Calpain inhibition impairs glycogen syntheses in HepG2 hepatoma cells without altering insulin signaling

Abstract

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 (t0), 5 (t5), 15 (t15), 30 (t30), 45 (t45), and 60 (t60) 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: t0, 4.90 ± 1.20%; t5, 5.90 ± 1.02%; t15, 5.29 ± 0.95%; t30, 5.60 ± 1.10%; t45, 5.52 ± 0.90%; t60, 5.67 ± 0.97%;+PD: t0, 4.56 ± 1.10%; t5, 6.16 ± 1.05%; t15, 7.52 ± 1.09%; t30, 7.68 ± 1.10%; t45, 8.28 ± 0.89%; t60, 7.69 ± 0.98%; P < 0.05). Incubation with PD151746 significantly increased the protein amount of PTPε in the cells after 12 h (−PD: t1, 0.85 ± 0.18 RU (Relative unit); t8, 0.87 ± 0.18 RU; t12, 0.9 ± 0.13 RU; +PD: t1, 0.92 ± 0.21 RU; t8, 1.1 ± 0.15 RU; t12, 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.