol. Chem. 233,
Cardiac natriuretic peptides act via p38 MAPK to induce 736–739.
the brown fat thermogenic program in mouse and human Chance, B. and Baltscheffsky, M. (1958b). Spectroscopic effects
adipocytes. J. Clin. Invest. 122, 1022–1036. of adenosine diphosphate upon the respiratory pigments of
Bota, D.A. and Davies, K.J. (2001). Protein degradation in rat-heart-muscle sarcosomes. Biochem. J. 68, 283–295.
mitochondria: implications for oxidative stress, aging and Choi, S.W., Gerencser, A.A., and Nicholls, D.G. (2009). Bioenergetic
disease: a novel etiological classification of mitochondrial analysis of isolated cerebrocortical nerve terminals on a
proteolytic disorders. Mitochondrion 1, 33–49. microgram scale: spare respiratory capacity and stochastic
Bota, D.A. and Davies, K.J. (2002). Lon protease prefer- mitochondrial failure. J. Neurochem. 109, 1179–1191.
entially degrades oxidized mitochondrial aconitase by an Chu, C.T. (2010). A pivotal role for PINK1 and autophagy in
ATP-stimulated mechanism. Nat. Cell Biol. 4, 674–680. mitochondrial quality control: implications for Parkinson
Boveris, A. and Chance, B. (1973). The mitochondrial generation of disease. Hum. Mol. Genet. 19, R28–37.
hydrogen peroxide. General properties and effect of hyperbaric Chuang, G.C., Yang, Z., Westbrook, D.G., Pompilius, M., Ballinger,
oxygen. Biochem. J. 134, 707–716. C.A., White, C.R., Krzywanski, D.M., Postlethwait, E.M., and
Boveris, A., Oshino, N., and Chance, B. (1972). The cellular Ballinger, S.W. (2009). Pulmonary ozone exposure induces
production of hydrogen peroxide. Biochem. J. 128, 617–630. vascular dysfunction, mitochondrial damage, and athero-
Brand, M.D. (1990). The proton leak across the mitochondrial inner genesis. Am. J. Physiol. Lung Cell Mol. Physiol. 297, L209–216.
membrane. Biochim. Biophys. Acta 1018, 128–133. Cooper, C.E. and Giulivi, C. (2007). Nitric oxide regulation of
Brand, M.D. and Nicholls, D.G. (2011). Assessing mitochondrial mitochondrial oxygen consumption II: Molecular mechanism
dysfunction in cells. Biochem. J. 435, 297–312. and tissue physiology. Am. J. Physiol. Cell Physiol. 292,
Briefel, R.R. and Johnson, C.L. (2004). Secular trends in dietary C1993–2003.
intake in the United States. Annu. Rev. Nutr. 24, 401–431. Cordain, L., Miller, J.B., Eaton, S.B., Mann, N., Holt, S.H., and Speth,
Brookes, P.S., Yoon, Y., Robotham, J.L., Anders, M.W., and Sheu, J.D. (2000). Plant-animal subsistence ratios and macronutrient
S.S. (2004). Calcium, ATP, and ROS: a mitochondrial love-hate energy estimations in worldwide hunter-gatherer diets. Am. J.
triangle. Am. J. Physiol. Cell Physiol. 287, C817–833. Clin. Nutr. 71, 682–692.
Brown, G.C. (1992). The leaks and slips of bioenergetic membranes. Coskun, P., Wyrembak, J., Schriner, S., Chen, H.W., Marciniack, C.,
FASEB J. 6, 2961–2965. Laferla, F., and Wallace, D.C. (2012). A mitochondrial etiology

