High-Fat Diet Causes Mitochondrial Dysfunction asa Result of Impaired ADP SensitivityPaula M. Miotto,1 Paul J. LeBlanc,2 and Graham P. Holloway1

Diabetes 2018;67:2199–2205 | https://doi.org/10.2337/db18-0417

Although molecular approaches altering mitochondrialcontent have implied a direct relationship between mi-tochondrial bioenergetics and insulin sensitivity, para-doxically, consumption of a high-fat (HF) diet increasesmitochondrial content while inducing insulin resistance.We hypothesized that despite the induction of mitochon-drial biogenesis, consumption of an HF diet would impairmitochondrial ADP sensitivity in skeletal muscle of miceand therefore manifest in mitochondrial dysfunction inthe presence of ADP concentrations indicative of skel-etal muscle biology. We found that HF consumption in-creased mitochondrial protein expression; however,absolute mitochondrial respiration and ADP sensitivitywere impaired across a range of biologically relevantADP concentrations. In addition, HF consumption atten-uated the ability of ADP to suppress mitochondrial H2O2

emission, further suggesting impairments in ADP sensi-tivity. The abundance of ADP transport proteins werenot altered, but the sensitivity to carboxyatractyloside-mediated inhibition was attenuated after HF con-sumption, implicating alterations in adenine nucleotidetranslocase (ANT) ADP sensitivity in these observa-tions. Moreover, palmitoyl-CoA is known to inhibitANT, and modeling intramuscular palmitoyl-CoA con-centrations that occur after HF consumption exacer-bated the deficiency in ADP sensitivity. Altogether,these data suggest that an HF diet induces mitochon-drial dysfunction secondary to an intrinsic impairmentin mitochondrial ADP sensitivity that is magnified bypalmitoyl-CoA.

Although the etiology of type 2 diabetes is poorly defined,chronic consumption of a high-fat (HF) diet is a majorcontributor to whole-body glucose intolerance (1) and

insulin resistance (2,3). The molecular explanation forthese responses is not fully understood; however, mito-chondrial dysfunction within skeletal muscle has receivedattention as a potential contributor because mitochondrialcontent is reduced in most reports of insulin-resistant/obese human skeletal muscle (4,5). Moreover, in variousmodels, the induction of mitochondrial biogenesis protectsagainst the development of insulin resistance (2,6), andgenetic approaches that decrease mitochondrial contentpredispose animals to HF diet–induced insulin resistance(7). These data suggest changes in mitochondrial contentare related to insulin sensitivity, but reductions in mito-chondrial oxidative capacity are not uniformly reported inthe literature with insulin resistance (8,9), and HF feedinghas been shown to induce glucose intolerance and insulinresistance despite increasing mitochondrial content(10–12). Together, these data suggest reductions in mito-chondrial respiratory capacity are not required for thedevelopment of insulin resistance.

Although short-term HF experiments challenge thenotion thatmitochondrial dysfunction contributes to insulin-resistance development, these data are difficult to rectifywith molecular approaches that alter mitochondrial con-tent and highlight a strong association with insulin sen-sitivity. An important consideration in this respect isthat previous examinations of mitochondrial functionhave been performed in the presence of saturating ADPconcentrations, which may not reflect the biological envi-ronment. An increase in mitochondrial content has beenpostulated to improve the sensitivity of oxidative metab-olism to ADP (13,14), and therefore, ADP responsivenessmay represent a key process in cellular homeostasis thatis not recapitulated in experiments conducted in the

1Department of Human Health and Nutritional Sciences, University of Guelph,Guelph, Ontario, Canada2Department of Health Sciences, Brock University, St. Catharines, Ontario, Canada

Corresponding author: Graham P. Holloway, ghollowa@uoguelph.ca, or Paula M.Miotto, pmiotto@uoguelph.ca.

Received 11 April 2018 and accepted 26 June 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0417/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

See accompanying article, p. 2152.

