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The Role of Mitochondria in the Pathogenesis ofType 2 Diabetes

Mary-Elizabeth Patti and Silvia Corvera

Research Division, Joslin Diabetes Center (M.-E.P.), and Harvard Medical School (M.-E.P.), Boston, Massachusetts02215; and Program in Molecular Medicine (S.C.), University of Massachusetts Medical School, Worcester,Massachusetts 01605

The pathophysiology of type 2 diabetes mellitus (DM) is varied and complex. However, the association of DMwith obesity and inactivity indicates an important, and potentially pathogenic, link between fuel and energyhomeostasisand theemergence of metabolic disease.Given thecentral rolefor mitochondriain fuelutilizationand energy production, disordered mitochondrial function at the cellular level can impact whole-body meta-bolic homeostasis. Thus, the hypothesis that defective or insufficient mitochondrial function might play apotentially pathogenic role in mediating risk of type 2 DM has emerged in recent years. Here, we summarize

current literature on risk factors for diabetes pathogenesis, on the specific role(s) of mitochondria in tissuesinvolved in its pathophysiology, and on evidence pointing to alterations in mitochondrial function in thesetissues that could contribute to thedevelopment of DM. We also review literature on metabolic phenotypes ofexistinganimalmodelsof impairedmitochondrialfunction. Weconclude that, whereasthe associationbetweenimpaired mitochondrial function and DM is strong, a causal pathogenic relationship remains uncertain. How-ever, we hypothesize that genetically determined and/or inactivity-mediated alterations in mitochondrial ox-idative activity may directly impact adaptive responses to overnutrition, causing an imbalance between oxida-tive activity and nutrient load. This imbalance may lead in turn to chronic accumulation of lipid oxidativemetabolites that can mediate insulin resistance and secretory dysfunction. More refined experimentalstrategies thataccurately mimic potential reductions in mitochondrial functionalcapacity in humans at riskfor diabetes will be required to determine the potential pathogenic role in human insulin resistance andtype 2 DM. (Endocrine Reviews 31: 364–395, 2010)

I. Type 2 Diabetes PathogenesisA. Risk factors associated with type 2 diabetes

II. General Overview of Mitochondrial BiologyA. The dynamic morphology of mitochondriaB. Mechanismsthatcontrol mitochondrialdensityand

capacityIII. Role of Mitochondria in Tissue-Specific Contexts

A. MuscleB. Adipose tissueC. LiverD. Pancreatic -cells

IV. Experimental Strategies to Explore the Relationship be-

tween Mitochondrial Function and DMA. PGC-1 and overexpressionB. PGC-1 knockout modelsC. Other mitochondrial function defects

V. Conclusions

I. Type 2 Diabetes Pathogenesis

Type 2 diabetes mellitus (DM) in the United States andaround the world has reached epidemic proportions.At present, 17.9 million people in the United States havebeen diagnosed with diabetes, with an additional 5.7 mil-lion undiagnosed (1). Together, this encompasses 8% of the population, and thus, diabetes is a major public healthissue. In addition, current data indicate that 57 millionAmericans suffer from prediabetes (defined as fastingblood glucose between 100 and 125 mg/dl) (1). Diabetesdisproportionately affects specific ethnic populations,with risk increased 1.8-fold in African-Americans, 1.7-fold in Mexican-Americans, and 2.2-fold in Native Amer-

ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2010 by The Endocrine Societydoi: 10.1210/er.2009-0027 Received July 2, 2009. Accepted December 24, 2009.First Published Online February 15, 2010

Abbreviations:BMI, Bodymassindex; CoA,coenzymeA; COX,cytochromeoxidase; CPT1,carnitine palmitoylotransferase 1; DM, diabetes mellitus; ERR, estrogen-related receptor;ETC, electron transport chain; FADH 2 , reduced flavin adenine dinucleotide; MIDD, mater-nally inherited diabetes and deafness; mtDNA, mitochondrial DNA; NADH, reduced nic-otinamide adenine dinucleotide; NASH, nonalcoholic steatohepatitis; NMR, nuclear mag-netic resonance;NRF, nuclearrespiratory factor;OXPHOS, oxidativephosphorylation; PGC,PPAR coactivator; PPAR,peroxisome proliferator-activatedreceptor; ROS,reactive oxygenspecies; RQ, respiratory quotient; TCA, tricarboxylic acid; UCP, uncoupling protein.

R E V I E W

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icans. In addition to the major health consequences toindividuals, including higher risk of death, heart disease,stroke, kidney disease, blindness, amputations, neuropa-thy,andpregnancy-relatedcomplications,diabetesand itscomplications result in a total cost of $174 billion in theUnited States (2). By far, the largest proportion is derivedfrom type 2 DM, which accounts for more than 90% of diabetes. Unfortunately, the incidence of diabetes hasmore than doubled in the past 25 yr, with 1.6 million newcases diagnosed in adults in 2007 (2) and a projected in-crease of 165% from 2000 to 2050 (4).

Intimately linked with the rise in diabetes prevalence istheburgeoning epidemic of obesity aroundthe world,par-ticularly in developed societies (5). In 2004, 17% of chil-dren in the United States between ages 2 and 19 yr wereoverweight,and 32% ofadults over age 20were obese (6).Both obesity and related inactivity are likely to contribute

to the pathogenesis of diabetes because the incidence of diabetescanbereducedbymodestweightlossandexercise(7–9).In lightof these findings,an importantpublic healthgoal should be to understand the complex pathophysiol-ogy of diabetes and to identify and target specific mech-anisms to prevent DM in at-risk individuals.

A. Risk factors associated with type 2 diabetesMultiple physiological abnormalities can be found in

individuals with established type 2 DM, defined on thebasis of elevations in fasting and/or postprandial glucose(2). These include insulin resistance in muscle andadiposetissue, -cell dysfunction leading to impaired insulin se-cretion, increased hepatic glucose production, abnormalsecretionandregulation of incretin hormones,andalteredbalance of central nervous system pathways controllingfood intake and energy expenditure. Given this diverseconstellation of abnormalities in multiple tissues and thesecondaryconsequencesof established hyperglycemiaandhyperlipidemia,it is difficultto identify theprimary eventsthat lead to the development of diabetes. To address thiskeyclinicalandscientific question, it is importantnotonlyto determine abnormalities associated with established

disease, but also to identify underlying metabolic char-acteristics preceding the onset of disease in at-riskindividuals.

Risk factors for the development of and/or progressionof type 2 DM include: 1) genetics (10–16), exemplified bythe high risk of type 2 DM in particular ethnic groups (17)and the high concordance rates in monozygotic twin pairs(18); and 2) both prenatal and postnatal environmentalfactors, including suboptimal intrauterine environment(19, 20), low birth weight (19, 21), obesity (22, 23), in-activity (24), gestational diabetes (25), and advancing age

(26). Several longitudinal studies have indicated that in-sulin resistance, measured as reduced insulin-stimulated

glucose disposal during the hyperinsulinemic euglycemicclamp or by iv glucose tolerance testing, is common inhigh-risk individuals years before the onset of type 2 DM(27–29). However, insulin resistance is not predictive of diabetes in individuals without a family history of diabe-tes, indicating that additional unidentified factors are nec-essary for disease progression (30).

Multiple mechanisms have recently emerged as poten-tial causes of insulin resistance and/or diabetes progres-sion, among them impaired mitochondrial capacityand/or function; altered insulin signaling due to cellularlipid accumulation, proinflammatory signals, and endo-plasmic reticulum stress; and reduced incretin-dependentand -independent -cell insulin secretion. In this review,we will focus on a critical assessment of the evidence link-ing mitochondrial function to diabetes pathogenesis, atboth a cellular and whole-body level.

II. General Overview of Mitochondrial Biology

Mitochondria are double-membrane organelles that servemultiple essential cellular functions (Fig. 1) mediated bythousands of mitochondrial-specific proteins encoded byboth the nuclear and mitochondrial genomes (31, 32).Although mitochondria are most often recognized fortheir role in generating the majority of cellular ATP viaoxidative phosphorylation (OXPHOS), other essentialmetabolic functions include the generation by the tricar-boxylic acid (TCA) cycle of numerous metabolites thatfunction in cytosolic pathways, oxidative catabolism of amino acids, ketogenesis, ornithine cycle activity (“ureacycle”), the generation of reactive oxygen species (ROS)with important signalingfunctions (33, 34), the control of cytoplasmic calcium (35, 36), and the synthesis of all cel-lular Fe/S clusters, protein cofactors essential for cellularfunctionssuchasprotein translationandDNA repair(37).Therate-limitingfirst step insteroidogenesisalso occurs inmitochondria, thus linking mitochondrial function toendocrine homeostasis (38–41). This multiplicity of organelle functions explains the variability in patho-physiology, severity, and age of onset of the increasingnumber of diseases recognized to arise from primary orsecondary alterations in specific mitochondrial path-ways (37, 42–44).

A. The dynamic morphology of mitochondriaIn the thin sections observed by electron microscopy

and shown in most textbooks, mitochondria appearas discrete, small, bean-shaped, double-membrane or-ganelles. However, more recent studies based on light mi-

croscopyin live cellshave revealed that mitochondriaexistas a reticulum that is in continuous communication

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through dynamic fusion and fission events, moving ac-tively to different regions of the cell through interactionswith the cytoskeleton (Fig. 2). The mitochondrial reticu-lum is composed of an outer and an inner membrane,

between which is the intermembrane space, and a matrixlimited by the inner membrane (Fig. 1). The area of theinnermembranecanbegreaterthanthatoftheoutermem-brane due to the presence of cristae, inner membrane in-vaginations that contain all the transmembrane proteinsof the electron transport chain (ETC) as well as the mito-chondrial ATPase (45–47). The mitochondrial matrixcontainsthecomponentsoftheTCAcycleandofthe -ox-idation pathway,whichprovide reducednicotinamide ad-enine dinucleotide (NADH) and reduced flavin adeninedinucleotide (FADH 2 ) to the ETC.

