Two weeks of high-intensity interval training increases skeletal muscle mitochondrial respiration via complex-specific remodeling in sedentary humans.

Aerobic training remodels the quantity and quality (function per unit) of skeletal muscle mitochondria to promote substrate oxidation, however, there remain key gaps in understanding the underlying mechanisms during initial training adaptations. We used short-term high-intensity interval training (HIIT) to determine changes to mitochondrial respiration and regulatory pathways that occur early in remodeling. Fifteen normal-weight sedentary adults started seven sessions of HIIT over 14 days and 14 participants completed the intervention. We collected vastus lateralis biopsies before and 48 h after HIIT to determine mitochondrial respiration, RNA sequencing, and Western blotting for proteins of mitochondrial respiration and degradation via autophagy. HIIT increased respiration per mitochondrial protein for lipid (+23% P = 0.020), complex I (+18%, P = 0.0015), complex I + II (+14%, P < 0.0001), and complex II (+24% P < 0.0001). Transcripts that increased with HIIT identified several gene sets of mitochondrial respiration, particularly for complex I, whereas transcripts that decreased identified pathways of DNA and chromatin remodeling. HIIT lowered protein abundance of autophagy markers for p62 (-19%, P = 0.012) and LC3 II/I (-20%, P = 0.004) in whole tissue lysates but not isolated mitochondria. Meal tolerance testing revealed HIIT increased the change in whole body respiratory exchange ratio and lowered cumulative plasma insulin concentrations. Gene transcripts and respiratory function indicate remodeling of mitochondria within 2 wk of HIIT. Overall changes are consistent with increased protein quality driving rapid improvements in substrate oxidation.NEW & NOTEWORTHY Aerobic training stimulates mitochondrial metabolism in skeletal muscle that is linked to improvements to whole body fuel metabolism. The mechanisms driving changes to the quantity and quality (function per unit) of mitochondria are less known. We used seven sessions of high-intensity interval training (HIIT) to determine functional changes and mechanisms of mitochondrial remodeling in skeletal muscle. HIIT increased mitochondrial respiration per mass for fatty acids, complex I, and complex II substrates. HIIT-induced remodeling pathways including gene transcripts for mitochondrial respiration (via RNA sequencing of muscle tissue) and proteins related to complex I respiration. We conclude that an early feature of aerobic training is increased mitochondrial protein quality via improved respiration and induction of mitochondrial transcriptional patterns.

Impact of 4 weeks of western diet and aerobic exercise training on whole-body phenotype and skeletal muscle mitochondrial respiration in male and female mice.

High dietary fat intake induces significant whole-body and skeletal muscle adaptations in mice, including increased capacity for fat oxidation and mitochondrial biogenesis. The impact of a diet that is high in fat and simple sugars (i.e., western diet [WD]), particularly on regulation of skeletal muscle mitochondrial function, is less understood. The purpose of the current study was to determine physiologic adaptations in mitochondrial respiratory capacity in skeletal muscle during short-term consumption of WD, including if adaptive responses to WD-feeding are modified by concurrent exercise training or may be sex-specific. Male and female C57BL/6J mice were randomized to consume low-fat diet (LFD) or WD for 4 weeks, with some WD-fed mice also performing concurrent treadmill training (WD + Ex). Group sizes were n = 4-7. Whole-body metabolism was measured using in-cage assessment of food intake and energy expenditure, DXA body composition analysis and insulin tolerance testing. High-resolution respirometry of mitochondria isolated from quadriceps muscle was used to determine skeletal muscle mitochondrial respiratory function. Male mice fed WD gained mass (p < 0.001), due to increased fat mass (p < 0.001), and displayed greater respiratory capacity for both lipid and non-lipid substrates compared with LFD mice (p < 0.05). There was no effect of concurrent treadmill training on maximal respiration (WD + Ex vs. WD). Female mice had non-significant changes in body mass and composition as a function of the interventions, and no differences in skeletal muscle mitochondrial oxidative capacity. These findings indicate 4 weeks of WD feeding can increase skeletal muscle mitochondrial oxidative capacity among male mice; whereas WD, with or without exercise, had minimal impact on mass gain and skeletal muscle respiratory capacity among female mice. The translational relevance is that mitochondrial adaptation to increases in dietary fat intake that model WD may be related to differences in weight gain among male and female mice.