Diabetes Volume 67, November 2018 2199

METABOLISM

presence or absence of saturating ADP. Importantly, themovement of ADP into the mitochondrial matrix andthe subsequent binding of ADP to F1F0 ATP synthasedecreases membrane potential and H2O2 production whilesimultaneously increasing substrate oxidation (15). In thismanner, ADP sensitivity may represent a key biologicalprocess influenced by changing mitochondrial contentbecause it can influence redox balance and reactive-lipidaccumulation. Intriguingly, mitochondrial ADP sensitivityis externally regulated and inhibited by reactive-lipidaccumulation (i.e., palmitoyl-CoA [P-CoA]); however, sur-prisingly, how mitochondrial ADP sensitivity is affected byHF consumption is not currently known. We hypothesizedthat mitochondrial ADP sensitivity would be impairedafter an HF diet, independent of reductions in mitochon-drial content. If accurate, this hypothesis would rectifya prominent discrepancy in the literature regarding thenotion that mitochondrial dysfunction occurs in parallelwith the development of insulin resistance.

RESEARCH DESIGN AND METHODS

Animals and EthicsAll experimental procedures were approved by the Uni-versity of Guelph Animal Care Committee and conformedto the Guide for the Care and Use of Laboratory Animals asindicated by the U.S. National Institutes of Health. Male

mice (10–12weeks of age) on a C57Bl6J backgroundwere feda control (CON; 10% lard) or HF (60% lard) diet ad libitumfor 4 weeks. All animals were anesthetized with an intraper-itoneal injection of sodium pentobarbital (60 mg/kg; MTCPharmaceuticals, Cambridge, Ontario, Canada) before redgastrocnemius muscle extraction for subsequent analyses.

Verification of HF Diet–Induced Glucose and InsulinIntoleranceIntraperitoneal glucose and insulin tolerance tests wereperformed on separate days as previously reported (16).On a separate day, animals were anesthetized, and the redgastrocnemius muscles were removed before or 15 minafter an intravenous injection of 1 unit/kg body weight ofinsulin (NovoRapid). The area under the curve (AUC)calculations were adjusted to account for baseline bloodglucose values.

Resting Whole-Body Metabolic MeasurementsResting VO2 and VCO2 were monitored in metabolic cages(Columbus Instruments, Columbus, OH) and used tocalculate total carbohydrate and fat oxidation and energyexpenditure as previously reported (17).

Mitochondrial BioenergeticsRespiration in red gastrocnemius permeabilized musclefibers was measured using high-resolution respirometry(Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria)

Figure 1—Verification of HF diet–induced glucose intolerance, insulin resistance, and reliance on fat oxidation. AUC during glucose tolerance(left) and insulin tolerance (right) testing (A), ratio of phosphorylated (Phospho)/total AKT (B), carbohydrate oxidation (C ), fat oxidation (D),respiratory exchange ratio (RER) (E), and energy expenditure (F) in mice fed the CON or HF diet. *P, 0.05 indicating a significant differencefrom CON. Data are expressed as mean 6 SEM; n = 8–11/group.

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at 37°C as previously reported (13). Briefly, ADP (0–12,000 mmol/L) was titrated in the presence of pyruvate(10 mmol/L) and malate (5 mmol/L), followed by gluta-mate (10 mmol/L) and succinate (10 mmol/L), in thepresence or absence of P-CoA (20 or 60 mmol/L). Inseparate experiments, P-CoA was titrated in the presenceof L-carnitine (2 mmol/L), malate (5 mmol/L), and ADP(5 mmol/L) in the absence or presence of malonyl-CoA(M-CoA) (7mmol/L). Carboxyatractyloside (0.2–1.6mmol/L)was used to inhibit ADP-supported respiration to inves-tigate changes in adenine nucleotide translocase (ANT)substrate sensitivity. Cytochrome c (10 mmol/L) did notstimulate respiration during experiments (data notshown). Succinate-supported (20 mmol/L) H2O2 emissionwas determined fluorometrically in the absence and pres-ence of ADP (100 mmol/L) as previously reported (13).

Citrate Synthase ActivityFrozen red gastrocnemius (6–10 mg) was homogenizedin Tris buffer (100 mmol/L, pH 8.3) and freeze-thawed tolyse mitochondria. Citrate synthase activity was measuredspectrophotometrically as previously reported (18).

Western Blot AnalysisThe red gastrocnemius was homogenized, and Westernblotting was performed as previously reported (17). SeeSupplementary Fig. 1 for a complete list of antibodies.