The ETC is composed of four large multisubunit com-plexes (complexes I to IV) with more than 85 individual

gene products. The ETC transports electrons from donors(NADH at complex I, FADH 2 at complex II) to a finalacceptor, molecular oxygen, forming H 2 O at complex IV.The transport of electrons is accompanied by release of

large amounts of free energy, most of which is harnessedfor the translocation of protons from the matrix to theintermembrane space; the remainder is dissipated as heat(Fig. 3). The energy contained in the proton electrochem-ical gradient generated by the ETC is then coupled to ATPproduction as protons flow back into the matrix throughthe mitochondrial ATPase. Thus, OXPHOS results fromelectron transport, the generation of a proton gradient,and subsequent proton flux coupled to the mitochondrialATPase. Each of these steps can vary in efficiency; forexample, the exact stoichiometry between electron flow

and proton pumping, or between proton pumping andATP synthesis varies depending on the probability of loss

FIG. 1. Basic structural and functional features of the mitochondrial reticulum (illustrated from left to right ). The mitochondrial reticulum iscomposed of an inner and outer membrane, between which lies the intermembrane space, and a matrix contained within the inner membrane.The surface of the inner membrane is folded into cristae. The organization and distribution of the mitochondrial reticulum is controlled byinteractions with cytoskeletal elements such as microtubules. The matrix contains the enzymatic machinery for fatty acid oxidation, whichgenerates acetyl-CoA from acyl chains, and reducing equivalents in the form of NADH and FADH 2 in the process. Acetyl-CoA fuels the TCA cycle,which also produces NADH and FADH 2 . These donate electrons to the ETC, leading to the generation of a proton gradient across the innermitochondrial membrane. Dissipation of this gradient through the mitochondrial ATPase generates ATP. Delay of electron transport by the ETCresults in the production of ROS, which can activate UCPs that dissipate the proton gradient without producing ATP. The electrochemical gradientalso causes cytoplasmic Ca to enter the matrix, buffering cytoplasmic Ca levels and promoting TCA cycle flux. Mitochondria are also crucialin the generation of iron-sulfur clusters that form the prosthetic group of numerous proteins involved in multiple cellular pathways. Themitochondrial reticulum undergoes continuous fusion and fission reactions that involve both the inner and outer mitochondrial membranes,allowing redistribution of matrix content, such as mtDNA, within the reticulum. The proteins that compose all mitochondrial machineries areencoded both by mtDNA and by nuclear DNA. The master transcription factor operating on mtDNA is TFAM, which is encoded in the nucleargenome. The expression of mitochondrial genes in the nucleus is driven by numerous transcription factors, which are in turn controlled by specificcoactivators and corepressors that respond to cellular energy demands.

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of electrons from the ETC before reaching complex IVand on non-ATPase-coupled proton leak through theinner mitochondrial membrane [ e. g ., via uncouplingproteins (UCPs)].

The high electronegative potential generated by theproton gradient also drives the rapid entry of Ca intothe mitochondrial matrix, buffering its concentration inthe cytoplasm. In the mitochondrial matrix, Ca canstimulate flux through the Krebs cycle by stimulatingdehydrogenase activities (36). The exit of Ca fromthe matrix is driven by electroneutral exchange withNa or H .

The ETC is also a potent source of ROS. Loss of elec-trons from the ETC can result in reduction of oxygen toform O 2 , which can be dismutated to H 2 O 2 and subse-quently converted to the hydroxyl radical, OH . Thesethree products constitute the major ROS formed duringrespiration. As the name implies, these species are highlyreactive, and acute, very high elevations, or more chronicelevations can be extremely damaging to the cell. ROSgeneration is more likely to occur when the proton gra-

dient is large andelectron carriers are highlyreduced, e. g .,when ADP is rate-limiting for ATP production or whenavailabilityof O 2 is limiting.Uncoupling proteins arecon-sideredto be natural regulators of this process, respondingto and controlling ROS production by mitigating the for-mation of a large proton gradient.

The mitochondrial matrix also contains the circularmitochondrial DNA (mtDNA) molecule, which encodesfor 37 genes (13 of which are subunits of the ETC). Trans-lation of these proteins occurs within the mitochondrialmatrix, utilizing mtDNA-encoded rRNA and tRNA.

Mitochondrial fission and fusion allow the transcrip-tionalproducts of mtDNA,as well as multiple metabolites

generatedinthemitochondrialmatrix, tobe sharedwithinthe entire mitochondrial reticulum. Although the molec-ular machinery of mitochondrial fusion and fission hasbeen elucidated (48), it has only recently been establishedthat mitochondrial fusionandfission also contribute mul-tiple other mitochondrial functions, including the controlof cellular calcium handling, ROS production, and ener-getic output (49–51). Moreover, human diseases arisingfrom mutations in conserved elements of the mitochon-drial fusion machinery have been identified, such asCharcot-Marie-Tooth type 2A caused by mutations inmitofusin 2, and autosomal dominant optic atrophy,caused by mutations in optic atrophy 1 (49). A role formitochondrial fusion machinery in metabolic control hasalso been suggested by the findings that mitofusin 2 levelsarecontrolledduringmuscledevelopmentandarereducedin both obesity and type 2 DM in parallel with insulinresistance (52).

B. Mechanisms that control mitochondrial densityand capacity

The term mitochondrial biogenesis is often used to de-scribe the generation of more mitochondria in response toincreased energy demands, or the multiplication of mito-chondrianecessary forcell growthand division.However,

the copy number of specific mitochondrial proteins andthe functional capacity of each distinct mitochondrial

FIG. 2. Visualization of the dynamic nature of the mitochondrialreticulum in a cultured muscle cell. C2C12 mouse myoblasts werestained with MitoTracker green and imaged in culture at 37 C. Imagesof a segment of the cell captured at 30-sec intervals are shown on theleft . The comparison between successive frames reveals scission events(arrowheads ), branching events ( V ), and fusion events ( brackets )occurring at 30-sec intervals.

FIG. 3. Coupled and uncoupled respiration. Electrons derived fromreduced donors NADH and FADH 2 are transported within the ETC tomolecular oxygen, producing water. The flow of electrons within theETC is coupled to translocation of protons due to the large amount offree energy released during electron transport. The remainder of thisfree energy is released as heat. The proton gradient thus produced isdissipated through the mitochondrial ATPase, and the consequentdecrease in free energy drives ATP synthesis. This process is known asOXPHOS, or coupled respiration. Under circumstances where NADHand FADH2 are available, but movement of electrons down therespiratory chain is slow, some of those electrons will be released fromthe respiratory chain and reduce molecular oxygen, forming thesuperoxide anion O 2 , hydrogen peroxide, and the hydroxyl radicalOH . These are the main ROS formed at steady state. Accumulation ofROS activates UCPs, which dissipate the proton gradient withoutproducing ATP, resulting in uncoupled respiration.



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pathway may be very variable between different tissuesand between different physiological conditions. Thus, theterm mitochondrial biogenesis can be ambiguous becausemultiple parameters, including mtDNA copy number,mitochondrial density, levels of specific mitochondrialproteins, and mitochondrial functional output may varyindependently of each other. For example, the prolifera-tion of mitochondria occurring to sustain hyperplasticgrowth is probably very different from that occurring tosupport hypertrophic growth in any given tissue, and theregulatorymechanisms controllingthese adaptivechangesare likely to be distinct.

1. Transcriptional control mechanismsAlthough we know very little about specific mecha-

nisms that control differentmodalitiesof mitochondrialbiogenesis, it is clear that these mechanisms require co-ordination between the nuclear and mitochondrial ge-nomes. Transcription of the mitochondrial genome isunder the control of a single transcription factor, Tfam,which is encoded by the nuclear genome. In turn, Tfamexpression is regulated by the transcription factors NRF(nuclear respiratory factor)-1 and NRF-2, which spe-cifically activate numerous nuclear-encoded genes in-volved in mitochondrial respiration (53, 54). Thus,through NRF-stimulated expression of Tfam, the tran-scription of the mitochondrial genome is stimulated incoordination with that of nuclear-encoded mitochondrialgenes. The expression of many other mitochondrialgenes is controlled by additional nuclear transcriptionfactors, including peroxisome proliferator-activatedreceptor (PPAR) , PPAR , estrogen-related receptor(ERR) / , and Sp1, which can induce expression of mitochondrial genes in a tissue-dependent and physio-logical context-dependent manner (55).

