Proteome Analysis Reveals Phosphorylation of ATP Synthase β-Subunit in Human Skeletal Muscle and Proteins with Potential Roles in Type 2 Diabetes

Abstract

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 ␤-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 ␤-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase ␤-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase ␤-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.Insulin resistance in skeletal muscle, defined as reduced insulin-stimulated glucose disposal, is a characteristic feature of type 2 diabetes mellitus (T2DM) 1 and is believed to be largely accounted for by reduced non-oxidative glucose metabolism (1-3). Furthermore, insulin stimulation of glucose oxidation and suppression of lipid oxidation is significantly impaired in patients with T2DM (2, 3). Conversely, in the basal, fasting state increased glucose oxidation and reduced lipid oxidation is seen in skeletal muscle of insulin resistant subjects, whether caused by T2DM or obesity alone (4). These defects suggest an impaired capacity to switch between carbohydrate and fat as oxidative energy sources in insulin-resistant subjects. Together with reports of reduced oxidative enzyme activity and dysfunction of mitochondria in skeletal muscle of patients with T2DM (4 -6) and the fact that mitochondrial DNA defects cause T2DM through impairment of oxidative phosphorylation (7, 8), these abnormalities in fuel metabolism have led to the hypotheses that perturbations in skeletal muscle mitochondrial metabolism (6, 9, 10) and defects in the signaling pathways of AMP-activated protein kinase (AMPK) are implicated in the pathogenesis of T2DM (11). That rates of fuel oxidation and mitochondrial function can affect glucose uptake and glycogen synthesis has been reported earlier (12, 13). In addition, both chronic activation of AMPK and induced expression of the transcriptional co-activator of peroxisom...

Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle

“…Here, we demonstrate that human ATPsyn-β is phosphorylated at multiple specific sites in vivo. These results extend our previous work [21] and that of several other studies that have used various phospholabelling techniques or phosphopeptide identification by MS, which have provided indirect evidence for serine/ threonine/tyrosine phosphorylation of ATPsyn-β in yeast, plants and mammalian tissues and cell lines [27][28][29][30][31][32][33][34][35].…”

“…However, although all sites were targeted in our analyses, of these we could only confirm the phosphorylation at Thr312 in human skeletal muscle. In addition to Thr213 [21] and Thr312 [27], we identified five novel phosphorylation sites at Thr475, Tyr230, Tyr269, Tyr361 and Tyr395, respectively. These data suggest the possibility that reversible phosphorylation of human ATPsyn-β at specific serine/threonine/tyrosine residues regulates the catalytic activity of ATP synthase, and hence ATP synthesis.…”

“…There is now evidence that a number of mitochondrial proteins including subunits in all respiratory complexes (I-V) of OxPhos are phosphorylated, and potentially regulated by several kinases and phosphatases localised to mitochondria [19,20]. Using a two-dimensional gel-based proteomic approach, we previously reported reduced protein content and altered phosphorylation of the catalytic beta subunit in the ATP synthase complex (ATPsyn-β) in muscle of patients with type 2 diabetes [21]. In that study, a single phosphopeptide was identified with the potential phosphorylation site, Thr213, in the nucleotide-binding region of ATPsyn-β.…”

Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

“…These studies suggest that HSP90 may serve to increase the amount of non-mutated relative to mutated insulin receptors. Interestingly, the level of HSP90 protein was recently reported to be increased in muscle of patients with type 2 diabetes [35]. Such an increase in HSP90 might antagonise the down-regulation of functional insulin receptors mediated by hyperinsulinaemia, and, as suggested by recent studies, preserve insulin signalling through Akt [36,37].…”

Mitochondrial dysfunction in Parkinson's disease

“…Recent investigations demonstrated that a number of the subunits of the respiratory chain enzyme complexes are phosphorylated, including several subunits of complex I. [45][46][47][48][49][50] As PINK1 protects cells against stress conditions that affect the mitochondrial membrane potential, which is maintained by the respiratory chain enzymes, it is tempting to speculate that PINK1 mediates its protective role through phosphorylation of certain respiratory chain enzyme subunits.…”