Statistical AnalysesMichaelis-Menten kinetics were determined by plottingdata points in GraphPad Prism 5 software to estimate theapparent Km as previously described (19). Unpaired two-tailed Student t tests were used to analyze data betweenCON- and HF-fed mice, and one-way ANOVAs were usedfor P-CoA–ADP sensitivity comparisons, followed byStudent-Newman-Keuls post hoc analyses where appropri-ate. Statistical significance was determined at P , 0.05.Data are expressed as mean 6 SEM.

RESULTS

Verification of HF Diet–Induced Glucose Intoleranceand Insulin ResistanceWe first verified the expected HF diet responses on bodymass and insulin-resistance. HF consumption resulted ingreater body weight (CON: 31 6 1.78 g, HF: 38.6 6 0.95 g;P , 0.05), greater epididymal fat mass (CON: 1.2 6 0.24 g,HF: 2.9 6 0.15 g; P , 0.05), greater AUC in response toa glucose tolerance test and insulin tolerance test (Fig. 1A),and lower skeletalmuscle insulin–stimulated AKT phosphor-ylation (Fig. 1B). Moreover, although mice exhibited theexpected diurnal changes in fuel metabolism (Fig. 1C–F),consumption of the HF diet resulted in lower carbohydrateoxidation (Fig. 1C), higher fat oxidation (Fig. 1D), lowerrespiratory exchange ratio (Fig. 1E), and greater energy

Figure 2—HF consumption increases markers of mitochondrial content in the absence of altered maximal respiratory capacity or CPT-Iregulation. Representative images and quantification of mitochondrial and electron transport chain proteins (A), citrate synthase activity (B),maximal ADP-stimulated respiration in the presence of complex (C) I- and II-linked substrates and respiratory control ratios (RCR) (C ),maximal P-CoA–supported respiration (D), P-CoA sensitivity in the absence ofM-CoA (E), and P-CoA sensitivity in the presence ofM-CoA (F )in mice fed the CON or HF diet. JO2, oxygen consumption. *P , 0.05, indicating a significant difference from CON. Data are expressed asmean6 SEM; n = 12–14/group for mitochondrial protein content; n = 8–11/group for respiration. PM, pyruvate1malate; PMD, PM1 ADP;PMDG, PMD 1 glutamate; PMDGS, PMDG 1 succinate.

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expenditure (Fig. 1F), further confirming the HF dietmodel.

Maximal Coupled Respiration RatesWith regard to potential mitochondrial changes influencedby HF consumption, we first examined markers of mito-chondrial content. HF consumption increased peroxisomeproliferator–activated receptor g coactivator (PGC-1a)and several markers of the electron transport chain, totalelectron transport chain abundance, and pyruvate dehy-drogenase E1a (PDHE1a), without altering carnitine pal-mitoyltransferase I (CPT-I), ATP synthase, and a subunit ofcytochrome c oxidase (Fig. 2A). Moreover, HF consump-tion increased citrate synthase activity in support ofa greater oxidative capacity (Fig. 2B). Despite the apparentHF diet–induced mitochondrial biogenesis, HF consump-tion did not alter state 2 respiration, maximal ADP-stim-ulated respiration, or respiratory control ratio values (Fig.2C). Similarly, HF consumption did not affect maximalCPT-I–dependent P-CoA–supported respiration (Fig. 2D)or P-CoA sensitivity (Fig. 2E). Moreover, the CPT-I in-hibitor M-CoA attenuated P-CoA sensitivity, but this wasnot affected by HF consumption (Fig. 2F). Although thesedata strongly suggest the absence of mitochondrial re-spiratory dysfunction after HF consumption, given thatskeletal muscle contains submaximal ADP concentrations,

we next examined mitochondrial respiration at physiolog-ical concentrations and ADP sensitivity.

Submaximal ADP-Stimulated Respiration Rates andADP SensitivityGiven the absence of changes in maximal ADP-stimulatedrespiration, we next examined mitochondrial respirationacross a range of ADP concentrations. The HF diet did notimpair respiration in the presence of.2,000 mmol/L ADP,but strikingly, respiration was attenuated ;30% at allbiologically relevant ADP concentrations (i.e., 100–1,000 mmol/L ADP) (Fig. 3A). In addition, mitochondrialADP sensitivity was decreased ;25% after HF consump-tion (i.e., greater apparent Km value) (Fig. 3B). Combined,despite the induction of mitochondrial biogenesis, thesedata strongly suggest the development of mitochondrialdysfunction with an HF diet secondary to impaired ADPsensitivity.

