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Regulation of intracellular protein degradation by insulin in cultured cells
Abstract
The mechanism by which insulin acts to inhibit intracellular protein degradation has been investigated in differentiating 3T3-L1 preadipocytes, Balb/c 3T3 fibroblasts, and SV40-transformed Balb/c 3T3 fibroblasts. Intracellular protein degradation in serum-free medium is measured as the rate of release of trichloroacetic acid-soluble radioactivity from cell monolayers prelabelled with {('3)H}leucine. During 3T3-L1 preadipocyte differentiation, protein degradation becomes more sensitive to inhibition by insulin in the physiological concentration range. Specific insulin binding per mg protein increases 1.5-fold during adipocyte conversion, but it does not correlate with the greater than 10-fold decrease in the insulin concentration which produces half-maximal inhibition of protein degradation. Evidence is presented which suggests that insulin suppresses protein degradation, and presumably autophagy, in 3T3-L1 adipocytes by a mechanism which is cyclic AMP-independent. The polypeptide hormone stimulates an accumulation of total cell protein based on its effect on protein turnover. Adrenocorticotropic hormone (ACTH) and the (beta)-adrenergic agonists, isoproterenol and epinephrine, also inhibit protein degradation in 3T3-L1 adipocytes, but by a mechanism which is cyclic AMP-dependent. In contrast to insulin, ACTH and the (beta)-adrenergic agonists do not stimulate protein labelling in adipocytes. The glucocorticoid analog, dexamethasone, stimulates protein degradation in both preadipocytes and adipocytes. Its effect is persistent and is greater in differentiated 3T3-L1 cells. Experiments with the calmodulin inhibitor, trifluoperazine, and the calcium ionophore A23187 indicate that calcium is not required for the effect of insulin on protein turnover. The removal of Na('+) from the medium, but not K('+), decreases protein degradation in Balb/c 3T3 and SV40-3T3 fibroblasts. The action of insulin on protein turnover in these two cell lines appears to involve a stimulation of K('+) uptake. The inhibition produced by insulin on protein degradation in SV40-3T3 fibroblasts is more sensitive to the removal of K('+) from the medium and to the ouabain-induced inhibition of the plasma membrane Na('+)/K('+)-ATPase. These results suggest that the Na('+)/K('+)-ATPase is more active in transformed cell lines compared to their contact-inhibited counterparts. This enhanced pump activity may give rise to the lower rates of protein breakdown and the increased sensitivity of protein breakdown to insulin in transformed cells.
Description
Typescript (photocopy).Subject
Biochemistry1983 Dissertation W874
Proteins
Metabolism
Insulin
Physiological effect
Cell culture
Collections
Citation
Wood, Carla Mauree (1983). Regulation of intracellular protein degradation by insulin in cultured cells. Texas A&M University. Texas A&M University. Libraries. Available electronically from https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -541474.
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