Reproducibility of an HPLC-ESI-MS/MS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R2 = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase134-143 peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase134-143 peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R2 = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase134-143 peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase134-143 peptide

Effects of acute exposure to increased plasma branched-chain amino acid concentrations on insulin-mediated plasma glucose turnover in healthy young subjects.

Plasma branched-chain amino acids (BCAA) are inversely related to insulin sensitivity of glucose metabolism in humans. However, currently, it is not known whether there is a cause-and-effect relationship between increased plasma BCAA concentrations and decreased insulin sensitivity.To determine the effects of acute exposure to increased plasma BCAA concentrations on insulin-mediated plasma glucose turnover in humans.Ten healthy subjects were randomly assigned to an experiment where insulin was infused at 40 mU/m2/min (40U) during the second half of a 6-hour intravenous infusion of a BCAA mixture (i.e., BCAA; N = 5) to stimulate plasma glucose turnover or under the same conditions without BCAA infusion (Control; N = 5). In a separate experiment, seven healthy subjects were randomly assigned to receive insulin infusion at 80 mU/m2/min (80U) in association with the above BCAA infusion (N = 4) or under the same conditions without BCAA infusion (N = 3). Plasma glucose turnover was measured prior to and during insulin infusion.Insulin infusion completely suppressed the endogenous glucose production (EGP) across all groups. The percent suppression of EGP was not different between Control and BCAA in either the 40U or 80U experiments (P > 0.05). Insulin infusion stimulated whole-body glucose disposal rate (GDR) across all groups. However, the increase (%) in GDR was not different [median (1st quartile - 3rd quartile)] between Control and BCAA in either the 40U ([199 (167-278) vs. 186 (94-308)] or 80 U ([491 (414-548) vs. 478 (409-857)] experiments (P > 0.05). Likewise, insulin stimulated the glucose metabolic clearance in all experiments (P 0.05).Short-term exposure of young healthy subjects to increased plasma BCAA concentrations does not alter the insulin sensitivity of glucose metabolism

Muscle Fiber Phenotype: A Culprit of Abnormal Metabolism and Function in Skeletal Muscle of Humans with Obesity

The proportion of the different types of fibers in a given skeletal muscle contributes to its overall metabolic and functional characteristics. Greater proportion of Type I muscle fibers is associated with favorable oxidative metabolism and function of the muscle. Humans with obesity have lower proportion of Type I muscle fibers. We discuss how lower proportion of Type I fibers in humans with obesity may explain metabolic and functional abnormalities reported in these individuals. These include lower glucose disposal rate, mitochondrial content, protein synthesis, and quality/contractile function at the muscle level, as well as increased risk for heart disease, lower levels of physical activity, and propensity for weight gain/resistance to weight loss. We delineate future research directions and the need to examine hybrid muscle fiber populations, which are indicative of a transitory state of fiber phenotype within skeletal muscle. We also describe methodologies for precisely characterizing muscle fibers and gene expression at the single muscle fiber level to enhance our understanding of the regulation of muscle fiber phenotype in obesity. By contextualizing research in the field of muscle fiber type in obesity, we lay a foundation for future advancements and pave the way for translation of this knowledge to address impaired metabolism and function in obesity

Reverting To A Healthier Diet Or Employing An Aerobic Exercise Regime Independently Restore Muscle Fiber Phenotype Disturbed By High-Fat Diet In Muscle Of Mice

Obesity affects roughly 42% of the US population. High fat/high sugar diets (HFHS) often referred to as “western diet” contributes to this prevalence. Diet-induced obesity results in impaired metabolic responses and associated disease states (i.e., Type 2 Diabetes). Metabolic impairments in diet-induced obesity are a result of changes in muscle metabolism, and changes in muscle fiber phenotype, which is determined by the isoform-content of the protein myosin heavy chain (MHC). Fast muscle fiber phenotype (i.e. type IIb in mice) is characterized by lower capacity for utilization of lipids, implicated in the pathogenesis of Type 2 Diabetes. Regular exercise shifts MHC proportions under healthy circumstances. However, exercise-driven fiber type shifts in diet-induced obesity are less understood. PURPOSE To determine the impact of exercise and diet on fiber-type proportions in mice. We hypothesized that exercise would shift the mouse gastrocnemius muscle phenotype induced by a HFHS diet away from IIb fast fiber types. METHODS 49 C57BL/6 mice were split into 4 groups: 1) a Control (n = 9) fed a standard chow diet and water for 24 weeks, 2) a HFHS, fed a HFHS diet (60% of calories from fat, high sugar/fructose: 42 g/L in drinking water) for 24 weeks, 3) a HFHS Control (n = 10) fed a HFHS diet for 12 weeks followed by a standard chow diet and water for the next 12 weeks (i.e., simulating traditional dieting approach), and 4) a HFHS + exercise group fed a HFHS diet for 24 weeks, and performed aerobic exercise (30 minutes of treadmill running 5 days/week) in the last 12 weeks. Gastrocnemius muscles were collected, homogenized, and analyzed for MHC isoforms using SDS-PAGE. Intensity of bands corresponding to MHC IIa, IIx, and IIb isoforms were quantified using Image J (bands for the IIa and IIx isoforms were analyzed as a single band). Paired sample t-tests were conducted for differences between the MHC isoforms across groups. RESULTS Proportions of MHC IIb isoform increased (91 +/- 3%) in HFHS compared to the Control (81 +/- 6.2%, p=0.195), HFHS Control (77 +/- 3%, p=0.004), and HFHS + exercise groups (79 +/- 5%, p=0.057). Additionally, MHC IIa/x proportions in the HFHS (8+/-3%) compared to the Control (17 +/- 5.9%, p=0.184), HFHS Control (20 +/- 2%, p=0.004), and the HFHS + exercise (18 +/- 4%, p=0.054) groups was reduced. CONCLUSION These data suggest HFHS diet increases the proportion of IIb fibers and reduces IIa/x fibers in mouse gastrocnemius muscle over 24 weeks. Importantly, performing aerobic exercise with a HFHS diet or switching to a healthier diet restores the muscle fiber phenotype in mouse gastrocnemius muscle. Thus, exercise and dietary interventions may be a good strategy to shift MHC isoforms away from the extreme fast fiber phenotype, which has lower capacity for lipid utilization. Future research should determine single muscle fiber phenotype shifts related to long-term diet changes and exercise in humans to better understand regulation of muscle fiber phenotype and its impact on human metabolism