Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

Sephadex (G-50 fine grade)-gel chromatography and trichloroacetic acid (TCA) precipitation were used to investigate the effects of chloroquine and bacitracin on the nature of cell-associated radioactivity in studies on the binding and degradation of 125I-insulin in cultured rat hepatocytes. Sephadex peak I, eluted with the void volume, increased with hepatocyte incubation time and comprised 6% of total cell-bound radioactivity at 120 min. However, all radioactivity in this peak was due to unspecific binding. Peak II, corresponding to intact insulin, represented 95% of specifically cell-associated label at 5 min and decreased to 77% at 120 min. Peak III, containing the final low-Mr degradation products, increased with incubation time (22% of specifically bound label at 120 min). The TCA-precipitable and TCA-soluble fractions of hepatocytes extracted with 0.1% SDS were within 4-7% of the proportions of radioactivity in peaks II and III respectively. Scatchard plots based on insulin-binding data from Sephadex chromatography or TCA precipitation were identical. Dissociation studies revealed that at least 75% of the intact insulin associated with the hepatocytes was bound to receptors at the cell surface. Bacitracin increased the proportion of cell-associated intact hormone and decreased that of ligand degraded when analysed by either Sephadex chromatography or TCA precipitation. The proportion of surface-bound to internalized intact hormone remained unaltered, indicating that bacitracin acted predominantly at the cell surface. In the presence of chloroquine, which dramatically increased the contribution of peak I to specific binding, 'intact' insulin was substantially overestimated when determined as the TCA-precipitable fraction. In addition, all peak I material and 50% of cell-associated label in peak II was trapped intracellularly, thereby pointing to the lysosomal or prelysosomal site of action of this drug.     

The degradation of semisynthetic tritiated insulin by perfused mouse livers.

Semisynthetic [3H]insulin was stored under various conditions for up to 180 days and the stability of the insulin under these conditions was assessed. A sample that had been stored for 180 days was repurified and shown to be degraded at the same rate as native insulin by perfused mouse livers, even at low physiological concentrations. After perfusion, intact insulin could be separated from degradation products, and the radioactivity associated with the insulin fraction could be used to determine the percentage degradation. The initial rate of degradation of insulin was a linear function of concentration over the range 360pM-1.9nM.     

Involvement of hormone processing in insulin-activated glucose transport by isolated cardiac myocytes.

Isolated muscle cells from adult rat heart were used to study the relationship between myocardial insulin processing and insulin action on 3-O-methylglucose transport at 37 degrees C. Internalization of the hormone as measured by determination of the non-dissociable fraction of cell-bound insulin increased linearly up to 10 min, reaching a plateau by 30-60 min at 3 nM-insulin. At this hormone concentration the onset of insulin action was found to be biphasic, with a rapid phase up to 8 min, followed by a much slower phase, reaching maximal insulin action by 30-60 min. Insulin internalization was totally blocked by phenylarsine oxide, whereas dansylcadaverine had no effect on this process. Initial insulin action (5 min) on glucose transport was not affected by chloroquine and dansylcadaverine, but was completely abolished by treatment of cardiocytes with phenylarsine oxide. This drug effect was partly prevented by the presence of 2,3-dimercaptopropanol. Under steady-state conditions (60 min), the stimulatory action of insulin was decreased by about 60% by both chloroquine and dansylcadaverine. This study, demonstrates that insulin action on cardiac glucose transport is mediated by processing of the hormone. The data suggest dual pathways of insulin action involving initial processing of hormone-receptor complexes and lysosomal degradation.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.     

Receptor-mediated insulin degradation and insulin-stimulated glycogenesis in cultured foetal hepatocytes.