A high level of transcriptional coordination is requiredto ensure coupling of mitochondrial activity to other met-abolic activities within thecell and to mediate appropriateparallel changes in all components of multiprotein com-plexes. This coordination is accomplished through the ac-

tion of transcriptional coactivators and corepressors. Thebest studied coactivators of mitochondrial gene transcrip-tion are members of the PPAR coactivator (PGC)family,including PGC-1 , PGC-1 (56, 57), and PPRC, a relatedserum-responsive coactivator (58). These respond to cel-lularenergy-requiringconditionssuchas cellgrowth, hyp-oxia, glucose deprivation, and exercise (55) to activatetranscription factors promoting mitochondrial remodel-ing and/or biogenesis, thus restoring cellular energetics.For example, PCG-1 is highly expressed in muscle, liver,andbrown fat, andexpression is further increased in these

tissues in response to exercise, fasting, and cold exposure,respectively. AlthoughPGC-1 and- donotappeartobe

required for mitochondrial biogenesis during develop-ment (59), they are necessary for the expression of the fullcomplement of proteins of mitochondrial OXPHOS andfatty acid -oxidation pathways in muscle and brown ad-ipose tissue(59– 69). Moreover,PGC-1 andPGC-1 arecrucial for the rapid bursts in mitochondrial proliferationthat accompany perinatal heart and brown adipose tissuedevelopment (59). These data support the concept thatmitochondrial adaptation to specific energy needs ismediated by PGC-1 and PGC-1 ; by contrast, mito-chondrial expansion during cell proliferation is morelikely to depend on serum-responsive coactivators suchas PPRC (70).

The role of corepressors in the transcriptional controlof energy metabolism genes is less extensively studied.However, evidence in cultured cells and in mouse modelspoints to a critical role of the corepressor RIP140 in con-

trolling important aspects of mitochondrial energy me-tabolism in both adipose tissue and muscle (71–75).RIP140 suppresses UCP1 through interaction with spe-cific enhancer elements and also suppresses expression of genes involved in -oxidation and respiratory chain as-sembly. RIP140 also interacts directly with many of thetranscription factors coactivated by PGC-1 (76). Themechanisms that control the balance between PGC-1 co-activators andRIP140 andothercorepressorsare notclearbut are likely to represent key regulatory mechanisms of energetic adaptation.

2. Posttranscriptional control mechanismsThe expansion of the mitochondrial reticulum requires

not only the expression of genes encoding mitochondrialproteins but also the import of these into the mitochon-drial space (77–80) and the coordinated expansion of mi-tochondrial membranes. Mitochondrial inner and outermembranes have distinct lipid compositions that differfrom that of other membrane-bound organelles and fromthe plasma membrane. Specific features of mitochondrialmembranes are their relative lack of cholesterol and the

high content of cardiolipin, which is unique to mitochon-drialmembranesand essentialfor theproper assemblyandfunction of the respiratory chain (81–83). Mitochondriallipids are most likely synthesized in the endoplasmicreticulum (the primary site of lipid biosynthesis in eu-karyotic cells) and transferred to mitochondria via as-yet unidentified mechanisms. However, recent workhas identified mechanisms regulating the synthesis of cardiolipin and phosphatidylethanolamine in mito-chondria inner membranes via the action of mitochon-drial prohibitins (84). In addition, cardiolipin synthesis

requires the mitochondrial translocator assembly andmaintenanceprotein Tam41, revealing a mechanism for



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the coordination of protein import and mitochondrialmembrane lipid assembly (85).

The area and composition of the mitochondrial innerand outer membranes must be tailored to accommodatethe specific components of mitochondria from differentcellsandtissues,whichareeachlikelytohaveoptimallipidcomposition and density. This essential requirement forspecific lipid composition is underscored by the morpho-logical and functional alterations in mitochondria seen inBarth syndrome, a disorder arising from mutations in alipid acyltransferase, tafazzin (41, 86). The resulting al-terations in cardiolipin structure cause profound changesin the assembly and distribution of respiratory chain com-ponents within mitochondrial cristae (84, 87, 88). Inter-estingly,lymphoblasts frompatients withBarth syndromecanproduceATPatnormallevelsbutdisplayanexpandedmitochondrial reticulum (89). These observations under-

score the existence of mechanisms that can compensate inpart for specific mitochondrial deficiencies.

Given thecomplex anddynamic structureof mitochon-dria and the diversity and physiological importance of their multiple functions, assessing the role of mitochon-dria in human pathology requires a comprehensive char-acterization not only of mitochondrial structure andabundance, but also of the pathways that compensatefor suboptimal mitochondrial capacity and functionaloutput—which may then modify disease severity andprogression. In the following sections, we will critically

analyze the findings that have suggested a role for mi-tochondrial function in the establishment of diabetesrisk and the gaps in our knowledge that must be filledto determine the merits of this hypothesis.

III. Role of Mitochondria inTissue-Specific Contexts

A. Muscle

1. Role of mitochondria in muscleMitochondria are particularly important for skeletal

muscle function, given the high oxidative demands im-posed on this tissue by intermittent contraction. Mito-chondria play a critical role in ensuring adequate levels of ATPneededfor contraction by themuscle sarcomere.Thishigh-level requirement for ATP by sarcomeres has likelycontributed to the distinct subsarcolemmal and sarco-mere-associated populations of mitochondria in muscle.Moreover, muscle cells must maintain metabolic flexibil-ity, defined as the ability to rapidly modulate substrateoxidation as a function of ambient hormonal and ener-getic conditions. For example, healthy muscle tissue pre-

dominantly oxidizes lipid in the fasting state, as evidencedby low respiratory quotient (RQ), with subsequent tran-

sition to carbohydrate oxidation (increased RQ) duringthe fed state. Availability of fuels, particularly lipids, andcapacity to oxidize them within mitochondria are alsocritical for sustained exercise. Thus, mitochondrial func-tional capacity is likely to directly affect muscle metabolicfunction and, because of its large contribution to totalbody mass, to have a significant impact on whole-bodymetabolism. This possibility is supported by the findingsof increasedmitochondrialcontent inskeletalmuscleinanindividual withhypermetabolismand resistance to weightgain (Luft syndrome) (90).

2. Potential mechanisms by which impaired musclemitochondrial oxidative function could result ininsulin resistance

Skeletal muscle is the largest insulin-sensitive organin humans, accounting for more than 80% of insulin-stimulated glucose disposal. Thus, insulin resistance inthis tissue has a major impact on whole-body glucosehomeostasis. Indeed, multiple metabolic defects havebeen observed in muscle from insulin-resistant but nor-moglycemic subjects at high risk for diabetes develop-ment, including: 1) reduced insulin-stimulated glycogensynthesis (27, 91, 92); 2) alterations in insulin signaltransduction (93); and 3) increased muscle lipid accu-mulation (94). Although it remains unclear whether anyof these defects play a causal role in insulin resistance,intramyocellular lipid excess strongly correlates withthe severity of insulin resistance, even after correctionfor the degree of obesity (94), and has been observed inmuscles of multiple fiber types (95). Moreover, lipidexcess has been linked experimentally to induction of insulin resistance (96) and alterations in insulin signaltransduction (97–99).

Thus, one possible mechanism by which impaired mi-tochondrial function might contribute to insulin resis-tance is via altered metabolism of fatty acids. Increasedtissue lipid load, as with obesity, and/or sustained inac-tivity, may lead totheaccumulation of fattyacyl coenzymeA (CoA), diacylglycerols, ceramides, products of incom-

plete oxidation, and ROS, all of which have been linkedexperimentally to reduced insulin signaling and action(96–102). Additional mechanisms potentially linking im-paired mitochondrial oxidative function to insulin re-sistance include: 1) reduced ATP synthesis for energy-requiring functions such as insulin-stimulated glucoseuptake; 2) abnormalities in calcium homeostasis (neces-sary for exercise-induced glucose uptake) (103–105); and3) reduced ATP production during exercise (106), poten-tially contributing to reduced aerobic capacity, musclefatigue, and decreased voluntary exercise over time—

further feeding a vicious cycle of inactivity-fueled insulinresistance.



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3. Evidence for reduced muscle mitochondrial oxidativefunction in DM

An important early clue suggesting that muscle mito-chondrial oxidative dysfunction may be associated withinsulin resistance in humanswas theseriesof observationsby Simoneau and Kelley that obesity is associated withreductions in citrate synthase, malate dehydrogenase, car-nitine palmitoylotransferase 1 (CPT1), and cytochromeoxidase (COX) activity in the fasting state (107, 108) andwith parallel increases in activity of the glycolytic enzymeshexokinase and phosphofructokinase (109). Moreover,oxidative activity ( e. g ., citrate synthase, acyl CoA dehy-drogenase) is a robust correlate of insulin sensitivity, evenbetter than either im triglycerides or long-chain fatty acylCoA (110). Furthermore, leg balance studies demon-strated that obesity-linked insulin resistance and diabetesare both associated with reduced fasting lipid oxidation,as indicated by higher RQ, as well as inability to suppresslipid oxidation and switch to carbohydrate oxidation inresponse to meals/insulinstimulation (111), a statetermed“metabolic inflexibility” (112). Impaired flexibility alsocorrelates with intramyocellular accumulation of lipids(107), and 24-h RQ can predict subsequent weight gain(110, 113). Together, these data suggest that an intrinsicdefectinmultiplecomponentsof oxidativemetabolism, oraltered regulation, may contribute to the development of both obesity and insulin resistance.