Authenticated
of Alzheimer and Parkinson disease. Biochim. Biophys. Acta Finck, B.N. and Kelly, D.P. (2007). Peroxisome proliferator-activated
1820, 553–564. receptor gamma coactivator-1 (PGC-1) regulatory cascade in
Cotan, D., Cordero, M.D., Garrido-Maraver, J., Oropesa-Avila, M., cardiac physiology and disease. Circulation 115, 2540–2548.
Rodriguez-Hernandez, A., Gomez Izquierdo, L., De la Mata, M., Fukuda, R., Zhang, H., Kim, J.W., Shimoda, L., Dang, C.V., and
De Miguel, M., Lorite, J.B., Infante, E.R., et al. (2011). Secondary Semenza, G.L. (2007). HIF-1 regulates cytochrome oxidase
coenzyme Q10 deficiency triggers mitochondria degradation by subunits to optimize efficiency of respiration in hypoxic cells.
mitophagy in MELAS fibroblasts. FASEB J. 25, 2669–2687. Cell 129, 111–122.
Cutting, W.L.T. and Tainter, M.L. (1933). Actions and uses of Garedew, A., Andreassi, C., and Moncada, S. (2012). Mitochondrial
dinitrophenol: promising metabolic applications. J. Am. Med. dynamics, biogenesis, and function are coordinated with the
Assoc. 101, 1472–1475. cell cycle by APC/C CDH1. Cell Metab. 15, 466–479.
De Benedictis, G., Rose, G., Carrieri, G., De Luca, M., Falcone, E., Gegg, M.E., Cooper, J.M., Chau, K.Y., Rojo, M., Schapira, A.H., and
Passarino, G., Bonafe, M., Monti, D., Baggio, G., Bertolini, Taanman, J.W. (2010). Mitofusin 1 and mitofusin 2 are ubiqui-
S., et al. (1999). Mitochondrial DNA inherited variants are tinated in a PINK1/parkin-dependent manner upon induction of
associated with successful aging and longevity in humans. mitophagy. Hum. Mol. Genet. 19, 4861–4870.
FASEB J. 13, 1532–1536. Geisler, S., Holmstrom, K.M., Skujat, D., Fiesel, F.C., Rothfuss, O.C.,
Diaz-Ruiz, R., Averet, N., Araiza, D., Pinson, B., Uribe-Carvajal, S., Kahle, P.J., and Springer, W. (2010). PINK1/Parkin-mediated
Devin, A., and Rigoulet, M. (2008). Mitochondrial oxidative mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell
phosphorylation is regulated by fructose 1,6-bisphosphate. A Biol. 12, 119–131.
possible role in Crabtree effect induction? J. Biol. Chem. 283, Gerencser, A.A., Neilson, A., Choi, S.W., Edman, U., Yadava, N.,
26948–26955. Oh, R.J., Ferrick, D.A., Nicholls, D.G., and Brand, M.D. (2009).
Diers, A.R., Broniowska, K.A., Chang, C.F., and Hogg, N. (2012). Quantitative microplate-based respirometry with correction for
Pyruvate fuels mitochondrial respiration and proliferation of oxygen diffusion. Anal. Chem. 81, 6868–6878.
breast cancer cells: effect of monocarboxylate transporter Gladden, J.D., Ahmed, M.I., Litovsky, S.H., Schiros, C.G., Lloyd, S.G.,
inhibition. Biochem. J. 444, 561–571. Gupta, H., Denney, T.S., Jr., Darley-Usmar, V., McGiffin, D.C.,
Ding, W.X., Ni, H.M., Li, M., Liao, Y., Chen, X., Stolz, D.B., Dorn, G.W., and Dell’Italia, L.J. (2011a). Oxidative stress and myocardial
2nd, and Yin, X.M. (2010). Nix is critical to two distinct phases remodeling in chronic mitral regurgitation. Am. J. Med. Sci.
of mitophagy, reactive oxygen species-mediated autophagy 342, 114–119.
induction and Parkin-ubiquitin-p62-mediated mitochondrial Gladden, J.D., Zelickson, B.R., Wei, C.C., Ulasova, E., Zheng, J.,
priming. J. Biol. Chem. 285, 27879–27890. Ahmed, M.I., Chen, Y., Bamman, M., Ballinger, S., Darley-
Ding, W.X., Li, M., and Yin, X.M. (2011). Selective taste of ethanol- Usmar, V., et al. (2011b). Novel insights into interactions
induced autophagy for mitochondria and lipid droplets. between mitochondria and xanthine oxidase in acute cardiac
Autophagy 7, 248–249. volume overload. Free Radic. Biol. Med. 51, 1975–1984.
Divakaruni, A.S. and Brand, M.D. (2011). The regulation and Gomes, L.C., Di Benedetto, G., and Scorrano, L. (2011). During
physiology of mitochondrial proton leak. Physiology autophagy mitochondria elongate, are spared from degradation
(Bethesda) 26, 192–205. and sustain cell viability. Nat. Cell Biol. 13, 589–598.
Donohue, T.M., Jr. (2009). Autophagy and ethanol-induced liver Gomez-Duran, A., Pacheu-Grau, D., Lopez-Gallardo, E.,
injury. World J. Gastroenterol. 15, 1178–1185. Diez-Sanchez, C., Montoya, J., Lopez-Perez, M.J., and
Dranka, B.P., Hill, B.G., and Darley-Usmar, V.M. (2010). Ruiz-Pesini, E. (2010). Unmasking the causes of multifactorial
Mitochondrial reserve capacity in endothelial cells: the impact disorders: OXPHOS differences between mitochondrial
of nitric oxide and reactive oxygen species. Free Radic. Biol. haplogroups. Hum. Mol. Genet. 19, 3343–3353.
Med. 48, 905–914. Gong, G., Liu, J., Liang, P., Guo, T., Hu, Q., Ochiai, K., Hou, M., Ye, Y.,
Dranka, B.P., Benavides, G.A., Diers, A.R., Giordano, S., Zelickson, Wu, X., Mansoor, A., et al. (2003). Oxidative capacity in failing
B.R., Reily, C., Zou, L., Chatham, J.C., Hill, B.G., Zhang, J., hearts. Am. J. Physiol. Heart Circ. Physiol. 285, H541–548.
et al. (2011). Assessing bioenergetic function in response to Green, D.R., Galluzzi, L., and Kroemer, G. (2011). Mitochondria and
oxidative stress by metabolic profiling. Free Radic. Biol. Med. the autophagy-inflammation-cell death axis in organismal
51, 1621–1635. aging. Science 333, 1109–1112.
Duffey, K.J. and Popkin, B.M. (2007). Shifts in patterns and Gutierrez, J., Ballinger, S.W., Darley-Usmar, V.M., and Landar, A.
consumption of beverages between 1965 and 2002. Obesity (2006). Free radicals, mitochondria, and oxidized lipids: the
(Silver Spring) 15, 2739–2747. emerging role in signal transduction in vascular cells. Circ.
Echtay, K.S., Esteves, T.C., Pakay, J.L., Jekabsons, M.B., Lambert, Res. 99, 924–932.
A.J., Portero-Otin, M., Pamplona, R., Vidal-Puig, A.J., Wang, S., Hafner, R.P., Brown, G.C., and Brand, M.D. (1990). Analysis of the
Roebuck, S.J., et al. (2003). A signalling role for 4-hydroxy-2- control of respiration rate, phosphorylation rate, proton leak rate
nonenal in regulation of mitochondrial uncoupling. EMBO. J. and protonmotive force in isolated mitochondria using the’top-
22, 4103–4110. down’ approach of metabolic control theory. FEBS 188, 313–319.
Elmore, S.P., Qian, T., Grissom, S.F., and Lemasters, J.J. (2001). The Hao, L.Y., Giasson, B.I., and Bonini, N.M. (2010). DJ-1 is critical for
mitochondrial permeability transition initiates autophagy in rat mitochondrial function and rescues PINK1 loss of function.
hepatocytes. FASEB J. 15, 2286–2287. Proc. Natl. Acad. Sci. USA 107, 9747–9752.
Ferrick, D.A., Neilson, A., and Beeson, C. (2008). Advances in Harper, M.E., Green, K., and Brand, M.D. (2008). The efficiency of
measuring cellular bioenergetics using extracellular flux. Drug. cellular energy transduction and its implications for obesity.
Discov. Today 13, 268–274. Annu. Rev. Nutr. 28, 13–33.

