Figure 3.

Prolonged adipokine contact induces insulin resistance by interfering with insulin receptor substrate 1 expression. (A,B) CHRF-288-11 cells lose insulin sensitivity upon prolonged contact with resistin. Cells were pre-incubated with resistin, leptin or IL-6 (72 h, 37°C) and insulin inhibition of thrombin-induced Ca²⁺ mobilization was measured in the absence and presence of the Ser phosphatase inhibitor cantharidin (CA, 1 μmol/L, 15 min, 37°C). Resistin and leptin but not IL-6 induce induce insulin resistance; cantharidin fails to rescue insulin sensitivity. (C,D) Leptin and resistin but not IL-6 induce degradation of insulin receptor substrate 1. After incubation with resistin, leptin, IL-6 or untreated controls (24 h, 37°C), expression of insulin receptor substrate 1, insulin receptor, G_iα₂ and insulin receptor substrate 2 was measured. Leptin and resistin (but not IL-6) induced a fall in insulin receptor substrate 1 expression but not in the other proteins. Data are means ± SEM, n=4. (E) A JAK2 inhibitor rescues insulin sensitivity. CHRF-288-11 cells were incubated with resistin, leptin and IL-6 without and with the JAK2 inhibitor AG490 (100 nM, 15 min, 37°C). (F) Leptin initiates upregulation of SOCS3. CHRF-288-11 cells were incubated with leptin for 0 – 32 h and expression of SOCS3 was measured. (G). Leptin induces ubiquitination of insulin receptor substrate 1. CHRF-288-11 cells were incubated with leptin, the proteasome inhibitor MG132 (10 μM) or both (1 or 2 h, 37°C) and expression of ubiquitinated insulin receptor substrate 1 was measured.