The diminished capacity for appropriate regulation of oxidative metabolism observed in the above studies couldbe linked to reduced mitochondrial function due to: 1)abnormal mitochondrial density and/or in vivo function;and/or 2) intrinsic defects in oxidative metabolism of lip-ids or other substrates. Multiple studies suggest that hu-maninsulin resistance is indeed accompanied by impairedin vivo mitochondrial oxidative function—in turn linked,at least in part, to reduced mitochondrial density. Ritov et al . (114) demonstrated that the enzymatic activity of OXPHOS complex I, as assessed by the activity of rote-

none-sensitive NADH:O 2 oxidoreductase, was reducedby about 40% in skeletal muscle biopsy samples fromindividuals with type 2 DM and by 20% in obese individ-uals. Similarly, Boushel et al . (115) found modest reduc-tions in ADP and succinate-stimulated oxygen consump-tion in permeabilized muscle fibers from obese individualswith type 2 DM. In each of these studies, differences inoxidative capacity did not remain after normalization formitochondrialmassby citrate synthaseactivityor mtDNAcontent, respectively, suggesting that reduced mitochon-drial mass might be a major contributor. This possibility

is consistent with electron microscopy demonstrating di-minished mitochondrial size in obesity anddiabetes (116),

particularly in subsarcolemmal fractions (114). Interest-ingly, this fraction is also characterized by even greaterreductions in OXPHOS activity (114).

Nuclear magnetic resonance (NMR) spectroscopy hasalso been used to assess mitochondrial function in vivo,with studies finding similar reductions in oxidative func-tion in both insulin resistance and type 2 DM. For exam-ple, rates of mitochondrial OXPHOS in offspring of type2 diabetic subjects, as assessed by 31 P spectroscopy, arereduced by 30% in the fasting state (117), and TCA cycleflux, modeled using rates of 4- 13 C-glutamate enrichmentduring infusion of 13 C-acetate, is reduced by 30% (118).The magnitude of these changes is strikingly similar to the38% lower muscle mitochondrial density, assessed byelectron microscopy, in this same population—again sug-gesting that decreased mitochondrial density might be animportant factor in reduced oxidative capacity in individ-uals with a family history of diabetes.

Alterations in intrinsic function of mitochondria havealsobeen identifiedin isolatedmitochondria fromhumanswith insulin resistance and DM. Mogensen et al . (119)observed decreases in maximal ADP-stimulated respira-tion (state 3, malate and pyruvate as substrates) inmitochondria isolated from obese subjects with DM ascompared with obesity alone; these differences persistedeven afternormalization to citrate synthase activity.Thus,these data suggest that in addition to decreased mitochon-

drial density, there is an additional intrinsic defect(s) inTCA, OXPHOS, membrane potential, or adenine nucle-otide transporters in mitochondria of individuals with es-tablished diabetes.

Such underlying functional defects may be subtle atbaseline but may be unmasked during acute energeticstress. For example, short-term exercise normally in-creases ATP synthesis rates. However, this adaptive re-sponse is completely mitigated/abolished in nonobesefirst-degree relatives of type 2 diabetics—despite normalbasal ATP synthesis rates (106). Similarly, insulin-stimu-

lated ATP synthesis is reduced bymore than90% innono-bese first-degree relatives of type 2 diabetics (120), morethan would be expected from the 30% decrease in mito-chondrial density and oxidative function observed in thesame population. Because these short-term experimentalprotocols(several hours in duration at most) would notbeexpected to alter mitochondrial density, DNA content, ornumber, these data strongly suggest that inability to ap-propriatelymodulateoxidativefunction in response to theprevailing energetic environment is a signature of insulinresistance and diabetes risk.

Analysis of global gene expression patterns has alsodemonstrated a 20–30% reduction in mRNA expression



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levels formultiple nuclear-encodedgenes of theOXPHOSpathway in humans with type 2 DM (121–123). Impor-tantly, similar reductions in OXPHOS gene expressionhave been observed in some, but not all, populations of insulin-resistant, but completely normoglycemic, individ-uals (122, 124). 1 These differences may reflect popula-tion-specific differences in obesity, physical fitness, orethnicity. Interestingly, a recent study of Asian Indiansubjects found no correlation between changes inOXPHOS gene expression and insulin resistance (125). Inthese individuals, expression of OXPHOS and TCA cyclegenes, mtDNA content, and ATP production rates wereactually higher in both nondiabetic and diabetic individ-uals compared with Northern European controls, despiteloweroverall insulinsensitivity.However,circulatingtrig-lycerides were significantly elevated in both nondiabeticand diabetic individuals of Asian Indian origin (125).

These results also raise the question of whether levels of OXPHOS gene expression and function must be consid-ered relative to theoxidativefuel load inan individual.Forexample, high OXPHOS expression in the populationmentioned above may still be inadequate for appropriateand complete oxidation of a chronic high load of circu-lating lipids, whereas lower OXPHOS levels may besufficient under conditions of a low circulating lipidload (see Fig. 5).

Such data also highlight the importance of consideringadditionalaspects of oxidativemitochondrial functionbe-

yond OXPHOS expression or capacity. For example, pri-mary myotubes isolated from obese humans with type 2DM display reduced basal lipid oxidation and insulin-stimulated glucose oxidation with no differences inOXPHOS gene expression (126). Thus, defects in lipidoxidation in DM can be significant contributors to disor-dered oxidative metabolism even in the absence of detect-able alterations in OXPHOS gene expression or function.

4. Factors affecting OXPHOS gene expression in muscleSeveral conditions associated with susceptibility to in-

sulin resistance, including obesity, lipid accumulation,and aging, have all been associated with reduced nuclear-encoded OXPHOS gene expression. Reduced OXPHOSgene expression has been observed in response to geneticand nutritional obesity (127), short-term high-fat feeding(even in humans) (128), lipid infusion (129), and lipidloading of myotubes (127). However, these responses arenotobserved in allstudies ofhigh-fat feeding; in fact, somestudies demonstrate that high-fat feeding is associatedwith increased numbers of mitochondrial protein and

DNA content, potentially mediated by chronic fatty acidactivation of PPAR nuclear receptors (130–132). Simi-larly, relatively short-term reductions in serum fatty acidsand intracellular fatty acyl CoA levels mediated by acipi-mox treatment in healthy humans are associated withreduced expression of nuclear-encoded mitochondrialox-idativegenes—inparallelwithenhanced insulinsensitivity(294). Together, these seemingly disparate data suggestthatgenetic background(127), ageatdietary intervention,specific dietary lipid composition, and duration of dietmay be important variables to consider when analyzingthe interaction between OXPHOS gene expression anddiet. Moreover, alterations in OXPHOS gene expressionmay be a secondary response to an underlying primarydefect in oxidative metabolism, reflecting attempts tocompensate for reductions in mitochondrial capacity (in-creased OXPHOS expression),or thedeleterious effects of lipid overload and accumulation on transcription of OXPHOS genes (decreased OXPHOS expression), or amixture of both. Additionally, because OXPHOS geneexpression is coordinately regulated, patterns of differen-tial OXPHOS expression may be more readily detectableindisease states, yetnotnecessarilymirrorother aspects of mitochondrial oxidative capacity.

Reduced physical fitness is associated with reducedmuscle OXPHOS gene expression. In humans, maximaloxygen uptake is robustly correlated with OXPHOS geneexpression (133). Similarly, in rats bred for low aerobiccapacity over multiple generations, expression of severalOXPHOS genes is markedly reduced, even in the absenceof obesity (134).Conversely, OXPHOS expression can beincreased with exercise training (133, 135), a potent in-sulin sensitizer.

Genetic and epigenetic modifications may also con-tribute to reduced expression of OXPHOS genes in type2 DM. For example, expression of COX7A1, a complexIV gene down-regulated in type 2 DM, is heritable (50–72% heritability, as assessed by analysis in monozy-gotic and dizygotic twins), indicating a strong geneticor shared familial environmental contribution (136).Similar patterns are observed for the complex I geneNDUFB6 (137) and the ATP synthase componentATP5O (138). Indeed, expression of nuclear-encodedOXPHOS genes is significantly more concordant be-tween monozygotic twins than expected and is the top-ranking gene set for concordance in pathway analysis of global gene expression. Mediators of mitochondrialbiogenesis, including ERR , may contribute to the

1 PattiME, LiuM, ZinW, LerinC, DreyfussJ, VokesM, Schroeder J,TatroE, ParkP, KohaneI, Kasif S, Goldfine AB, submitted. Transcriptome analysis reveals parallel dysregulation ofoxidative metabolism and inflammation in muscle and adipose tissue with progression ofinsulin resistance in humans.



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strong heritability of OXPHOS components. 2 Interest-ingly, epigenetic mechanisms may also contribute tothese patterns because reduced expression parallels in-creased DNA methylation of both the COX7A1 pro-moter (136) and NDUFB6 (137, 139).

Aging is also linked to impaired oxidative function(140) in parallel with reductions in OXPHOS gene ex-pression, including COX7A1, NDUFB6, and ATP50(136–138). It is unclear at this time whether this is a directeffect of aging per se or related to reduced physical fitness,increased tissue lipid accumulation, or other factors ac-companying typical patterns of aging. Genetic polymor-phisms may also influence age-dependent reductions inexpression (137).

A key question is whether the changes in OXPHOSgene expression observed in type 2 DM are secondaryfeatures of the diabetes metabolic environment such ashyperglycemia or insulin resistance. Reductions inOXPHOS gene expression in patients with establishedtype 2 DM can be partially normalized by insulin treat-ment (123).Expression of multiple OXPHOSgenes is alsomarkedly reduced in mice made insulin deficient by treat-ment with the -cell toxin streptozotocin, and can benormalized by insulin (141). Similarly, withdrawal of insulin in individuals with type 1 diabetes reduces muscleOXPHOS gene expression and ATP production rates(142). Short-term experimental induction of acute hyper-glycemia in humans does not fully mirror this pattern of

gene expression (143), suggesting that the response to in-sulin deficiency is not completely due to resultant hyper-glycemia. Moreover, experimental insulin therapy doesnot modulate mitochondrial respiration (144), so mech-anisms linking insulin action with OXPHOS gene expres-sion remain unclear.

