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

Abstract

Aims/hypothesis Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-β) and determine protein abundance of ATPsyn-β and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. Methods Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n=10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-β using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. Results Seven phosphorylation sites were identified on ATPsyn-β purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a ∼30% higher phosphorylation of ATPsyn-β at Tyr361 and Thr213 (within the nucleotidebinding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-β protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-β by ∼50% in lean and healthy, but not insulinresistant, individuals. Conclusions/interpretation These data demonstrate that ATPsyn-β is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-β together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-β may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

“…Moreover, significant positive correlations were also observed for summative spot volumes (sum of volumes of all spots with the same identified protein) relating to cytoplasmic malate dehydrogenase (MDH1), an enzyme controlling TCA cycle pool size and providing contractile function [27], and the mitochondrial enzymes ACO2, ATP5A1, ATP5B and GBAS. These results, suggesting reduced TCA cycle and mitochondrial protein content in insulin resistance, are consistent with previous findings of reduced enzyme activities [10,28,29], protein expression [16,17,30], altered phosphorylation [16,31,32], altered transcript levels [10,33,34] or altered flux through mitochondrial ATP synthase [8].…”

Reduction of non-esterified fatty acids improves insulin sensitivity and lowers oxidative stress, but fails to restore oxidative capacity in type 2 diabetes: a randomised clinical trial

“…Despite the positive correlation between mitochondrial oxidative capacity and insulin sensitivity, the results suggest that insulin sensitivity and mitochondrial function are not per se causally related, but rather represent independent parallel features associated with type 2 diabetes. However, lipids are known to stimulate muscle ROS production [13], which has also been implicated in insulin resistance [30] in both type 1 and type 2 diabetes [18]. In the present paper, we explore in more detail the predominant role of complex I of the electron transport chain (ETC) in lipidinduced ROS production in humans with type 2 diabetes.…”

“…This effect seems to be blunted or absent in patients with type 2 diabetes [7,12], FDRs [8] and patients with type 1 diabetes [9]. Insulin has been shown to increase specific phosphorylation sites of ATP synthase by ∼50% in lean and healthy, but not insulinresistant, individuals [13]. Some investigators suggest that the insulin-resistant state may limit glucose uptake or substrate oxidation or reduce substrate transport into mitochondria during hyperinsulinaemia [11,12].…”

Common variation in oxidative phosphorylation genes is not a major cause of insulin resistance or type 2 diabetes

“…Based on the substantial evidence that mitochondrial dysfunction is linked to insulin resistance and beta cell dysfunction, and is present in multiple tissues relevant to the pathogenesis of type 2 diabetes [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22], we hypothesised that common variation in OxPhos genes contributes to an increased risk of type 2 diabetes or influences metabolic traits related to the disease. OxPhos gene variants showing nominal association with type 2 diabetes in a meta-analysis of three GWA studies that were part of the DIAGRAM Study [28] were selected for follow-up in an independent cohort of Danish individuals.…”

“…Recent studies have provided strong evidence of a link between insulin resistance and mitochondrial dysfunction in human skeletal muscle [7]. Global approaches such as transcriptional profiling and proteomics have demonstrated a co-ordinated downregulation of mRNA levels and protein content of nuclearencoded genes involved in oxidative phosphorylation (OxPhos) in muscle of patients with type 2 diabetes and of high-risk individuals [8][9][10][11]. Moreover, a reduced content and impaired functional capacity of muscle mitochondria may also contribute to mitochondrial dysfunction in type 2 diabetes and obesity [12][13][14].…”