Oral combined contraceptives induce liver mitochondrial reactive oxygen species and whole-body metabolic adaptations in female mice.

Compared to age-matched men, pre-menopausal women show greater resilience against cardiovascular disease (CVD), hepatic steatosis, diabetes and obesity - findings that are widely attributed to oestrogen. However, meta-analysis data suggest that current use of oral combined contraceptives (OC) is a risk factor for myocardial infarction, and OC use further compounds with metabolic disease risk factors to increase CVD susceptibility. While mitochondrial function in tissues such as the liver and skeletal muscle is an emerging mechanism by which oestrogen may confer its protection, effects of OC use on mitochondria and metabolism in the context of disease risk remain unexplored. To answer this question, female C57Bl/6J mice were fed a high fat diet and treated with vehicle or OCs for 3, 12 or 20 weeks (n = 6 to 12 per group) at a dose and ratio that mimic the human condition of cycle cessation in the low oestrogen, high progesterone stage. Liver and skeletal muscle mitochondrial function (respiratory capacity, H2 O2 , coupling) was measured along with clinical outcomes of cardiometabolic disease such as obesity, glucose tolerance, hepatic steatosis and aortic atherosclerosis. The main findings indicate that regardless of treatment duration, OCs robustly increase hepatic mitochondrial H2 O2 levels, likely due to diminished antioxidant capacity, but have no impact on muscle mitochondrial H2 O2 . Furthermore, OC-treated mice had lower adiposity and hepatic triglyceride content compared to control mice despite reduced wheel running, spontaneous physical activity and total energy expenditure. Together, these studies describe tissue-specific effects of OC use on mitochondria as well as variable impacts on markers of metabolic disease susceptibility. KEY POINTS: Oestrogen loss in women increases risk for cardiometabolic diseases, a link that has been partially attributed to negative impacts on mitochondria and energy metabolism. To study the effect of oral combined contraceptives (OCs) on hepatic and skeletal muscle mitochondria and whole-body energy metabolism, we used an animal model of OCs which mimics the human condition of cessation of hormonal cycling in the low oestrogen, high progesterone state. OC-treated mice have increased hepatic mitochondrial oxidative stress and decreased physical activity and energy expenditure, despite displaying lower adiposity and liver fat at this time point. These pre-clinical data reveal tissue-specific effects of OCs that likely underlie the clinical findings of increased cardiometabolic disease in women who use OCs compared to non-users, when matched for obesity.

Rats with high aerobic capacity display enhanced transcriptional adaptability and upregulation of bile acid metabolism in response to an acute high-fat diet.

Rats selectively bred for the high intrinsic aerobic capacity runner (HCR) or low aerobic capacity runner (LCR) show pronounced differences in susceptibility for high-fat/high sucrose (HFHS) diet-induced hepatic steatosis and insulin resistance, replicating the protective effect of high aerobic capacity in humans. We have previously shown multiple systemic differences in energy and substrate metabolism that impacts steatosis between HCR and LCR rats. This study aimed to investigate hepatic-specific mechanisms of action via changes in gene transcription. Livers of HCR rats had a greater number of genes that significantly changed in response to 3-day HFHS compared with LCR rats (171 vs. 75 genes: >1.5-fold, p < 0.05). HCR and LCR rats displayed numerous baseline differences in gene expression while on a low-fat control diet (CON). A 3-day HFHS diet resulted in greater expression of genes involved in the conversion of excess acetyl-CoA to cholesterol and bile acid (BA) synthesis compared with the CON diet in HCR, but not LCR rats. These results were associated with higher fecal BA loss and lower serum BA concentrations in HCR rats. Exercise studies in rats and mice also revealed higher hepatic expression of cholesterol and BA synthesis genes. Overall, these results suggest that high aerobic capacity and exercise are associated with upregulated BA synthesis paired with greater fecal excretion of cholesterol and BA, an effect that may play a role in protection against hepatic steatosis in rodents.

Substrate-Specific Respiration of Isolated Skeletal Muscle Mitochondria after 1 h of Moderate Cycling in Sedentary Adults.