Given that ADP binding to F1F0 ATP synthase is knownto suppress H2O2 emission rates (15), we also examinedmitochondrial H2O2 emissions in the absence and presenceof ADP to further solidify this relationship. The maximalcapacity for H2O2 emission did not change after the HFdiet (Fig. 3C), but mice fed the HF diet had an impairedability to suppress H2O2 emission rates via ADP transportinto the mitochondria, as indicated by H2O2 production in

Figure 3—HF consumption impairs submaximal ADP-stimulated respiration, ADP sensitivity, and ADP suppression of H2O2 emissionindependent of substrate transporter protein expression. ADP-stimulated respiration (A), apparent Km for ADP and Michaelis-Menten kinetic curve(B), maximal H2O2 emission (C), H2O2 emission in the presence of 100mmol/L ADP (D), percentage suppression of H2O2 emission for ADP (E), Westernblots for proteins implicated in ADP handling/transport and redox stress (F), and ADP-stimulated respiration in the presence of carboxyatractyloside(CTA) (G) inmice fed theCONorHFdiet. 4HNE, 4-hydroxynonenal; JO2, oxygenconsumption;Mi-CK,mitochondrial creatine kinase;MM-CK, cytosoliccreatine kinase; SOD2, superoxidedismutase2;UCP3, uncouplingprotein 3. *P,0.05 indicatinga significant difference fromCON.Dataare expressedas mean 6 SEM; n = 12214/group for H2O2 emission; n = 8214/group for Western blots; n = 8210/group for carboxyatractyloside experiments.

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the presence of ADP that was approaching significance(Fig. 3D) and a lower percentage suppression of H2O2

emission rates by ADP (Fig. 3E). Further, mice fed theHF diet had greater antioxidant expression (catalase andsuperoxide dismutase 2) and 4-hydroxynonenal adductsthan mice fed the CON diet (Fig. 3F), supporting anincrease in oxidative stress/damage and stimulation ofantioxidant adaptation during HF consumption. Theimpairment in ADP sensitivity occurred independent ofchanges in protein expression of ADP transporters (i.e.,voltage-dependent anion channel [VDAC] and ANT), mi-tochondrial creatine kinase, cytosolic creatine kinase, anduncoupling protein 3 (Fig. 3F). We therefore examined theability of carboxyatractyloside to inhibit ADP-stimulatedrespiration to gain insight into the possibility that post-translational modifications on ANT contribute to theattenuated ADP responsiveness after the HF diet. Al-though 1.6 mmol/L carboxyatractyloside inhibited respi-ration;90% regardless of diet (data not shown), respirationwas higher after the HF diet in the presence of 0.2 mmol/Lcarboxyatractyloside (Fig. 3G), suggesting a decreased abilityfor carboxyatractyloside to interact with the ADP-bindingmotif on ANT. Altogether, these data suggest an HF dietattenuates ANT sensitivity to ADP, likely contributing tothe apparent induction in mitochondrial dysfunction.

P-CoA–Mediated Inhibition on ADP SensitivityAlthough these data strongly suggest impaired ADP re-sponsiveness after the HF diet, these experiments wereconducted in optimal conditions. However, long-chainfatty acid CoAs (e.g., P-CoA) are known to increase afterHF consumption (20), and this lipid derivative has beenshown to inhibit ANT and reduce ADP sensitivity in

permeabilized muscle fibers (13,17). Therefore, giventhe insensitivity to carboxyatractyloside, we next exam-ined P-CoA–mediated inhibition of ADP sensitivity inCON- and HF-fed mice. To do this, we repeated experi-ments on mitochondrial ADP sensitivity in the absenceand presence of P-CoA concentrations that better reflectCON (20 mmol/L) (21) and HF diet intramuscular sit-uations (60 mmol/L) (20). P-CoA concentrations thatreflected CON conditions did not alter respiration ratesat any ADP concentration (data not shown), but thepresence of 60 mmol/L P-CoA attenuated respiration atmost ADP concentrations (Fig. 4A) and further attenuatedADP sensitivity after the HF diet (i.e., increased theapparent ADP Km) (Fig. 4B). Combined, these data indicatethe presence of mitochondrial respiratory dysfunctionafter the HF diet is exacerbated in the presence of in-creased intramuscular P-CoA concentrations.

