Tumor necrosis factor (TNF)-alpha is postulated to play a major role in the pathogenesis of obesity-linked insulin resistance, probably resulting from an interaction with insulin signaling pathways. This cross talk has now been investigated in human adipocytes at the level of phosphatidylinositol (PI) 3-kinase, and the TNF receptors (TNFRs) mediating these processes have been identified. Equilibrium binding studies using human adipocytes from mammary tissue indicated the presence of two populations of TNFR with apparent affinity constants of 13 pmol/l and 1.6 nmol/l, respectively. Interaction of TNF-alpha with insulin signaling was determined by quantification of insulin receptor substrate (IRS)-1-associated PI 3-kinase activity. Under control conditions, PI 3-kinase was activated about 10-fold in response to insulin (10[-7] mol/l, 5 min). Preincubation of adipocytes with 5 nmol/l TNF-alpha for 15 min resulted in a 60-70% reduction of insulin action, reaching a stable inhibition (40%) after longer incubation with the cytokine. The inhibitory action of TNF-alpha was dose-dependent, already detectable at 10 pmol/l, and was correlated to inhibition of tyrosine phosphorylation of IRS-1 with an unaltered autophosphorylation of the insulin receptor beta-subunit. The modulation of insulin signaling by TNF-alpha was found to be paralleled by a comparable inhibition of insulin-stimulated glucose transport. An agonistic TNFR1 antibody completely mimicked the inhibitory action of TNF-alpha on insulin signaling, whereas at 100 pmol/l TNF-alpha, a nonagonistic p80 TNFR antibody, was shown to ameliorate the inhibitory action of the cytokine. These findings indicate that in human adipocytes, low concentrations of TNF-alpha induce a rapid inhibition of insulin signaling at the level of PI 3-kinase. We suggest that under these conditions, the p80 TNFR is essential for initiating the intracellular cross talk that involves signaling by the p60 TNFR.

OBJECTIVE: Increased levels of hypothalamic neuropeptide-Y (NPY) are thought to contribute to the manifestation of the obese phenotype. The aim of this study was to characterize the interactions between leptin, insulin and NPY in the pathogenesis of polygenic obesity. DESIGN AND METHODS: A polygenic obese animal model, the New Zealand obese mouse (NZO) and its age-matched control, was used to assess the hypothalamic mRNA expression of NPY, the insulin receptor (IR) and the leptin receptor (Ob-Rb), by semiquantitative polymerase chain reaction. Experiments were performed early (at eight weeks old) and late (at 40 weeks old) in the life of these animals. RESULTS: Serum glucose was signi®cantly elevated in obese mice. Serum insulin levels were not different between obese and lean mice, whereas serum leptin levels were signi®cantly elevated in obese mice and increased continuously during life [reaching extremely high values at 40 weeks (41.5 AE 4.1 vs 3.4 AE 0.25 ngaml for obese and lean, respectively). The hypothalamic expression of NPY mRNA was signi®cantly higher in NZO mice compared to controls at both eight weeks (2.3-fold) and 40 weeks (1.9-fold), respectively, whereas expression of IR and Ob-Rb remained unaffected. CONCLUSION: Increased hypothalamic expression of NPY due to leptin resistance, may be involved in the development of polygenic obesity. Unchanged Ob-Rb expression suggests that either a defective hypothalamic uptake or defects in Ob-Rb signalling underly this process.

Freshly isolated adult rat ventricular cardiomyocytes have been used to characterize the action profile of the new thiazolidinedione antidiabetic drug MCC-555. Preincubation of cells with the compound (100 microM for 30 min or 10 microM for 2 h) did not modify basal 3-O-methylglucose transport, but produced a marked sensitizing effect (2- to 3-fold increase in insulin action at 3 x 10(-11) M insulin) and a further enhancement of maximum insulin action (1.8-fold). MCC-555 did not modulate autophosphorylation of the insulin receptor and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). However, insulin action (10(-10) and 10(-7) M) on IRS-1-associated phosphatidylinositol (PI) 3-kinase activity was enhanced 2-fold in the presence of MCC-555. Association of the p85 adapter subunit of PI 3-kinase to IRS-1 was not modified by the drug. Immunoblotting experiments demonstrated expression of the peroxisomal proliferator-activated receptor-gamma in cardiomyocytes reaching about 30% of the abundance observed in adipocytes. The insulin-sensitizing effect of MCC-555 was lost after inhibition of protein synthesis by preincubation of the cells with cycloheximide (1 mM; 30 min). Cardiomyocytes from obese Zucker rats exhibited a completely blunted response of glucose transport at 3 x 10(-11) M insulin. MCC-555 ameliorates this insulin resistance, producing a 2-fold stimulation of glucose transport, with maximum insulin action being 1.6-fold higher than that in control cells. This drug effect was paralleled by a significant dephosphorylation of IRS-1 on Ser/Thr. In conclusion, MCC-555 rapidly sensitizes insulin-stimulated cardiac glucose uptake by enhancing insulin signaling resulting from increased intrinsic activity of PI 3-kinase. Acute activation of protein expression leading to a modulation of the Ser/Thr phosphorylation state of signaling proteins such as IRS-1 may be underlying this process. It is suggested that MCC-555 may provide a causal therapy of insulin resistance by targeted action on the defective site in the insulin signaling cascade.