Equilibrium model for insulin-induced receptor down-regulation. Regulation of insulin receptors in differentiated BC3H-1 myocytes

The mechanism of insulin-induced down-regulation of surface membrane insulin receptors was studied in the muscle cell line BC3H-1. Down-regulation for the differentiated myocytes is dose- and time-dependent with a half-maximum response at 0.5 nM insulin and a maximum decrease of 50% in the number of surface insulin receptors following exposure to 20 nM insulin for 18 h at 37 degrees C, as confirmed by Scatchard analysis. These receptors were fully recoverable upon lysis of the down-regulated myocyte with Triton X-100, demonstrating that down-regulation is mediated solely by insulin-induced receptor internalization without detectable receptor degradation. Phospholipase C treatment of intact down-regulated cells and Triton X-100 treatment after subcellular fractionation showed that no cryptic or masked receptors were detectable within the plasma membrane. Insulin-induced receptor internalization was dependent upon cellular energy production, protein synthesis, and endocytosis, but was insensitive to agents which primarily affect lysosomal, cytoskeletal, or transglutaminase activities. The magnitude of insulin-induced down-regulation and the kinetics of down-regulation and recovery of cell surface receptors indicate that the surface and internal receptor pools are in dynamic equilibrium with each other. The kinetic data are accommodated by separate internalization rate constants for the unoccupied (0.01 h-1) and occupied (0.11 h-1) surface receptors and a single recycling rate constant (0.11 h-1) for the internalized receptors. This model also explains the previous apparently paradoxical finding in several other systems that down-regulation is more sensitive to hormone than hormone-receptor binding under physiologic conditions. Down-regulation in BC3H-1 myocytes, therefore, appears to be mediated solely by an insulin-induced increase in the receptor internalization rate constant and a consequent shift in the dynamic equilibrium between the surface and internalized receptor pools, resulting in a 50% decrease in the number of cell surface receptors. In other systems where the internalized hormone receptor is a substrate for rapid degradation, the essential role of this shift in mediating the down-regulation process may be obscured.     

The effects of insulin and tunicamycin on insulin receptors of cultured fibroblasts

The effect of down-regulation on the intracellular pool of insulin receptors and the role of glycosylation in recovery from down-regulation have been studied in fibroblastic cultures from the skin of non-diabetic mice. In control cultures, 55% of the total specific [¹²⁵I]insulin-binding activity was in the intracellular compartment. Insulin caused a time- and concentration-dependent decrease in the number of cell surface insulin receptors, with no significant change in total insulin receptors. This decrease in surface receptors was accompanied by an increase in the specific binding of [¹²⁵I]insulin in the intracellular compartment. Removal of insulin from down-regulated cells resulted in a time-dependent increase in the binding of [¹²⁵I]insulin to surface receptors, reaching 90% of that in controls by 12 h. The recovery of surface insulin receptors after removal of insulin was blocked by incubation of cultures with tunicamycin, but not by cycloheximide. These results indicate that down-regulation of surface insulin receptors by insulin is associated with translocation of receptors into the intracellular pool and suggest that protein glycosylation is important in insulin receptor recycling and externalization.     

Lack of insulin effect on its own receptors in fetal rat hepatocytes

To determine the effect of insulin on its receptor concentrations in hepatocytes of fetal and adult rats, these cells were preincubated in the presence or absence of insulin. The reduced [125I]-insulin binding observed in adult hepatocytes was dependent on the concentration of insulin and on the duration of exposure, while in fetal hepatocytes insulin did not induce any reduction in insulin binding. In contrast, glucagon receptors were unaffected by preincubation with insulin. The modifications observed in insulin binding were accounted for by changes in receptor concentrations rather than any change in receptor affinity for the hormone. Studies on the kinetic properties of the insulin receptors of fetuses and adult rats revealed that association and dissociation rates were undistinguishable. These results indicate an absence of insulin receptor down-regulation in the fetus, which could favour anabolic processes during intrauterine life.     

