The Potential Role of SOCS-3 in the Interleukin-1β-Induced Desensitization of Insulin Signaling in Pancreatic Beta-Cells

IL-1β inhibits the insulin-induced IRS/p85^PI3K complex formation and IRS-associated PI3K activity in RINm5F cells. RINm5F cells were starved overnight then pretreated or not with IL-1β (10 ng/ml) for 2 h. Cells were then stimulated or not with insulin (100 nmol/l) for 5 min and lysed. IRS proteins were immunoprecipitated and then subjected to Western blot or PI3K activity analysis. A: The association of PI3K p85 regulatory subunit with IRS proteins was assessed by an anti-p85^PI3K Western blot. Quantification of the presence of PI3K p85 regulatory subunit in IRS immunocomplex is expressed as a mean ± SEM from three independent experiments. **P < 0.01 B: PI3K assay was performed with IRS immunoprecipitates as described in research design and methods.

The inhibitory effect of IL-1β on insulin signaling pathway is not dependent on NO production. RINm5F cells were starved overnight and then submitted to several treatments before insulin stimulation. Cells were pretreated with IL-1β (10 ng/ml) for 2 h in lanes 3–6. In lanes 5 and 6, IL-1β was coincubated with NAME (2 mmol/l). As a control, cells were incubated with NAME alone for 2 h (lanes 7 and 8). Cells were preincubated with SNAP (100 μmol/l) for 1 h (lanes 9 and 10) or 2 h (lanes 11 and 12). Cells were then stimulated or not with insulin (100 nmol/l) for 5 min and lysed. IRS proteins were immunoprecipitated and tyrosine phosphorylation was assessed by an antiphosphotyrosine Western blot.

IL-1β induces SOCS-3 mRNA expression and SOCS-3/IR complex formation in RINm5F cells. RINm5F cells were starved overnight and then stimulated or not with IL-1β (10 ng/ml) for 30 min, 1 h, 2 h, 3 h, or 4 h. A: Total RNA was extracted and analyzed by Northern blot using SOCS-3 as a probe. RNA loading and integrity were verified with an 18S ribosomal probe. Quantification of SOCS-3 mRNA (arbitrary units). B: After 2 h of treatment, total RNA was extracted and analyzed by real-time quantitative PCR using SOCS-3 primers. mRNA expression was normalized using hypoxanthine guanine phosphoribosyl transferase (HPRT) RNA levels. Results are expressed as a mean ± SEM from three independent experiments. C: RINm5F cells were starved overnight then pretreated or not with IL-1β (10 ng/ml) for 2 h. Cells were then stimulated or not with insulin (100 nmol/l) for 5 min and lysed. IRβ subunits were immunoprecipitated with an anti-IRβ antibody. An anti-SOCS-3 Western blot was then performed to analyze the coimmunoprecipitation of SOCS-3 with IRβ subunits. The expression levels of IRβ subunits was monitored by anti-IRβ Western blot of total lysate.

Ectopically expressed SOCS-3 associates with IRβ subunits and decreases insulin-dependent IR autophosphorylation and IRS tyrosine phosphorylation in RINm5F cells. RINm5F cells were infected or not with comparable amounts of GFP-AdV or SOCS-3-AdV. Forty-eight hours after infection, cells were starved overnight then stimulated or not with insulin (100 nmol/l) for 5 min and lysed. A: IRβ subunits were immunoprecipitated with anti-IRβ antibody. Anti-SOCS-3 and antiphosphotyrosine Western blot were then performed. B: IRS proteins were immunoprecipitated with an anti-IRS antibody recognizing both IRS-1 and IRS-2. Tyrosine phosphorylation of these proteins was assessed by an antiphosphotyrosine Western blot. Quantification of IRS tyrosine phosphorylation is expressed as a mean ± SEM from five independent experiments. *P < 0.05. The expression levels of IRβ subunits, IRS-1, and IRS-2 were monitored by anti-IRβ, anti-IRS-1, and anti-IRS-2 Western blot of total lysate, respectively. Ectopically expressed SOCS-3 was revealed after an immunoprecipitation with a rabbit polyclonal anti-SOCS-3 antibody followed by a Western blot with a goat polyclonal anti-SOCS-3 antibody.