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Abstract Context Humans with obesity and insulin resistance exhibit lipid accumulation in skeletal muscle, but the underlying biological mechanisms responsible for the accumulation of lipid in the muscle of these individuals remain unknown. Objective We investigated how plasma insulin modulates the extraction of circulating triglycerides (TGs) and nonesterified fatty acids (NEFAs) from ingested and endogenous origin in the muscle of lean, insulin-sensitive humans (Lean-IS) and contrasted these responses to those in humans with obesity and insulin resistance (Obese-IR). Methods The studies were performed in a postprandial state associated with steady-state plasma TG concentrations. The arterio-venous blood sampling technique was employed to determine the extraction of circulating lipids across the forearm muscle before and after insulin infusion. We distinguished the kinetics of TGs and NEFAs from ingested origin from those from endogenous origin across muscle by incorporating stable isotope-labeled triolein in the ingested fat. Results Insulin infusion rapidly suppressed the extraction of plasma TGs from endogenous but not ingested origin in the muscle of the Lean-IS, but this response was absent in the muscle of the Obese-IR. Furthermore, in the muscle of the Lean-IS, insulin infusion decreased the extraction of circulating NEFAs from both ingested and endogenous origin; however, this response was absent for NEFAs from ingested origin in the muscle of the Obese-IR subjects. Conclusion Partitioning of circulating lipids away from the skeletal muscle when plasma insulin increases during the postprandial period is impaired in humans with obesity and insulin resistance.

We recently discovered persistent downregulation of the H + ‐ATP synthase catalytic subunit (β‐F1‐ATPase) in skeletal muscle of obese individuals, which was linked to comorbid hyperlipidemia. β‐F1‐ATPase expression is primarily regulated post‐transcription, i.e. miRNAs, but the precise mechanism impeding expression are unclear. We tested the hypothesis that hyperlipidemia reduces β‐F1‐ATPase translational efficiency and is mediated by miR‐127‐5p, a miRNA known to control β‐F1‐ATPase translation. Biopsies isolated from the vastus lateralis of lean (BMI蠄25; n=7) and obese (BMI蠅30; n=6) subjects were evaluated for mRNA/miRNA expression via qRT‐PCR, protein by Western blot, and translation efficiency was calculated as the mRNA/protein ratio. Our results confirmed β‐F1‐ATPase translation was lower in obese individuals (P<0.01; 0.25±0.05%) compared to healthy weight controls (0.59±0.09%), and correlated with miR‐127‐5p expression (P<0.01; r=‐0.67). Bisulfite sequencing analysis of the miR‐127‐5p promoter revealed two hypomethylated sites in obese individuals, which may sustain an upregulation. Moreover, we found miR‐127‐5p present in muscle‐derived blood exosomes suggesting putative involvement in intracellular cross‐talk. In primary myotubes from a lean volunteer, prolonged fatty acid exposure increased miR‐127‐5p expression. Free Fatty Acid (mEq/L) Unsaturated/ Saturated mRNA (2 ‐ DD Ct ) Protein (AU) Translation (%) CTRL 0.347 ‐ 1.70±0.113 0.216±0.062 0.130±0.040 Lo Fat 0.185 0.55 1.60±0.094 0.202±0.038 0.123±0.008 Hi Fat 0.251 2.29 1.35±0.133* 0.095±0.006* 0.073±0.010 Hi Fat 0.269 0.54 1.66±0.145 0.093±0.003* 0.050±0.004* * P <0.05; One‐way ANOVA, Tukey9s posttest Inhibiting miR‐127‐5p with locked nucleic acids dose‐dependently improved β‐F1‐ATPase translation. Inhibitor (nM) mRNA (2 ‐ DD Ct ) Protein (AU) Translation (%) 0 1.84±0.314 0.22±0.062 0.647±0.120 10 1.93±0.387 1.24±0.135* 0.500±0.148 50 2.00±0.498 1.24±0.242** 1.483±0.233* 100 1.76±0.207 1.54±0.086* 0.892±0.096 * P <0.05; ** P <0.01; *** P <0.001; 1‐way ANOVA, Tukey9s posttest Our results provide evidence implicating hyperlipidemia‐induced miR‐127‐5p expression in suppressing β‐F1‐ATPase translation in human obesity. Funding from NIH/NIDDK grant DK094062 to CSK