Time dependent effects of dexamethasone on serum insulin level and insulin receptors in rat liver and erythrocytes

The effects of glucocorticoid excess on regulation of insulin receptors were investigated in dexamethasone-treated rats. Glucocorticoid excess was produced by administration of dexamethasone (0.5 mg/100 g b.w.) 30 min, 4, 12, 18, 24, 42 or 70 h before experiments. This treatment caused time-dependent changes of glucose and insulin concentration in blood, as well as in amounts of specific insulin binding and insulin receptors of liver cells and erythrocytes. The time intervals in which dexamethasone produced the increase in insulin concentration were accompanied with decrease in insulin binding to receptors in membranes of liver cells, while significant changes in insulin binding to receptors of erythrocytes were not observed under the same experimental conditions. The effect is maximal 18 and 42 h after dexamethasone treatment that increase insulin blood level by about 85% and 60%, respectively. Receptor analysis revealed that changes in specific binding of insulin could be due to significant changes in amount of binding sites on cell surface rather than to mild alteration in receptor affinity. These findings suggest that besides the changes in insulin level, the alterations in insulin receptor number and affinity may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess.     

Insulin induces a similar reduction in the concentrations of its own receptor and of an insulin-sensitive glycosyl-phosphatidylinositol in isolated rat hepatocytes

We have used isolated rat hepatocytes to study whether the insulin-induced reduction of its own receptors may modify the transduction of hormone signals by changes in the content of a glycosyl-phosphatidylinositol. Both subsequent insulin binding and glycosyl-phosphatidylinositol concentrations markedly decreased as a function of time and insulin concentration during preincubation of hepatocytes with insulin. The modifications observed in insulin binding were due to changes in receptor concentration. These results show that insulin regulates both the number of its own receptors and glycosyl-phosphatidylinositol concentrations in target cells, which may be of interest in many pathophysiological situations.     

Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation

Three major functional characteristics of the insulin receptor are negative cooperativity, down-regulation, and beta-subunit tyrosine kinase activity. To investigate the inter-relationships among these functions we studied four antibodies to the insulin receptor alpha-subunit. These monoclonal antibodies competitively inhibited 125I-insulin binding to the insulin receptor of human IM-9 and HEP-G2 cells. When the antibodies were radiolabeled, insulin competed strongly with two antibodies (MA-10 and MA-51) for binding to the insulin receptor, but competed weakly with the two others (MA-5 and MA-20). Antibodies MA-10 and MA-51, like insulin, accelerated the dissociation of bound 125I-insulin from receptors; in contrast, MA-5 and MA-20 strongly inhibited 125I-insulin dissociation. Antibodies MA-10 and MA-51 induced down-regulation of insulin receptors with a potency similar to that of insulin. In contrast, MA-5 and MA-20 were more potent than insulin. None of the antibodies either alone or in combination influenced autophosphorylation of the insulin receptor beta-subunit. These data indicate, therefore, that two major epitopes can be identified on the alpha-subunit of the insulin receptor by the use of monoclonal antibodies. One epitope, recognized by antibodies MA-10 and MA-51, is close to or near the insulin-binding site and mimics insulin-induced negative cooperatively and down-regulation. The other epitope, recognized by antibodies MA-5 and MA-20, is at some distance from the insulin-binding site, and only mimics down-regulation. These data suggest, therefore, that: negative cooperativity and down-regulation may not be inter-related and both processes are independent of insulin receptor tyrosine kinase activity.     

Preparation and characterization of a colloidal gold-insulin complex with binding and biological activities identical to native insulin

We studied the binding and biological activities of gold-insulin complexes to develop a complex with properties identical to native insulin. Stabilizing amounts of insulin absorbed to 5-, 10-, or 15-nm gold particles resulted in complexes with 40-327 insulin molecules per gold particle and 4-111 times the biological activity of unlabeled insulin, based on the molar concentration of gold complex. These data suggested that these complexes behaved as multivalent ligands. Gold-insulin complexes were prepared with 5% of the stabilizing insulin concentration and were stabilized with bovine serum albumin. This resulted in a complex with 5-7 insulin molecules per 10-nm gold particle, which stimulated glucose oxidation in rat adipocytes and competed with [125I]-insulin for binding to the insulin receptor identically to unlabeled insulin on an equimolar basis. The organization and distribution of insulin receptors occupied by this monovalent-behaving gold-insulin complex were virtually identical to previous observations using monomeric ferritin-insulin. Since multivalent ligands may affect receptor binding, re-distribution, and intracellular processing, the use of electron-dense probes that resemble the unlabeled ligand in biological and binding properties is appropriate when studying receptor dynamics of in vivo or in vitro biological systems. The gold-insulin complex developed in this study should serve this function.     