DISCUSSION

We provide compelling evidence that consumption of anHF diet induced mitochondrial dysfunction as a result ofimpaired ADP sensitivity. Specifically, we show that de-spite the induction of mitochondrial biogenesis and un-altered maximal ADP-stimulated respiration, respirationat physiological ADP concentrations was impaired after theHF diet. In addition, the ability of ADP to stimulate respi-ration (apparent Km) and attenuate mitochondrial H2O2

emission were decreased after the HF diet, whereas modelingreactive lipid levels that occur with HF consumption exac-erbated the apparent mitochondrial dysfunction. Combined,these data strongly indicate impaired mitochondrial bioen-ergetics after an HF diet due to impaired ADP sensitivity.

Figure 4—HF consumption in the presence of reflective P-CoA concentrations exacerbated impairments on mitochondrial respiration andsensitivity for ADP. ADP-stimulated respiration (A) and apparent ADP sensitivity (B) in CON- or HF-fed mice in the absence or presence of60 mmol/L P-CoA. JO2, oxygen consumption. *P , 0.05 indicating a significant difference from CON, #P , 0.05 indicates a significantdifference from HF. Data are expressed as mean 6 SEM; n = 8212/group.

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Whereas genetic models and interventions that increasemitochondrial content typically have improved insulinsensitivity and glucose tolerance (1,2,6), consumption ofthe HF diet in the current study, and in others (10,11),paradoxically resulted in increased mitochondrial contentdespite the induction of glucose intolerance and insulinresistance. Therefore, the notion that mitochondrial dys-function contributes to the development of insulin re-sistance has waned in recent years. Our data, however,implicate HF consumption in impairing respiration andincreasing mitochondrial H2O2 emission as a result ofimpaired ADP sensitivity. These derangements appear tobe amplified in the presence of higher P-CoA concentra-tions indicative of insulin-resistant muscle, suggesting thepresence of mitochondrial ADP insensitivity in skeletalmuscle after HF consumption and insulin resistance. Theconstant VDAC/ANT and ATP synthase protein abundancesuggest altered regulation of these proteins may lead tomitochondrial dysfunction. Whereas an impairment of ATPsynthase may also exist after an HF diet, the insensitivity tocarboxyatractyloside suggests that ANT is less sensitive toADP binding, a response likely amplified by increases inintramuscular P-CoA concentrations (22,23).

Our data show that impaired mitochondrial ADP sen-sitivity plays an important role in HF diet–induced mito-chondrial dysfunction. Although speculative, the inductionof mitochondrial biogenesis likely represents a compensa-tory mechanism to improve ADP sensitivity and poten-tially mitigate HF diet–induced redox stress (24); however,this appears inadequate to maintain cellular homeostasis.Whether the reduction in ADP sensitivity directly contrib-utes to the metabolic inflexibility observed in HF mice isunknown. However, it is also possible that this response,similar to the induction of mitochondrial biogenesis, rep-resents a compensatory adaptation to preserve somecarbohydrate utilization in the presence of increased in-tramuscular lipid availability, because a rise in cytosolicADP could in theory promote carbohydrate metabolism. Inaddition to the potential influence on fuel selection, be-cause the movement of ADP into the mitochondrial matrixcan bind to ATP synthase to stimulate respiration andattenuate mitochondrial-derived H2O2 emission (15), animpairment in ADP transport may represent a nexus pointinfluencing several models implicated in the developmentof insulin resistance.

Funding. P.M.M. is supported by a Natural Sciences and Engineering ResearchCouncil of Canada graduate scholarship. This work was supported by the NaturalSciences and Engineering Research Council of Canada (03656 to G.P.H.). In-frastructure was purchased with assistance from the Canadian Foundation forInnovation/Ontario Research Fund.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. P.M.M. conducted experiments, analyzed data,and drafted the manuscript. P.M.M., P.J.L., and G.P.H. interpreted results andedited the manuscript. P.M.M. and G.P.H. designed the study. G.P.H. isguarantor of this work and, as such, had full access to all the data in the

study and takes responsibility for the integrity of the data and the accuracyof the data analysis.

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