In an attempt to address the complex and clinically challenging question of the causes of muscle wasting in patients with cachexia, we have developed a primary adult human skeletal muscle cell model. The cultured cells were characterised by immunocytochemistry using antibodies to the myofibrillar protein constituents desmin and titin. Myotube formation was confirmed biochemically by a fourfold increase in the activity of the muscle-specific enzyme creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive retinoblastoma protein dephosphorylation. Having successfully confirmed the phenotype of these adult human muscle cells, we assessed their interaction with the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, like insulin, is a potent anabolic agent in the regulation of protein metabolism. We have shown that IGF-I stimulated both replication and differentiation of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the IGF system during the process of terminal differentiation, we found that both myoblasts and myotubes expressed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also produced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFBP-4 and abundant IGFBP-3, which has not been shown to be produced by any other skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrations. Insulin was more potent than IGF-I: use of the IGF analogue long R 3 IGF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. In summary, we have developed and characterised a clinically relevant in vitro model with which to address the aetiology of muscle wasting associated with chronic catabolic conditions, and we anticipate that future work will enable the development of novel, effective therapeutic interventions.

Aims/Introduction Glucose metabolic disorder is the main cause for type 2 diabetes progression. Exploring the molecular mechanisms of metabolic disorder are crucial for type 2 diabetes treatment. Materials and Methods Micro ribonucleic acid (miR)‐363, NOTCH1 and forkhead box C2 (FOXC2) expressions in high glucose (HG)‐treated HepG2 cells and the livers of type 2 diabetes mellitus rats were assessed using quantitative polymerase chain reaction. Protein levels of NOTCH1, FOXC2 and phosphatidylinositol 3‐kinase (PI3K)/serine/threonine protein kinase (Akt)‐related proteins were evaluated using western blot. Lipid accumulation was determined using Oil Red O staining. Then glucose consumption, blood glucose level and glycogen content were detected using kits. Finally, dual luciferase reporter assay was used to verify the binding relationship between miR‐363 and NOTCH1, and the binding relationship between miR‐363 and FOXC2. Results MiR‐363 was significantly upregulated in the livers of diabetic rats and HG‐induced HepG2 cells, whereas NOTCH1 and FOXC2 were downregulated. In HG‐induced HepG2 cells, miR‐363 inhibitor markedly increased glucose consumption and uptake, and reduced accumulation of lipid droplets. Then NOTCH1 and FOXC2 were identified as downstream targets of miR‐363. NOTCH1 overexpression or FOXC2 overexpression could ameliorate glucose and lipids metabolism disorder in type 2 diabetes model cells. In addition, we found that FOXC2 inhibition abolished the effect of NOTCH1 overexpression on HG‐induced HepG2 cells. Finally, we proved that the PI3K/Akt pathway was the downstream pathway of FOXC2. Conclusion MiR‐363 was considered as a key regulator of glucose and lipids metabolism in type 2 diabetes mellitus, which regulated PI3K/Akt axis by targeting NOTCH1 and FOXC2, thus leading to hepatic glucose and lipids metabolism disorder in type 2 diabetes.