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 H 2 O 2 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.

mitochondrial protein synthesis helps maintain the mitochondrial proteome of skeletal muscle and is regulated in part by insulin (2, 38). The development of insulin resistance with obesity is associated with decreased skeletal muscle insulin signaling and may have negative impacts on mitochondrial protein synthesis. It has been reported that compared with lean controls, obese adults had lower basal mitochondrial protein synthesis and a blunted response to insulin infusion (14). Furthermore, adults with type 2 diabetes had lower content of gene transcripts for mitochondrial proteins compared with lean adults (30). Lower protein synthesis may contribute to the decreased mitochondrial protein abundance and decreased respiratory capacity that has been reported in adults with obesity or type 2 diabetes (7, 40).

Other findings have challenged the notion of mitochondrial declines with insulin resistance. Compared with low-fat diet controls, rats with diet-induced obesity and insulin resistance had greater mitochondrial protein abundance by Western blot and palmitate oxidation capacity (16). Mice consuming a high-fat diet also had greater mitochondrial density by electron microscopy and greater respiration of isolated mitochondria using substrates for complexes I or II despite having lower insulin sensitivity than mice consuming a low-fat diet (23). Such gains may be protective against developing insulin resistance with high-fat diet (31). Accordingly, mitochondrial protein synthesis was reported to be increased in obese humans (15) and rats (10) compared with lean controls. It is therefore not clear whether mitochondrial protein synthesis is altered following changes to insulin sensitivity and contributes to changes in mitochondrial protein abundance or respiratory capacity.

Longer-duration labeling approaches may allow insight into the regulation of protein synthesis with insulin resistance that is not captured over acute time frames. Use of deuterium oxide (D 2 O) labeling can provide a measurement of protein synthesis over days to several weeks (26, 27, 39). The synthesis rate represents an average over periods of feeding, fasting, and physical activity, which all influence mitochondrial protein synthesis rates (6, 48). D 2 O is provided in drinking water to enrich the body water pool with 2H atoms. Amino acids, including alanine, become endogenously labeled with 2H and are subsequently incorporated into proteins (9). Previous work using D 2 O labeling for 24 h demonstrated that obese rats had similar rates of mitochondrial protein synthesis at rest and a blunted response to exercise compared with lean controls (32, 33), suggesting that mitochondrial protein synthesis may develop resistance to anabolic stimuli. Understanding cumulative rates of protein synthesis over longer periods provides insight into the regulation of mitochondrial proteins following changes to insulin sensitivity.

Our purpose was to investigate whether mitochondrial protein synthesis, using D 2 O labeling for 14 days, is impaired with insulin resistance and then subsequently restored with an insulin-sensitizing treatment, pioglitazone (PIO). We hypothesized that compared with lean controls, mice with diet-induced obesity would have decreased rates of long-term skeletal muscle protein synthesis, including mitochondrial protein synthesis, and decreased mitochondrial respiratory capacity. The secondary hypothesis was that restoring insulin sensitivity with PIO would increase protein synthesis and mitochondrial respiratory capacity. Contrary to our hypothesis, mitochondrial protein synthesis and energetics were not impaired following induction of insulin resistance. Instead, a high-fat diet appeared to have a stronger effect on mitochondrial adaptations than did changes to insulin sensitivity. The increased mitochondrial protein synthesis provides a possible mechanism for increased mitochondrial lipid oxidation as an adaptation to a high-fat diet.

