Calpains are a family of non-lysosomal cytoplasmatic cysteine proteases. Since calpain 10 (CAPN10), a member of the calpain family of proteases, has been found to represent a putative diabetes susceptibility gene, it was argued that calpains may be involved in the development of type 2 diabetes. The functional role of calpains in insulin signaling and/or insulin action is, however, not clear. We investigated the effects of the calpains 1 and 2 inhibitor PD151746 on insulin signaling and insulin action in human hepatoma G2 cells (HepG2). HepG2 cells were incubated without (−PD) or with (+PD) 5.33 μmol/l PD151746 for different times and then stimulated with 100 nmol/l insulin for 0 (t ₀), 5 (t ₅), 15 (t ₁₅), 30 (t ₃₀), 45 (t ₄₅), and 60 (t ₆₀) min. After solubilization of the cells, insulin receptor kinase activity, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1-associated phosphatidylinositol-3 kinase (PI3-kinase), PI3-kinase activity, Thr³⁰⁸ phosphorlyation of Akt, amount of protein tyrosine phosphatase-ε (PTPε), and glycogen synthase activity were determined. Incubation with PD151746 resulted in a significant reduction of insulin-stimulated glycogen synthesis compared with cells not pre-incubated with the calpain inhibitor (−PD: t ₀, 4.90 ± 1.20%; t ₅, 5.90 ± 1.02%; t ₁₅, 5.29 ± 0.95%; t ₃₀, 5.60 ± 1.10%; t ₄₅, 5.52 ± 0.90%; t ₆₀, 5.67 ± 0.97%;+PD: t ₀, 4.56 ± 1.10%; t ₅, 6.16 ± 1.05%; t ₁₅, 7.52 ± 1.09%; t ₃₀, 7.68 ± 1.10%; t ₄₅, 8.28 ± 0.89%; t ₆₀, 7.69 ± 0.98%; P < 0.05). Incubation with PD151746 significantly increased the protein amount of PTPε in the cells after 12 h (−PD: t ₁, 0.85 ± 0.18 RU (Relative unit); t ₈, 0.87 ± 0.18 RU; t ₁₂, 0.9 ± 0.13 RU; +PD: t ₁, 0.92 ± 0.21 RU; t ₈, 1.1 ± 0.15 RU; t ₁₂, 1.34 ± 0.16 RU; P < 0.05). Calpain inhibition with PD151746 had no effect on the insulin stimulation of the investigated insulin signaling parameters. These results in HepG2 cells suggest that calpains play a role in the hepatic regulation of insulin-stimulated glycogen synthesis independent of the PI3-kinase/Akt signaling pathway.

The melanocortin (MC) system is a pivotal component of the hypothalamo-pituitary–adrenal (HPA) stress axis and plays an important role in the pathogenesis of obesity and the metabolic syndrome. Adipose dysfunction is implicated in the pathogenesis of these disorders. We investigated direct ACTH effects on adipose functions in immortalised murine white and brown adipocytes. MC receptor types 2 and 5 were expressed at the mRNA and protein levels and were strongly up-regulated during differentiation. Chronic ACTH stimulation did not affect adipogenesis. Insulin-induced glucose uptake in white adipocytes was acutely and transiently reduced by 45% upon ACTH treatment. Visfatin and adiponectin gene expression was reduced by about 50% in response to ACTH, while interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were acutely up-regulated by 2100 and 60% respectively. Moreover, IL-6 secretion was increased by 1450% within 4 h of ACTH treatment. In brown adipocytes, stimulation with ACTH caused a 690% increase in uncoupling protein (UCP)-1 mRNA levels within 8 h, followed by a 470% increase in UCP-1 protein concentrations after 24 h. Consistently, p38 mitogen-activated protein kinase (MAPK) phosphorylation was acutely increased by 1800% in response to ACTH stimulation, and selective inhibition of p38 MAPK abolished the ACTH-mediated UCP-1 protein increase. Taken together, ACTH acutely promotes an insulin-resistant, pro-inflammatory state and transiently enhances energy combustion. In conditions characterised by a dysregulation of the HPA stress axis such as the metabolic syndrome, direct MC interaction with adipocytes may contribute to dysregulated energy balance, insulin resistance and cardiometabolic complications.