Kinome Profiling Reveals Abnormal Activity of Kinases in Skeletal Muscle From Adults With Obesity and Insulin Resistance.

CONTEXT: Obesity-related insulin resistance (OIR) is one of the main contributors to type 2 diabetes and other metabolic diseases. Protein kinases are implicated in insulin signaling and glucose metabolism. Molecular mechanisms underlying OIR involving global kinase activities remain incompletely understood. OBJECTIVE: To investigate abnormal kinase activity associated with OIR in human skeletal muscle. DESIGN: Utilization of stable isotopic labeling-based quantitative proteomics combined with affinity-based active enzyme probes to profile in vivo kinase activity in skeletal muscle from lean control (Lean) and OIR participants. PARTICIPANTS: A total of 16 nondiabetic adults, 8 Lean and 8 with OIR, underwent hyperinsulinemic-euglycemic clamp with muscle biopsy. RESULTS: We identified the first active kinome, comprising 54 active protein kinases, in human skeletal muscle. The activities of 23 kinases were different in OIR muscle compared with Lean muscle (11 hyper- and 12 hypo-active), while their protein abundance was the same between the 2 groups. The activities of multiple kinases involved in adenosine monophosphate-activated protein kinase (AMPK) and p38 signaling were lower in OIR compared with Lean. On the contrary, multiple kinases in the c-Jun N-terminal kinase (JNK) signaling pathway exhibited higher activity in OIR vs Lean. The kinase-substrate-prediction based on experimental data further confirmed a potential downregulation of insulin signaling (eg, inhibited phosphorylation of insulin receptor substrate-1 and AKT1/2). CONCLUSIONS: These findings provide a global view of the kinome activity in OIR and Lean muscle, pinpoint novel specific impairment in kinase activities in signaling pathways important for skeletal muscle insulin resistance, and may provide potential drug targets (ie, abnormal kinase activities) to prevent and/or reverse skeletal muscle insulin resistance in humans.

Quantitative proteomics reveals novel interaction partners of Rac1 in pancreatic ß-cells: Evidence for increased interaction with Rac1 under hyperglycemic conditions.

Rac1, a small G protein, regulates physiological insulin secretion from the pancreatic ß-cell. Interestingly, Rac1 has also been implicated in the onset of metabolic dysfunction of the ß-cell under the duress of hyperglycemia (HG). This study is aimed at the identification of interaction partners of Rac1 in ß-cells under basal and HG conditions. Using co-immunoprecipitation and UPLC-ESI-MS/MS, we identified 324 Rac1 interaction partners in INS-1832/13 cells, which represent the largest Rac1 interactome to date. Furthermore, we identified 27 interaction partners that exhibited increased association with Rac1 in ß-cells exposed to HG. Western blotting (INS-1832/13 cells, rat islets and human islets) and co-immunoprecipitation (INS-1832/13 cells) further validated the identity of these Rac1 interaction partners including regulators of GPCR-G protein-effector coupling in the islet. These data form the basis for future investigations on contributory roles of these Rac1-specific signaling pathways in islet ß-cell function in health and diabetes.

Quantitative proteomics reveals novel protein interaction partners of PP2A catalytic subunit in pancreatic ß-cells.

Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases. We hypothesize that PP2A regulates signaling cascades in pancreatic ß-cells in the context of glucose-stimulated insulin secretion (GSIS). Using co-immunoprecipitation (co-IP) and tandem mass spectrometry, we globally identified the protein interaction partners of the PP2A catalytic subunit (PP2Ac) in insulin-secreting pancreatic ß-cells. Among the 514 identified PP2Ac interaction partners, 476 were novel. This represents the first global view of PP2Ac protein-protein interactions caused by hyperglycemic conditions. Additionally, numerous PP2Ac partners were found involved in a variety of signaling pathways in the ß-cell function, such as insulin secretion. Our data suggest that PP2A interacts with various signaling proteins necessary for physiological insulin secretion as well as signaling proteins known to regulate cell dysfunction and apoptosis in the pancreatic ß-cells.