Down-regulation and recycling of insulin receptors. Effect of monensin on IM-9 lymphocytes and U-937 monocyte-like cells

Receptor down-regulation is the result of various cellular processes including receptor internalization, new synthesis, and recycling. Monensin, a monocarboxylic acid ionophore, has been used to characterize the role of recycling in the metabolism of insulin receptors on two cultured human cell lines, U-937 and IM-9, which have different rates of internalization. The U-937 monocyte-like cell internalizes insulin receptors readily. Incubation with monensin at low doses (10(-6) to 10(-7) M) for 2 h did not affect subsequent surface insulin binding. However, the drug markedly enhanced insulin-induced down-regulation. Monensin had little effect on ligand internalization in this cell line as demonstrated by quantitative morphometric analysis. The IM-9 lymphocyte, a slow internalizer, was less sensitive to monensin exposure. Prolonged exposure (12 h) to this compound of either cell line resulted in apparent inhibition of insertion into the surface membrane of both newly synthesized and recycled receptors. When solubilization was used to quantitate total cell receptors, there was essentially no difference in intact cell binding (i.e. surface receptors) and total cell binding in IM-9 cells when insulin-induced down regulation alone was compared to insulin and monensin. By contrast for the U-937 cells there was only a small further decrease in binding when monensin was added to insulin in the solubilized cells compared to the marked augmentation of down-regulation when monensin was added to insulin in intact cells. These data demonstrate that cells with a rapid internalization rate have an associated active recycling process. By contrast cells with a slow internalization rate have a similarly slow recycling rate. This is consistent with relatively equal rates of receptor biosynthesis and plasma membrane insertion in both cell types.     

A simple computer model for insulin-receptor interactions and insulin dependent glucose uptake by adipocytes

A simple computer model is described for the simulation of insulin binding to cell surface receptors on adipocytes and the subsequent stimulation of glucose uptake. The model is based on the currently accepted physiology and biochemistry of insulin action. The model successfully simulated changes in sensitivity to insulin with changes in receptor numbers seen with in vitro experiments; it is also consistent with the proposal that an increased rate of insulin-receptor complex internalisation should lead to an insulin-resistant state. The model also suggests that such an insulin-resistant state should not be affected by a subsequent increase in the rate of return of internalised receptors to the outer cell surface.     

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.     

Effects of growth and insulin treatment on the levels of insulin receptors and their mRNA in Hep G2 cells

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.     

Properties and Regulation of the T Lymphocyte Insulin Receptor

Abstract

Primary human T lymphocytes that have been mitogen activated in chemically defined medium express cell surface insulin receptors. The receptor is identical to other mammalian insulin receptors in binding properties, including: pH dependency, ligand affinity, hormone specificity, and cooperative interactions. Scatchard plots are curvilinear and a ligand-induced increase in dissociation, the property normally associated with “negative cooperativity”, is kinetically demonstrable. In vitro insulin treatment of the receptor-negative, resting T lymphocyte slightly enhances the degree of insulin binding which emerges following cellular activation. Insulin treatment of receptor-positive lymphoblasts results in insulin receptor “down-regulation”. These findings indicate that T lymphoblast insulin receptor concentrations are not significantly influenced by insulin before their emergence but are dramatically regulated by insulin following their appearance at the cell surface.     

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.     

Insulin receptors in dog lymphocytes

The circulating lymphocyte because of its easy accessibility provides a useful model for the study of insulin receptors in dog. Consequently, we have studied the binding of insulin to lymphocytes prepared from Ficoll gradient. Our results demonstrate that circulating dog lymphocytes bind 125I-Insulin and this binding is inhibited by unlabeled insulin. This binding is function of the cell concentration and a saturable function of 125I-Insulin concentration. At 4 degrees C the equilibrium conditions are reached in 30 mn. Scatchard analysis revealed a cirvilinear plot. The number of receptors per cell was calculated as 1000 to 1500. This study have also revealated a decrease in the number of sites in diabetic dogs. We believe that the study of insulin receptor in lymphocyte's dog is important to understand some aspects of pathological states in diabetes.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

Effects of a long-lasting hyperinsulinemia induced by insulin infusion on the subcellular distribution of liver insulin receptors in the rat

