Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.     

Glucose transporter expression in human skeletal muscle fibers

The present study was initiated to investigate GLUT-1 through -5 expression in developing and mature human skeletal muscle. To bypass the problems inherent in techniques using tissue homogenates, we applied an immunocytochemical approach, employing the sensitive enhanced tyramide signal amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas after birth, the characteristic subcellular localization is as seen in adult muscle fibers. Our results show that GLUT-1, GLUT-3, and GLUT-4 seem to be of importance during muscle fiber growth and development. GLUT-5 protein was undetectable in fetal and adult skeletal muscle fibers. In adult muscle fibers, only GLUT-4 was expressed at significant levels. GLUT-1 immunoreactivity was below the detection limit in muscle fibers, indicating that this glucose transporter is of minor importance for muscle glucose supply. Thus we hypothesize that GLUT-4 also mediates basal glucose transport in muscle fibers, possibly through constant exposure to tonal contraction and basal insulin levels.     

Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect

In myotubes established from patients with type 2 diabetes (T2D), lipid oxidation and insulin-mediated glucose oxidation are reduced, whereas in myotubes from obese non-diabetic subjects, exposure to palmitate impairs insulin-mediated glucose oxidation. To determine the underlying mechanisms of these metabolic malfunctions, we studied mitochondrial respiration, uncoupled respiration and oxidative enzyme activities (citrate synthase (CS), 3-hydroxy-acyl-CoA-dehydrogenase activity (HAD)) before and after acute exposure to insulin and/or palmitate in myotubes established from healthy lean and obese subjects and T2D patients. Basal CS activity was lower (14%) in diabetic myotubes compared with myotubes from lean controls (P=0.03). Incubation with insulin (1 microM) for 4 h increased the CS activity (26-33%) in myotubes from both lean (P=0.02) and obese controls (P<0.001), but not from diabetic subjects. Co-incubation with palmitate (0.6 mM) for 4 h abolished the stimulatory effect of insulin on CS activity in non-diabetic myotubes. No differences were detected in mitochondrial respiration and HAD activity between myotubes from non-diabetic subjects and T2D patients, and none of these measures responded to high levels of insulin and/or palmitate. These results provide evidence for an intrinsic defect in CS activity, which may play a role in the pathogenesis of T2D. Moreover, the data suggest that insulin resistance at the CS level can be induced by exposure to high free fatty acid levels.     

The basal kinetic parameters of glycogen synthase in human myotube cultures are not affected by chronic high insulin exposure.

There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV(0.1)) of GS, increased the sensitivity of GS to the allosteric activator glucose 6-phosphate (A(0.5)) and increased the sensitivity of GS to its substrate UDPG (K(m(0.1))) when myotubes were precultured at low insulin with/without high glucose conditions. However, this effect of acute insulin stimulation was abolished in myotubes precultured at high insulin with or without high glucose. Furthermore, we found significant correlations between the fractional velocities FV(0.1) of GS and K(m(0.1)) (rho=-0.72, P<0.0001), between FV(0.1) and A(0.5) (rho=-0.82, P<0.0001) and between K(m(0.1)) and A(0.5) values (rho=0.71, P<0.0001). Our results show that chronic exposure of human myotubes to high insulin with or without high glucose did not affect the basal kinetic parameters but abolished the reactivity of GS to acute insulin stimulation. We suggest that insulin induced insulin resistance of GS is caused by a failure of acute insulin stimulation to decrease A(0.5) and K(m(0.1)) in human skeletal muscle.     

The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3alpha activity in diabetic myotubes

The mechanism responsible for the diminished activation of glycogen synthase (GS) in diabetic myotubes remains unclear, but may involve increased activity and/or expression of glycogen synthase kinase-3 (GSK-3). In myotubes established from type 2 diabetic and healthy control subjects we determined GS activity ratio, protein expression, and activity of GSK-3alpha and beta under basal and insulin-stimulated conditions when precultured in increasing insulin concentrations. In myotubes precultured at low insulin concentrations acute insulin stimulation increased GS activity more in control than in diabetic subjects, whereas the corresponding GSK-3alpha but not GSK-3beta activity was significantly reduced by acute insulin treatment in both groups. However, in myotubes precultured at high insulin concentrations the effect of insulin on GS and GSK-3alpha activity was blunted in both groups. The protein expression of GSK-3alpha or beta was unaffected. In conclusion, myotubes with a primary defect in GS activity express insulin responsive GSK-3alpha, suggesting that failure of insulin to decrease GS phosphorylation involves abnormal activity of another kinase or phosphatase.     

Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene

It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine triphosphate in 2'-specific nucleotidyl transfer reactions to synthesize 2',5'-oligoadenylates, which activate latent ribonuclease, resulting in degradation of viral RNA and inhibition of virus replication. We showed elsewhere that constitutive (basal) activity of 2'5'AS is correlated with virus-stimulated activity. In the present study, we asked whether constitutive activity is genetically determined and, if so, by which variants. Analysis of 83 families containing two parents and two children demonstrated significant correlations between basal activity in parent-child pairs (P<.0001) and sibling pairs (P=.0044), but not spousal pairs, suggesting strong genetic control of basal activity. We next analyzed association between basal activity and 15 markers across the OAS gene cluster. Significant association was detected at multiple markers, the strongest being at an A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (AG or AA) of the OAS1 gene. At this unusual polymorphism, allele G had a higher gene frequency in persons with high enzyme activity than in those with low enzyme activity (0.44 vs. 0.20; P=3 x 10(-11)). Enzyme activity varied in a dose-dependent manner across the GG, GA, and AA genotypes (tested by analysis of variance; P=1 x 10(-14)). Allele G generates the previously described p46 enzyme isoform, whereas allele A ablates the splice site and generates a dual-function antiviral/proapoptotic p48 isoform and a novel p52 isoform. This genetic polymorphism makes OAS1 an excellent candidate for a human gene that influences host susceptibility to viral infection.     

Induction of GLUT-1 protein in adult human skeletal muscle fibers

Prompted by our recent observations that GLUT-1 is expressed in fetal muscles, but not in adult muscle fibers, we decided to investigate whether GLUT-1 expression could be reactivated. We studied different stimuli concerning their ability to induce GLUT-1 expression in mature human skeletal muscle fibers. Metabolic stress (obesity, non-insulin-dependent diabetes mellitus), contractile activity (training), and conditions of de- and reinnervation (amyotrophic lateral sclerosis) could not induce GLUT-1 expression in human muscle fibers. However, regenerating muscle fibers in polymyositis expressed GLUT-1. In contrast to GLUT-1, GLUT-4 was expressed in all investigated muscle fibers. Although the significance of GLUT-1 in adult human muscle fibers appears limited, GLUT-1 may be of importance for the glucose supplies in immature and regenerating muscle.     

Reference intervals for glucose, beta-cell polypeptides, and counterregulatory factors during prolonged fasting

To establish reference intervals for the pancreatic beta-cell response and the counterregulatory hormone response to prolonged fasting, we studied 33 healthy subjects (16 males, 17 females) during a 72-h fast. Glucose, insulin, C-peptide, and proinsulin levels decreased (P < 0.001), and the levels of counterregulatory factors increased during the fast [P < 0.05; glucagon and free fatty acids (FFA) with a linear increase and epinephrine, norepinephrine, and cortisol with a clear underlying circadian rhythm]. Growth hormone secretion increased from the first to third day of fasting (P < 0.05) but actually decreased from the second to third day of fasting (P = 0.03). Males had higher glucose and glucagon levels and lower FFA levels during the fast (P < 0.05), whereas no effect of gender on beta-cell polypeptides was observed. A high body mass index resulted in higher insulin and C-peptide levels during the fast (P < 0.05). In conclusion, we have provided reference intervals for glucoregulatory factors during a 72-h fast. We observed a diminished beta-cell response concomitant with an increased secretion of counterregulatory hormones. These results should be of clinical and scientific value in the investigation of hypoglycemic disorders.     

Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods

Skeletal muscle is a key tissue site of insulin resistance in type 2 diabetes. Human myotubes are primary skeletal muscle cells displaying both morphological and biochemical characteristics of mature skeletal muscle and the diabetic phenotype is conserved in myotubes derived from subjects with type 2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery strategy using isobaric peptide tags for relative and absolute quantification (iTRAQ) and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring to identify, quantify, and validate changes in protein abundance among human myotubes obtained from nondiabetic lean, nondiabetic obese, and type 2 diabetic subjects, respectively. Using an optimized protein precipitation protocol, a total of 2832 unique proteins were identified and quantified using the iTRAQ strategy. Despite a clear diabetic phenotype in diabetic myotubes, the majority of the proteins identified in this study did not exhibit significant abundance changes across the patient groups. Proteins from all major pathways known to be important in type 2 diabetic subjects were well-characterized in this study. This included pathways like the trichloroacetic acid (TCA) cycle, lipid oxidation, oxidative phosphorylation, the glycolytic pathway, and glycogen metabolism from which all but two enzymes were found in the present study. None of these enzymes were found to be regulated at the level of protein expression or degradation supporting the hypothesis that these pathways are regulated at the level of post-translational modification. Twelve proteins were, however, differentially expressed among the three different groups. Thirty-six proteins were chosen for further analysis and validation using selected reaction monitoring based on the regulation identified in the iTRAQ discovery study. The abundance of adenosine deaminase was considerably down-regulated in diabetic myotubes and as the protein binds propyl dipeptidase (DPP-IV), we speculate whether the reduced binding of adenosine deaminase to DPP-IV may contribute to the diabetic phenotype in vivo by leading to a higher level of free DPP-IV to bind and inactivate the anti-diabetic hormones, glucagon-like peptide-1 and glucose-dependent insulintropic polypeptide.