METHODS Study design. The study was approved by the Animal Care and Use Committees at the Mayo Clinic (no. 385-14) and Oregon State University (no. 4788). Eight- to ten-week-old male C57BL/6J mice (Jackson Laboratories) were housed 3–5 per cage in 12:12-h light-dark cycle at 22°C with free access to food and water. Mice consumed either a low-fat diet (LFD) or a high-fat diet (HFD) for 4 wk, and then diets were enriched in PIO or not (CON) for 8 additional weeks. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed at baseline (week 0), following induction of insulin resistance (week 4), and then at week 10. Metabolic assessment by continuous laboratory animal monitoring system (CLAMS; Columbus Instruments, Columbus, OH) was performed at week 10 along with body composition by EchoMRI. D 2 O labeling was performed between weeks 10 and 12 for skeletal muscle protein synthesis. At week 12, mice were fasted for 4 h and then anesthetized by sodium pentobarbital overdose for tissue collection. Diet composition. Diets were purchased as pellets and matched for sucrose (LFD, D12450J; HFD, D12492; Research Diets, New Brunswick, NJ). The percent kilocalories from total fat/carbohydrates/protein for the LFD were 10/70/20 and for the HFD were 60/20/20. PIO (TCI America, Portland, OR) was mixed into diets at 53 mg PIO/4,057 kcal of diet. PIO was provided in the diet to avoid stress on the mice associated with a regular intraperitoneal injection. The dose provided ~2 mg·kg body wt−1·day−1 for HFD and 6 mg·kg body wt−1·day−1 for LFD, which was comparable to an intraperitoneal dose of 10 mg·kg body wt−1·day−1 that was effective for improving insulin sensitivity in mice consuming a HFD (43). D 2 O labeling. On week 10, all mice had an intraperitoneal injection of D 2 O (~99%; Cambridge Isotope Laboratories, Tewksbury, MA) at 30 μl/g body wt to raise body water enrichment to ~5% (assuming 60% water weight) followed by continued consumption of D 2 O enriched at 8% in drinking water (11). Plasma samples collected at euthanasia were analyzed for D 2 O enrichment to calculate precursor enrichment. Body water enrichment was determined from plasma using an Agilent 7890A gas chromatograph coupled to a 5975C mass spectrometer (39). Quadriceps muscle was fractionated by differential centrifugation to subcellular fractions enriched with myofibrillar, sarcoplasmic, and mitochondrial proteins (11, 26). Protein fractions were hydrolyzed to amino acids, derivatized, and analyzed on an Agilent 7890B gas chromatograph coupled to a 5977A mass spectrometer. The newly synthesized fraction (f) of muscle proteins was calculated from the enrichment of labeled alanine bound to muscle proteins divided by the true precursor enrichment (p). The precursor enrichment was calculated from D 2 O enrichment in plasma, which was adjusted using mass isotopomer distribution analysis calculations (9). The fraction then was divided by time and multiplied by 100 to obtain the fractional synthesis rate (FSR). Glucose and insulin tolerance testing. GTT and ITT were performed in individual mice in fresh cages. Blood samples were collected into a microhematocrit capillary tube from a tail nick. Mice were not restrained during testing or blood collection. For GTT, food was removed at 0700 to induce a 6-h fast. A blood sample was collected for fasting plasma insulin (Alpco, Salem, NH), and then glucose was provided as an intraperitoneal injection of 2 g/kg body wt of 20% sucrose. Blood glucose concentrations were measured by a handheld glucometer at 0, 5, 15, 30, 60, and 120 min. For ITT, food was removed at 0700 to induce a 4-h fast. Insulin (Humulin R; Eli Lilly, Indianapolis IN) was injected intraperitoneally at 0.5 IU/kg body wt. Blood glucose concentrations were measured at 0, 15, 30, 60, and 90 min. We compared PIO and CON treatment within each diet group to avoid comparing mice with large differences in body weights and therefore different injection volumes (25). High-resolution respirometry. Mitochondria were isolated from quadriceps muscle using differential centrifugation (24). High-resolution respirometry was performed