Authenticated
Haslam, D.W. and James, W.P. (2005). Obesity. Lancet 366, Kant, A.K. and Graubard, B.I. (2004). Eating out in America,
1197–1209. 1987–2000: trends and nutritional correlates. Prev. Med. 38,
He, C., Bassik, M.C., Moresi, V., Sun, K., Wei, Y., Zou, Z., An, Z., 243–249.
Loh, J., Fisher, J., Sun, Q., et al. (2012). Exercise-induced Kant, A.K. and Graubard, B.I. (2006). Secular trends in patterns of
BCL2-regulated autophagy is required for muscle glucose self-reported food consumption of adult Americans: NHANES
homeostasis. Nature 481, 511–515. 1971–1975 to NHANES 1999–2002. Am. J. Clin. Nutr. 84,
Higdon, A.N., Benavides, G.A., Chacko, B.K., Ouyang, X., Johnson, 1215–1223.
M.S., Landar, A., Zhang, J., and Darley-Usmar, V.M. (2012). Karbowski, M. and Neutzner, A. (2012). Neurodegeneration as
Hemin causes mitochondrial dysfunction in endothelial cells a consequence of failed mitochondrial maintenance. Acta
through promoting lipid peroxidation: the protective role of Neuropathol. 123, 157–171.
autophagy. Am. J. Physiol Heart Circ. Physiol. 302, H1394–1409. Kauppinen, R.A. and Nicholls, D.G. (1986). Synaptosomal
Hill, B.G., Dranka, B.P., Zou, L., Chatham, J.C., and Darley-Usmar, bioenergetics. The role of glycolysis, pyruvate oxidation and
V.M. (2009). Importance of the bioenergetic reserve capacity responses to hypoglycaemia. Eur. J. Biochem. 158, 159–165.
in response to cardiomyocyte stress induced by 4-hydrox- Kim, I. and Lemasters, J.J. (2011). Mitochondrial degradation by
ynonenal. Biochem. J. 424, 99–107. autophagy (mitophagy) in GFP-LC3 transgenic hepatocytes
Hirsch, G.A., Bottomley, P.A., Gerstenblith, G., and Weiss, R.G. during nutrient deprivation. Am. J. Physiol. Cell Ph. 300,
(2012). Allopurinol acutely increases adenosine triphospate C308–317.
energy delivery in failing human hearts. J. Am. Coll. Cardiol. 59, Kim, I., Rodriguez-Enriquez, S., and Lemasters, J.J. (2007). Selective
802–808. degradation of mitochondria by mitophagy. Arch. Biochem.
Hori, O., Ichinoda, F., Tamatani, T., Yamaguchi, A., Sato, N., Ozawa, Biophys. 462, 245–253.
K., Kitao, Y., Miyazaki, M., Harding, H.P., Ron, D., et al. (2002). Kim, K.Y., Stevens, M.V., Akter, M.H., Rusk, S.E., Huang, R.J., Cohen,
Transmission of cell stress from endoplasmic reticulum to A., Noguchi, A., Springer, D., Bocharov, A.V., Eggerman, T.L.,
mitochondria: enhanced expression of Lon protease. J. Cell et al. (2011). Parkin is a lipid-responsive regulator of fat uptake
Biol. 157, 1151–1160. in mice and mutant human cells. J. Clin. Invest. 121, 3701–3712.
Hu, F.B., Li, T.Y., Colditz, G.A., Willett, W.C., and Manson, J.E. (2003). Kopecky, J., Rossmeisl, M., Hodny, Z., Syrovy, I., Horakova, M., and
Television watching and other sedentary behaviors in relation Kolarova, P. (1996). Reduction of dietary obesity in aP2-Ucp
to risk of obesity and type 2 diabetes mellitus in women. J. Am. transgenic mice: mechanism and adipose tissue morphology.
Med. Assoc. 289, 1785–1791. Am. J. Physiol. 270, E776–786.
Ingwall, J.S. (2009). Energy metabolism in heart failure and Korzeniewski, B., Harper, M.E., and Brand, M.D. (1995). Proportional
remodelling. Cardiovasc. Res. 81, 412–419. activation coefficients during stimulation of oxidative
Irwin, J.A., Saunier, J.L., Niederstatter, H., Strouss, K.M., Sturk, phosphorylation by lactate and pyruvate or by vasopressin.
K.A., Diegoli, T.M., Brandstatter, A., Parson, W., and Parsons, Biochim. Biophys. Acta 1229, 315–322.
T.J. (2009). Investigation of heteroplasmy in the human Kovsan, J., Bluher, M., Tarnovscki, T., Kloting, N., Kirshtein, B.,
mitochondrial DNA control region: a synthesis of observations Madar, L., Shai, I., Golan, R., Harman-Boehm, I., Schon, M.R.,
from more than 5000 global population samples. J. Mol. Evol. et al. (2011). Altered autophagy in human adipose tissues in
68, 516–527. obesity. J. Clin. Endocrinol. Metab. 96, E268–277.
Jaber, N., Dou, Z., Chen, J.-S., Catanzaro, J., Jiang, Y.-P., Ballou, L.M., Krebiehl, G., Ruckerbauer, S., Burbulla, L.F., Kieper, N., Maurer, B.,
Selinger, E., Ouyang, X., Lin, R.Z., Zhang, J., et al. (2012). Class Waak, J., Wolburg, H., Gizatullina, Z., Gellerich, F.N., Woitalla,
III PI3K Vps34 plays an essential role in autophagy and in heart D., et al. (2010). Reduced basal autophagy and impaired
and liver function. Proc. Natl. Acad. Sci. USA 109, 2003–2008. mitochondrial dynamics due to loss of Parkinson’s disease-
Jaeschke, H., Gores, G.J., Cederbaum, A.I., Hinson, J.A., Pessayre, associated protein DJ-1. PLoS One 5, e9367.
D., and Lemasters, J.J. (2002). Mechanisms of hepatotoxicity. Krysko, D.V., Agostinis, P., Krysko, O., Garg, A.D., Bachert, C.,
Toxicol. Sci. 65, 166–176. Lambrecht, B.N., and Vandenabeele, P. (2011). Emerging
James, A.M., Wei, Y.H., Pang, C.Y., and Murphy, M.P. (1996). Altered role of damage-associated molecular patterns derived from
mitochondrial function in fibroblasts containing MELAS or mitochondria in inflammation. Trends Immunol. 32,
MERRF mitochondrial DNA mutations. Biochem. J. 318 ( Pt 2), 157–164.
401–407. Krzywanski, D.M., Moellering, D.R., Fetterman, J.L., Dunham-Snary,
Jenner, P. (2003). Oxidative stress in Parkinson’s disease. Ann. K.J., Sammy, M.J., and Ballinger, S.W. (2011). The mitochondrial
Neurol. 53 Suppl 3, S26–36; discussion S36–28. paradigm for cardiovascular disease susceptibility and cellular
Jia, W. and He, Y.W. (2011). Temporal regulation of intracellular function: a complementary concept to Mendelian genetics.
organelle homeostasis in T lymphocytes by autophagy. J. Lab. Invest. 91, 1122–1135.
Immunol. 186, 5313–5322. Lee, J., Giordano, S., and Zhang, J. (2012). Autophagy, mitochondria
Jin, S.M. and Youle, R.J. (2012). PINK1- and Parkin-mediated and oxidative stress: cross-talk and redox signalling. Biochem.
mitophagy at a glance. J. Cell. Sci. 125, 795–799. J. 441, 523–540.
Johnson-Cadwell, L.I., Jekabsons, M.B., Wang, A., Polster, B.M., Lemasters, J.J. (2005). Selective mitochondrial autophagy, or
and Nicholls, D.G. (2007).‘Mild Uncoupling’ does not decrease mitophagy, as a targeted defense against oxidative stress,
mitochondrial superoxide levels in cultured cerebellar mitochondrial dysfunction, and aging. Rejuv. Res. 8, 3–5.
granule neurons but decreases spare respiratory capacity Lemasters, J.J. (2007). Modulation of mitochondrial membrane
and increases toxicity to glutamate and oxidative stress. J. permeability in pathogenesis, autophagy and control of
Neurochem. 101, 1619–1631. metabolism. J. Gastroen. Hepatol. 22 Suppl 1, S31–37.

