Kras, Katon A.

AbstractObjectiveThis study tested the hypothesis that expression of insulin‐like growth factor 1 (IGF‐1) protein and mRNA splice variants is lower in skeletal muscle of humans with obesity who have a lower mixed‐muscle protein fractional synthesis rate (MMP‐FSR) when compared with individuals without obesity.MethodsThe study included nine participants with obesity (OB, mean [SD], BMI = 35 [3] kg/m2, MMP‐FSR = 0.06%/h [0.02%/h]) and nine participants without obesity (W‐OB, BMI = 24 [3] kg/m2, MMP‐FSR = 0.08%/h [0.02%/h]; for both BMI and MMP‐FSR p < 0.05). MMP‐FSR and mitochondrial protein FSR were measured following an overnight fast.ResultsAlong with lower MMP‐FSR, OB participants displayed lower mitochondrial protein FSR (p = 0.03) compared with W‐OB participants. Expression of IGF‐1 (p = 0.04) and IGF‐1 receptor (p < 0.01) proteins was lower in muscle of OB participants. In addition, OB participants had lower (p < 0.05) mRNA expression of IGF1 variants Eb and Ec. This study demonstrates that lower protein synthesis in muscle of humans with obesity occurs concurrently with lower expression of muscle IGF‐1 and IGF‐1 receptor proteins, as well as lower mRNA expression of the IGF1 splice variants.ConclusionsThese findings indicate that lower protein synthesis observed in muscle of humans with obesity may result from diminished muscle IGF1 gene expression.

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.

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

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.

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.