Tumor necrosis factor-alpha (TNF-alpha) 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-alpha 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-alpha-mediated inhibition of IR signaling, whereas transfected 32D cells that express IRS-1 were very sensitive to this effect of TNF-alpha. An inhibitory form of IRS-1 was observed in muscle and fat tissues from obese rats. These results indicate that TNF-alpha induces insulin resistance through an unexpected action of IRS-1 to attenuate insulin receptor signaling.

Previously, we have isolated and characterized an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice. The key regulator of this enhancer is a cell type-restricted nuclear factor termed ARF6. Target sequences for ARF6 in the aP2 enhancer exhibit homology to a direct repeat of hormone response elements (HREs) spaced by one nucleotide; this motif (DR-l) has been demonstrated previously to be the preferred binding site for heterodimers of the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). We have cloned a novel member of the peroxisome proliferator-activated receptor family designated mPPART2, and we demonstrate that a heterodimeric complex of mPPAR~/2 and RXR~ constitute a functional ARF6 complex. Expression of mPPAR,/2 is induced very early during the differentiation of several cultured adipocyte cell lines and is strikingly adipose-specific in vivo. mPPAR-/2 and RXRc~ form heterodimers on ARF6-binding sites in vitro, and antiserum to RXRc~ specifically inhibits ARF6 activity in adipocyte nuclear extracts. Moreover, forced expression of mPPAR~/2 and RXR~ activates the adipocyte-specific aP2 enhancer in cultured fibroblasts, and this activation is potentiated by peroxisome proliferators, fatty acids, and 9-cis retinoic acid. These results identify mPPAR~/2 as the first adipocyte-specific transcription factor and suggest mechanisms whereby fatty acids, peroxisome proliferators, 9-cis retinoic acid, and other lipids may regulate adipocyte gene expression and differentiation.

Insulin resistance is an important metabolic abnormality often associated with infections, cancer, obesity, and especially non-insulin-dependent diabetes mellitus (NIDDM). We have previously demonstrated that tumor necrosis factor-cr produced by adipose tissue is a key mediator of insulin resistance in animal models of obesity-diabetes. However, the mechanism by which TNF-a interferes with insulin action is not known. Since a defective insulin receptor (IR) tyrosine kinase activity has been observed in obesity and NIDDM, we measured the IR tyrosine kinase activity in the Zucker (fa/fa) rat model of obesity and insulin resistance after neutralizing TNF-a with a soluble TNF receptor (TNFR) -lgG fusion protein. This neutralization resulted in a marked increase in insulin-stimulated autophosphorylation of the IR, as well as phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle and fat tissues of the fa/fa rats, restoring them to near control (lean) levels. In contrast, no significant changes were observed in insulin-stimulated tyrosine phosphorylations of IR and IRS-1 in liver. The physiological significance of the improvements in IR signaling was indicated by a concurrent reduction in plasma glucose, insulin, and free fatty acid levels. These results demonstrate that TNF-a participates in obesity-related systemic insulin resistance by inhibiting the IR tyrosine kinase in the two tissues mainly responsible for insulin-stimulated glucose uptake: muscle and fat. (J. Clin. Invest. 1994Invest. . 94:1543Invest. -1549

Previously, we identified a novel transcription factor, ARF6, as a key regulator of the tissue-specific adipocyte P2 (aP2) enhancer. In order to identify the proteins which comprise the adipocyte ARF6 complex, we have purified this DNA binding activity from a cultured adipocyte cell line. We have developed a system for growth and differentiation of HIB-1B brown adipocytes in suspension culture that facilitates the production of large quantities of adipocyte nuclear extract. ARF6 was purified from HIB-1B nuclear extract by a combination of conventional and sequence-specific DNA affinity chromotography. Chemical sequencing and mass spectral analysis of tryptic peptides derived from the purified polypeptides identifies the ARF6 complex as a heterodimer of the retinoid X receptor alpha (RXR alpha) and the murine peroxisome proliferator activated receptor gamma (PPAR gamma). Of the known PPAR gamma isoforms, PPAR gamma is the predominant form expressed in adipose tissue. These results suggest that PPAR gamma 2 serves a unique function among PPAR family members as an important regulator of adipocyte-specific gene expression.