Authenticated
Lemasters, J.J., Theruvath, T.P., Zhong, Z., and Nieminen, A.L. mitochondrial clustering but not mitophagy; VDAC1 is
(2009). Mitochondrial calcium and the permeability transition dispensable for both. Autophagy 6, 1090–1106.
in cell death. Biochim. Biophys. Acta 1787, 1395–1401. Narendra, D.P., Jin, S.M., Tanaka, A., Suen, D.F., Gautier, C.A., Shen,
Li, B., Nolte, L.A., Ju, J.S., Han, D.H., Coleman, T., Holloszy, J.O., J., Cookson, M.R., and Youle, R.J. (2010b). PINK1 is selectively
and Semenkovich, C.F. (2000). Skeletal muscle respiratory stabilized on impaired mitochondria to activate Parkin. PLoS
uncoupling prevents diet-induced obesity and insulin Biol. 8, e1000298.
resistance in mice. Nat. Med. 6, 1115–1120. Nascimben, L., Ingwall, J.S., Lorell, B.H., Pinz, I., Schultz, V.,
Liao, R., Nascimben, L., Friedrich, J., Gwathmey, J.K., and Ingwall, Tornheim, K., and Tian, R. (2004). Mechanisms for increased
J.S. (1996). Decreased energy reserve in an animal model glycolysis in the hypertrophied rat heart. Hypertension 44,
of dilated cardiomyopathy. Relationship to contractile 662–667.
performance. Circ. Res. 78, 893–902. Nicholls, D.G., Darley-Usmar, V.M., Wu, M., Jensen, P.B., Rogers,
Liu, L., Feng, D., Chen, G., Chen, M., Zheng, Q., Song, P., Ma, G.W., and Ferrick, D.A. (2010). Bioenergetic profile experiment
Q., Zhu, C., Wang, R., Qi, W., et al. (2012). Mitochondrial using C2C12 myoblast cells. J. Vis. Exp. 46, 2511.
outer-membrane protein FUNDC1 mediates hypoxia-induced Nielsen, S.J. and Popkin, B.M. (2003). Patterns and trends in food
mitophagy in mammalian cells. Nat. Cell. Biol. 14, 177–185. portion sizes, 1977–1998. J. Am. Med. Assoc. 289, 450–453.
Marcil, M., Ascah, A., Matas, J., Belanger, S., Deschepper, C.F., and Nisoli, E., Clementi, E., Paolucci, C., Cozzi, V., Tonello, C., Sciorati,
Burelle, Y. (2006). Compensated volume overload increases C., Bracale, R., Valerio, A., Francolini, M., Moncada, S., et al.
the vulnerability of heart mitochondria without affecting their (2003). Mitochondrial biogenesis in mammals: the role of
functions in the absence of stress. J. Moll. Cell. Cardiol. 41, endogenous nitric oxide. Science 299, 896–899.
998–1009. Nobes, C.D., Lakin-Thomas, P.L., and Brand, M.D. (1989). The
Matsuda, N., Sato, S., Shiba, K., Okatsu, K., Saisho, K., Gautier, contribution of ATP turnover by the Na+/K+-ATPase to the rate
C.A., Sou, Y.S., Saiki, S., Kawajiri, S., Sato, F., et al. (2010). of respiration of hepatocytes. Effects of thyroid status and fatty
PINK1 stabilized by mitochondrial depolarization recruits acids. Biochim. Biophys. Acta 976, 241–245.
Parkin to damaged mitochondria and activates latent Parkin for Nobes, C.D., Hay, W.W., Jr., and Brand, M.D. (1990). The mechanism
mitophagy. J. Cell Biol. 189, 211–221. of stimulation of respiration by fatty acids in isolated
Menzies, R.A. and Gold, P.H. (1971). The turnover of mitochondria in hepatocytes. J. Biol. Chem. 265, 12910–12915.
a variety of tissues of young adult and aged rats. J. Biol. Chem. Okamoto, K. and Kondo-Okamoto, N. (2012). Mitochondria and
246, 2425–2429. autophagy: Critical interplay between the two homeostats.
Michel, S., Wanet, A., De Pauw, A., Rommelaere, G., Arnould, T., Biochim. Biophys. Acta 1820, 595–600.
and Renard, P. (2012). Crosstalk between mitochondrial (dys) Okatsu, K., Saisho, K., Shimanuki, M., Nakada, K., Shitara, H.,
function and mitochondrial abundance. J. Cell Physiol. 227, Sou, Y.S., Kimura, M., Sato, S., Hattori, N., Komatsu, M., et al.
2297–2310. (2010). p62/SQSTM1 cooperates with Parkin for perinuclear
Mitra, K. and Lippincott-Schwartz, J. (2010). Analysis of clustering of depolarized mitochondria. Genes Cells 15,
mitochondrial dynamics and functions using imaging 887–900.
approaches. Curr. Protoc. Cell Biol. Chapter 4, Unit 4.2521. Oliva, C.R., Nozell, S.E., Diers, A., McClugage, S.G., 3rd, Sarkaria,
Mitra, K., Wunder, C., Roysam, B., Lin, G., and Lippincott-Schwartz, J.N., Markert, J.M., Darley-Usmar, V.M., Bailey, S.M., Gillespie,
J. (2009). A hyperfused mitochondrial state achieved at G1-S G.Y., Landar, A., et al. (2010). Acquisition of temozolomide
regulates cyclin E buildup and entry into S phase. Proc.Natl. chemoresistance in gliomas leads to remodeling of
Acad. Sci. USA 106, 11960–11965. mitochondrial electron transport chain. J. Biol. Chem. 285,
Moreno-Loshuertos, R., Acin-Perez, R., Fernandez-Silva, P., Movilla, 39759–39767.
N., Perez-Martos, A., Rodriguez de Cordoba, S., Gallardo, Osiewacz, H.D. (2011). Mitochondrial quality control in aging and
M.E., and Enriquez, J.A. (2006). Differences in reactive oxygen lifespan control of the fungal aging model Podospora anserina.
species production explain the phenotypes associated with Biochem. Soc. Trans. 39, 1488–1492.
common mouse mitochondrial DNA variants. Nat. Genet. 38, Pacher, P., Beckman, J.S., and Liaudet, L. (2007). Nitric oxide and
1261–1268. peroxynitrite in health and disease. Physiol. Rev. 87, 315–424.
Murphy, M.P. (2009). How mitochondria produce reactive oxygen Pan-Zhou, X.R., Cui, L., Zhou, X.J., Sommadossi, J.P., and Darley-
species. Biochem. J. 417, 1–13. Usmar, V.M. (2000). Differential effects of antiretroviral
Nair, K.S., Bigelow, M.L., Asmann, Y.W., Chow, L.S., Coenen- nucleoside analogs on mitochondrial function in HepG2 cells.
Schimke, J.M., Klaus, K.A., Guo, Z.K., Sreekumar, R., and Irving, Antimicrob. Agents Chemother. 44, 496–503.
B.A. (2008). Asian Indians have enhanced skeletal muscle Pankiv, S., Clausen, T.H., Lamark, T., Brech, A., Bruun, J.A., Outzen,
mitochondrial capacity to produce ATP in association with H., Overvatn, A., Bjorkoy, G., and Johansen, T. (2007). p62/
severe insulin resistance. Diabetes 57, 1166–1175. SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of
Narendra, D.P. and Youle, R.J. (2011). Targeting mitochondrial ubiquitinated protein aggregates by autophagy. J. Biol. Chem.
dysfunction: role for PINK1 and Parkin in mitochondrial quality 282, 24131–24145.
control. Antioxid. Redox. Signal. 14, 1929–1938. Patel, M.S. and Korotchkina, L.G. (2006). Regulation of the pyruvate
Narendra, D., Tanaka, A., Suen, D.F., and Youle, R.J. (2008). Parkin dehydrogenase complex. Biochem. Soc. Trans. 34, 217–222.
is recruited selectively to impaired mitochondria and promotes Petros, J.A., Baumann, A.K., Ruiz-Pesini, E., Amin, M.B., Sun, C.Q.,
their autophagy. J. Cell Biol. 183, 795–803. Hall, J., Lim, S., Issa, M.M., Flanders, W.D., Hosseini, S.H., et al.
Narendra, D., Kane, L.A., Hauser, D.N., Fearnley, I.M., and Youle, (2005). mtDNA mutations increase tumorigenicity in prostate
R.J. (2010a). p62/SQSTM1 is required for Parkin-induced cancer. Proc. Natl. Acad. Sci. USA 102, 719–724.