INTRODUCTION: Skeletal muscle mitochondria have dynamic shifts in oxidative metabolism to meet energy demands of aerobic exercise. Specific complexes oxidize lipid and nonlipid substrates. It is unclear if aerobic exercise stimulates intrinsic oxidative metabolism of mitochondria or varies between substrates. METHODS: We studied mitochondrial metabolism in sedentary male and female adults (n = 11F/4M) who were free of major medical conditions with mean ± SD age of 28 ± 7 yr, peak aerobic capacity of 2.0 ± 0.4 L·min-1, and body mass index of 22.2 ± 2 kg·m-2. Biopsies were collected from the vastus lateralis muscle on separate study days at rest or 15 min after exercise (1 h cycling at 65% peak aerobic capacity). Isolated mitochondria were analyzed using high-resolution respirometry of separate titration protocols for lipid (palmitoylcarnitine, F-linked) and nonlipid substrates (glutamate-malate, N-linked; succinate S-linked). Titration protocols distinguished between oxidative phosphorylation and leak respiration and included the measurement of reactive oxygen species emission (H2O2). Western blotting determined the protein abundance of electron transfer flavoprotein (ETF) subunits, including inhibitory methylation site on ETF-ß. RESULTS: Aerobic exercise induced modest increases in mitochondrial respiration because of increased coupled respiration across F-linked (+13%, P = 0.08), N(S)-linked (+14%, P = 0.09), and N-linked substrates (+17%, P = 0.08). Prior exercise did not change P:O ratio. Electron leak to H2O2 increased 6% increased after exercise (P = 0.06) for lipid substrates but not for nonlipid. The protein abundance of ETF-α or ETF-ß subunit or inhibitory methylation on ETF-ß was not different between rest and after exercise. CONCLUSION: In sedentary adults, the single bout of moderate-intensity cycling induced modest increases for intrinsic mitochondrial oxidative phosphorylation that was consistent across multiple substrates.

Short-Term High-Fat Feeding Does Not Alter Mitochondrial Lipid Respiratory Capacity but Triggers Mitophagy Response in Skeletal Muscle of Mice.

Lipid overload of the mitochondria is linked to the development of insulin resistance in skeletal muscle which may be a contributing factor to the progression of type 2 diabetes during obesity. The targeted degradation of mitochondria through autophagy, termed mitophagy, contributes to the mitochondrial adaptive response to changes in dietary fat. Our previous work demonstrates long-term (2-4 months) consumption of a high-fat diet increases mitochondrial lipid oxidation capacity but does not alter markers of mitophagy in mice. The purpose of this study was to investigate initial stages of mitochondrial respiratory adaptations to high-fat diet and the activation of mitophagy. C57BL/6J mice consumed either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 3 or 7 days. We measured skeletal muscle mitochondrial respiration and protein markers of mitophagy in a mitochondrial-enriched fraction of skeletal muscle. After 3 days of HFD, mice had lower lipid-supported oxidative phosphorylation alongside greater electron leak compared with the LFD group. After 7 days, there were no differences in mitochondrial respiration between diet groups. HFD mice had greater autophagosome formation potential (Beclin-1) and greater activation of mitochondrial autophagy receptors (Bnip3, p62) in isolated mitochondria, but no difference in downstream autophagosome (LC3II) or lysosome (Lamp1) abundance after both 3 and 7 days compared with the LFD groups. In cultured myotubes, palmitate treatment decreased mitochondrial membrane potential and hydrogen peroxide treatment increased accumulation of upstream mitophagy markers. We conclude that several days of high-fat feeding stimulated upstream activation of skeletal muscle mitophagy, potentially through lipid-induced oxidative stress, without downstream changes in respiration.

Skeletal Muscle ACSL Isoforms Relate to Measures of Fat Metabolism in Humans.

