Insulin Receptor Turnover Research Articles

Effects of anti-insulin receptor antibodies (AIRA) on downregulation and turnover of insulin receptors on cultured hepatocytes.

We have studied the effects of two polyclonal anti-insulin receptor antibodies (AIRA) on insulin receptor downregulation and turnover in rat hepatocytes in primary culture. Downregulation was determined by measurement of insulin binding after acid washing of cells to remove AIRA. Insulin receptor turnover was estimated by measurement of insulin binding after inhibition of synthesis of functional receptors with tunicamycin (0.5 micrograms/ml). Exposure of hepatocytes to AIRA (both sera were of comparable effectiveness) resulted in progressive, time- and dose-dependent losses of insulin binding (maximal loss was about 55% after 24 h of incubation with AIRA diluted 1:25). Cycloheximide (100 microM) prevented AIRA-mediated downregulation. The t1/2 of disappearance of cell surface insulin binding capacity determined with tunicamycin was 8.0 h. Addition of insulin (1000 ng/ml) or AIRA to tunicamycin reduced the t1/2 to 2.6 h (insulin), 2.2 h (patient B10), and 2.0 h (patient 1). These data suggest that AIRA downregulated insulin receptors on cultured hepatocytes by accelerating their rate of disappearance, inhibition of protein synthesis prevented AIRA-mediated downregulation, and downregulation by AIRA of insulin binding may be partially responsible for the desensitization of target cells to some of the insulin-like actions of these autoantibodies.

Dexamethasone stimulates insulin receptor synthesis in cultured rat hepatocytes.

The ability of the glucocorticoid dexamethasone to modulate the insulin receptor was examined directly in primary cultures of hepatocytes prepared from adult male rats. Hepatocytes were cultured in a defined medium in the presence and absence of dexamethasone, 0.1 microM. The exposure of hepatocytes to dexamethasone resulted in a time-dependent (steady state by 32 h) increase in insulin binding in both intact hepatocytes and Triton X-100-soluble extracts (total insulin receptor content). The enhanced insulin binding found in soluble extracts of dexamethasone-treated hepatocytes was the result of an increase in insulin receptor number without a change in receptor affinity. In order to assess the mechanism by which dexamethasone "up-regulates" the insulin receptor, the heavy isotope density-shift technique was used to analyze insulin receptor turnover in control and dexamethasone-treated hepatocytes. Hepatocytes were initially cultured for 32 h in standard culture media containing only "light" (14C, 12C, 1H) amino acids. In hepatocytes exposed to dexamethasone, a 417% increase in insulin binding in Triton X-100-soluble extracts was observed. After 32 h, when steady state binding is achieved in dexamethasone-treated cultures, parallel cultures of hepatocytes incubated in the absence and presence of dexamethasone were washed and subsequently cultured in media containing "heavy" amino acids (15N, 13C, 2H). The time-dependent disappearance of light insulin receptor (receptor degradation) and appearance of heavy insulin receptor (receptor synthesis) were monitored using CsCl gradients to resolve the two density species of receptor. At steady state, the rate of receptor synthesis (k8) was 2.94 and 0.62 fmol of insulin bound h-1 in dexamethasone-treated and control hepatocytes, respectively. In contrast to this large increase in the rate of receptor synthesis observed in dexamethasone-treated cells, the first order rate constant for decay (k d) was the same in dexamethasone-treated (0.074 h-1) and in control (0.077 h-1) hepatocytes. We therefore conclude that glucocorticoid-induced up-regulation of the insulin receptor in the liver is due to stimulation of insulin receptor synthesis.

Cycloheximide causes accumulation of insulin receptors at the cell surface of cultured fibroblasts.

Incubation of confluent cultures of mouse fibroblasts with cycloheximide caused a time-dependent increase in the binding of 125I-insulin. The increase was concentration-dependent between 0.05 and 3.5 microM cycloheximide and showed a high correlation (r = 0.97) between the increase in 125I-insulin binding and the inhibition of protein synthesis. In the presence of 3.5 microM cycloheximide, insulin binding increased to 236% of control and incorporation of [3H]valine into proteins fell to 10-20% of control. Scatchard analysis of the binding data indicated that cycloheximide-treated cultures had a total of 6.6 X 10(4) binding sites/cell compared to 3.9 X 10(4) sites/cell in untreated cultures. No significant changes in affinity were observed. Other protein synthesis inhibitors also caused an increase in 125I-insulin binding. With 25 mM ethionine and 2 mM sodium fluoride, binding was 155 and 245% of control and incorporation of [3H]leucine into proteins was decreased to 41 and 47% of control, respectively. These results suggest that the accumulation of insulin receptors at the cell surface following treatment with cycloheximide results from inhibition of synthesis of proteins involved in insulin receptor turnover.

Role of glycosylation and protein synthesis in insulin receptor metabolism by 3T3-L1 mouse adipocytes.

The roles of glycosylation and protein synthesis in the maintenance of insulin receptor levels and turnover rates in 3T3-L1 adipocytes were investigated. The heavy isotope density-shift technique was employed to determine the effects of inhibitors of these processes on the rates of synthesis and degradation of cellular insulin receptors. Inhibitors of protein synthesis--i.e., cycloheximide and puromycin--markedly decreased the rate of degradation of the insulin receptor, the half-life for receptor decay increasing from 7.5 hr without to 25 hr with inhibitor. The continued synthesis of a short-lived protein appears to be necessary for normal insulin receptor turnover. Tunicamycin, a potent inhibitor of core oligosaccharide addition in the formation of N-glycosidically linked glycoproteins, caused the depletion of cell-surface and total cellular detergent-extractable insulin receptors. This inhibitor totally prevented the formation of functional newly synthesized insulin receptor, yet receptor degradation was affected minimally. Thus, glycosylation of the receptor appears to be required for its activation after translation.