Authenticated
Piantadosi, C.A. and Suliman, H.B. (2012). Transcriptional control of Scaglioni, F., Ciccia, S., Marino, M., Bedogni, G., and Bellentani, S.
mitochondrial biogenesis and its interface with inflammatory (2011). ASH and NASH. Dig. Dis. 29, 202–210.
processes. Biochim.Biophys. Acta 1820, 532–541. Schapira, A.H. and Gegg, M. (2011). Mitochondrial contribution
Pilsl, A. and Winklhofer, K.F. (2012). Parkin, PINK1 and mitochondrial to Parkinson’s disease pathogenesis. Parkinsons Dis. 2011,
integrity: emerging concepts of mitochondrial dysfunction in 159160.
Parkinson’s disease. Acta Neuropathol. 123, 173–188. Schneider, L., Giordano, S., Zelickson, B.R., Johnson, M.S., Benavides,
Popkin, B.M., Armstrong, L.E., Bray, G.M., Caballero, B., Frei, B., G.A., Ouyang, X., Fineberg, N., Darley-Usmar, V.M., and Zhang, J.
and Willett, W.C. (2006). A new proposed guidance system for (2011). Differentiation of SH-SY5Y cells to a neuronal phenotype
beverage consumption in the United States. Am. J. Clin. Nutr. changes cellular bioenergetics and the response to oxidative
83, 529–542. stress. Free Redic. Biol. Med. 51, 2007–2017.
Pua, H.H., Guo, J., Komatsu, M., and He, Y.W. (2009). Autophagy is Schrauwen-Hinderling, V.B., Kooi, M.E., Hesselink, M.K., Moonen-
essential for mitochondrial clearance in mature T lymphocytes. Kornips, E., Schaart, G., Mustard, K.J., Hardie, D.G., Saris,
J. Immunol. 182, 4046–4055. W.H., Nicolay, K., and Schrauwen, P. (2005). Intramyocellular
Rakovic, A., Grunewald, A., Kottwitz, J., Bruggemann, N., lipid content and molecular adaptations in response to a
Pramstaller, P.P., Lohmann, K., and Klein, C. (2011). Mutations 1-week high-fat diet. Obes. Res. 13, 2088–2094.
in PINK1 and Parkin impair ubiquitination of Mitofusins in Schrauwen-Hinderling, V.B., Hesselink, M.K., Moonen-Kornips, E.,
human fibroblasts. PLoS One 6, e16746. Schaart, G., Kooi, M.E., Saris, W.H., and Schrauwen, P. (2006).
Ramachandran, A., Ceaser, E., and Darley-Usmar, V.M. (2004). Chronic Short-term training is accompanied by a down regulation of
exposure to nitric oxide alters the free iron pool in endothelial ACC2 mRNA in skeletal muscle. Int. J. Sports Med. 27, 786–791.
cells: role of mitochondrial respiratory complexes and heat shock Shiva, S., Oh, J.Y., Landar, A.L., Ulasova, E., Venkatraman, A., Bailey,
proteins. Proc. Natl. Acad. Sci. USA 101, 384–389. S.M., and Darley-Usmar, V.M. (2005). Nitroxia: the pathological
Rautou, P.E., Mansouri, A., Lebrec, D., Durand, F., Valla, D., and consequence of dysfunction in the nitric oxide-cytochrome c
Moreau, R. (2010). Autophagy in liver diseases. J. Hepatol. 53, oxidase signaling pathway. Free Radic. Biol. Med. 38, 297–306.
1123–1134. Singh, R., Xiang, Y., Wang, Y., Baikati, K., Cuervo, A.M., Luu, Y.K.,
Robinson, T.N. (1999). Reducing children’s television viewing to Tang, Y., Pessin, J.E., Schwartz, G.J., and Czaja, M.J. (2009).
prevent obesity: a randomized controlled trial. J. Am. Med. Autophagy regulates adipose mass and differentiation in mice.
Assoc. 282, 1561–1567. J. Clin. Invest. 119, 3329–3339.
Roger, V.L., Go, A.S., Lloyd-Jones, D.M., Benjamin, E.J., Berry, J.D., Smith, D.E., Lewis, C.E., Caveny, J.L., Perkins, L.L., Burke, G.L., and Bild,
Borden, W.B., Bravata, D.M., Dai, S., Ford, E.S., Fox, C.S., et al. D.E. (1994). Longitudinal changes in adiposity associated with