Changes in the levels of OXPHOS and other oxidativegenes must occur in response to cellular energetic andmet-abolic needs,andin a coordinated manner that ensures thestoichiometric assembly of the products of distinct genesinto functional complexes. As in other tissues, the coor-

dination of OXPHOS gene expression in muscle is medi-ated in part by theactionof coactivatorsandcorepressors.PGC-1 has been recognized as an important coactivatorin skeletal muscle, contributing to fiber type determina-tion, glucose uptake, and oxidative capacity (see SectionIV. A ). Moreover, alterations in muscle PGC-1 and -mRNA expression are observed in humans with insulinresistance—being reduced by nearly 50% in muscle fromindividuals with diabetes (122, 145) and in some popula-tions of normoglycemic insulin-resistant humans (121,

124, 137). In turn, PGC-1 expression may also be re-duced as a consequence of promoter methylation (146) orcaused by insulin itself (145), obesity (126), and sustainedlipid exposure (126). For example, saturated fatty acidsreduce PGC-1 promoter transcriptional activity and ex-pression in cultured myotubes, in parallel with reducedOXPHOS expression and O 2 consumption (127). PGC-1activity can also be modulated at the level of translationand by posttranscriptional changes, including inhibitoryGCN5-mediatedacetylation (147)and stimulatory sirtuin1 mediated deacetylation (148). These multiple modes of PGC-1 regulation are likely to have evolved from theneed to adapt mitochondrial energy metabolism in re-sponse to increasingly diverse inputs.

In summary,insulin resistancehasbeen associated withalterations in skeletal muscle mitochondrial oxidativefunction and its transcriptional regulatory pathways.

However, several lines of evidence suggest that this maynot be a causal relationship in all situations. First, oxida-tive dysfunction is not observed in all insulin resistant in-dividuals (125). Second, oxidative activity is determinedby the need to generate energy to meet cellular demands,e. g ., contraction and ion transport; thus oxidative capac-ity is not likely to be limiting in the resting state in muscle(3).Rather, alterations in relative utilization of substrates,an imbalance between fuel load and cellular energy re-quirements, and/or differential thresholds for generationof or resolution of oxidative stress in this setting may con-

tribute to differential susceptibility to insulin resistance inmuscle. These concepts are examined more fully in theconclusion ( Section V ).

B. Adipose tissue

1. Roles of mitochondria in adipose tissueThe role of adipose tissue mitochondria is most appar-

ent in brown adipose tissue, where flux through the ETCgenerates heat in the process of thermogenesis, a poten-tially important mechanism regulating systemic metab-olism even in adult humans (149–152). In this tissue,electron transport is greatly accelerated due to tissue-spe-cificexpression of themitochondrialUCP1.UCP1 hindersthe establishment of, or dissipates, a proton gradient of sufficient magnitude to sustain thesyntheticactivity of themitochondrial ATPase (150, 153–155), thus driving con-tinuous accelerated electron transport. UCP1-mediateduncoupling alone, however, cannot fully account for thelarge thermogenic capacity of brown adipocytes in theabsence of mechanisms that ensure continuous substratedelivery to theETC. Thus, brown adipocytemitochondria

2 Stender-Petersen KL,Poulsen P,ButteA, Jensen CB,Yee J, LeykinI, Vaag A, PedersenO,Patti ME,manuscript underreview. Geneexpressionanalysisin monozygotic twinsrevealsheritable contributions to PGC-1/ERR pathways.



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also contain high levels of CPT1b, which is critical for theentry of fatty acids into themitochondria for -oxidation.

-Oxidation, in turn, generates large amounts of reducingequivalents for the ETC.

White adipocytes have been described to contain lowlevels of mitochondria, which is indeed the case whencompared with brown adipocytes or muscle. However,mitochondrial density increases dramatically, and mi-tochondrial remodeling occurs during white adipocytedifferentiation (156–158), suggesting that mitochondrialfunctions are required to support the multiple biologicalroles of mature white adipocytes. Interestingly, a recentcompendium of mitochondrial proteins from 14 differentmousetissues indicates thatwhite adipocytemitochondriacontain a more diverse protein repertoire than mitochon-dria from heart,skeletalmuscle, or brain (31).Thus, whiteadipocyte mitochondria appear to be equipped for a

broader array of functions compared with mitochondriain tissues that must sustain rapid bursts of energy-requir-ing processes. Among the mitochondrial functions thatmay be relevant for white adipose tissue function are theanaplerotic generation of metabolic intermediates forfatty acid synthesis and esterification (159), the mainte-nance of a robust pathway for the folding and secretion of high abundance circulating proteins such as adiponectin(160), and interactions between mitochondrial functionand components of the insulin signaling pathway (161).

2. Potential mechanisms by which impaired adipose tissuemitochondrial oxidative capacity could result ininsulin resistance

The large capacity of brown adipose tissue mitochon-dria to oxidize fatty acids results in a measurable impacton whole-body metabolism; increased brown adipose tis-sue abundance correlates negatively with fuel storage andweight gain in rodents, and vice versa (162). The role of brown adipose tissue in human metabolism has typicallybeen thought to be minor. However, recent work has ledto reconsideration of this notion, noting that humans pos-sess adipose tissue depots that are cold-sensitive and hy-

permetabolic, as assessed by their very high uptake of labeled glucose (152, 163). Such depots appear to be lessactive as a function of aging and/or obesity (151, 164–167). Thus, impaired mitochondrial capacity in brownadipose tissue might be functionally linked to impairedthermogenesis andenergyexpenditure, and thusincreasedsusceptibility to obesity-linked insulin resistance.

The relevance of white adipocyte mitochondria towhole-body metabolism and metabolic disease may de-pend on the extent to which mitochondrial respiratorycapacity and/or the total mass of white adipose tissue

would be sufficient to impact circulating free fatty acidlevels. White adipocytes display a high degree of plasticity

(168),and regionaldifferences inmetabolic activitycanbelinked to varying mitochondria densities (169). Highermitochondrial density and even UCP1 can be induced inresponse to pharmacological or genetic alterations of white adipocytes (170–177), suggesting that white adi-pose tissue could potentially be induced to acquire moreoxidativemetabolicphenotypes,promotingincreasedfuelconsumption and thus energy expenditure. Whether re-spiratory chain uncoupling mediated through the induc-tion of UCP1 in white adipocytes alone could reduce freefatty acid release, or whether an additional increase inmitochondrial oxidative capacity would be required, isdebated (178–182).

Gain-of-function studies in mice where ectopic expres-sion of UCPs mitigate diet-induced obesity support thenotion that uncoupling could be sufficient (183, 184).However, UCP1 expression in adipocytes driven by the

aP2promoterfailedtosignificantlyraiserestingmetabolicrate (185). Moreover, in cultured adipocytes, ectopic ex-pression of UCP1 impairs fatty acid synthesis (186, 187).These results suggest that, in theabsenceof mechanisms toensure continuouslyelevated fuel oxidation, such as thosepresent in brown fat, uncoupling of white adipose tissuemitochondria may decrease ATP levels and impair ana-bolic flux (183).

In addition to effects on fuel utilization, decreased mi-tochondrial capacity in adipocytes may also alter adipo-cyte insulin sensitivity and/or function due to the high

energetic requirements for fatty acid storage, adipokinesecretion (160), insulin signaling (161), and glucose up-take. Interestingly, in cultured adipocytes, impairment of respiratory chain function through depletionof Tfam dur-ing adipocyte differentiation results in impaired insulin-stimulated glucose transport (161);data in animal modelsare necessary to determine the physiological relevance of this finding.

3. Evidence for reduced adipose tissue mitochondrial capacity in DM

White adipocyte mitochondrial content is decreased in

both rodent and human obesity (177, 188–191) and cor-relates withinsulin resistancethataccompanies obesity. Inhumans, white adipocyte mtDNA copy number is in-versely correlated with age and BMI and directly corre-lated with basal and insulin-induced lipogenesis (192).Thus, reduced mtDNA content could reduce adipocytecapacity for lipid storage, promoting ectopic lipid accu-mulation in peripheral tissues such as muscle and liver. Inparallel,expressionof nuclear-encoded OXPHOS genesisdown-regulated in visceral adipose tissue of humans withtype 2 DM (193). Administration of thiazolidinediones

induces changes in mitochondrial content andremodelingin white adipocytes concomitantly with an improvement



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in insulin sensitivity (170, 173, 177, 190, 194–198). Mi-tochondrial levels in white adipocytes arealso increased inresponse to adrenergic stimulation, -3 agonists, andCB1blockade in mice (195, 199, 200), again in parallel withenhanced insulin sensitivity.

Whether changes in mitochondrial density are a causeor consequence of changes in insulin sensitivity is unclear.However, some evidence suggests that lack of insulin sig-naling does not reduce mitochondrial capacity in adiposetissue. For example, mice with adipose tissue-specific ab-lation of the insulin receptor (FIRKO mice) display highlevels of mitochondrial genes involved in fatty acid oxi-dation and OXPHOS over the lifespan of the animals(201). Thus, mechanisms that induce and maintain activemitochondria in adipocytes can bypass defects in insulinsignaling, and indeed, insulin signaling may repress mito-chondrial gene expression and/or function.