Insulin-stimulated glycogenesis and insulin degradation were studied simultaneously at 37 degrees C in cultured foetal hepatocytes grown for 2-3 days in the presence of cortisol. Degradation of cell-associated insulin, as measured by trichloroacetic acid precipitation, was significant after 4 min in the presence of 1-3 nM-125I-labelled insulin. This process became maximal (30% of insulin degraded) after 20 min, a time when binding-state conditions were achieved. No insulin-degradative activity was detected in a medium that had been exposed to cells. At steady-state, the appearance of insulin degradation products in the medium was linearly dependent on time (1.5 fmol/min per 10(6) cells at 1nM-125I-labelled insulin). Chloroquine (3-50 microM), bacitracin (0.1-10 mM) and NH4Cl (1-10 mM) inhibited insulin degradation as soon as this became detectable and caused an increase in the association of insulin to hepatocytes after 20 min. Lidocaine and dansylcadaverine had similar effects, whereas N-ethylmaleimide, aprotinin, phenylmethanesulphonyl fluoride and leupeptin were found to be ineffective. Chloroquine, and also bacitracin, at concentrations that inhibited insulin degradation, decreased the insulin-stimulated incorporation of [14C]glucose into glycogen over 2 h. This effect of chloroquine was specific, since it did not modify the basal glycogenesis, or the glycogenic effect of a glucose load in the absence of insulin. It therefore appears that the receptor-mediated insulin degradation (or some associated pathway) is functionally related to the glycogenic effect of insulin in foetal hepatocytes.     

Internalization of insulin and its receptor in the isolated rat adipose cell. Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37 degrees C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [125I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [125I]insulin to these same membrane fractions. At 37 degrees C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20-30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16 degrees C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a direct effect of bacitracin on cell-mediated insulin degradation in isolated hepatocytes

In freshly isolated hepatocytes, in which extracellular degradation of insulin was very low, the degradation velocity was first-order with respect to the amount of insulin bound at steady state. The addition of bacitracin decreased the degradation velocity considerably, so that a higher proportion of cell-associated radioactivity remained intact. The results demonstrate that bacitracin affects the mechanism of insulin processing by intact hepatocytes.     

Internalization of the neonatal brain insulin receptor

Using 10-15 day neonatal rabbit brain cells, we studied the internalization (n = 6) and intracellular degradation (n = 8) of specifically bound 125I-insulin. In addition we investigated the association between the internalization of the specifically bound 125I-insulin and the metabolic effects of insulin such as glucose (n = 13) and amino-acid (leucine) uptake (n = 6). Phenylarsine oxide (10 microM), an agent that inhibits the internalization of the insulin receptor (n = 6) decreased the specifically bound 125I-insulin in the intact and trypsin-resistant (inside) part of the brain cells by 50% (p less than 0.05). On the other hand chloroquine (100 microM), a lysosomotropic agent that interferes with the intracellular degradation of the insulin receptor (n = 8) increased two-fold the 125I-insulin specifically bound to the intact and trypsin resistant part of the cells (p less than 0.05). Both these agents did not alter the time-dependent basal glucose uptake by the brain cells. Glucose alone regulated its own uptake (n = 4) whereas 1 X 10(-6) M insulin did not augment the glucose uptake (n = 11+13) above basal. Similarly leucine regulated the leucine uptake (n = 4) but insulin did not alter this basal uptake by the brain cells (n = 6). In summary we observed no associated glucose or leucine uptake along with the presence of internalization and intracellular degradation of specifically bound 125I-insulin in the brain cells.     

Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting

We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.     

Shunting of insulin from a retroendocytotic pathway to a degradative pathway by sodium vanadate