(2012). Heart disease and stroke statistics–2012 update: a report pregnancy. The CARDIA Study. Coronary Artery Risk Development
from the American Heart Association. Circulation 125, e2–e220. in Young Adults Study. J. Am. Med. Assoc. 271, 1747–1751.
Rose, G., Passarino, G., Carrieri, G., Altomare, K., Greco, V., Speakman, J.R. (2008). Thrifty genes for obesity, an attractive but
Bertolini, S., Bonafe, M., Franceschi, C., and De Benedictis, G. flawed idea, and an alternative perspective: the’drifty gene’
(2001). Paradoxes in longevity: sequence analysis of mtDNA hypothesis. Int. J. Obesity 32, 1611–1617.
haplogroup J in centenarians. Eur. J. Hum. Genet. 9, 701–707. Sridharan, V., Guichard, J., Li, C.Y., Muise-Helmericks, R., Beeson,
Rothwell, N.J. and Stock, M.J. (1983). Luxuskonsumption, C.C., and Wright, G.L. (2008). O(2)-sensing signal cascade:
diet-induced thermogenesis and brown fat: the case in favour. clamping of O(2) respiration, reduced ATP utilization, and
Clin. Sci. (Lond) 64, 19–23. inducible fumarate respiration. Am. J. Physiol. Cell Physiol.
Ruiz-Pesini, E., Mishmar, D., Brandon, M., Procaccio, V., and Wallace, 295, C29–37.
D.C. (2004). Effects of purifying and adaptive selection on Strauer, B.E. (1987). Cardiac energetics in clinical heart disease.
regional variation in human mtDNA. Science 303, 223–226. Basic Res. Cardiol. 82 Suppl 2, 389–402.
Sako, E.Y., Kingsley-Hickman, P.B., From, A.H., Foker, J.E., and Suen, D.F., Narendra, D.P., Tanaka, A., Manfredi, G., and Youle, R.J.
Ugurbil, K. (1988). ATP synthesis kinetics and mitochondrial (2010). Parkin overexpression selects against a deleterious
function in the postischemic myocardium as studied by 31P mtDNA mutation in heteroplasmic cybrid cells. Proc. Natl.
NMR. J. Biol. Chem. 263, 10600–10607. Acad. Sci. USA 107, 11835–11840.
Sansbury, B.E., Jones, S.P., Riggs, D.W., Darley-Usmar, V.M., and Sun, F., Cui, J., Gavras, H., and Schwartz, F. (2003). A novel class
Hill, B.G. (2011a). Bioenergetic function in cardiovascular of tests for the detection of mitochondrial DNA-mutation
cells: the importance of the reserve capacity and its biological involvement in diseases. Am. J. Hum. Genet. 72, 1515–1526.
regulation. Chem. Biol. Interact. 191, 288–295. Tanaka, A., Cleland, M.M., Xu, S., Narendra, D.P., Suen, D.F.,
Sansbury, B.E., Riggs, D.W., Brainard, R.E., Salabei, J.K., Jones, S.P., Karbowski, M., and Youle, R.J. (2010). Proteasome and p97
and Hill, B.G. (2011b). Responses of hypertrophied myocytes mediate mitophagy and degradation of mitofusins induced by
to reactive species: implications for glycolysis and electrophile Parkin. J. Cell Biol. 191, 1367–1380.
metabolism. Biochem. J. 435, 519–528. Thorsness, P.E. and Fox, T.D. (1990). Escape of DNA from
Sarti, P., Giuffre, A., Barone, M.C., Forte, E., Mastronicola, D., and mitochondria to the nucleus in Saccharomyces cerevisiae.
Brunori, M. (2003). Nitric oxide and cytochrome oxidase: Nature 346, 376–379.
reaction mechanisms from the enzyme to the cell. Free Radic. Thorsness, P.E., White, K.H., and Fox, T.D. (1993). Inactivation of
Biol. Med. 34, 509–520. YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of
Sato, M. and Sato, K. (2011). Degradation of paternal mitochondria putative ATPase-encoding genes, causes increased escape
by fertilization-triggered autophagy in C. elegans embryos. of DNA from mitochondria in Saccharomyces cerevisiae. Mol.
Science 334, 1141–1144. Cell. Biol. 13, 5418–5426.