INTRODUCTION: Evidence from model systems implicates long-chain acyl-coenzyme A synthetase (ACSL) as key regulators of skeletal muscle fat oxidation and fat storage; however, such roles remain underexplored in humans. PURPOSE: We sought to determine the protein expression of ACSL isoforms in skeletal muscle at rest and in response to acute exercise and identify relationships between skeletal muscle ACSL and measures of fat metabolism in humans. METHODS: Sedentary adults (n = 14 [4 males and 10 females], body mass index = 22.2 ± 2.1 kg·m-2, V˙O2max = 32.2 ± 4.5 mL·kg-1⋅min-1) completed two study visits. Trials were identical other than completing 1 h of cycling exercise (65% V˙O2max) or remaining sedentary. Vastus lateralis biopsies were obtained 15-min postexercise (or rest) and 2-h postexercise to determine ACSL protein abundance. Whole-body fat oxidation was assessed at rest and during exercise using indirect calorimetry. Skeletal muscle triacylglycerol (TAG) was measured via lipidomic analysis. RESULTS: We detected protein expression for four of the five known ACSL isoforms in human skeletal muscle. ACSL protein abundances were largely unaltered in the hours after exercise aside from a transient increase in ACSL5 15-min postexercise (P = 0.01 vs rest). Skeletal muscle ACSL1 protein abundance tended to be positively related with whole-body fat oxidation during exercise (P = 0.07, r = 0.53), when skeletal muscle accounts for the majority of energy expenditure. No such relationship between ACSL1 and fat oxidation was observed at rest. Skeletal muscle ACSL6 protein abundance was positively associated with muscle TAG content at rest (P = 0.05, r = 0.57). CONCLUSION: Most ACSL protein isoforms can be detected in human skeletal muscle, with minimal changes in abundance after acute exercise. Our findings agree with those from model systems implicating ACSL1 and ACSL6 as possible determinants of fat oxidation and fat storage within skeletal muscle.

Mitochondrial adaptations to exercise do not require Bcl2-mediated autophagy but occur with BNIP3/Parkin activation.

Understanding the mechanisms regulating mitochondrial respiratory function and adaptations to metabolic challenges, such as exercise and high dietary fat, is necessary to promote skeletal muscle health and attenuate metabolic disease. Autophagy is a constitutively active degradation pathway that promotes mitochondrial turnover and transiently increases postexercise. Recent evidence indicates Bcl2 mediates exercise-induced autophagy and skeletal muscle adaptions to training during high-fat diet. We determined if improvements in mitochondrial respiration due to exercise training required Bcl2-mediated autophagy using a transgenic mouse model of impaired inducible autophagy (Bcl2AAA ). Mitochondrial adaptations to a treadmill exercise training protocol, in either low-fat or high-fat diet fed mice, did not require Bcl2-mediated autophagy activation. Instead, training increased protein synthesis rates and basal autophagy in the Bcl2AAA mice, while acute exercise activated BNIP3 and Parkin autophagy. High-fat diet stimulated lipid-specific mitochondrial adaptations. These data demonstrate increases in basal mitochondrial turnover, not transient activation with exercise, mediate adaptations to exercise and high-fat diet.

Diet and Exercise Training Influence Skeletal Muscle Long-Chain acyl-CoA Synthetases.

INTRODUCTION: Long-chain acyl-CoA synthetases (ACSL) are implicated as regulators of oxidation and storage of fatty acids within skeletal muscle; however, to what extent diet and exercise alter skeletal muscle ACSL remains poorly understood. PURPOSE: This study aimed to determine the effects of diet and exercise training on skeletal muscle ACSL and to examine relationships between ACSL1 and ACSL6 and fat oxidation and fat storage, respectively. METHODS: Male C57BL/6J mice consumed a 60% high-fat diet (HFD) for 12 wk to induce obesity compared with low-fat diet (LFD). At week 4, mice began aerobic exercise (EX-Tr) or remained sedentary (SED) for 8 wk. At week 12, the protein abundance of five known ACSL isoforms and mRNA expression for ACSL1 and ACSL6 were measured in gastrocnemius muscle, as was skeletal muscle lipid content. Fat oxidation was measured using metabolic cage indirect calorimetry at week 10. RESULTS: Of the five known ACSL isoforms, four were detected at the protein level. HFD resulted in greater, yet nonsignificant, ACSL1 protein abundance (+18%, P = 0.13 vs LFD), greater ACSL6 (+107%, P < 0.01 vs LFD), and no difference in ACSL4 or ACSL5. Exercise training resulted in greater ACSL6 protein abundance in LFD mice (P = 0.05 LFD EX-Tr vs SED), whereas ACSL4 was lower after exercise training compared with sedentary, regardless of diet. Under fasted conditions, skeletal muscle ACSL1 protein abundance was not related to measures of whole-body fat oxidation. Conversely, skeletal muscle ACSL6 protein abundance was positively correlated with intramyocellular lipid content (P < 0.01, r = 0.22). CONCLUSION: We present evidence that ACSL isoforms 1, 4, and 6 may undergo regulation by HFD and/or exercise training. We further conclude that increased skeletal muscle ACSL6 may facilitate increased intramyocellular fat storage during HFD-induced obesity.