Acute hyperinsulinemia in rats have been shown to cause enhanced endocytosis of liver insulin receptors with little or no change in the total receptor number. To determine whether a similar phenomenon occurs in long-lasting hyperinsulinemia, the subcellular distribution of liver insulin receptors has been studied in rats infused continuously with insulin (0.4 and 0.2 U/h) for 4 days. In rats in which plasma insulin concentration was maintained at 15-20 ng/ml, there was, from 3 to 24 h, a 2-fold decrease in insulin binding to plasma membranes (PM), along with 2 to 4-fold increase in insulin binding to the light (GEI), intermediate (GEi) and heavy (GEh) Golgi-endosomal fractions; concomitantly, there was a 10-fold increase in the insulin content of Golgi-endosomal fractions. After 24 h, the changes in insulin binding to PM and GEI were maintained, but the increase in both insulin binding activity and insulin content of GEi and GEh became progressively less marked, although plasma insulin concentration remained elevated. Throughout infusion, insulin binding to the total particulate fraction was unchanged. In rats, in which plasma insulin was maintained at 6-8 ng/ml, insulin binding to PM was decreased to a lesser degree and insulin binding to Golgi-endosomal fractions was unchanged (GEh) or decreased (GEI and GEi), although the insulin content of these fractions remained high. These results suggest that, while an enhanced receptor endocytosis accounts for the decrease in cell surface receptors observed at an early stage of the hyperinsulinemia, additional regulatory mechanisms are probably involved at a later stage.     

Insulin receptor desensitization correlates with attenuation of tyrosine kinase activity, but not of receptor endocytosis.

A model of insulin-receptor down-regulation and desensitization has been developed and described. In this model, both insulin-receptor down-regulation and functional desensitization are induced in the human HepG2 cell line by a 16 h exposure of the cells to 0.1 microM-insulin. Insulin-receptor affinity is unchanged, but receptor number is decreased by 50%, as determined both by 125I-insulin binding and by protein immunoblotting with an antibody to the beta-subunit of the receptor. This down-regulation is accompanied by a disproportionate loss of insulin-stimulated glycogen synthesis, yielding a population of cell-surface insulin receptors which bind insulin normally but which are unable to mediate insulin-stimulated glycogen synthesis within the cell. Upon binding of insulin, the desensitized receptors are internalized rapidly, with characteristics indistinguishable from those of control cells. In contrast, this desensitization is accompanied by a loss of the insulin-sensitive tyrosine kinase activity of insulin receptors isolated from these cells. Receptors isolated from control cells show a 5-25-fold enhancement of autophosphorylation of the beta-subunit by insulin; this insulin-responsive autophosphorylation is severely attenuated after desensitization to a maximum of 0-2-fold stimulation by insulin. Likewise, the receptor-mediated phosphorylation of exogenous angiotensin II, which is stimulated 2-10-fold by insulin in receptors from control cells, is completely unresponsive to insulin in desensitized cells. These data provide evidence that the insulin-receptor tyrosine kinase activity correlates with insulin stimulation of an intracellular metabolic event. The data suggest that receptor endocytosis is not sufficient to mediate insulin's effects, and thereby argue for a role of the receptor tyrosine kinase activity in the mediation of insulin action.     

Lack of desensitization of muscarinic receptor-mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase.

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor down-regulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.     

Effects of insulin on lipoprotein secretion in rat hepatocyte cultures. The role of the insulin receptor

Insulin inhibits the secretion of lipoprotein components such as triglyceride, phospholipid, and apolipoproteins B and E in primary rat hepatocyte cultures. The aim of this study was to determine whether these hormonal effects are related to the interaction of insulin with its receptor on the surface of cultured hepatocytes. Half-maximal inhibition of secretion of apolipoprotein E and triglyceride occurred at 6 ng/ml porcine insulin, equivalent to a 20% receptor occupancy. When compared to porcine insulin, both guinea pig insulin and desoctapeptide insulin were 60 times less inhibitory on triglyceride and apolipoprotein secretion. These analogs were also 60 times less effective in competing with porcine 125I-insulin for receptor binding. Anti-insulin receptor IgG inhibited binding of porcine insulin to cells in a dose-dependent fashion. However, similar to the hormone itself, it reduced the secretion of triglyceride and apolipoproteins E and B. Preincubation of cells with 200 ng/ml porcine insulin for 15 h caused a 2.5-fold reduction of surface receptor number. These cells were less sensitive to the inhibitory effect of porcine insulin on secretion of triglyceride and apolipoproteins B and E. We conclude that the effects of insulin on lipoprotein processing by hepatocytes in culture are receptor-mediated, can be imitated by antibodies, to the insulin receptor, and are subject to control by receptor down-regulation.