Authenticated
Tseng, Y.H., Cypess, A.M., and Kahn, C.R. (2010). Cellular vascular oxidative stress and mitochondrial damage in
bioenergetics as a target for obesity therapy. Nat. Rev. Drug non-human primates. Cardiovasc. Toxicol. 10, 216–226.
Discov. 9, 465–482. Williamson, J.R. and Cooper, R.H. (1980). Regulation of the citric
Turrens, J.F. (2003). Mitochondrial formation of reactive oxygen acid cycle in mammalian systems. FEBS Lett 117 Suppl, K73–85.
species. J. Physiol. 552, 335–344. Williamson, D.F., Madans, J., Anda, R.F., Kleinman, J.C., Kahn, H.S.,
Twig, G., Elorza, A., Molina, A.J., Mohamed, H., Wikstrom, J.D., Walzer, and Byers, T. (1993). Recreational physical activity and ten-year
G., Stiles, L., Haigh, S.E., Katz, S., Las, G., et al. (2008a). Fission weight change in a US national cohort. Int. J. Obes. Relat.
and selective fusion govern mitochondrial segregation and Metab. Disord. 17, 279–286.
elimination by autophagy. EMBO J. 27, 433–446. Wu, H., Kanatous, S.B., Thurmond, F.A., Gallardo, T., Isotani, E.,
Twig, G., Hyde, B., and Shirihai, O.S. (2008b). Mitochondrial Bassel-Duby, R., and Williams, R.S. (2002). Regulation of
fusion, fission and autophagy as a quality control axis: the mitochondrial biogenesis in skeletal muscle by CaMK. Science
bioenergetic view. Biochim. Biophys. Acta 1777, 1092–1097. 296, 349–352.
Ugarte, N., Petropoulos, I., and Friguet, B. (2010). Oxidized Wu, M., Neilson, A., Swift, A.L., Moran, R., Tamagnine, J., Parslow,
mitochondrial protein degradation and repair in aging and D., Armistead, S., Lemire, K., Orrell, J., Teich, J., et al. (2007).
oxidative stress. Antiox. Redox. Signal. 13, 539–549. Multiparameter metabolic analysis reveals a close link
Ulasova, E., Gladden, J.D., Chen, Y., Zheng, J., Pat, B., Bradley, W., between attenuated mitochondrial bioenergetic function and
Powell, P., Zmijewski, J.W., Zelickson, B.R., Ballinger, S.W., enhanced glycolysis dependency in human tumor cells. Am. J.
et al. (2011). Loss of interstitial collagen causes structural Physiol. Cell. Physiol. 292, C125–136.
and functional alterations of cardiomyocyte subsarcolemmal Wu, D., Wang, X., Zhou, R., and Cederbaum, A. (2010). CYP2E1
mitochondria in acute volume overload. J. Mol. Cell. Cardiol. enhances ethanol-induced lipid accumulation but impairs
50, 147–156. autophagy in HepG2 E47 cells. Biochem. Biophys. Res. Comm.
van der Windt, G.J., Everts, B., Chang, C.H., Curtis, J.D., Freitas, T.C., 402, 116–122.
Amiel, E., Pearce, E.J., and Pearce, E.L. (2012). Mitochondrial Xu, Y.N., Cui, X.S., Sun, S.C., Lee, S.E., Li, Y.H., Kwon, J.S., Lee, S.H.,
respiratory capacity is a critical regulator of CD8+ T cell Hwang, K.C., and Kim, N.H. (2011). Mitochondrial dysfunction
memory development. Immunity 36, 68–78. influences apoptosis and autophagy in porcine parthenotes
Van Humbeeck, C., Cornelissen, T., Hofkens, H., Mandemakers, W., developing in vitro. J. Reprod. Dev. 57, 143–150.
Gevaert, K., De Strooper, B., and Vandenberghe, W. (2011). Yadava, N. and Nicholls, D.G. (2007). Spare respiratory capacity
Parkin interacts with Ambra1 to induce mitophagy. J. Neurosci. rather than oxidative stress regulates glutamate excitotoxicity
31, 10249–10261. after partial respiratory inhibition of mitochondrial complex I
Venkatraman, A., Landar, A., Davis, A.J., Chamlee, L., Sanderson, T., with rotenone. J. Neurosci. 27, 7310–7317.
Kim, H., Page, G., Pompilius, M., Ballinger, S., Darley-Usmar, Yang, Z., Harrison, C.M., Chuang, G.C., and Ballinger, S.W. (2007). The
V., et al. (2004a). Modification of the mitochondrial proteome role of tobacco smoke induced mitochondrial damage in vascular
in response to the stress of ethanol-dependent hepatotoxicity. dysfunction and atherosclerosis. Mutation Research 621,
J. Biol. Chem. 279, 22092–22101. 61–74.
Venkatraman, A., Landar, A., Davis, A.J., Ulasova, E., Page, G., Youle, R.J. and Narendra, D.P. (2011). Mechanisms of mitophagy.
Murphy, M.P., Darley-Usmar, V., and Bailey, S.M. (2004b). Nature reviews Mol. Cell. Biol. 12, 9–14.
Oxidative modification of hepatic mitochondria protein Yun, K.L., Rayhill, S.C., Niczporuk, M.A., Fann, J.I., Derby, G.C.,
thiols: effect of chronic alcohol consumption. Am. J. Physiol.– Daughters, G.T., Ingels, N.B., Jr., and Miller, D.C. (1992). Left
Gastrointest. Liver Physiol. 286, G521–527. ventricular mechanics and energetics in the dilated canine
Venkatraman, A., Shiva, S., Wigley, A., Ulasova, E., Chhieng, D., heart: acute versus chronic mitral regurgitation. J. Thorac.
Bailey, S.M., and Darley-Usmar, V.M. (2004c). The role of Cardiovasc. Surg. 104, 26–39.
iNOS in alcohol-dependent hepatotoxicity and mitochondrial Zakhari, S. and Li, T.K. (2007). Determinants of alcohol use and
dysfunction in mice. Hepatology 40, 565–573. abuse: Impact of quantity and frequency patterns on liver
Wallace, D.C. (2005). A mitochondrial paradigm of metabolic disease. Hepatology 46, 2032–2039.
and degenerative diseases, aging, and cancer: a dawn for Zatloukal, K., French, S.W., Stumptner, C., Strnad, P., Harada,
evolutionary medicine. Annu. Rev. Genet. 39, 359–407. M., Toivola, D.M., Cadrin, M., and Omary, M.B. (2007). From
Wallace, D.C. (2010). Mitochondrial DNA mutations in disease and Mallory to Mallory-Denk bodies: what, how and why? Exp. Cell
aging. Environ. Mol. Mutagen. 51, 440–450. Res. 313, 2033–2049.
Wallace, D.C. (2011). Bioenergetic Origins of Complexity and Zelickson, B.R., Benavides, G.A., Johnson, M.S., Chacko, B.K.,
Disease. Cold Spring Harb. Symp. Quant. Biol. 76, 1–16. Venkatraman, A., Landar, A., Betancourt, A.M., Bailey, S.M.,
Wang, Y.C., Bleich, S.N., and Gortmaker, S.L. (2008). Increasing and Darley-Usmar, V.M. (2011). Nitric oxide and hypoxia
caloric contribution from sugar-sweetened beverages and exacerbate alcohol-induced mitochondrial dysfunction in
100% fruit juices among US children and adolescents, hepatocytes. Biochim. Biophys. Acta 1807, 1573–1582.
1988–2004. Pediatrics 121, e1604–1614. Zhang, J. and Ney, P.A. (2009). Role of BNIP3 and NIX in cell death,
Wasko, M.C., Hubert, H.B., Lingala, V.B., Elliott, J.R., Luggen, M.E., autophagy, and mitophagy. Cell Death Differ. 16,
Fries, J.F., and Ward, M.M. (2007). Hydroxychloroquine and risk 939–946.
of diabetes in patients with rheumatoid arthritis. J. Am. Med. Zhang, Y., Goldman, S., Baerga, R., Zhao, Y., Komatsu, M., and Jin, S.
Assoc. 298, 187–193. (2009). Adipose–specific deletion of autophagy-related gene 7
Westbrook, D.G., Anderson, P.G., Pinkerton, K.E., and Ballinger, (atg7) in mice reveals a role in adipogenesis. Proc. Natl. Acad.
S.W. (2010). Perinatal tobacco smoke exposure increases Sci. USA 106, 19860–19865.

