Gegham Barseghian

Decreased expression of liver epidermal growth factor receptors in rats with alloxan and streptozotocin diabetes

Male rats (200 g) were rendered diabetic with one intraperitoneal injection of alloxan (150 mg/kg) or streptozotocin (60 mg/kg). In hyperglycemic animals within 3 hours after the injection, the binding of EGF to liver membranes decreased by 43-52%; the maximal drop was by 70% and persisted for the 20 days of the experiment. EGF receptors decreased in number with almost no changes in their affinity. Autophosphorylation of the receptors decreased parallel to the ligand binding. In animals that received lower doses and did not develop diabetes and in animals in whom diabetes was prevented by the injections of glucose (before alloxan) or nicotinamide (before streptozotocin) the binding of EGF to liver receptors remained normal. We conclude that the decreased expression of EGF receptors was caused by diabetes and not by the toxic effects of the diabetogenic compounds on the liver.

Effect of Triton X-100 on insulin and epidermal growth factor receptor binding and autophosphorylation in Golgi fractions and partially purified receptors from rat liver.

Triton X-100 strongly affects the receptor binding and autophosphorylation of insulin and epidermal growth factor (EGF) in rat liver Golgi fractions and partially purified microsomal receptors. At concentration 0.05% Triton X-100 decreased the insulin receptor binding by 15% and the EGF receptor binding by 70% as compared to controls. In contrast, 0.05% Triton X-100 increased insulin-stimulated receptor autophosphorylation by more than 370% as compared to 87% in the control. Similarly, the same concentration of Triton X-100 increased the EGF-stimulated receptor phosphorylation by 65% as compared to 14% in the control. EGF receptor binding was more sensitive to the treatment of Triton. At Triton concentrations 0.2% or more, the EGF receptor binding was totally abolished while the insulin receptor binding was decreased by 50%. On the other hand, the activity of ligand-stimulated receptor phosphorylation of both insulin and EGF receptors was only slightly decreased in the presence of 0.2% Triton.

Ethanolamine mimics insulin effects invitro

The comparative effects of insulin and ethanolamine on 14CO2 production and lipid synthesis from [U-14C]-D-glucose in isolated rat adipocytes were studied. Ethanolamine (10 mM) increased 14CO2 production (glucose oxidation) about 5-fold and lipogenesis about 3-fold as compared to the control. Ethanolamine was more efficient than 25 microU/ml insulin regarding both parameters, but it was less efficient than 200 microU/ml insulin in glucose oxidation, and equally potent in lipogenesis. The combination of ethanolamine and insulin was more active than insulin alone. The mechanisms of ethanolamine action include facilitation of glucose transport and increase of pyruvate dehydrogenase activity.

Binding of epidermal growth factor (EGF) and insulin to human liver microsomes and Golgi fractions

Microsomes and Golgi fractions were isolated from 13 human liver samples without local malignancy. Binding of insulin to microsomes (per cent per 0.5 mg protein) was 14.4 +/- 7.9% with two classes of receptors: K1 = 1.4 nM, R1 = 0.28 pmol/mg; K2 = 8.1 nM, R2 = 0.62 pmol/mg. The binding was insignificantly lower than in rats. Binding of EGF was only 3.4 +/- 1.7% with two classes of receptors: K1 = 1.4 nM, R1 = 0.06 pmol/mg; K2 = 10.8 nM, R2 = 0.22 pmol/mg; the binding was much lower than in rats (26.3 +/- 5.8%). Binding of insulin to Golgi fraction (per cent per 0.1 mg protein) was 5.5 +/- 0.4% with straight line Scatchard plot; Kd = 5.6 nM, Ro = 3.06 pmol/mg; it was only half of that found in rats. In one case of hepatoma, the binding of insulin to microsomes was normal but that of EGF very low.

Absence of Down-Regulation of Insulin Receptors in Human Breast Cancer Cells (MCF-7) Cultured in Serum-Free Medium: Comparison with Epidermal Growth Factor

MCF-7 cells were cultured either in RPMI-1640 medium containing 10% fetal calf serum (FCS) or in a serum-free (SF) medium supplemented with insulin, epidermal growth factor (EGF) and transferrin. Binding studies were performed with 125I-insulin or 125I-EGF. In the FCS containing culture, down-regulation was seen for insulin receptors (47%), and for the EGF receptors (75%). Using cells grown in the serum-free medium we could not demonstrate down-regulation of the insulin receptors, while the EGF receptors were down-regulated to the same extent (74%). The number of binding sites per cell was about twice as much in cells cultured in FCS as that in SF medium. No significant differences were observed for receptor affinity of insulin or EGF in cells grown in both media. The rate of internalization of insulin or EGF into cells was similar in both culture conditions. The mechanism in which only EGF but not insulin demonstrated receptor down-regulation in SF medium remains unknown.

Fenfluramine inhibits insulin secretion and potentiates glucagon release by the perfused rat pancreas

Fenfluramine, an anorectic used in the treatment of obesity, in a final concentration of 1 mM strongly inhibited both phases of insulin release by the perfused rat pancreas. Insulin secretion resumed promptly after cessation of the drug infusion. This concentration of the drug markedly increased glucagon output. The blockade of alpha-adrenergic receptors and the use of antiserotonin agents did not alter the inhibitory effect of fenfluramine on insulin secretion. It is concluded that in the perfused rat pancreas 1 mM fenfluramine acutely inhibits glucose-induced insulin secretion and potentiates glucagon output. The direct effect of fenfluramine on insulin secretion is not related to alpha-adrenergic activity, nor is it mediated by serotonin.

In vitro effects of ethanolamine on insulin secretion

The effects of ethanolamine on insulin secretion by the perfused rat pancreas were examined. During the second phase of glucose-induced insulin secretion 5-minute perfusions of ethanolamine at final concentrations of 0.1, 1 and 10 mM inhibited insulin release in a dose-related manner. When given throughout the experiment the highest dose of ethanolamine markedly suppressed both phases of glucose-induced insulin release. The inhibitory effect of ethanolamine was blunted in the presence of phentolamine. It is concluded that ethanolamine inhibits glucose-induced insulin secretion by the perfused rat pancreas and that alpha-adrenergic receptors play a role in its actions on insulin output.