using two Oxygraph-2k units (Oroboros Instruments, Innsbruck, Austria) with MiR05 respiration buffer (0.5 mM EGTA, 3 mM MgCl 2 -6H 2 O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose, and 1 g/l bovine serum albumin). One Oxygraph measured oxygen consumption and H 2 O 2 production using 10 μM Amplex red, 5 U/ml superoxide dismutase, and 1 U/ml horseradish peroxidase and calibrated using injections of H 2 O 2 . The titration sequence was 50 μl of mitochondrial suspension (Mitos), 10 mM glutamate + 2 mM malate, 5 mM ADP (OxPHOS GM , where OxPHOS stands for oxidative phosphorylation), 10 mM succinate (OxPHOS GMS ), 0.5 μM rotenone (OxPHOS GMS+Rot ), 2 μg/ml oligomycin (Leak GMS ), sequential additions of 0.05 mM FCCP to plateau (ETS GMS+Rot , where ETS stands for electron transport system), and 2.5 mM antimycin for residual oxygen consumption (ROX GMS ). A second Oxygraph measured oxygen consumption during fatty acid oxidation (FAO) using palmitoyl-l-carnitine with addition of 50 μl of mitochondrial suspension (Mitos), 0.005 mM palmitoyl-l-carnitine + 2 mM malate, 5 mM ADP (OxPHOS FAO ), 2 μg/ml oligomycin (Leak FAO ), sequential additions of 0.05 mM FCCP to plateau (ETS FAO ), and 2.5 mM antimycin (ROX FAO ) as described previously (23). Membrane integrity was verified by adding 10 μM cytochrome c and observing little to no change in OxPHOS FAO [average change −2.7 ± 2.9% (SD)]. Cytochrome c is not compatible with Amplex red and was not added to the glutamate-malate-succinate titrations. Protein concentration of the mitochondrial preparation was measured by bicinchoninic assay (Bio-Rad, Hercules, CA). Western blotting. Quadriceps muscle (~30 mg) was homogenized 1:10 wt/vol in buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 , and 1× Sigma Protease Inhibitor Cocktail no. P8340) and then incubated at 4°C for 20 min and centrifuged at 10,000 g for 10 min at 4°C. Approximately 30 μg of protein from the supernatant were separated on 10 or 12% bis-Tris gels and then transferred to nitrocellulose membranes. Equal loading was verified using Ponceau staining of membranes and tubulin as a loading control. Gels included a negative and positive control cell lysate plus repeated internal control. The average intensities for all three controls were used to normalize intensities between gels. Membranes were blocked in 5% bovine serum albumin in Tris-buffered saline with Tween (TBST) and then incubated overnight at 4°C in primary antibodies (1:1,000). Membranes were washed 3 × 10 min in TBST and then incubated for 1 h at room temperature in secondary antibody diluted in blocking buffer. Images were generated using infrared imaging (Odyssey; Li-Cor, Lincoln NE). Primary antibodies were microtubule-associated light chain 3 (LC3; no. 12741; Cell Signaling Technology), p62 (no. 7695; Cell Signaling Technology), tubulin (no. 926-42213; Li-Cor), pSer473 Akt (no. 9271; Cell Signaling Technology), total Akt (no. 2920; Cell Signaling Technology), OxPHOS cocktail (no. 110413; Abcam), hydroxyacyl-CoA dehydrogenase (HADH; PA5-28203; Thermo Fisher Scientific), and voltage-dependent anion channel (VDAC; no. 9412; Cell Signaling Technology). Secondary antibodies were anti-rabbit-700 (1:10,000; no. 926-68071; Li-Cor) and anti-mouse-800 (1:50,000; no. 926-32212; Li-Cor). Oxidative damage was measured using a kit (Oxyblot; Abcam, Cambridge, MA). Statistics. Data generated at death were analyzed with a two-way analysis of variance model (ANOVA) by diet and pioglitazone treatment. Post hoc comparisons were made using Tukey’s honestly significant difference. Energy expenditure was analyzed using analysis of covariance (ANCOVA) of lean body mass and pioglitazone treatment (44). A lack of interaction for lean body mass × pioglitazone was verified before ANCOVA. The results of ITT and GTT between PIO and CON were compared by unpaired t-test within diet groups. Statistical significance was set at P = 0.05. Statistics were performed using JMP Pro version 12.0 (SAS Institute, Cary, NC). Graphs were generated using Prism version 6 (GraphPad Software, La Jolla, CA). Data are presented as means ± SD.