Authenticated
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.

Authenticated
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.

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DOI 10.1515/hsz-2012-0198 Biol. Chem.

, 2012; 393(12): 1485–1512

Integration of cellular bioenergetics with

Keywords: cardiovascular disease; haplotypes;

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in the dynamic context of cell function. Fission

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Oligomycin capacity value is subtracted from other OCR measurements to deter-

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A Cardiomyocytes Hepatocytes Endothelial cells

Oxygen consumption rate

Oxygen consumption rate

ATP-linked Proton leak Reserve capacity Non-mitochondrial

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

Figure 3 Cellular bioenergetics in different cell types.

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High: a) ATP turnover in

Low: a) ATP demand has

Figure 4 Interpretation tree for the mitochondrial bioenergetic profile.

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Basal oxygen consumption rate Maximal OCR

The addition of oligomycin is assumed to completely elimi-

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Oligo Anti-A/R KA or 2-DG

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High ATP demand

Low energy demand

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Figure 7 General pathway for activation of autophagy.

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Role of fission and fusion in mitophagy

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Normal mito irreversible oxidative protein modification to metabolic

shown that CD8+ memory T-cells possessed a substan-

tial reserve capacity that was responsive to interleukin-15

(IL-15), which regulated the stability of memory T-cells

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Given the high energy demands of the cardiovascular

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phenylephrine (Sansbury et al., 2011b). This resulted in

and failing hearts, where failure was shown to deplete XO

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Obesity and diabetes overexpression of UCP1 in adipose tissue (Kopecky

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Brought to you by | Universidad de Chile

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Louis Dell’Italia received his MD and subsequent Internal Medicine

Jack R. Lancaster received his PhD in Biochemistry from the

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Victor M. Darley-Usmar is presently Professor of Pathology

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