guanosine-5--o-(3-thiotriphosphate) and Diabetes-Mellitus

Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

Topics: Adenosine Triphosphate; Adult; Awards and Prizes; Calcium; Carbachol; Cytoplasm; Diabetes Mellitus; Europe; Glucagon; Glucose; Guanosine 5'-O-(3-Thiotriphosphate); History, 20th Century; Humans; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Membrane Potentials; Models, Biological; Patch-Clamp Techniques; Signal Transduction; Societies, Medical; Sweden

A novel pathway for physiological "cross-talk" between the insulin receptor and the regulatory Gi-protein has been demonstrated. We tested the hypothesis that a coupling defect between Gi and the insulin receptor is present in the liver of obese patients with and without type II diabetes. Insulin 1 x 10(-9) M (approximately ED50) and 1 x 10(-7) M (Max) inhibited pertussis toxin-catalyzed ADP ribosylation of Gi in human liver plasma membranes from lean and obese nondiabetic patients. However, 1 x 10(-7) M insulin was without effect in membranes from patients with type II diabetes. This coupling defect was not intrinsic to Gi, since Mg2+ and GTP gamma S inhibited pertussis toxin-catalyzed ADP ribosylation in both diabetic and nondiabetic patients. Binding of insulin of the alpha-subunit and activation of the tyrosine kinase intrinsic to the beta-subunit of the insulin receptor are not responsible for the coupling defect. 125I insulin binding is the same in obese patients with or without diabetes. Tyrosine kinase of the insulin receptor is decreased in diabetes. However, a monoclonal antibody to the insulin receptor (MA-20) at equimolar concentrations with insulin equally inhibits pertussis toxin-catalyzed ADP ribosylation of Gi without activating tyrosine kinase or insulin receptor autophosphorylation. Immunodetection of G-proteins suggested that Gi3 alpha was normal in diabetes and Gi1-2 alpha was decreased by 40% in the diabetic group as compared to the obese nondiabetic group but was normal when compared to the lean non diabetic group. We conclude that the novel pathway of insulin signaling involving the regulatory Gi proteins via biochemical mechanisms not directly involving the tyrosine kinase of the insulin receptor is altered in obese type II diabetes and offers a new target for the search of the mechanism(s) of insulin resistance.

Topics: Adenosine Diphosphate Ribose; Antibodies, Monoclonal; Cholera Toxin; Cytosol; Diabetes Mellitus; Diabetes Mellitus, Type 2; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Insulin; Insulin Resistance; Liver; Membrane Proteins; Obesity; Pertussis Toxin; Poly(ADP-ribose) Polymerases; Receptor Protein-Tyrosine Kinases; Receptor, Insulin; Signal Transduction; Virulence Factors, Bordetella