4. Factors affecting mitochondrial OXPHOS expression and function in adipose tissue

The genetic program leading to brown adipose tissuedevelopment, and potentially to the high abundance of mitochondria, is initiated by the zinc-finger proteinPRDM16(202–204).Currentreportssupportthehypoth-esis that brown adipocytes and myocytes share a commoncellular lineage, potentially explaining their similaritywith regard to containing mitochondriaspecialized in fueloxidation. In addition, the transcriptional coactivatorsPGC-1 and -1 (56) play a critical role in the expansionof the mitochondrial reticulum and in the induction of UCP1 andthe brown adipose tissue thermogenic programduring the perinatal period (59).

Adipocyte mitochondrial density and OXPHOS activ-ity can be regulated in response to factors that affect lipidmetabolism. For example, Toh et al. (176) andNishino, et al . (205) find that mice deficient in Fsp27, a lipid dropletprotein that promotes lipid storage in white and brownadipocytes, have increased whole-body energy expendi-ture, resistance to diet-induced obesity, and enhanced in-sulin sensitivity. This apparent paradoxical result (high

insulin sensitivity despite deficiency in lipid storage), ap-pears to be due to the increased mitochondrial density andactivity in white adipocytes, which are brown-like in theirincreased capacity to oxidize large quantities of fatty ac-ids. Nitricoxide production bytheendothelialnitricoxidesynthase has also been linked to enhanced adipose tissuemitochondrial biogenesis and prevention of high-fat diet-induced obesity (200). Conversely, both genetic and diet-induced obesity result in decreased mitochondrial densityand OXPHOS activity in adipose tissue (127, 177,189–191), potentially contributing to adipose tissue dys-

function andexacerbation of insulin resistance.The mech-anisms whereby obesity results in a reduction in adipose

mitochondrial density are not known but could be medi-ated by decreased expression of PGC-1 , as observed inobese humans (206).

C. LiverThe liver plays a central, unique role in carbohydrate,

protein, and fat metabolism. It is critical for maintainingglucose homeostasis (1) during fuel availability, via stor-ageof glucose as glycogen or conversion to lipid forexportand storage in adipose tissue, and (2) in the fasting state,via catabolism of glycogen, synthesis of glucose fromnoncarbohydrate sources such as amino acids (gluconeo-genesis), and ketogenesis. In turn, these responses are reg-ulated by the key hormones insulin and glucagon, whichmodulate signaling pathways and gene expression, lead-ing to inhibition or stimulation of glucose production,respectively.

Recent human data have highlighted the importance of disordered hepatic metabolism, including inappropriatelyincreased hepatic glucose production, hyperlipidemia,and lipid accumulation, in both obesity and type 2 DM(207). Similarly, rodent data also support an importantrole for the liver in diabetes pathogenesis. For example,liver-specific insulin receptor knockout (LIRKO) mice de-velop insulin resistance, glucose intolerance, impaired in-sulin suppression of hepatic glucose production, andaltered patterns of hepatic gene expression (208). Inter-estingly, these mice are also dyslipidemic and susceptible

to atherosclerosis (209).

1. Role of mitochondria in liver Given the diverse array of unique metabolic functions

centered in the liver, it is not surprising that ultrastructureand function of hepatic mitochondria are distinct fromthat of muscle. Electron microscopy demonstrates thatmitochondrial area is 44% lower in liver than in heart(210) with smaller size, fewer cristae, and lower matrixdensity. Protein expression of multiple OXPHOS compo-nents and Tfam (expressed per milligram of protein) and

citrate synthase activity are also lower in liver ( e. g ., 7%that of cardiac muscle) (211). Similarly, patterns of geneexpression are distinct in liver (32). Functionally, isolatedhepatic mitochondria have relative reductions inOXPHOS proteins, respiratory chain cytochromes, andmaximal activity of complexes III and IV (211). Despitelower OXPHOS capacity, state 3 respiration and respira-tory control ratio are equivalent in liver and muscle, in-dicating differences in relative substrate concentrationsandlower “excesscapacity” in liver.Recentapplication of 31 P NMR tothe liver inhumans demonstrates that rates of

ATP synthesis are 3-fold higher in liver than in muscle(212). By contrast, the content of mtDNA, expressed ei-



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ther per gram of tissue or per mitochondrion, is actuallyhigher in liver than in other tissues. Together, these dataagain emphasize differences in protocols assessing mi-tochondrial abundance, capacity, and function andhighlight tissue diversity of mitochondrial structure andfunction, which may contribute to tissue-specific diseasesusceptibility.

2. Potential mechanisms by which impaired hepatic mitochondrial function could influence hepatic insulin sensitivity

Impairments in mitochondrial number and/or oxida-tive function could potentially affect multiple cellularfunctions within hepatocytes, both directly ( e. g ., reducedATP generation, alterations in oxidative stress, reducedcapacity forfatty acid oxidation) andindirectly,via effectson energy-requiringprocesses, includinggluconeogenesis,synthesis of urea, bile acids,cholesterol, andproteins, anddetoxification. Because accumulation of lipid withinhepatocytesisakeymarkerofinsulinresistanceinhumans(207) and a major contributor to nonalcoholic fatty liverdisease, nonalcoholic steatohepatitis (NASH),and cirrho-sis, we will first consider relationships between hepaticlipid metabolism and insulin resistance, and in SectionIII.C.3 will review evidence linking DM and hepatic ste-atosis to alterations in fatty acid metabolism or moreglobal mitochondrial dysfunction.

Hepatic lipid accumulation may result when adiposelipid storage capacity is exceeded, as in obesity or adi-pocyte dysfunction ( e. g ., lipodystrophy) (213). Alter-natively, lipid accumulation may reflect an additionalimbalance between de novo hepatic lipogenesis and mi-tochondrial oxidative metabolism. Although the relativerolesofeachofthesepossibilitiesisincompletelyunderstood,hepatic lipid accumulation is associated with obesity in hu-mans, particularly central (abdominal) in location (214,215), and in parallel with low adiponectin levels (216).Interestingly, hepatic lipid accumulation is also a robustpredictor of not only hepatic, but also muscle and adiposeinsulin sensitivity [better than intraabdominal fat, body

mass index (BMI), or other obesity measures] (217, 218).Conversely, modest weight loss (about 8 kg) normalizesintrahepatic lipid in subjects with type 2 DM, in parallelwith normalization of hepatic insulin sensitivity, even inthe absence of changes in intramyocellular lipid accumu-lation or circulating adipocytokines (215).

Although these data highlight an intimate relationshipbetween obesity, intrahepatic lipid metabolism, and insu-lin sensitivity in humans, mechanisms responsible fortheselinksremainunclear.Onepossibilityisthatexcessivehepatic lipid accumulationmayplay a central, pathogenic

role in insulin resistance. Support for this hypothesiscomes from experimental lipid loading, which can induce

hepatic insulin resistance. Transgenic mice expressing li-poprotein lipase in the liver have a 2-fold increase in he-patic triglyceride content and are insulin resistant (219).At a cellular level, incubation of hepatocytes with satu-rated long-chain fatty acids induces insulin resistance byreducing insulin-stimulated tyrosine phosphorylation of the insulin receptor and its downstream substrates (220,221). These effects in the liver appear to be mediated viareduced expressionof the insulin receptor (221). Althoughthese effects could be mediated by accumulation of fattyacyl CoA, diacylglycerols, and ceramides (as in muscle;Section III.A ), it is intriguing that effects of fatty acids inliver cells can be prevented by inhibition of CPT1, indi-catinga critical role for mitochondrial oxidation in induc-ing lipid-mediatedinsulin resistance,perhaps viaproductsof incomplete oxidation and/or generation of ROS (220).Fattyacidscanalsoalterexpressionand/orfunctionofkey

regulatory transcription factors in the liver ( e. g ., PGC-1 ,PPAR , hepatic nuclear factor 4 ) (127, 222–224) orposttranscriptional regulation of mRNA stability (225).Fatty acid-induced reductions in insulin receptor numberand function in the liver (211) may also reduce hepaticinsulin clearance (226), causing systemic hyperinsulin-emia, itself a contributor to both insulin resistance andreduced mitochondrial function (214, 227, 228).

A second possibility is that hepatic insulin resistanceitself contributesto alterations in mitochondrialoxidativecapacity. Indeed, a recent paper demonstrated that mice

with hepatic insulin resistance due to deletions of themajor insulin receptor substrates (IRS-1 and IRS-2) haveimpaired mitochondrial function and biogenesis, as dem-onstrated by reduced NADH oxidation, reduced ATPproduction rates, reduced numbers of mitochondria percell, reduced fatty acid oxidation, and increased hepatictriglyceride accumulation (229). Mitochondrial dysfunc-tion was reversed by deletion of Foxo1. These data indi-cate that normal insulin signaling, which inhibits Foxo1,is requiredformaintenance ofnormal mitochondrialfunc-tion in this model. It remains unclear whether additionalcomponents of the in vivo environment, such as glucoseintolerance and hyperinsulinemia, contribute to mito-chondrial dysfunction in these mice. However, morebroadly, these data indicate that hepatic insulin resistancecan cause mitochondrial dysfunction, at least in mice.

3. Evidence for impaired liver mitochondrial functionin diabetes and NASH

Although human liver studies have been limited due tolack of tissue biopsy samples from otherwise healthy in-dividuals, twogroupshave examined hepatic gene expres-sion related to mitochondrial function in both obesity and

type 2 DM (230–232). In the first (232), severe obesity(mean BMI 52 kg/m 2 ) was associated with reduced ex-



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pression of seven of 25 genes encoding OXPHOS genes;expression of these genes was inversely correlated withhepatic lipid accumulation and paralleled by reduced ex-pression of PGC-1 and genes known to be regulated bythyroid hormone. Similar patterns were observed in obesesubjects with established type 2 DM. Interestingly, re-duced expression of OXPHOS genes ( e. g ., COX7C,ATP5C1) was also observed in mice fed a high-fat diet andnormalized by acute therapy with thyroid hormone T 3 —suggesting that functional hepatic thyroid hormone resis-tance couldcontribute to reduced expression ofmitochon-drial oxidative genes in this context (232).