Whether obesity influences content and functional capacity of skeletal muscle mitochondria (MITO) remains unresolved. In this study, biopsies of vastus lateralis from lean and obese human subjects were used to isolate subsarcolemmal (SS) and intermyofibrillar (IMF) MITO. IMF MITO were liberated using a protease. The SS and IMF were pelleted separately, using differential centrifugation. Skeletal muscle from obese subjects gave higher MITO protein yield, per g wet muscle vs. skeletal muscle from lean subjects in the SS but not in the IMF. There was a trend for higher isolated yields of citrate synthase (CS) activity, a marker for MITO content, in the muscle of obese subjects vs. lean subjects in SS, but not in IMF MITO. O 2 consumption ( J O ) and ATP production rates measured with NAD‐linked substrates (malate+pyruvate+glutamate), were not different in obese vs. lean in either SS or IMF MITO expressed either per mg MITO protein or per unit CS activity. The similarities in MITO function observed in SS and IMF from both lean and obese human muscle, suggests comparable functional capacity per unit MITO, regardless of the location within the myocyte, or whether the MITO reside in skeletal muscle from lean or obese subjects. However, higher total yield of both protein and CS activities in the SS MITO from the muscle of obese subjects, point to the possibility for greater content of SS MITO in muscle of obese individuals. This conclusion must be qualified; these results may be due to a greater fractional yield of MITO during the isolation procedure of skeletal muscle from obese subjects.

The amino acid enrichment in blood (E b ) is often used in clinically relevant stable isotope studies to calculate muscle protein fractional synthesis rates (FSR), but it underestimates the FSR compared to methods using intracellular enrichment in muscle (E i ). We compared the FSR calculated with intravenous infusion of the novel tracer L‐[2,3,3,4,5,5,5,6,6,6‐ 2 H 10 ]leucine ([ 2 H 10 ]leu) to infusion of the traditional tracer L‐[ 13 C 6 ]phenylalanine ([ 13 C 6 ]phe) using E b and E i . Because [ 2 H 10 ]leu is intracellularly metabolized to [ 2 H 9 ]leu prior to reentry into the blood, we hypothesize that using [ 2 H 10 ]leu will minimize the discrepancy between FSR values calculated using E b or E i compared to [ 13 C 6 ]phe‐calculated FSR values using the corresponding E b or E i . To test our hypothesis, six healthy subjects received simultaneous infusions of the two stable‐isotope tracers following standard protocols. The [ 2 H 9 ]leu‐calculated FSR (%h ‐1 ) was 0.08±0.01 based on E b and 0.18±0.01 based on E i . The corresponding [ 13 C 6 ]phe‐calculated FSR values were 0.06±0.01 and 0.25±0.02, respectively. Moreover, the difference in the [ 2 H 9 ]leu‐calculated FSR values using E i vs E b was significantly less ( P <0.01; 0.10±0.01) compared to the difference in the corresponding [ 13 C 6 ]phe‐calculated FSR values (0.19±0.02). We conclude that, compared to [ 13 C 6 ]phe, the [ 2 H 10 ]leu tracer offers a more accurate approach to calculating FSR when E b is sought as an alternative to the E i. Grant Funding Source : Supported by NIH/NIDDK Grant# R01 DK094062; NCATS Grant# UL1 TR000135

Multiple conditions are linked to perturbed skeletal muscle protein (SMP) metabolism, including excess adiposity and insulin resistance. Here we delineate the precise roles of body fat and insulin sensitivity on SMP metabolism, and test the hypothesis that hyperaminoacidemia can eliminate the influence of these factors. Six healthy individuals underwent a DEXA scan to determine percent body fat (%BF; 16.3‐29.4%) and an oral glucose tolerance test (OGTT) to evaluate insulin sensitivity via Matsuda index (MI; 1.21‐12.0). Subjects received a primed, continuous infusion of L‐[2,3,3,4,5,5,5,6,6,6‐ 2 H 10 ]leucine to determine SMP fractional synthesis rate (FSR) in biopsies from the vastus lateralis . Myostatin (MSTN) mRNA, a marker of muscle wasting, was quantified in similar biopsies, and MSTN activation was assessed in plasma as the percent of active over latent protein. To evaluate the effect of hyperaminoacidemia, samples were collected before and during infusions of high amino acids (AA). In our subjects, %BF was not correlated with MI ( P =0.11). The %BF was only associated with MSTN mRNA ( P =0.02; r=0.81). However, MI was a key determinant in FSR ( P =0.02; r=0.83) and MSTN activation ( P <0.01; r=‐0.94). AA infusion eliminated all discrepancies in FSR and MSTN quantitations. Thus, SMP metabolism is more strongly linked to insulin sensitivity than body fat, but the influence is attenuated with hyperaminoacidemia. Grant Funding Source : Supported by NIH/NIDDK Grant# R01 DK094062; NCATS Grant# UL1 TR000135