DISCUSSION The major conclusion of our study is that mitochondrial protein synthesis is increased rather than impaired with HFD-induced insulin resistance. This conclusion is based on greater long-term rates of mitochondrial protein synthesis in mice consuming a HFD compared with LFD, regardless of restoration of insulin sensitivity using PIO treatment. The results do not support that insulin resistance leads to declines in mitochondrial respiratory capacity as there were no measured impairments to mitochondrial respiration or H 2 O 2 emission following induction of insulin resistance and improving insulin sensitivity with PIO did not influence these outcomes. Instead, mice consuming a HFD had higher mitochondrial lipid oxidation capacity and whole body lipid oxidation. Our results provide a mechanism by which mitochondrial lipid oxidation is increased with HFD and support the notion that changes in mitochondrial respiration are a primary response to HFD and not a direct result of insulin resistance. Insulin contributes to mitochondrial protein turnover through stimulation of protein synthesis when amino acid concentrations are maintained (38) and suppression of degradation (37); therefore, it could be expected that the development of insulin resistance may decrease mitochondrial protein turnover. In support, declines in whole body protein turnover have been reported in patients with type 2 diabetes compared with lean or obese controls (34). Yet, studies have indicated that protein synthesis was not impaired with insulin resistance when measured at the whole body or regional level over several hours (4, 41, 46). Furthermore, mitochondrial protein synthesis was higher in obese adults than in lean when measured over several hours (15). Our results using a long-term labeling approach indicate that protein synthesis rates of either mitochondrial or other muscle fractions were not decreased as a result of insulin resistance. A potential mechanism for increased mitochondrial protein synthesis with a HFD may be a response to high circulating concentrations of insulin and fatty acids. Hyperinsulinemia stimulates mitochondrial protein synthesis when essential amino acids are maintained by exogenous infusion (38). Lipids can signal through the peroxisome proliferator-activated receptor (PPAR) family of transcription factors, which regulate the transcription of many genes involved with mitochondrial metabolism, particularly for fatty acid oxidation (12). Thus increased mitochondrial protein synthesis with a HFD may result from a combination of translational activation by hyperinsulinemia and transcriptional activation in response to high lipids. Previous work using D 2 O labeling for 24 h demonstrated similar mitochondrial protein synthesis rates between obese Zucker rats and controls at rest (32). Compared with lean rats, obese rats did not stimulate mitochondrial protein synthesis as measured by the average synthesis rate during the 24 h following resistance exercise, indicating that mitochondrial protein synthesis may develop resistance to an anabolic stimulus (33). Our approach measured protein synthesis over 14 days as the average across feeding, fasting, and physical activity periods. Any potential resistance to acute anabolic stimuli did not develop into differences over the longer-term labeling period. The increase in mitochondrial protein synthesis with a HFD was matched by increased lipid oxidation capacity, regardless of improving insulin sensitivity with PIO. The mouse model of diet-induced obesity with varying degrees of insulin sensitivity indicates that changes in mitochondrial respiration were in response to diet and not dependent on insulin sensitivity. Others have also demonstrated gains in mitochondrial respiratory capacity in models of insulin resistance, as rodents on high-fat diets have increased markers of mitochondrial content (16), respiration (23), and capacity for fatty acid oxidation compared with low-fat diet controls (45). Thus higher mitochondrial protein synthesis may contribute to increased mitochondrial protein abundance observed with a HFD (16). Our results indicated that insulin resistance was not accompanied by impairments to mitochondrial respiratory capacity and, instead, demonstrated increased capacity for lipid oxidation in the mitochondria and consistently greater whole body fat oxidation during feeding and fasting periods. There is a strong precedent for decreased mitochondrial content and respiration with obesity, insulin resistance, or type 2 diabetes (19, 29, 30, 36, 40). Lower mitochondrial content and respiratory capacity are suggested to contribute to an accumulation of excess fuels, particularly as intramuscular lipids (35), and impaired insulin signaling. Yet data generated from adults across a span of ages and body compositions showed no relationship between mitochondrial respiratory capacity and insulin sensitivity measured by insulin clamp (22). Instead of decreased