In contrast, studies in Japanese individuals with estab-lished DM and modest obesity (BMI 27 kg/m 2 ) observeda modestly increased expression of multiple genes withinall complexes of OXPHOS complexes, in parallel withBMI and insulin resistance (measured by homeostasismodelassessmentof insulin resistance,HOMA-IR)(231).Up-regulation of these OXPHOSgeneswasalso positivelyassociated with expression of several genes linked to mi-tochondrial biogenesis ( e. g ., PGC-1 , ERR , NRF, thy-roid hormone receptor) and both ROS generation ( e. g .,NADPH oxidase) and attenuation ( e. g ., glutathione per-oxidase). Thus, increased ROS related to increased fattyacid oxidation and/or hyperglycemia might contribute toup-regulation of OXPHOS gene expression in coexistingobesity and type 2 DM. Although these two data sets ap-peartobediscordant( i.e., obesity-linkeddown-regulation

of mitochondrial oxidative gene expression in the first,andup-regulation in thesecond), several differences in thestudy population mayaccount for these findings: 1) muchgreater degree of adiposity and hepatic steatosis in thefirst; 2) differences in ethnicity (Caucasian-Americans vs. Japanese); and 3) differences in insulin sensitivity and gly-cemia(insulinsensitive vs. resistantcomparison in thefirststudy, coexisting DM in the second).

Studies of individuals with NASH provide additionalopportunities to identify potential interactions betweenhepatic lipid accumulation, insulin resistance, and mito-

chondrial function in humans. Indeed, enzymatic activityof complexes I-V is reduced in liver extracts from patientswith NASH and is inversely correlated with BMI andHOMA-IR (233, 234). Moreover, NASH is characterizedby prominent abnormalities in mitochondrial ultrastruc-ture, with increased size, loss of cristae, and paracrystal-line inclusion bodies similar to those observed in somemitochondrial myopathies (235). Although these datacannot address whether such changes are indeed patho-genic, it is interesting that reduced OXPHOS activity inthis setting is accompanied by increased tissue long-chain

acylcarnitines and reduced short-chain acylcarnitines, de-spite normal CPT1 activity and increased expression

of -oxidation genes (230, 236). Similarly, circulating-hydroxybutyrate levels are increased in NASH (235).

Together, these data suggest excessive, but incomplete,fatty acid oxidation, potentially limited by reducedavailability of NAD and FAD. Byproducts of incom-plete fatty acid oxidation could act in concert with ad-ipose tissue-derived inflammatory signals ( e. g ., TNF ),and altered expression and activation of proinflamma-tory (e. g ., IL-1R family) and profibrotic genes ( e. g .,TGFB1, FGFR2), to increase production of ROS andultimately contribute to the development of NASH andcirrhosis (235).

In summary, available data indicate that hepatic lipidaccumulation and insulin resistance are intimately linkedwith mitochondrial oxidative dysfunction. We hypothe-size that modest obesity may be associated with compen-satory up-regulation of OXPHOS gene expression in

response to sustained lipid load and/or functional defectsin complete fatty acid oxidation. Up-regulation of PGC-1 in this context may contribute to increased glu-coneogenesisandhyperlipidemia, in part viacoactivationof sterolregulatory element binding transcriptionfactor 1,as observed in high-fat diet-fed mice (223). With aging,chronic ROS exposure, and/or the development of insulinresistance related to obesity or sustained lipid accumula-tion, OXPHOS expression may fall. Although this may bean appropriate response, limiting oxidative stress, it mayalso contribute to a vicious cycle of further impairments in

oxidative capacity, increased lipid accumulation, andprogressive insulin resistance. To test this hypothesis, lon-gitudinal measurements of gene expression, oxidativefunction, and lipid accumulation in humans with progres-sive obesity and evolution of insulin resistance would berequired—but are unlikely to be performed due to the in-vasive nature of serial liver biopsies in humans.

D. Pancreatic -cells

1. Roles of mitochondria in -cellsMitochondrial capacity is central to the keyfunction of

the pancreatic -cell—regulated insulin secretion. Bothrapid (first phase) and more prolonged (second phase)insulin secretion (237) are dependent on glucose metab-olism and mitochondrial oxidative capacity; glucose ox-idation increases the ATP/ADP ratio, inhibiting plasmamembrane K-ATP channels and allowing voltage-gatedcalcium channels to open. Increased cytoplasmic calciumthen triggers exocytosis of plasma-membrane docked in-sulin granules (first phase). Subsequent recruitment of granules to the plasma membrane (second phase) appearsto depend on mitochondrial metabolites produced by

anaplerosis (238). Mitochondrial metabolism is also re-quired for the transient, controlled production of ROS,



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which is required for the mitochondrial signaling path-ways that trigger granule exocytosis (239, 240).

2. Evidencefor reduced -cellmitochondrialcapacity in DM Given the crucial role of mitochondrial ATP genera-

tion, anaplerosis, and ROS production in insulin secre-tion, mitochondrial dysfunction in -cells would beexpected to reduce insulin secretion and thus promote thedevelopment of DM. Consistent with this possibility,

-cell specific deletion of Tfam reduces insulin secretorycapacity and -cellmass,yielding so-called mitochondrialDM (241).Moreover,Tfam hasrecentlybeenshownto bedirectly downstream of PDX1, a key transcription factorfor -cell development (242).

In humans, the key role of -cell mitochondria is ex-emplified by the development of diabetes in familiesharboring mutations in mtDNA. Of these,the best stud-ied is the 3243A G mutation in the mtDNA-encodedtRNALeu, UURgene, which is associated with maternallyinherited diabetes and deafness (MIDD) (243, 244). An-other example is mutation 14577 T C, a missense sub-stitutionin theNADH dehydrogenase 6 gene (245).In thiscase, mitochondrial respiratory chain complex I activityand O 2 consumption rates are decreased by 65 and 62%,respectively, in hybrid cell lines derived from probands.

Interestingly, mitochondrial diabetes only developsupon aging, with an average age of onset between 35 and40yr for MIDD and48 yrfor14577T C. This contrasts

with the early childhood onset of diabetes in syndromessuch as maturity-onsetdiabetes of theyoung 2 (MODY2),in which a mutation in glucokinase, the first step of gly-colysis, results in attenuated glucose-stimulated ATP gen-eration and insulin secretion. These data suggest thatmitochondrial diabetes is more likely to result from agradual deterioration of -cell function, rather thanfrom an acute functional impairment due to insufficientATP production (246).

One of the mechanisms by which mtDNA mutationsmight lead to a gradual deterioration in -cell function,

and not to an acute failure of insulin secretion due to de-creased ATP levels, could be the stress imposed by an in-crease in metabolicflux to compensate for inefficiencies intheETC.Consistentwith this view,clonalcytosolic hybridcells harboring mitochondria derived from MIDD pa-tients exhibit impaired calcium handling and elevatedROS under metabolic stress (247, 248). Chronically in-creased ROS production could also induce -cell deathand result in gradual onset of diabetes (249–253).

3. Factors affecting mitochondrial function in -cells

Mitochondrial function in -cells is highlyregulatedbythe levels and activities of UCPs, in turn regulated by ROS

producedbytheactivityoftheETC.LowlevelsofROSarenecessary for insulin secretion, but chronic, high mito-chondrialROS production canhave a deleterious effecton

-cell function (254–256). Thus, the activation of UCP2protects the -cell from the deleterious effects of excessROS(257)by dissipating theprotongradient anddecreas-ing ROS production in a controlled negative feedbackmanner (Fig. 4). However, it also leads to decreased ATPproduction, which impairs insulin secretion. Thus, UCPsmust uncouple respiration sufficiently to mitigate toxiclevels of ROS, but not enough to decrease ATP and ROSbelow the levels necessary for insulin secretion. This del-icate balance in which UCP2 is desirable for -cell pro-tection, but undesirable for glycemic control, probablyunderlies the discrepancy in results between two reportson the phenotype of UCP2 knockout mice. In a mixedbackground, UCP2 knockout improves glycemic control

in ob/ob mice (258), whereas in a pure C57BL6/J back-ground, UCP2 knockout accelerates -cell failure and di-abetes (259).

The levels and activity of UCP2 and the rate of ROSproduction are both increased by high-fat diet and hy-perphagia, possibly through the actions of nonesterifiedfatty acids and their ceramide derivatives (260). It is likelythat decreased ATP production due to unbalanced acti-vation of UCPs by direct actions of fatty acids and theirderivatives, in addition to excessive ROS, could underliethe accumulation of -cell damage that precedes type 2

DM (Fig. 4).

FIG. 4. Hypothesized mechanism by which free fatty acid (FFA) excessimpairs insulin secretion. A, As described above, the activity of the ETCleads to the synthesis of ATP and the generation of a small amount ofROS. In the -cell, both ATP and ROS are signals that trigger insulinsecretion. Excessive accumulation of ROS is mitigated normally by theactivation of UCP2, which dissipates the proton gradient, decreasingboth ATP and ROS production. The presence of this normal negativefeedback loop suggests that the control of excessive ROS generation isimperative in the -cell, even if it occurs at the expense of decreasingATP synthesis. B, In the presence of excess FFA, this normal feedbackloop is compromised by a direct activation of UCP2 by FFA, as well asan effect of FFA to increase the amount of UCP2. Thus, uncouplingoccurs to an excessive degree, compromising ATP synthesis enough toimpair insulin secretion and -cell fitness.



