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V P Knutson

Publications and source records attributed to V P Knutson.

25 records · Page 2Linked to original sources
PubMed1983

Rapid, reversible internalization of cell surface insulin receptors. Correlation with insulin-induced down-regulation.

Chronic treatment of 3T3-C2 fibroblasts with insulin causes the slow (t1/2 = 3-4 h) down-regulation of cellular insulin receptor to a new steady state level by accelerating receptor decay (Knutson, V.P., Ronnett, G.V., and Lane, M.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In the present investigation, the synthesis and turnover of the receptor during the transition to the down-regulated state was examined by the heavy isotope density-shift method. It was observed that within two h after insulin addition, receptor decay increased abruptly for several hours then gradually declined until the "down-regulated" rate was achieved. The abrupt increase in receptor decay induced by insulin was preceded by a more rapid (t1/2 less than or equal to 10 min) translocation of cell surface receptor to an "intracellular" trypsin-resistant compartment. Thus, upon exposure to ligand, insulin receptor rapidly redistributes from the cell surface to an intracellular compartment, without an initial net loss of cellular receptors. The translocation process was rapidly reversed (t1/2 less than or equal to 20 min) upon removal of insulin. With prolonged exposure to insulin, the initial rapid translocation of receptor was followed by a slower inactivation of receptor apparently in the intracellular compartment. Cycloheximide, which lengthens receptor half-life by blocking a step in receptor inactivation, had no effect on receptor internalization. Internalization of insulin receptor and its bound ligand were, however, rapidly (less than 10 min) blocked by phenylarsine oxide. These results support the following sequence of events. Upon exposure to ligand, insulin receptors are translocated from the cell surface to an intracellular site which results in accelerated receptor decay and ultimately to a lower steady state cellular receptor level.

Animals↗
PubMed1983

Kinetics of insulin receptor transit to and removal from the plasma membrane.

The heavy isotope density shift method, in combination with a procedure for labeling cell surface insulin receptors, was used to determine the rate of transit of receptor to the cell surface from their site of synthesis and to follow the net rate of receptor removal from the plasma membrane in 3T3-L1 adipocytes. To label surface receptors, 125I-insulin was bound to cells at 4 degrees C and then covalently cross-linked to the receptors with disuccinimidyl suberate. The identity of the surface-labeled product as insulin receptor was established by immunoprecipitation with antireceptor antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fully differentiated 3T3-L1 adipocytes were shifted to medium containing heavy (greater than 95% 15N, 13C and 2H) amino acids. The rates of appearance of newly synthesized heavy receptor at the cell surface and the loss of previously synthesized light receptor from the cell surface were followed by resolving labeled heavy and light surface receptors in CsCl density gradients and quantitating labeled receptor subunits by gel electrophoresis. It was shown that 2.5-3.0 h are required for newly synthesized insulin receptor to reach and become functional in the plasma membrane. Insulin-induced down-regulation of cellular insulin receptor level had no effect on the time required for the newly synthesized receptors to reach the cell surface. Down-regulation, however, increased the first order rate constants for the inactivation of cell surface insulin receptors from 0.046 to 0.10 h-1. The fact that the rate constants for inactivation of cell surface and total cellular insulin receptors were identical in the up-regulated state (0.046 and 0.044 h-1, respectively) or in the down-regulated state (0.10 and 0.096 h-1, respectively) suggests that the rate-limiting step in the receptor inactivation pathway occurs at the cell surface.

Adipose Tissue↗
PubMed1982

Electric pulse-induced fusion of 3T3 cells in monolayer culture.

Swiss mouse 3T3-C2 fibroblasts, grown to confluence in monolayer culture, are shown to fuse when exposed to electric fields. Exposure to five repetitive electric pulses of about 1 kilovolt per centimeter with a duration of 50 microseconds caused approximately 20 percent of the cells to become fused (multinucleate) when 1 millimolar magnesium was present in the medium. The effects of minimum thresholds of field strength, pulse duration, and number of pulses were determined. Cell disruption was observed when the electric field exceeded 2.0 kilovolts per centimeter or the pulse was of longer duration than 120 microseconds.

