FIG. 3.

Chronic insulin (Ins) exposure reduces IRS1 and IRS2 and attenuates Akt→Foxo1 phosphorylation in cardiomyocytes. A: Immunoblot analysis of IRS, Akt, and Foxo1 phosphorylation after 100 nmol/L insulin treatment for 0.5, 3, and 24 h in NRVMs. *P < 0.05 vs. noninsulin treatment from three independent experiments. B: Detection of insulin sensitivity in NRVMs after chronic insulin treatment. Cells were treated with 100 nmol/L insulin for 24 h (Pre-Ins 24h) and washed with DMEM, followed by adding 100 nmol/L insulin for another 0.5 h, and cellular protein lysates were prepared for immunoblotting. Graphs indicate the relative expression levels of phosphorylation of Akt at S⁴⁷³ (p-Akt) and Foxo1 at S²⁵³ (p-Foxo1), which were normalized to total Akt and Foxo1 protein, respectively, from three different experiments. *P < 0.05 vs. noninsulin treatment. ⁺P < 0.05 vs. 0.5 h insulin treatment in each group. C: Detection of insulin sensitivity after chronic high-glucose treatment in NRVMs. Cells were treated with 25 mmol/L glucose (HG), with or without 100 nmol/L insulin for 24 h, and then washed and treated with the same dose insulin for another 0.5 h, and cell protein lysates were prepared for immunoblotting. *P < 0.05 vs. noninsulin treatment; ⁺P < 0.05 vs. HG plus 0.5 h insulin treatment. D: Overexpression of IRS1 or IRS2 rescued insulin sensitivity after chronic insulin treatment in NRVMs. Cells were infected with IRS1 or IRS2 adenovirus (Ad-IRS1or Ad-IRS2, 75 MOI) for 8 h, starved 8 h with serum-free medium before insulin (Pre-Ins) 24 h, and then washed and treated with insulin for another 0.5 h. IRS, Akt, and Foxo1 phosphorylation were estimated by immunoblotting. ⁺P < 0.05 vs. Ad-GFP with 0.5 h insulin treatment.