Katsanos, Christos S.

Nagarse (N) is traditionally used to isolate mitochondria (mito) from skeletal muscle (SM). We investigated the effects of N on functional parameters of mechanically liberated mito. Mito from mouse (n = 7) gastrocnemius muscle (119‐175 mg tissue) was isolated using a glass on glass homogenizer followed by differential centrifugation. The 800g supernatant from the first centrifugation, containing mito, was divided into two equal volumes. One was exposed to N and the other was not. In each volume, mito were isolated by standard differential centrifugation. State 3 max ADP‐stimulated O 2 consumption rates (J o ) were measured using polarography with malate + pyruvate + glutamate. N increased state 3 J o , 16.1 ± 1.1 vs. 11.9 ± 1.4 nmol O 2 / ml mito suspension / min; P < 0.004, while it decreased protein concentration in the final mito suspension (typically used to adjust final J o values), 1.9 ± 0.1 vs. 2.5 ± 0.1 mg / ml; P < 0.004. Citrate synthase activity in the final mito suspension, indicative of the available mito, was not different in the preparation treated with N vs. that not treated with N (0.067 ± 0.01 vs. 0.065 ± 0.01μmol / min; P = 0.8). These findings suggest that N treatment: 1) hydrolyzes non‐mito contaminating proteins in the final mito pellet, and 2) induces apparent qualitative changes in mito function. Therefore, calculated functional parameters of mito treated with N are not directly comparable to mito not treated with N. Grant Funding Source : Supported by NIH/NIDDK RO1 DK094062

Abstract Background: Various pathophysiological conditions alter protein metabolism in skeletal muscle, with obesity being one of them. Obesity impairs regeneration of skeletal muscle, and the same biological mechanism(s) may adversely affect protein metabolism in the muscle of these individuals. Methods: We used C2C12 cell line to evaluate the effects of the anabolic hormone insulin on the expression of protein syncytin-1, which regulates regeneration of muscle, and in the presence of fatty acids whose metabolism is altered in obesity. We used muscle biopsy samples from obese humans with lower muscle protein synthesis and lean controls to evaluate expression of syncytin-1 in obesity and its correlation with protein synthesis in muscle. Results: Insulin upregulated syncytin-1 expression in C2C12 cells and this response was impaired in the presence of the fatty acid palmitate, but not oleate. Expression of the protein 4E-BP1, which signals increase in protein synthesis in muscle, showed response similar to that of syncytin-1. Humans with obesity characterized by lower muscle protein synthesis had higher expression of syncytin-1 in muscle compared to lean humans (P < 0.01). The rate of synthesis of protein in skeletal muscle across humans subjects correlated inversely (r = -0.51; P = 0.03) with the expression of syncytin-1 in muscle. Conclusions: Our studies provide novel insights in the regulation of syncytin-1 in skeletal muscle, and describe potential link between syncytin-1 expression and protein metabolism in skeletal muscle of humans. Altered syncytin-1 expression in muscle may mediate lower protein turnover in muscle of humans with obesity.

Mitochondria from skeletal muscle of humans with obesity often display alterations with respect to their morphology, proteome, biogenesis, and function. These changes in muscle mitochondria are considered to contribute to metabolic abnormalities observed in humans with obesity. Most of the evidence describing alterations in muscle mitochondria in humans with obesity, however, lacks reference to a specific subcellular location. This is despite data over the years showing differences in the morphology and function of subsarcolemmal (found near the plasma membrane) and intermyofibrillar (nested between the myofibrils) mitochondria in skeletal muscle. Recent studies reveal that impairments in mitochondrial function in obesity with respect to the subcellular location of the mitochondria in muscle are more readily evident following exposure of the skeletal muscle to physiological stimuli. In this review, we highlight the need to understand skeletal muscle mitochondria metabolism in obesity in a subpopulation-specific manner and in the presence of physiological stimuli that modify mitochondrial function in vivo. Experimental approaches employed under these conditions will allow for more precise characterization of impairments in skeletal muscle mitochondria and their implications in inducing metabolic dysfunction in human obesity.

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.

ABSTRACT Introduction Current evidence indicates mitochondrial dysfunction in humans with obesity. Acute exercise appears to enhance mitochondrial function in the muscle of nonobese humans, but its effects on mitochondrial function in muscle of humans with obesity are not known. We sought to determine whether acute aerobic exercise stimulates mitochondrial function in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in humans with obesity. Methods We assessed maximal adenosine triphosphate production rate (MAPR) and citrate synthase (CS) activity in isolated SS and IMF mitochondria from subjects with body mass index < 27 kg·m −2 (median age, 25 yr; interquartile range, 22–39 yr) and subjects with body mass index > 32 kg·m −2 (median age, 29 yr; interquartile range, 20–39 yr) before and 3 h after a 45-min cycling exercise at an intensity corresponding to 65% HR reserve. The SS and IMF mitochondria were isolated from muscle biopsies using differential centrifugation. Maximal adenosine triphosphate production rate and CS activities were determined using luciferase-based and spectrophotometric enzyme-based assays, respectively. Results Exercise increased MAPR in IMF mitochondria in both nonobese subjects and subjects with obesity ( P < 0.05), but CS-specific activity did not change in either group ( P > 0.05). Exercise increased MAPR supported by complex II in SS mitochondria, in both groups ( P < 0.05), but MAPR supported by complex I or palmitate did not increase by exercise in the subjects with obesity ( P > 0.05). Citrate synthase–specific activity increased in SS mitochondria in response to exercise only in nonobese subjects ( P < 0.05). Conclusions In nonobese humans, acute aerobic exercise increases MAPR in both SS and IMF mitochondria. In humans with obesity, the exercise increases MAPR in IMF mitochondria, but this response is less evident in SS mitochondria.

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.