Context. Skeletal muscle insulin resistance is one of the primary contributors of type 2 diabetes (T2D). Metformin is the first-line drug for the treatment of T2D. The primary effects of metformin include decreasing glucose production in the liver and decreasing insulin resistance in the skeletal muscle. However, the molecular mechanism of metformin’s action in skeletal muscle is not well understood. Protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, plays a pivotal role in cellular processes, such as signal transduction, cell proliferation, and apoptosis, and acts through dephosphorylating key signaling molecules such as AKT and AMPK. However, whether PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells remains to be elucidated. Objective. To investigate if PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells. Participants. Eight lean insulin-sensitive nondiabetic participants (4 females and 4 males; age: 21.0 ± 1.0 years; BMI: 22.0 ± 0.7   kg / m 2 ; 2-hour OGTT: 97.0 ± 6.0   mg / dl ; HbA1c: 5.3 ± 0.1 % ; fasting plasma glucose: 87.0 ± 2.0   mg / dl ; M value; 11.0 ± 1.0   mg / kgBW / min ). Design. A hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects, and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies that included 8 lean insulin-sensitive participants. Cultured cells were expanded, differentiated into myotubes, and treated with 50 μM metformin for 24 hours before harvesting. PP2Ac activity was measured by a phosphatase activity assay kit (Millipore) according to the manufacturer’s protocol. Results. The results indicated that metformin significantly increased the activity of PP2A in the myotubes for all 8 lean insulin-sensitive nondiabetic participants, and the average fold increase is 1.54 ± 0.11 ( P < 0.001 ). Conclusions. These results provided the first evidence that metformin can activate PP2A in human skeletal muscle cells derived from lean healthy insulin-sensitive participants and may help to understand metformin’s action in skeletal muscle in humans.

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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.

The endothelium is the frontline target of multiple metabolic stressors and pharmacological agents. As a consequence, endothelial cells (ECs) display highly dynamic and diverse proteome profiles. We describe here the culture of human aortic ECs from healthy and type 2 diabetic donors, the treatment with a small molecular coformulation of trans-resveratrol and hesperetin (tRES+HESP), followed by proteomic analysis of whole-cell lysate. A number of 3666 proteins were presented in all of the samples and thus further analyzed. We found that 179 proteins had a significant difference between diabetic ECs vs. healthy ECs, while 81 proteins had a significant change upon the treatment of tRES+HESP in diabetic ECs. Among them, 16 proteins showed a difference between diabetic ECs and healthy ECs and the difference was reversed by the tRES+HESP treatment. Follow-up functional assays identified activin A receptor-like type 1 and transforming growth factor β receptor 2 as the most pronounced targets suppressed by tRES+HESP in protecting angiogenesis in vitro . Our study has revealed the global differences in proteins and biological pathways in ECs from diabetic donors, which are potentially reversible by the tRES+HESP formula. Furthermore, we have identified the TGFβ receptor as a responding mechanism in ECs treated with this formula, shedding light on future studies for deeper molecular characterization.