Activation of SOCS-3 by resistin

Abstract

Resistin is an adipocyte hormone that modulates glucose homeostasis. Here we show that in 3T3-L1 adipocytes, resistin attenuates multiple effects of insulin, including insulin receptor (IR) phosphorylation, IR substrate 1 (IRS-1) phosphorylation, phosphatidylinositol-3-kinase (PI3K) activation, phosphatidylinositol triphosphate production, and activation of protein kinase B/Akt. Remarkably, resistin treatment markedly induces the gene expression of suppressor of cytokine signaling 3 (SOCS-3), a known inhibitor of insulin signaling. The 50% effective dose for resistin induction of SOCS-3 is approximately 20 ng/ml, close to levels of resistin in serum. Association of SOCS-3 protein with the IR is also increased by resistin. Inhibition of SOCS function prevented resistin from antagonizing insulin action in adipocytes. SOCS-3 induction is the first cellular effect of resistin that is independent of insulin and is a likely mediator of resistin's inhibitory effect on insulin signaling in adipocytes.

FIG. 1.

Resistin inhibits insulin-stimulated tyrosine phosphorylation of IR and IRS-1 in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with resistin (30 ng/ml) for 2 h prior to insulin stimulation (10 nM) for 2 min, and then phosphorylation of IR and IRS-1 and total protein levels were determined by immunoblot analysis. (a) Effects of resistin on IR tyrosine phosphorylation (p-tyr). *, P = 0.002. (b) Effects of resistin on IRS-1 tyrosine phosphorylation. *, P = 0.008. A representative Western blot and quantitation of three independent experiments are shown.

FIG. 2.

Resistin inhibits insulin-stimulated IRS-1-associated PI3K and PIP₃ generation in living cells. 3T3-L1 adipocytes were treated with resistin (30 ng/ml) for 2 h prior to insulin stimulation (10 nM) for 2 min. (a) Effects of resistin on IRS-1-associated PI3K activity. The fold increase in PI3K activity relative to the basal levels is noted in parentheses. Data are represented as the percent of insulin effect in control cells and the average of three independent experiments. *, P < 0.05. (b) Effects of resistin on in vivo PIP₃ accumulation at the plasma membrane. 3T3-L1 adipocytes were infected with an adenovirus expressing a PIP₃ binding protein, GFP-GRP1-PH, 1 day prior to resistin and insulin treatment as in panel a. Fluorescence confocal laser microscopy was used to image the living cells before and after insulin stimulation (5 min). An arrow denotes a representative cell which upon insulin stimulation results in the accumulation of PIP₃ at the plasma membrane in response to insulin. A movie of these experiments is available (see Video S1 in the supplemental material). (c) Quantitation of the change in fluorescence intensity of the plasma membrane (pm) relative to the cytosol (cyt) calculated as [(pm − cyt)/(pm + cyt)] following insulin stimulation for 5 min (Final) minus values prior to insulin stimulation (Initial). Data are expressed as the mean values of 10 cells ± SEM. *, P = 0.05.

FIG. 3.

Resistin decreases insulin-stimulated serine and threonine phosphorylation of Akt in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with resistin (30 ng/ml) for 2 h prior to insulin stimulation (10 nM) for 2 min, and then phosphorylation of Akt on serine 473 (pSer-473) and threonine 307 (pThr-308) and total Akt protein levels were measured by immunoblot analysis. A representative Western blot and quantitation of three independent experiments are shown. *, P < 0.05.

FIG. 4.

Resistin treatment does not result in serine phosphorylation of IRS-1. Serum-starved 3T3-L1 adipocytes were either untreated (UNT) or treated with vehicle control or resistin (30 ng/ml) for 2 h prior to insulin stimulation for 15 min. Immunoblot (IB) analysis using IRS-1 and phosphospecific antibodies generated to specific serine residues (Ser 307 and 626/639) is shown. IP, immunoprecipitation.

FIG. 5.

Time course of resistin-induced insulin resistance. 3T3-L1 adipocytes were treated with resistin (30 ng/ml) for various times prior to insulin stimulation (10 nM) for 2 min. In vitro PI3K activity is represented as a percentage of the insulin effect in control cells and is the average of three independent experiments. *, P < 0.05 as compared to control cells treated with insulin.

FIG. 6.

Resistin activates SOCS-3 in a time- and dose-dependent manner in 3T3-L1 adipocytes. (a) Effect of 2-h treatment with resistin (30 ng/ml) on SOCS-3 mRNA relative to 36B4. (b) Time course of effects of resistin (30 ng/ml) on SOCS-3 mRNA. *, P < 0.02. (c) Dose response for SOCS-3 mRNA induction after resistin treatment for 2 h. The average values ± SEM of three to five independent experiments are shown. *, P < 0.05.

FIG. 7.

SOCS-3 induced by resistin associates with the IR. 3T3-L1 adipocytes were treated with resistin (30 ng/ml) or TNF-α (30 ng/ml) for the indicated times. (a) Treatment with TNF-α, but not resistin, reduces IRS-1 protein levels. Actin immunoblot analysis was used as a loading control. (b) Resistin stimulates IR association of SOCS-3. Cell extracts were immunoblotted (IB) for IR and SOCS-3 following immunoprecipitation (IP) of IR. (c) Time course of resistin enhancement of IR association with SOCS-3.

FIG. 8.

Resistin activates SOCS-3 in vivo. Resistin (1.5 μg) or vehicle was administered i.p. to male C57BL/6J mice following an overnight fast, epididymal adipose tissue was isolated at the indicated time points, and SOCS-3 and 36B4 mRNA was measured by qPCR. SOCS-3 expression was normalized to that of 36B4, and the data shown are the fold activation in resistin-treated mice relative to vehicle-treated mice. The average values ± SEM of three independent experiments are shown. *, P < 0.04 compared to time zero.

FIG. 9.

Dominant-negative SOCS protein prevents resistin antagonism of insulin signaling in adipocytes. 3T3-L1 adipocytes were infected with adenovirus expressing EGFP or SOCSF59D and then treated with resistin (30 ng/ml) for 2 h prior to insulin stimulation (10 nM) for 2 min, and then phosphorylation of IR and total IR protein levels were determined by immunoblot analysis.