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. 2013 Oct 18;62(11):3887–3900. doi: 10.2337/db13-0095

FIG. 6.

FIG. 6.

Chronic insulin exposure blocks the effect of insulin on metabolic gene expression and induces IRS1-S636 phosphorylation and IRS2 ubiquitylation in cardiomyocytes. A: Chronic insulin exposure disrupted insulin-induced gene expression in cells. NRVMs were treated with 100 nmol/L insulin for 24 h (Pre-Ins 24 h) and then washed and treated with or without 100 nmol/L insulin for another 18 h, before harvesting of cellular RNA for real-time PCR determination. Graphs indicate relative expression of genes. Data are expressed as the mean ± SEM from at least three different experiments. *P < 0.05 vs. noninsulin treatment. B: Insulin induced phosphorylation of IRS1 at S636 and S612 in NRVMs. Cells were treated with 100 nmol/L insulin for 0.5, 3, and 24 h, and S636, S612, and Y608 of IRS1 were determined by immunoblotting. C: p38-WT and p38-DN differentially induced IRS1 serine phosphorylation at S636 and S612 in cells. NRVMs were infected with adenovirus as indicated (75 MOI), and cellular protein lysates were prepared for immunoblotting. D and E: Chronic insulin or p38 overexpression mediated IRS2 degradation in a 26S proteasome-dependent manner in cells. NRVMs were treated with 10 µmol/L MG132 for 0.5 h before 100 nmol/L insulin treatment for 24 h, and cellular proteins were then prepared for immunoblotting. E: For adenovirus infection, the cells were treated with MG132 for 0.5 h, and then adenovirus was added for 8 h, after which cells were washed and protein lysates prepared for immunoblotting. Graphs indicate relative IRS1 and IRS2 protein expression, and data are expressed as the mean ± SEM from three different experiments. *P < 0.05 vs. DMSO (D) or Ad-GFP (E). F: Dose effect of chronic insulin exposure decreased IRS1 and IRS2 protein expression. NRVMs were treated with 0, 1, 10, or 100 nmol/L insulin for 24 h, and cellular protein lysates were prepared for immunoblotting. Data are expressed as the mean ± SEM from at least three different experiments. *P < 0.05 vs. noninsulin treatment. N.S., no significant difference.