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Although the tissues reviewed above are consideredcentral to the pathophysiology of DM, other tissues suchas gut, brain, kidney, neuronal tissues, and endotheliumare also likely to be implicated in a primary or secondarymanner in the pathophysiology of DM and /or its com-plications. The aspects of mitochondrial function uniqueto each of these tissues and the consequences of their po-tential dysfunction in relation to DM pathophysiologyarerelativelylessexploredareasandarethusoutsidethescopeof this review.

IV. Experimental Strategies to Explore theRelationship between Mitochondrial Functionand DM

Although available data demonstrate links between mito-

chondrialoxidative function and phenotypes linked to in-sulin resistance and diabetes, it remains unclear whetherthese are simply associations or whether oxidative dys-function can contribute to insulin resistance and diabetesrisk. To address this question, we will examine availabledata from experimental models in which OXPHOSfunction has been altered. Such studies have shed lighton the basic mechanisms underlying mitochondrial bio-genesisandontheconsequencesofdisruptionofnormalmitochondrial homeostatic mechanisms on cell andwhole-body oxidative metabolism. A summary of thesestudies is presented in Tables 1 and 2 and is discussed inSection IV.A and B.

A. PGC-1 and overexpressionPGC-1 and related coactivators are critical for the

regulation of mitochondrial oxidative capacity, as dem-onstrated by the approximately 2-fold increases inmtDNA and oxygen consumption and a 50% increasein mitochondrial density in myotubes overexpressingPGC-1 (56). To address whether this family plays thesame functional role in vivo, several different models of PGC-1 transgenic expression have been generated, each

of which differs in tissue selectivity, levels of overexpres-sion achieved, and resulting metabolic phenotype (Ta-ble 1). The lowest level of PGC-1 overexpression wasachieved in rat muscle by means of electroporation (261).This resulted in modest up-regulation of mitochondrialproteins, increased palmitate oxidation, and increased in-sulin-stimulated glucose uptake. Similarly, transgenicmice expressing human PGC-1 driven by its own pro-moter displayed a modest (30% higher than basal) in-crease in mRNA expression of several OXPHOS genes,fiber type switching, and enhanced muscle insulin sensi-

tivity (262). Importantly, this modest PGC-1 overex-pressionalso wasaccompaniedby decreasedlevels ofROS

and inflammatory signaling. However, these same ani-mals displayed increased liver gluconeogenic enzyme lev-els and impaired insulin suppression of hepatic glucoseproduction,thus nullifyingthe potentially beneficialeffectof modest muscle PGC-1 overexpression on whole-bodyglucose homeostasis.

Higher levels of overexpression of PGC-1 achievedthrough actin promoter-driven expression in transgenicanimals display a strikingly different phenotype (263,264). In these animals, mitochondrial density andOXPHOS gene expression are more than double basallevels, and large increases in UCP2 gene expression arealso observed. Mitochondrial energetics are impaired,with 60% decreases in ATP levels in muscle homogenatesand concomitant increases in AMP kinase activation,probably as a compensatory response to decreased mito-chondrial functionality. Muscle function appears com-promised, as evidenced by decreased voluntary exercise,muscle atrophy, and decreased insulin sensitivity.

Inducible overexpression of PGC-1 in skeletal muscle(265) results in increased mitochondrial density and a ro-bust increase in expression of OXPHOS genes and genesnecessary for fatty acid oxidation. In these animals, bothmuscle glucose uptake and glycogen deposition were in-creased. Although low-intensity exercise performance didnot differ in this model, high-performance exercise wasimpaired, in parallel with failure to mobilize stored gly-cogen. Similarly, inducible expression in cardiac muscle

can have deleterious effects. When higher levels of PGC-1 overexpression are restricted to heart duringearly life (266), increased neonatal mitochondrial prolif-eration is observed, but it is accompanied by myofibrillardisplacement. In adults, PGC-1 induction led to a moremodest mitochondrial proliferation, which was neverthe-less surprisingly accompanied by cardiomyopathy. Thus,whole-body and inducible skeletal muscle- or cardiac-spe-cific overexpression of PGC-1 can produce deleteriouseffects on muscle structure and function.

When PGC-1 overexpression is restricted to skeletal

muscle but overexpressedthroughout development by theuse of the creatine kinase gene promoter, very large in-creases in mitochondrial mass, OXPHOS, and fatty acidoxidation genes are observed (267–269). In this model,ATP synthesis rate and exercise performance are in-creased, and fatty acid oxidation is also enhanced. De-spite these effects, insulin sensitivity is normal inPGC-1 transgenic mice fed a chow diet and, surpris-ingly, is reduced during high-fat feeding. Insulin resis-tance was paralleled by accumulation of triglyceridesand long-chain acyl CoA (270).

Although PGC-1 -mediated gene expression andfunc-tion appear to overlap considerably with that of PGC-1 ,



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T A B L E 1

. S t u d i e s o f P G C - 1

t r a n s g e n i c e x p r e s s i o n m o d e l s

F i r s t a u t h o r , y e a r

( R e f

. )

M o d e l

M i t o D N A

M i t o d e n s i t y

( E M )

O X P H O S

m R N A / p r o t e i n

N o n - O

X P H O S

m R N A / p r o t e i n

M i t o e n e r g e t i c s

E x e r c i s e c a p a c i t y

I n s u l i n

s e n s i t i v i t y

O t h e r

B e n t o n , 2

0 0 8 ( 2 6 1 )

P G C - 1

e l e c t r o p o r a t i o n

1

1 1 3 %

1 8 0 % 1

C O X I V

p r o t e i n

1

m u s c l e

1

A M P k i n a s e

a c t i v i t y

W a r

d , 2 0 0 9 ( 2 6 2 )

P C G - 1

w h o l e

- b o d y

o w n p r o m o t e r

1 3 0 % 1

m R N A

1

m u s c l e

H e p a t i c i n s u

l i n

r e s i s t a n c e

M i u r a , 2 0 0 3

, 2 0 0 6

( 2 6 3

, 2 6 4 )

P G C - 1

w h o l e

- b o d y

- a c t i n p r o m o t e r

1

2 0 0 – 3

0 0 %

1

n u m

b e r

1 5 0 – 2

0 0 %

1

m R N A

3 0 0 % 1

i n U C P 2

m R N A

6 0 %

2

A T P l e v e

l s i n

h o m o g e n a t e s

2

v o l u n t a r y

2

w h o l e

- b o d y

1

A M P k i n a s e

a c t i v i t y

R u s s e l

l , 2 0 0 4 ( 2 6 6 )

P G C - 1

i n d u c i

b l e

h e a r t

1

3 5 0 %

M y o

f i b r i l

l a r

d i s o r g a n i z a t i o n

c a r d i a c

f a i l u r e

W e n

d e , 2

0 0 7 ( 2 6 5 )

P G C - 1

i n d u c i

b l e

s k e l e t a l m u s c l e

1

1 5 0 – 2

5 0 %

1

m R N A

1 5 0 % 1

F A O g e n e

m R N A

N o c h a n g e i n l o w i n t e n s i t y

,

2

p e r f o r m a n c e a t

h i g h

i n t e n s i t y

1

g l u c o s e u p t a k e

,

g l y c o g e n

d e p o s i t i o n ,

d e c r e a s e

d

g l y c o l y s i s

L i n , 2 0 0 2 ( 2 6 8 ) ;

S a n d r i , 2 0 0 6

( 2 6 9 ) ; C a l v o ,

2 0 0 8 ( 2 6 7 ) ;

C h o i , 2 0 0 8 ( 2 7 0 )

P G C - 1

M C K

p r o m o t e r

1

1 6 6 – 2

5 0 %

1

2 5 0 % i n E D L

1 7 0 – 3

0 0 %

1

m R N A

2 0 0 –

4 0 0 % 1

i n

F A O g e n e m R N A

5 0 –

6 0 % 1

A T P

s y n t

h e s i s

b y N M R

1

e x e r c i s e p e r f o r m a n c e ;

2

f a t i g u e

i n

v i t r o ,

p r o t e c t i o n

f r o m

d e n e r v a t i o n - i n d u c e

d

a t r o p h y

2

m u s c l e a n

d

w h o l e

- b o d y

o n l y i n h i g h -

f a t d i e t

N o c h a n g e i n A M P

k i n a s e a c t i v i t y

A r a n y , 2 0 0 7 ( 2 7 1 )

P G C - 1

M C K

p r o m o t e r

1

2 0 0 – 5

0 0 %

1

m R N A a n

d

p r o t e i n

2 0 0 – 5

0 0 % 1

F A O

g e n e m R N A

1 2 0 – 1

3 0 % 1

e n d u r a n c e

K a m e i , 2

0 0 3 ( 2 7 2 )

P G C 1 -

w h o l e

- b o d y

- a c t i n p r o m o t e r

1

w h o l e

- b o d y

M i t o , M i t o c h o n

d r i a l ; E M

, e l e c t r o n m i c r o s c o p y ; M C K , m

u s c l e c r e a t i n e

k i n a s e ;

2

, d e c r e a s e ;

1

, i n c r e a s e ; E D L , e x t e n s o r

d i g i t o r u m

l o n g u s ; F A O

, f a t t y a c i d o x i d a t i o n .


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