Animals↗
PubMed1982

Insulin-induced down-regulation of insulin receptors in 3T3-L1 adipocytes. Altered rate of receptor inactivation.

Fully differentiated 3T3-L1 adipocytes, maintained in the presence of insulin, exhibit up-regulation of insulin-binding capacity when insulin is removed from the culture medium. Both cell surface and total cellular insulin receptors increase by 1.8- to 2.0-fold during the 24-h period following the removal of insulin. When up-regulated 3T3-L1 cells are exposed to 10(-8) M insulin down-regulation of insulin receptors occurs with a t1/2 of 2-3 h. Down-regulation was complete after a 10-h exposure to insulin and resulted in a 50-60% decrease in levels of cell surface and total cellular insulin-binding capacities, respectively. Scatchard analysis revealed that these changes in insulin binding are due to an alteration of receptor number and not insulin-binding affinity. To clarify the mechanism(s) by which the regulation of insulin receptor level occurs, rates of receptor synthesis and degradation were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of up-regulation or down-regulation. Up-regulation, however, caused an increase in receptor half-life from 8.1 h in the control cells to 14.8 h. Subsequent down-regulation brought about a return of receptor half-life to 6.9 h. These results indicate that insulin-dependent regulation of insulin receptor level in 3T3-L1 adipocytes involves a change in the rate of receptor degradation. Further studies indicated that regulation of insulin receptor level has physiological significance, since up-regulated cells exhibit an increased responsiveness of 2-deoxyglucose uptake to insulin compared to down-regulated cells.

Adipose Tissue↗
PubMed1982

Formation and isolation of delta 5-3-ketosteroids using a purified rat liver alcohol dehydrogenase.

3 beta-Hydroxy-5-androsten-17-one is converted to 5-androstene-3, 17-dione by rat liver alcohol dehydrogenase (ADH). We have reported on the purity of the enzyme which is eluted with pyrazole as a single homogeneous protein using an AMP-agarose affinity column. Rat liver ADH can oxidize hydroxyl groups not only at 3 beta-, but also at 3 alpha-, and 17 beta-positions to a lesser extent; thus it is a pure mammalian enzyme with multifunctional activity for steroids. Since it does not contain delta 5-isomerase activity, the reaction of the dehydrogenase to form the delta 5-ketosteroid intermediate can be observed at pH 7.0, 25 degrees C. Similarly, intermediary product, 5-pregnene-3,20-dione, can be isolated in the conversion of pregnenolone by ADH to progesterone. With buffer alone in a cuvette, a non-enzymatic isomerization of the delta 5-3-ketone occurs at a slow rate (t 1/2 = 6 hrs) but occurs rapidly during isolation procedures. The delta 5-3-ketosteroid intermediates were identified by their behavior on TLC plates with UV light and by their characteristic spectra in the NMR.

Alcohol Dehydrogenase↗
PubMed1982

Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation.

Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down- and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in total receptor level are due to changes in cell-surface receptor level is indicated by the fact that the level of trypsin-insensitive, presumably intracellular, insulin binding sites does not change appreciably upon down- and up-regulation. The effects of insulin-induced down-regulation and dexamethasone-induced up-regulation on the rates of insulin receptor synthesis and decay were assessed by the heavy-isotope density-shift technique. Cells were shifted to medium containing heavy (2H, 13C, 15N) amino acids and, at various times after the shift, light and heavy receptors solubilized from total cellular membranes were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that the insulin- and dexamethasone-induced alterations in insulin receptor level were due entirely to changes in the rate constant for receptor inactivation. The decrease in the first-order rate constant for receptor decay caused by dexamethasone is unexpected in view of the known action of steroid hormones in the induction of the synthesis of specific proteins.

Animals↗