Insulin labeled with iodine-125 binds to receptors on isolated rat hepatocytes. At low temperatures initial binding is restricted to the plasma membrane as detected by direct quantitative autoradiographic analysis with the electron microscope. With increasing time and temperature of incubation there is a systematic and progressive translocation of autoradiographic grains to a highly limited area of the cell periphery representing no more than 15% of the radius of the cell.
From mutagenized Chinese hamster ovary (CHO) cells we have isolated, in a single step, 11 independent mutants resistant to the growth-inhibitory effects of 8-Br-cyclic AMP, cholera toxin, and methylisobutylxanthine. Two major classes and several subclasses of mutants were obtained. Mutants from all classes have a normal doubling time. None of the mutants respond to cyclic AMP treatment with increased flattening and elongation as do the parental cells. Members of the first class have an altered protein kinase activity which has either an increased Ka for cyclic AMP or an absent response to cyclic AMP. Most of those mutations which result in a protein kinase with increased Ka for cyclic AMP (6/11) are dominant in somatic cell hybrids. Those mutations which result in a protein kinase with little or no response to cyclic AMP (3/11) are recessive. Members of the second major class (2/11) have normal levels of basal and cyclic AMP-dependent protein kinase activity. One is recessive and one is dominant by genetic tests. The basis for the defect in this second class of mutants has not been determined.
Summary.By quantitative electron microscopic autoradiographic technique, we have previously shown that I2sI-insulin initially localizes to the plasma membrane of isolated rat hepatocytes and is subsequently internalized in a limited region of the peripheral cytoplasm. In the present study, we have shown that when cells are incubated at 20 ~ steady state binding is reached by 60 minutes and maintained up until 120 minutes of incubation while at 37 ~ steady state binding is reached by 10 minutes and maintained for 30 minutes. Under both of these conditions, internalization of the labelled material occurs as a constant function of the binding. These data suggest that under normal conditions the binding of the ligand is an important rate limiting determinant of the internalization process.Key words: Insulin receptor, internalization, endocytosis, hepatocytes, binding, insulin.The initial interaction of 125I-insulin with hepatocytes occurs through specific, saturable receptors on the surface of the plasma membrane. More recently, it has been demonstrated in isolated hepatocytes [1,2,3,4] and in hepatocytes from intact liver [5,6] that the interaction of insulin with its cell surface receptor results in the internalization of the ligand. While initial binding is primarily controlled by the affinity of insulin for the receptor (K) and the number of receptor sites (Ro), the factors controlling internalization are unknown. In the present study, we have investigated the relationship between the binding of the hormone and its internalization. Materials and Methods Cells and ReagentsHepatocytes were isolated from normal 6 to 8 weeks old Wistar rats fed ad libitum using a modification of the method described by Seglen [7]. IasI-insulin was prepared at a specific activity of 250 vCi/gg by a modification of the chloramine-T-method [8]. The labelled insulin was purified by filtration on G-50 Sephadex at 4 ~ prior to each experiment. Incubation ConditionsHepatocytes at a final concentration of 1 • 10 6 eens/rnl were incubated in duplicate in 0.5 ml of modified Krebs Ringer bicarbonate (KRB) (pH 7.7), containing 25 mg/ml bovine serum albumin (Fraction V) and 0.8 mg/ml of bacitracin, with 5 • 10-1~ ~25I-insulin at 20 ~ and 37 ~ for varying periods of time. Bacitracin was added in order to prevent insulin degradation in the medium during exposure to hepatocytes [9]. Identical incubations were carried out in the presence of 2.7 • 10-5mol/1 unlabelled insulin to determine non-specific binding (i. e. cell associated radioactivity in the presence of an excess of unlabelled hormone). At the times indicated, 1 ml of chilled buffer was added to each tube, immediately followed by centrifugation for 20 seconds at 50 • g. The supematant was discarded and the cell pellet quickly resuspended in I ml of chilled buffer and centrifuged for 90 seconds at about 500 • g in a Beckman plastic microfuge tube. Cell pellets were further washed (without resuspension) by chilled buffer containing 100 mg/ml of sucrose. At the end of the wash procedure, 4% g...
ATP-dependent potassium (K ATP) channels occupy a key position in the control of insulin release from the pancreatic beta cell since they couple cell polarity to metabolism. These channels close when more ATP is produced via glucose metabolism. They are also controlled by sulfonylureas, a class of drugs used in type 2 diabetic patients for triggering insulin secretion from beta cells that have lost part of their sensitivity to glucose. We have demonstrated the existence of endogenous counterparts to sulfonylureas which we have called 'endosulfines.' In this review, we describe the discovery, isolation, cloning, and biological features of the high-molecular-mass form, alpha-endosulfine, and discuss its possible role in the physiology of the beta cell as well as in pathology.
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