oxidative capacity as a contributor to insulin resistance, evidence indicates that increased fat oxidation with a HFD is associated with the accumulation of lipid metabolites that impair insulin signaling (21), although the mechanism of how lipid metabolites interfere with insulin signaling remains to be fully elucidated. Our data are consistent with a HFD resulting in greater flux through lipid oxidation pathways, regardless of insulin-sensitizer treatment, when measured across the whole body or isolated mitochondria. Mitochondrial H 2 O 2 emissions with a HFD are implicated in the development of insulin resistance (1). Higher rates of mitochondrial H 2 O 2 emissions during ADP-limited respiration have been detected in obese adults and were restored to a lean phenotype following aerobic training (20). We measured H 2 O 2 emission simultaneously during ADP-saturating conditions and did not detect any differences with insulin resistance or treatment with PIO. We cannot exclude the possibility that maximal H 2 O 2 emission is different during ADP-limited respiration. Autophagy is a primary pathway to degrade mitochondria and was increased with a HFD when measured in whole cell lysates, regardless of PIO treatment. Such activation may explain why the abundance of specific mitochondrial proteins did not change despite the increased mitochondrial protein synthesis with the HFD. The degradation of individual proteins may vary given that specific mitochondrial proteins were increased (e.g., HADH) but not others (e.g., oxidative phosphorylation subunits). Previous work has demonstrated differences in degradation products of individual mitochondrial proteins during insulin deprivation (37). Insulin signaling through Akt has a suppressive effect on autophagy, yet Akt phosphorylation in the fasted state was increased in HFD mice and may be attributed to higher circulating insulin than in LFD mice. An important consideration is that autophagy and Akt were assessed in a fasted state, whereas defects may occur in response to greater nutrient availability. For example, other studies have reported that insulin resistance was associated with blunted Akt phosphorylation during a hyperinsulinemic clamp (42). Autophagy promotes cellular function in part by degrading proteins that have accumulated damage as irreversible posttranslational modification, such as oxidative modifications (50). We did not detect any differences in global oxidative damage to proteins between diet or PIO treatment groups but cannot exclude that individual proteins may accumulate oxidative damage (18). Our present results of increased autophagy are consistent with a previous study showing increased skeletal muscle protein degradation in adults with insulin resistance (3). Our approach induced insulin resistance with a HFD and then treated mice with PIO to model disease development and treatment. Weighed-food intake indicated that mice on the HFD received a lower dose per body weight than those on the LFD, yet the dose of PIO achieved the goal of improving insulin sensitivity during the HFD. PIO can disrupt complex I assembly and inhibit mitochondrial respiration (13), and such inhibition appears to be concentration dependent (8). Our dose of PIO did not appear to inhibit respiration with GMS or palmitoyl-l-carnitine. All mitochondrial measurements occurred after the induction or treatment of insulin resistance. We cannot determine whether changes in mitochondria preceded the development of insulin resistance or may result from longer durations of insulin resistance. Our results provide a mechanism of increased mitochondrial protein synthesis as a contributor to increased lipid respiration with a HFD. Extended exposure to increased flux through lipid oxidation pathways may contribute to insulin resistance (21). In conclusion, long-term rates of mitochondrial protein synthesis and respiratory capacity were not impaired with HFD-induced insulin resistance. Instead, mitochondrial protein synthesis was increased and may account for increased mitochondrial lipid oxidation capacity in mice consuming a HFD, regardless of differences in insulin sensitivity with PIO treatment. Collectively, our results indicate that changes to mitochondria with a HFD may be due to nutrient availability and not intrinsic defects that contribute to insulin resistance.

GRANTS Funding was provided by National Institute of Diabetes and Digestive and Kidney Diseases Grant 103829 to M. M. Robinson.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS M.M.R. conceived and designed research; M.M.R. performed experiments; S.A.N., B.F.M., K.L.H., S.E.E., H.D.S., and M.M.R. analyzed data; S.A.N., B.F.M., K.L.H., S.E.E., H.D.S., and M.M.R. interpreted results of experiments; M.M.R. prepared figures; M.M.R. drafted manuscript; S.A.N., B.F.M., K.L.H., S.E.E., H.D.S., and M.M.R. edited and revised manuscript; S.A.N., B.F.M., K.L.H., S.E.E., H.D.S., and M.M.R. approved final version of manuscript.