Adipocytes route internalized insulin through two major pathways, a degradative pathway and a retroendocytotic pathway. To examine whether sorting of incoming insulin-receptor complexes can be altered, we assessed the effect of vanadate on the intracellular processing of both insulin and insulin receptors. After cells were pretreated with vanadate (1 mM for 30 min at 37 degrees C), 125I-insulin was loaded into the cell interior. When the net efflux of insulin from cells into the medium was then monitored, vanadate was found to slow the efflux of insulin from a t1/2 of 6.2 min (controls) to 11 min. Since efflux reflects both the rapid extrusion of intact insulin and the slower release of degradative products, we proposed that vanadate diverts more insulin into the degradative pathway. Further evidence in support of this idea included the following: 1) when intracellular degradation of insulin was impaired by chloroquine, undegraded insulin accumulated faster within vanadate-treated cells, consistent with greater flux through a degradative pathway; 2) vanadate increased the percentage of degraded insulin released from cells from 61 and 72%; and 3) under steady-state binding conditions, more insulin resided in the cell interior of vanadate-treated cells (44.8% versus 34.5%), and the time required for the intracellular pool to reach equilibrium was prolonged (t1/2 of 5.5 min versus 4.0). Neither insulin internalization nor degradation was impaired by vanadate alone. In related studies Tris was found to inhibit insulin-mediated receptor recycling by only 10%, whereas in the presence of vanadate (plus Tris) almost all incoming insulin receptors were prevented from recycling. Vanadate alone had no effect on the ability of insulin receptors to recycle. Based on these results we conclude that: 1) vanadate shunts incoming insulin from a more rapid retroendocytotic pathway to a slower degradative pathway and diverts insulin receptors from a Tris-insensitive recycling pathway to one that can be completely inhibited by Tris; 2) these effects are selective, in that vanadate impairs neither insulin degradation nor receptor uptake and recycling. Considered together, these findings support the idea that a sorting mechanism exists for the intracellular routing of incoming insulin-receptor complexes.     

Turnover of adenosine 3'':5''-cyclic monophosphate in chicken erythrocytes.

Turnover of cyclic AMP was studied in intact chicken erythrocytes. Production of cyclic AMP was stimulated by adrenaline and then blocked by propranolol. The decline in the cyclic AMP concentration under these conditions is solely due to its intracellular degradation, whereas efflux of the nucleotide, although existing in these cells, does not contribute significantly to the change in its concentration. Intracellular degradation of cyclic AMP follows a first-order kinetics with a half-life of about 6 min. Similar half-lives were obtained at widely different adrenaline concentrations or when the ration of propranolol to adrenaline was varied by 25-fold. Theoretical equations were applied to calculate the rates of cyclic AMP synthesis and degradation in the intact cells under different experimental conditions. Maximal adrenaline concentrations raise the rate of cyclic AMP synthesis and its steady-state concentration by about 10-fold. The addition of caffeine causes a further 33% increase in intracellular concentration of the nucleotide, which is in good agreement with the theoretical increase computed from its slowed-down degradation.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

Renal uptake and degradation of trapped-label insulin

In order to study the kinetics of insulin degradation in the kidneys and liver, insulin was labelled by a trapped-label procedure and injected into rats. In contrast to conventional 125I-insulin, the trapped-label preparation allows quantitative measurements of the extent of degradation in vivo because the final degradation products do not leave the cells. One hour after injection, the amount of radioactivity in the kidneys from a trace dose of trapped-label insulin was 10 times higher that from conventionally labelled insulin; over 80% of the increase was due to low molecular weight degradation products which were retained in the kidneys. The amount of acid-precipitable radioactivity in the blood was the same for both labelled preparations, indicating that their rates of clearance were similar. In the kidney, we detected no degradation products of molecular weight intermediate between intact insulin and the end products of proteolysis. After 2 h, 33% of the injected dose remained in the kidneys and only 13% in the liver. Over 80% of the renal radioactivity was sedimentable in an isotonic density gradient, indicating that intact insulin, as well as degradation products in the cells, were enclosed within membrane-bound vesicles.     

Internalization of insulin and its receptor in the isolated rat adipose cell: Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37°C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [¹²⁵I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [¹²⁵I]insulin to these same membrane fractions. At 37°C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20–30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16°C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Intracellular proteolysis in rat cardiac and skeletal muscle cells in culture.