Treatment with Nitrate, but Not Nitrite, Lowers the Oxygen Cost of Exercise and Decreases Glycolytic Intermediates While Increasing Fatty Acid Metabolites in Exercised Zebrafish.

BACKGROUND: Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood. OBJECTIVES: We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production. METHODS: We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise. RESULTS: Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., ß-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts. CONCLUSIONS: Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.

Robust intrinsic differences in mitochondrial respiration and H2O2 emission between L6 and C2C12 cells.

Rat L6 and mouse C2C12 cell lines are commonly used to investigate myocellular metabolism. Mitochondrial characteristics of these cell lines remain poorly understood despite mitochondria being implicated in the development of various metabolic diseases. To address this need, we performed high-resolution respirometry to determine rates of oxygen consumption and H2O2 emission in suspended myoblasts during multiple substrate-uncoupler-inhibitor titration protocols. The capacity for oxidative phosphorylation supported by glutamate and malate, with and without succinate, or supported by palmitoyl-l-carnitine was lower in L6 compared with C2C12 myoblasts (all P < 0.01 for L6 vs. C2C12). Conversely, H2O2 emission during oxidative phosphorylation was greater in L6 than C2C12 myoblasts (P < 0.01 for L6 vs. C2C12). Induction of noncoupled respiration revealed a significantly greater electron transfer capacity in C2C12 compared with L6 myoblasts, regardless of the substrate(s) provided. Mitochondrial metabolism was also investigated in differentiated L6 and C2C12 myotubes. Basal rates of oxygen consumption were not different between intact, adherent L6, and C2C12 myotubes; however, noncoupled respiration was significantly lower in L6 compared with C2C12 myotubes (P = 0.01). In summary, L6 myoblasts had lower respiration rates than C2C12 myoblasts, including lesser capacity for fatty acid oxidation and greater electron leak toward H2O2. L6 cells also retain a lower capacity for electron transfer compared with C2C12 following differentiation to form fused myotubes. Intrinsic differences in mitochondrial metabolism between these cell lines should be considered when modeling and investigating myocellular metabolism.

Insulin-stimulated Rac1-GTP binding is not impaired by palmitate treatment in L6 myotubes.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is required for normal insulin-stimulated glucose transport in skeletal muscle and evidence indicates Rac1 may be negatively regulated by lipids. We investigated if insulin-stimulated activation of Rac1 (i.e., Rac1-GTP binding) is impaired by accumulation of diacylglycerols (DAG) and ceramides in cultured muscle cells. Treating L6 myotubes with 100 nmol/L insulin resulted in increased Rac1-GTP binding that was rapid (occurring within 2 min), relatively modest (+38 ± 19% vs. basal, P < 0.001), and short-lived, returning to near-basal levels within 15 min of continuous treatment. Incubating L6 myotubes overnight in 500 µmol/L palmitate increased the accumulation of DAG and ceramides (P < 0.05 vs. no fatty acid control). Despite significant accumulation of lipids, insulin-stimulated Rac1-GTP binding was not impaired during palmitate treatment (P = 0.39 vs. no fatty acid control). Nevertheless, phosphorylation of Rac1 effector protein p21-activated kinase (PAK) was attenuated in response to palmitate treatment (P = 0.02 vs. no fatty acid control). Palmitate treatment also increased inhibitory phosphorylation of insulin receptor substrate-1 and attenuated insulin-stimulated phosphorylation of Akt at both Thr308 and Ser473 (all P < 0.05 vs. no fatty acid control). Such signaling impairments resulted in near complete inhibition of insulin-stimulated translocation of glucose transporter protein 4 (GLUT4; P = 0.10 vs. basal during palmitate treatment). In summary, our finding suggests that Rac1 may not undergo negative regulation by DAG or ceramides. We instead provide evidence that attenuated PAK phosphorylation and impaired GLUT4 translocation during palmitate-induced insulin resistance can occur independent of defects in insulin-stimulated Rac1-GTP binding.

