The effects of CP 68722 (racemic englitazone) were examined in ob/ob mice, in adipocytes and soleus muscles from ob/ob mice, and in 3T3-L1 adipocytes. Administration of englitazone at 5-50 mg.kg-1.day-1 lowered plasma glucose and insulin dose dependently without producing frank hypoglycemia in either the diabetic or nondiabetic lean animals. The glucose-lowering effect in ob/ob mice preceded the reduction in hyperinsulinemia. On cessation of drug, plasma insulin returned to untreated levels within 48 h, whereas plasma glucose rose slowly over 5 days. Englitazone (50 mg/kg) for 11 days lowered plasma glucose (22.2 +/- 1.4 to 14.0 +/- 1.9 mM), insulin (7.57 +/- 0.67 to 1.64 +/- 0.60 nM), nonesterified fatty acids (1813 +/- 86 to 914 +/- 88 microM), glycerol (9.20 +/- 0.98 to 4.94 +/- 0.03 mM), triglycerides (1.99 +/- 0.25 to 1.03 +/- 0.11 g/L), and cholesterol (6.27 +/- 0.96 to 3.87 +/- 0.57 mM), but no effects were observed 3 h after a single dose. Basal and insulin-stimulated lipogenesis were enhanced in adipocytes from ob/ob mice treated with 50 mg/kg englitazone for 11 days compared with lipogenesis in cells from vehicle-treated controls. Treatment of ob/ob mice with 50 mg/kg englitazone reversed the defects in insulin-stimulated glycolysis (from [3-3H]glucose) and glycogenesis and basal glucose oxidation (from [1-14C]glucose) in isolated soleus muscles. Englitazone (30 microM) stimulated 2-deoxy-D-glucose transport in 3T3-L1 adipocytes from 0.37 +/- 0.03 to 0.65 +/- 0.06 and 1.53 nmol.min-1.mg-1 protein at 24 and 48 h, respectively. Thus, englitazone has 1) insulinomimetic and insulin-enhancing actions in vitro and 2) glucose-, insulin-, triglyceride-, and cholesterol-lowering properties in an animal model of non-insulin-dependent diabetes mellitus (NIDDM) in which sulfonylureas have little or no effect. Thus, this new agent may have beneficial effects including a reduced risk of hypoglycemia in patients with NIDDM.

Human A431 epidermoid carcinoma cells in culture exhibit epidermal growth factor (EGF)-induced "down-regulation" of cell-surface and total cellular (Triton X-100 extractable) EGF receptors caused entirely by an enhanced rate (4-fold) of receptor inactivation [Krupp, M. N., Connolly, D. T. & Lane, M. D. (1982) J. Biol. Chem. 257, 11489-11496]. The following observations show that this enhanced rate of EGF receptor inactivation is closely correlated with an increased cellular activity of plasminogen activator (PA), a serine protease. First, EGF-induced down-regulation of cell-surface and total cellular EGF receptors and the concomitant increase in cellular PA activity occur with identical kinetics, the tl/2 for both processes being 3-3.5 hr.Second, the EGF dose-response curves for down-regulation of total cellular EGF receptor and increased PA activity are similar. The EGF concentrations for half-maximal responses of both processes are 10-15 nM and 20 nM, respectively. Third, the removal of EGF from previously down-regulated cells results in the recovery of total cellular EGF binding activity with a concurrent loss of cellular PA activity. Fourth, blocking PA synthesis or activity with cycloheximide or dexamethasone prevents down-regulation of the EGF receptor. Fifth, the addition of leupeptin, an inhibitor of PA and plasmin action, blocks EGF-induced receptor downregulation as well as the increase of PA activity. That EGF receptor down-regulation is independent of plasminogen per se in the culture medium suggests that PA-mediated events may initiate the rapid inactivation of the EGF receptor that occurs during downregulation.The interaction of epidermal growth factor (EGF), a polypeptide hormone, with specific cell-surface receptors initiates numerous biochemical events (1) in target cells, most notably mitogenesis (2). Such factors as the period of exposure to EGF, the concentration of EGF, and the level of functional receptors at the plasma membrane affect the magnitude of ligand-receptor interactions and, thus, the cellular response to EGF (3-5). It has been established that cells can modulate their level of surface EGF receptors in response to EGF and thereby alter the biological response to this potent mitogen (3-5).Like other receptors for polypeptide hormones (6), the EGF receptor exhibits ligand-induced "down-regulation" of cell-surface EGF binding capacity (3-5). This negative modulation of EGF receptor level in vitro is rapid, EGF concentration dependent, and EGF specific and occurs in many cell types (1, 3, 5). Reduced EGF binding capacity is the result of a lowered EGF receptor level rather than an altered affinity of the EGF receptor for its ligand (3,5 Although the mechanism by which ligand-induced downregulation promotes EGF receptor inactivation is unknown, there is some evidence with another cell-surface receptor that proteolysis modulates receptor metabolism. When proteolysis in chicken myogenic cultures is induced by chemical or viral transformation, both the steady-state concentratio...

The components of fat cell membranes responsible for the binding of insulin were solubilized by treatment with the nonionic detergent Triton X-100. By using a polyethylene glycol precipitation method to assay specific insulin binding, the The binding of insulin to target cells has been extensively studied in many laboratories (1-5). Evidence from these studies suggests that this interaction is a complex process and not a simple bimolecular association of the hormone with one class of binding sites. This nonclassical behavior is indicated primarily by the curvilinear Scatchard plot (6) obtained from analysis of insulin-binding data and the accelerated dissociation of bound 125I-labeled insulin (l25I-insulin) after the addition of native hormone (7). Several models have been proposed to explain these findings. Initially, the curvilinear Scatchard plot was attributed to the presence of two or more classes of binding sites that have different affinities for insulin (8). However, this model alone cannot accommodate the results of the dissociation studies. More recently, DeMeyts and coworkers (7, 9), who first described the effect of native hormone on the dissociation of labeled insulin, proposed a negative cooperative model that involves insulin binding to a homogeneous class of empty highaffinity sites (10). These sites then undergo conformational changes through site-site interactions that result in their transformation to a low-affinity state. A third model, the mobile receptor hypothesis, has been advanced by Jacobs and Cuatrecasas (11), by Boeynaems and Dumont (12), and by De HienThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2593(13); it is based on the possible existence in the membrane of an equilibrium between receptors and receptor-effector complexes that differ in their affinities for insulin. Both the negative cooperativity model and the mobile receptor hypothesis provide explanations for the shape of the Scatchard plots and for the dissociation data.Support for the validity of these or other possible models requires more detailed information on the insulin binding structure(s) than is now available. In the present report, we show evidence that two distinct insulin-binding components exist in detergent-solubilized material prepared from membranes of adipocytes, a well-established target cell for insulin. The two species have significantly different insulin-binding characteristics, which may explain some of the features of the insulin-adipocyte interaction.

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