Treatment of adult rats with dexamethasone resulted in an increase in cardiac muscle weight but a decrease in skeletal muscle weight. The different response of skeletal and cardiac muscles to the glucocorticoid was also reflected by a dexamethasone-induced enhancement of myofibrillar protease activity in the gastrocnemius muscle and an inhibition of a similar proteolytic activity in the heart. Newborn rats also exhibit the same, tissue-specific response to the glucocorticoid hormone. Consequently, the difference between cardiac and skeletal muscle responsiveness to conditions of wasting was investigated in culture. Average rates of degradation of intracellular proteins were determined in cultured cells derived from rat skeletal and cardiac muscle by following the release of radioactivity from cells prelabelled with ¹⁴C-phenylalanine. The release of label into the TCA soluble medium as measured during 12 hours of incubation, conformed to a first-order reaction and both cell types were found to degrade intracellular proteins at a similar rate. After 12 hours of incubation in a complete Ham F-10 medium supplemented with serum approximately 18% of total cellular protein was degraded. Incubation in a minimal medium or serum-deprivation enhanced the average rate of proteolysis to a value of 29% degradation at 12 hours indicating that intracellular proteolysis in these cells is responding to nutritional deprivation by increased activity. However, addition of glucose (22.2 nM) or dexamethasone (10^?6M) to the incubation medium failed to affect the rate of net protein degradation. Under no experimental condition could a difference be found between the proteolytic response of skeletal muscle cells to that of cardiac muscle cells and both cell types displayed similar changes in rates of protein degradation under various nutritional and hormonal conditions in culture. Thus, protein sparing in the heart of intact animals under catabolic conditions which enhance protein loss in skeletal muscle can probably not be ascribed to intrinsic differences in the direct response of cellular proteases to the tested hormones and nutrients. Rather, an extracellular factor(s) is apparently required for induction of the differential response of these tissues in the intact animal to protein wasting conditions. Alternatively, cells in culture might have lost the property of differential degradative response which operates in vivo.     

Bacitracin enhances intracellular accumulation of insulin in rat adipocytes

Bacitracin (1 mg/ml) markedly increased (approx. 75%) the cell-associated specifically bound 125I-labelled insulin without altering the affinity of the binding sites. Bacitracin also exerted a modest inhibitory effect on the degradation of insulin in the incubation medium determined as radioactivity not precipitated by trichloroacetic acid (from 9.6 to 4.8%). The effect on insulin binding was about 5-times as sensitive as the effect on degradation. The increased binding was due to intracellular accumulation of radioactivity which could not be removed by treating the cells with trypsin. This increase was not seen when the internalization process was reduced by ATP-depletion or low temperature. Since the trypsin-sensitive fraction of cell-associated radioactivity was apparently not altered, it is suggested that bacitracin, in addition to its well-known inhibition of extracellular degradation, also inhibits the intracellular degradation of insulin.     

Receptor- and non-receptor-mediated uptake and degradation of insulin by hepatocytes

Native insulin inhibits the binding and degradation of ¹²⁵I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of ¹²⁵I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of ¹²⁵I-labelled insulin (1nm) with the insulin receptor. In contrast, 20μm-glucagon inhibits 75% of the uptake of insulin (0.1μm) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37°C. During dissociation, fragments of ¹²⁵I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione–insulin transhydrogenase, with which both insulin and glucagon interact.     

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.     

Evidence that dissociation, not intracellular degradation, is the major pathway for removal of receptor-bound 125I-human chorionic gonadotropin in cultured rat luteal cells

The nature of the labeled products released by cultured rat luteal cells pulse-labeled with 125I-human chorionic gonadotropin (hCG) was examined. After pulse labeling in a 3-h incubation, the cells containing receptor-bound 125I-hCG were incubated in fresh medium in the absence of 125I-hCG up to 48 h. The medium was collected at different time intervals and analyzed to determine the extent of degradation of 125I-hCG. The amounts of radioactivity remaining associated with the cells at these time intervals were also determined. Most of the released radioactivity could be precipitated with 10% trichloracetic acid and was identical in molecular weight to intact 125I-hCG as determined by gel filtration chromatography. After 20 h of reincubation, only less than 50% of the initially bound hormone remained on the cells. At this time point the cells were capable of rebinding 125I-hCG at levels comparable to the original when incubated with a fresh dose of the labeled hormone. The rebinding ability was not a result of de novo receptor synthesis since cycloheximide had no effect on this process. The results indicate that dissociation is the major pathway for release of hCG bound to cultured rat luteal cells and that receptors become functional again after dissociation of the hormone by a cycloheximide-independent process.