Skeletal muscle autophagy remains responsive to hyperinsulinemia and hyperglycemia at higher plasma insulin concentrations in insulin-resistant mice.

Skeletal muscle autophagy is suppressed by insulin, but it is not clear if such suppression is altered with insulin resistance. We investigated if the inhibitory action of insulin on autophagy remains intact despite insulin resistance to glucose metabolism. C57BL/6J mice consumed either a low-fat (10% fat) diet as control or high-fat (60% fat) diet for 12 weeks to induce insulin resistance. Following a 5-hour fast, mice underwent either hyperinsulinemic-euglycemic, hyperinsulinemic-hyperglycemic, or saline infusion to test the effect of insulin on autophagy markers in the quadriceps muscle (n = 8-10 per diet and clamp condition). Mice were anesthetized by sodium pentobarbital for tissue collection after 2 h of infusion. Despite the high-fat group having lower insulin-stimulated glucose uptake, both low-fat and high-fat groups had similar autophagosome abundance during hyperinsulinemic conditions. The lipidation of microtubule-associated proteins 1A/1B light chain 3B (LC3II/LC3I) was decreased in hyperinsulinemia versus saline control (P < 0.01) in low-fat (-54%) and high-fat groups (-47%), demonstrating similar suppression of autophagy between diet groups. Mitochondrial-associated LC3II was greater in the high-fat compared to the low-fat group (P = 0.045) across clamp conditions, suggesting a greater localization of autophagosomes with mitochondria. L6 myotubes were treated with insulin and rapamycin to determine the role of mechanistic target of rapamycin complex-1 (mTORC1) in insulin-mediated suppression of autophagy. Inhibition of mTORC1 blunted the decline of LC3II/LC3I with insulin by 40%, suggesting mTORC1 partially mediates the insulin action to suppress autophagy. Collectively, autophagy remained responsive to the suppressive effects of insulin in otherwise insulin-resistant and obese mice.

Remodeling of skeletal muscle mitochondrial proteome with high-fat diet involves greater changes to ß-oxidation than electron transfer proteins in mice.

Excess fat intake can increase lipid oxidation and expression of mitochondrial proteins, indicating remodeling of the mitochondrial proteome. Yet intermediates of lipid oxidation also accumulate, indicating a relative insufficiency to completely oxidize lipids. We investigated remodeling of the mitochondrial proteome to determine mechanisms of changes in lipid oxidation following high-fat feeding. C57BL/6J mice consumed a high-fat diet (HFD, 60% fat from lard) or a low-fat diet (LFD, 10% fat) for 12 wk. Mice were fasted for 4 h and then anesthetized by pentobarbital sodium overdose for tissue collection. A mitochondrial-enriched fraction was prepared from gastrocnemius muscles and underwent proteomic analysis by high-resolution mass spectrometry. Mitochondrial respiratory efficiency was measured as the ratio of ATP production to O2 consumption. Intramuscular acylcarnitines were measured by liquid chromatography-mass spectrometry. A total of 658 mitochondrial proteins were identified: 40 had higher abundance and 14 had lower abundance in mice consuming the HFD than in mice consuming the LFD. Individual proteins that changed with the HFD were primarily related to ß-oxidation; there were fewer changes to the electron transfer system. Gene set enrichment analysis indicated that the HFD increased pathways of lipid metabolism and ß-oxidation. Intramuscular concentrations of select acylcarnitines (C18:0) were greater in the HFD mice and reflected dietary lipid composition. Mitochondrial respiratory ATP production-to-O2 consumption ratio for lipids was not different between LFD and HFD mice. After the 60% fat diet, remodeling of the mitochondrial proteome revealed upregulation of proteins regulating lipid oxidation that was not evident for all mitochondrial pathways. The accumulation of lipid metabolites with obesity may occur without intrinsic dysfunction to mitochondrial lipid oxidation.

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H2O2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.