Tumor necrosis factor-α (TNF-α) is an important mediator of insulin resistance in obesity and diabetes through its ability to decrease the tyrosine kinase activity of the insulin receptor (IR). Treatment of cultured murine adipocytes with TNF-α was shown to induce serine phosphorylation of insulin receptor substrate 1 (IRS-1) and convert IRS-1 into an inhibitor of the IR tyrosine kinase activity in vitro. Myeloid 32D cells, which lack endogenous IRS-1, were resistant to TNF-α-mediated inhibition of IR signaling, whereas transfected 32D cells that express IRS-1 were very sensitive to this effect of TNF-α. An inhibitory form of IRS-1 was observed in muscle and fat tissues from obese rats. These results indicate that TNF-α induces insulin resistance through an unexpected action of IRS-1 to attenuate insulin receptor signaling.

The SOCS proteins are induced by several cytokines and are involved in negative feedback loops. Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2. Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression. Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter. Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity. Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif. First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds. Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor. This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960. Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3. We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation. By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr⁹⁶⁰ of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-α (TNF-α), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-α induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-α target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.

Tumor necrosis factor (TNF)-alpha plays a central role in the state of insulin resistance associated with obesity. It has previously been shown that one important mechanism by which TNF-alpha interferes with insulin signaling is through the serine phosphorylation of insulin receptor substrate-1 (IRS-1), which can then function as an inhibitor of the tyrosine kinase activity of the insulin receptor (IR). However, the receptors and the signaling pathway used by TNF-alpha that mediate the inhibition of IR activity are unknown. We show here that human TNF-alpha, which binds only to the murine p55 TNF receptor (TNFR), is as effective at inhibiting insulin-dependent tyrosine phosphorylation of IR and IRS-1 in adipocytes and myeloid 32D cells as murine TNF-alpha, which binds to both p55 TNFR and p75 TNFR. Likewise, antibodies that are specific agonists for p55 TNFR or p75 TNFR demonstrate that stimulation of p55 TNFR is sufficient to inhibit insulin signaling, though a small effect can also be seen with antibodies to p75 TNFR. Exogenous sphingomyelinase and ceramides, known to be formed by activation of p55 TNFR, inhibit IR and IRS-1 tyrosine phosphorylation and convert IRS-1 into an inhibitor of IR tyrosine kinase in vitro. Myeloid 32D cells expressing IR and IRS-1 are sensitive to this inhibition, but cells expressing IR and IRS-2 are resistant, pointing to an important difference in the biological function between IRS-1 and IRS-2. These data strongly suggest that TNF-alpha inhibits insulin signaling via stimulation of p55 TNFR and sphingomyelinase activity, which results in the production of an inhibitory form of IRS-1.

OBJECTIVE—Obesity is characterized by an overgrowth of adipose tissue that leads to the formation of hypoxic areas within this tissue. We investigated whether this phenomenon could be responsible for insulin resistance by studying the effect of hypoxia on the insulin signaling pathway in adipocytes. RESEARCH DESIGN AND METHODS—The hypoxic signaling pathway was modulated in adipocytes from human and murine origins through incubation under hypoxic conditions (1% O2) or modulation of hypoxia-inducible factor (HIF) expression. Insulin signaling was monitored through the phosphorylation state of several key partners of the pathway and glucose transport. RESULTS—In both human and murine adipocytes, hypoxia inhibits insulin signaling as revealed by a decrease in the phosphorylation of insulin receptor. In 3T3-L1 adipocytes, this inhibition of insulin receptor phosphorylation is followed by a decrease in the phosphorylation state of protein kinase B and AS160, as well as an inhibition of glucose transport in response to insulin. These processes were reversible under normoxic conditions. The mechanism of inhibition seems independent of protein tyrosine phosphatase activities. Overexpression of HIF-1α or -2α or activation of HIF transcription factor with CoCl2 mimicked the effect of hypoxia on insulin signaling, whereas downregulation of HIF-1α and -2α by small interfering RNA inhibited it. CONCLUSIONS—We have demonstrated that hypoxia creates a state of insulin resistance in adipocytes that is dependent upon HIF transcription factor expression. Hypoxia could be envisioned as a new mechanism that participates in insulin resistance in adipose tissue of obese patients.

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein α and peroxisome proliferator-activated receptor (PPAR) γ2 expression, whereas PPARγ1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte-specific markers, and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells. Disclosure of potential conflicts of interest is found at the end of this article.