New Findings What is the central question of this study? Humans with obesity have lower ATP synthesis in muscle along with lower content of the β‐subunit of the ATP synthase (β‐F1‐ATPase), the catalytic component of the ATP synthase. Does lower synthesis rate of β‐F1‐ATPase in muscle contribute to these responses in humans with obesity? What is the main finding and its importance? Humans with obesity have a lower synthesis rate of β‐F1‐ATPase and ATP synthase specific activity in muscle. These findings indicate that reduced production of subunits forming the ATP synthase in muscle may contribute to impaired generation of ATP in obesity. AbstractThe content of the β‐subunit of the ATP synthase (β‐F1‐ATPase), which forms the catalytic site of the enzyme ATP synthase, is reduced in muscle of obese humans, along with a reduced capacity for ATP synthesis. We studied 18 young (37 ± 8 years) subjects of which nine were lean (BMI = 23 ± 2 kg m−2) and nine were obese (BMI = 34 ± 3 kg m−2) to determine the fractional synthesis rate (FSR) and gene expression of β‐F1‐ATPase, as well as the specific activity of the ATP synthase. FSR of β‐F1‐ATPase was determined using a combination of isotope tracer infusion and muscle biopsies. Gene expression of β‐F1‐ATPase and specific activity of the ATP synthase were determined in the muscle biopsies. When compared to lean, obese subjects had lower muscle β‐F1‐ATPase FSR (0.10 ± 0.05 vs. 0.06 ± 0.03% h−1; P < 0.05) and protein expression (P < 0.05), but not mRNA expression (P > 0.05). Across subjects, abundance of β‐F1‐ATPase correlated with the FSR of β‐F1‐ATPase (P < 0.05). The specific activity of muscle ATP synthase was lower in obese compared to lean subjects (0.035 ± 0.004 vs. 0.042 ± 0.007 arbitrary units; P < 0.05), but this difference was not significant after the activity of the ATP synthase was adjusted to the β‐F1‐ATPase content (P > 0.05). Obesity impairs the synthesis of β‐F1‐ATPase in muscle at the translational level, reducing the content of β‐F1‐ATPase in parallel with reduced capacity for ATP generation via the ATP synthase complex.

ObjectiveObesity alters protein metabolism in skeletal muscle, but consistent evidence is lacking. This study compared muscle protein synthesis in adults with obesity and in lean controls in the fasted state and during an amino acid infusion.MethodsTen subjects with obesity (age: 36 ± 3 years; BMI: 34 ± 1 kg/m2) and ten controls (age: 35 ± 3 years; BMI: 23 ± 1 kg/m2) received an infusion of L‐[2,3,3,4,5,5,5,6,6,6‐2H10]leucine (0.15 μmol/kg fat‐free mass/min) to measure muscle protein synthesis after an overnight fast and during amino acid infusion.ResultsDespite greater muscle mammalian target of rapamycin phosphorylation (P ≤ 0.05), fasted‐state mixed‐muscle and mitochondrial protein synthesis were lower in subjects with obesity (P ≤ 0.05). However, the change in mixed‐muscle protein synthesis during the amino acid infusion was 2.7‐fold greater in subjects with obesity (P ≤ 0.05), accompanied by a greater change in S6 kinase‐1 phosphorylation (P ≤ 0.05). The change in mitochondrial protein synthesis did not differ between groups (P > 0.05).ConclusionsAdults with obesity have reduced muscle protein synthesis in the fasted state, but this response is compensated for by a greater change in overall muscle protein synthesis during amino acid infusion.

Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change ( P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased ( P < 0.01) whole body phenylalanine rate of appearance (μmol·kg−1·min−1), indicating suppression of muscle proteolysis, in both the control (1.02 ± 0.04 vs 0.76 ± 0.04) and the BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments ( P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA.