Intracellular Processing Of Insulin Research Articles

Sequential processing of insulin by cultured kidney cells.

It is now well established that in kidney, as in liver, endosomes participate in the degradation of insulin. Degradation in this compartment involves the action of the insulin-degrading enzyme or a similar enzyme with the formation of large intermediate products. The role of lysosomes is less clear, for although earlier studies suggested that they are the major or sole site of degradation, this has been increasing questioned. More recently, it has been concluded that endosomes, at least in liver, are the major site of insulin degradation and that classic lysosomes are only involved in the latter stages of degradation if at all. As intracellular insulin processing varies among cell types, we set out to examine directly the processing of insulin within cultured proximal-like opossum kidney cells. By means of analytical subcellular fractionation and reverse phase HPLC analysis of products, we established the following sequence of events. After internalization, [125I]A14-insulin is partially degraded in endosomes. The formed products together with intact insulin, which accounts for most of the radioactive material in the endosomes, are then directed to the lysosomal compartment, where degradation proceeds rapidly to completion. Bacitracin inhibited degradation in both compartments and, although not eliminating insulin trafficking, may impair the transfer of insulin from endosomes to lysosomes. This study establishes a major role for lysosomes in kidney cell insulin degradation.

Degradation of insulin by isolated rat renal cortical endosomes.

It has been widely accepted that in kidney, degradation of insulin occurs in lysosomes. It is thought that after internalization into the cell, insulin dissociates from its receptor, which then recycles to the plasma membrane, while the hormone is transported in endosomes to the lysosomes, where it is degraded. However, earlier studies from this laboratory have suggested that insulin may also be degraded in an extralysosomal site, most likely endosomes. Indeed, studies in other tissues, most notably liver, have shown that insulin degradation does take place in endosomes. Since the intracellular processing of insulin differs between different tissues and cell types, and as the kidney is a major site of insulin degradation, we set out to determine directly whether endosomes degrade internalized insulin in the kidney. Rats were injected with [125I]monoiodoinsulin, labeled at either the A14 or B26 tyrosine. After killing, the kidney cortex was excised, and heavy endosomes were prepared by differential and isopycnic centrifugation. The isolated [125I]insulin-loaded endosomes were incubated for up to 60 min in intracellular medium, and degradation of [125I] insulin was estimated by means of precipitation in trichloroacetic acid. In the presence of ATP (10 mM), the percent degraded was increased over the control value (no ATP present), but under these circumstances, degradation was greater when the endosomes contained internalized 125I-labeled [B26]insulin than with A14-labeled [125I]insulin (26% vs. 13% degraded/h). In the absence of ATP, the percent degraded increased when the pH of the incubation medium was lowered. Radiolabeled material was extracted from endosomes, and Sephadex G-50 analysis revealed the presence of high mol wt, insulin-size, and low mol wt material. Reverse phase HPLC analysis of the insulin-size material revealed the presence of intact insulin and a number of degradation products. The elution profiles of some of these products were consistent with that reported to arise from the action of the insulin-degrading enzyme. Western blot analysis with the antiinsulin-degrading enzyme monoclonal antibody 9B12 confirmed the presence of the enzyme in endosomal preparations. We conclude that degradation of insulin does occur in kidney cortical endosomes, probably involves the insulin-degrading enzyme, and results in the formation of relatively large intermediate products as well as low mol wt products.

Degradation of insulin by isolated rat renal cortical endosomes

It has been widely accepted that in kidney, degradation of insulin occurs in lysosomes. It is thought that after internalization into the cell, insulin dissociates from its receptor, which then recycles to the plasma membrane, while the hormone is transported in endosomes to the lysosomes, where it is degraded. However, earlier studies from this laboratory have suggested that insulin may also be degraded in an extralysosomal site, most likely endosomes. Indeed, studies in other tissues, most notably liver, have shown that insulin degradation does take place in endosomes. Since the intracellular processing of insulin differs between different tissues and cell types, and as the kidney is a major site of insulin degradation, we set out to determine directly whether endosomes degrade internalized insulin in the kidney. Rats were injected with [125I]monoiodoinsulin, labeled at either the A14 or B26 tyrosine. After killing, the kidney cortex was excised, and heavy endosomes were prepared by differential and isopycnic centrifugation. The isolated [125I]insulin-loaded endosomes were incubated for up to 60 min in intracellular medium, and degradation of [125I] insulin was estimated by means of precipitation in trichloroacetic acid. In the presence of ATP (10 mM), the percent degraded was increased over the control value (no ATP present), but under these circumstances, degradation was greater when the endosomes contained internalized 125I-labeled [B26]insulin than with A14-labeled [125I]insulin (26% vs. 13% degraded/h). In the absence of ATP, the percent degraded increased when the pH of the incubation medium was lowered. Radiolabeled material was extracted from endosomes, and Sephadex G-50 analysis revealed the presence of high mol wt, insulin-size, and low mol wt material. Reverse phase HPLC analysis of the insulin-size material revealed the presence of intact insulin and a number of degradation products. The elution profiles of some of these products were consistent with that reported to arise from the action of the insulin-degrading enzyme. Western blot analysis with the antiinsulin-degrading enzyme monoclonal antibody 9B12 confirmed the presence of the enzyme in endosomal preparations. We conclude that degradation of insulin does occur in kidney cortical endosomes, probably involves the insulin-degrading enzyme, and results in the formation of relatively large intermediate products as well as low mol wt products.

The dissociation and degradation of internalized insulin occur in the endosomes of rat hepatoma cells.

We have studied the intracellular processing of insulin in the rat hepatoma cell line Fao. Fao cells internalized cohorts of surface-bound 125I-insulin or 125I-insulin-like growth factor II within 3-5 min. Degraded 125I-insulin-like growth factor II did not appear in the medium until 20-30 min after uptake, consistent with a time course of lysosomal delivery. In contrast, internalized insulin was completely degraded within 7-10 min. The half-times for dissociation and degradation of internalized insulin were identical at 37 degrees C (3 min), suggesting that the two processes occurred in the same compartment. Subcellular fractionation of Fao cells showed that a pulse of internalized insulin was largely intact after 3 min and associated with a light membrane fraction devoid of lysosomal markers. After an additional 4 min, the amount of insulin in this compartment decreased by 40%, with an increase in degraded insulin in the cytosol; no transfer of intact insulin to lysosomes or the cytosol was detected. The relationship between insulin-receptor dissociation and insulin degradation was further studied with inhibitors of insulin processing. Monensin blocked both dissociation and degradation of internalized insulin, as did incubation of the cells at 20 degrees C, suggesting that both endosomal acidification and endosomal fusion were required for insulin processing. At 25 degrees C, dissociation (+ t 1/2 = 12.9 min) preceded degradation (+ t 1/2 = 15.8 min). Inhibitors of lysosomal proteases were without effect on the half-time for either process. In contrast, bacitracin, an inhibitor of insulin degradation, caused a 2-fold increase in the half-times for both dissociation and degradation. Thus, intracellular insulin dissociation and degradation are tightly coupled endosomal processes in Fao cells, and insulin degradation facilitates the dissociation of insulin from its receptor inside the cell.

Improvement With Metformin in Insulin Internalizaron and Processing in Monocytes From NIDDM Patients

This study investigated the relative effect of obesity alone and in combination with non-insulin-dependent diabetes mellitus (NIDDM) on the intracellular processing of insulin and evaluated the effect of metformin therapy on this process. Monocytes from 11 obese hyperinsulinemic subjects, 13 obese hyperinsulinemic NIDDM patients, and 7 nondiabetic control subjects were incubated with A14-125I-labeled insulin for 60 min at 37 degrees C, and intracellular insulin degradation was characterized by high-performance liquid chromatography. Total cell-associated insulin (insulin binding) and internalized and degraded insulin were decreased in obese subjects and significantly decreased in obese NIDDM patients compared with nondiabetic control subjects. In NIDDM patients, intracellular insulin degradation was inversely correlated with fasting plasma glucose (P less than 0.01). Eight obese subjects and 9 obese NIDDM patients were restudied after 4 wk of therapy with metformin (850 mg twice a day). Plasma levels of the drug were superimposable in the two groups. Metformin therapy did not change glucose and insulin levels in obese subjects but caused a decrease in blood glucose in obese NIDDM patients. Total cell-associated radioactivity (insulin binding) significantly increased in both groups (P less than 0.01). On the contrary, internalized radioactivity increased (0.83 +/- 0.3 vs. 1.31 +/- 0.35%, P less than 0.01), and similarly, insulin degradation was enhanced (54.6 +/- 8.9 vs. 74.22 +/- 9.15%, P less than 0.01) only in monocytes from obese NIDDM patients. However, the levels of these parameters were still lower than in control subjects (internalization, 2.94 +/- 0.68%; degradation, 93.03 +/- 3.7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of a human insulin receptor with a COOH-terminal truncation. I. Insulin binding, autophosphorylation, and endocytosis.

In order to test the contribution of the insulin receptor COOH terminus to insulin action, a truncation of 43 COOH-terminal amino acids was engineered by cDNA-based deletion mutagenesis. This cDNA (HIR delta CT), as well as cDNA encoding the complete receptor (HIRc) was transfected into Rat 1 fibroblasts. Cells expressing 6.4 X 10(3) and 1.25 X 10(6) normal receptors and 2.5 X 10(5) HIR delta CT receptors, as well as control Rat 1 fibroblasts were selected for further analysis. All cell lines exhibited insulin binding of similar affinity. Partial tryptic digestion and immunoprecipitation by region-specific antibodies verified that the HIR delta CT receptors were truncated at the COOH terminus. Purified HIRc and HIR delta CT receptors underwent autophosphorylation with similar insulin and ATP sensitivity, although the HIR delta CT receptors were slightly more active in the absence of insulin. Transfected HIRc and HIR delta CT receptors undergo endocytosis in a normal fashion. Insulin internalization and degradation in both HIRc and HIR delta CT cells is increased in proportion to receptor number. Intracellular insulin processing, degradation, and release were qualitatively comparable among the transfected cell lines. Complete and truncated receptors internalize, recycle, and down-regulate normally. We conclude the following: 1) the COOH-terminal portion of the insulin receptor is not necessary for partial autophosphorylation or endocytosis; 2) following internalization the intracellular itinerary of the receptor and ligand appear normal with the truncated receptor; and 3) truncation of the COOH terminus does not impair recycling of the receptor or retroendocytosis of internalized ligand.

Effect of bacitracin on binding and processing of insulin by established renal cell line.

The effect of bacitracin on the binding and processing of 125I-labeled insulin was studied in a proximal tubular epithelium-like opossum kidney cell line. This cultured cell line handles insulin in a manner comparable to the in vivo situation, which requires membrane binding, internalization, and intracellular degradation. The addition of bacitracin inhibited insulin degradation significantly and delayed the time of appearance of products in the medium (22 min) compared with control cells (14 min). Maximum total cell-associated radioactivity increased from 1.5 +/- 0.19% in the control cells to 2.5 +/- 0.17% in the treated cells. Separation of cell membrane from internalized radioactivity was achieved by acid washing and showed no change in membrane-bound radioactivity or rate of internalization, but a significant increase in intracellular radioactivity was noted. Gel-filtration chromatography revealed that this was due to an accumulation of chromatographically intact insulin. Accordingly, we conclude that bacitracin inhibits insulin degradation in cultured kidney cells by perturbing the intracellular processing of insulin, not by altering the binding or internalization of the hormone or by inhibiting the release of small degradation products. Because of the multiple actions of this agent, the exact site in these kidney cells at which intracellular degradation is inhibited remains to be established. However, in contrast to studies with lysosomes isolated from cells of other tissues, this study showed that when lysosomes isolated from rat renal cortex were exposed to bacitracin, insulin degradation was inhibited markedly (81%).

Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus.

We studied total cell-associated A14-[125I]insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients [2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity; P less than 0.01]. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity; P less than 0.01). Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-[125I] insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-[125I]insulin, [125I]iodide, or [125I]tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity (P less than 0.01) after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% (P less than 0.01) after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients.

The effect of insulin concentration on retroendocytosis in isolated rat adipocytes.

After internalization by isolated rat adipocytes, insulin can be degraded or released intact from the cell, a process termed retroendocytosis. To determine whether the amount of ligand entering the cell could modulate its ultimate intracellular disposition, adipocytes were incubated with 0.4-25 ng/ml radiolabeled insulin for 20 min at 37 C to reach steady state binding and internalization. After this, surface bound insulin was removed by acid extraction and the cells were reincubated in insulin-free 37 C buffer. The fractional rate of release of internalized cell associated radioactivity was similar at all insulin concentrations. However, insulin enhanced the appearance of trichloroacetic acid (TCA)-precipitable material in a dose-dependent manner reaching a maximum at an insulin concentration of 10 ng/ml. At 0.4 ng/ml insulin, 18 +/- 2% of the released radioactivity was TCA precipitable whereas 36 +/- 3% was precipitable at 25 ng/ml. Sephadex G-50 gel chromatography and reverse phase HPLC analysis of the reincubation medium confirmed TCA-precipitable material was intact insulin. To further investigate the dual pathways of intracellular insulin processing, adipocytes were incubated with 0.4 and 25 ng/ml insulin for 20 min, acid extracted to remove surface receptor insulin, and solubilized. Sephadex G-50 and HPLC analysis revealed that proportionately less insulin intermediates and low molecular weight degradation products are found in cells incubated at the higher insulin concentrations. In conclusion, as adipocytes internalize more insulin, less is converted into insulin intermediates and low molecular weight degradation products and more is diverted to retroendocytosis.

Demonstration of insulin receptors and modulation of alkaline phosphatase activity by insulin in rat osteoblastic cells.

Osteoporosis is a known complication of diabetes mellitus, suggesting a role for insulin in bone homeostasis. We studied insulin receptors and insulin action in the osteoblast-like rat osteogenic sarcoma cell line ROS 17/2.8. These cells share many common features with the osteoblast, such as 1,25-dihydroxyvitamin D3 receptors, PTH receptors, and 1,25-dihydroxyvitamin D3-induced modulation of alkaline phosphatase activity and osteocalcin. Competition binding studies revealed high affinity insulin receptors, with an ED50 for insulin of 1 nM. The receptors were highly specific for insulin, with 60% inhibition of insulin binding by an antireceptor antibody, no competition by epidermal growth factor, and an ED50 of 300 nM for proinsulin. Steady state maximal insulin binding was obtained by 40 min at 37 C, and insulin degradation, as measured by trichloroacetic acid solubility, was 1%/h at 37 C. ROS cells readily internalized insulin, and under steady state binding conditions at 37 C, 56% of the cell-associated radioactivity consisted of intracellular material. Chloroquine (100 microM) inhibited intracellular processing of insulin, leading to a 300% increase in cell-associated insulin by 2 h (37 C). Photoaffinity labeling of the insulin receptor with the photosensitive analog of insulin, B2 (2-nitro-4-azidophenyl-acetyl)des-pheB1-insulin, followed by solubilization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed specific bands of 125K and 430K mol wt under reducing and nonreducing conditions, respectively. Thus, the structure of insulin receptors in ROS cells appears comparable to that of insulin receptors of known target tissues. Insulin action was also examined. Insulin did not stimulate [2-3H]deoxyglucose uptake or [1-14C]leucine incorporation into protein. In contrast, physiological concentrations of insulin inhibited alkaline phosphatase activity in nonconfluent cells. After exposure to insulin for 24 h, alkaline phosphatase activity was decreased compared to basal by 39.5% and 50% with 5 and 50 ng/ml insulin, respectively. In conclusion, ROS cells bind insulin to specific receptors that are similar to insulin receptors on other target tissues; receptors internalize insulin, which is then processed through a chloroquine-sensitive pathway; insulin does not affect membrane substrate transport; and insulin does inhibit the activity of an enzyme that is important in bone metabolism. ROS cells represent a model for studying insulin effects on bone.

Insulin internalization and degradation in adipocytes from normal and type II diabetic subjects.

We studied the ability of isolated adipocytes from normal and type II diabetic subjects to internalize and process [125I]insulin. Adipocytes were incubated with [125I]insulin at 16 or 37 C, and at various times total cell-associated, surface-bound, and intracellular insulin were quantitated using an acid-barbital extraction technique which quickly removes cell surface insulin, leaving behind the intracellular insulin. Insulin internalization was slow in normal adipocytes at 16 C, such that only 13% of total cell-associated insulin was intracellular after 2 h of incubation. In contrast, internalization was rapid at 37 C, such that the intracellular pool of insulin was near maximal by 30 min and accounted for approximately 40% of the total cell-associated insulin. Sephadex G-50 column chromatography of the intracellular insulin demonstrated that more than 95% of this pool coeluted with native insulin. In adipocytes from the diabetic subjects, approximately 45% of total cell-associated insulin was intracellular after 30 min of incubation at 37 C. After 60 min of incubation at 37 C, the percentages of total cell-associated and surface-bound insulin were significantly lower in adipocytes from diabetic compared to normal subjects [1.81 +/- 0.31% (+/- SEM) vs. 2.92 +/- 0.24% (P less than 0.05) and 0.97 +/- 0.14% vs. 1.72 +/- 0.15% (P less than 0.01), respectively]. The percentage of insulin in the intracellular compartment was also slightly lower in adipocytes from diabetic compared to normal subjects (0.84 +/- 0.19% vs. 1.20 +/- 0.16%; P greater than 0.05). The lysosomotropic agent chloroquine increased total cell-associated insulin, and this was due entirely to an increase in intracellular insulin. In adipocytes from normal subjects, chloroquine increased intracellular insulin by 32% at 30 min, by 89% at 60 min, by 140% at 90 min, and by 178% at 120 min. In comparison to the normal adipocytes, the chloroquine-mediated increase in intracellular insulin was lower in adipocytes from the diabetic subjects (-8.1% at 30 min, 37% at 60 min, 58% at 90 min, and 63% at 120 min; P less than 0.05 at all time points). These results indicate that insulin is rapidly internalized in human adipocytes at 37 C such that approximately half of total cell-associated insulin is intracellular the intracellular insulin is largely intact; and intracellular processing of insulin by a chloroquine-sensitive pathway(s) is impaired in adipocytes from type II diabetic subjects.

Degradative processing of internalized insulin in isolated adipocytes.

Based on the distribution of 125I-insulin between the cell surface and the cell interior, it was found that insulin rapidly binds (t 1/2 = 0.4 min) to surface receptors at 37 degrees C, and after an initial lag period of about 1 min, accumulates intracellularly until steady state is reached (t 1/2 = 3.5 min). At this time about 40% of the total cell-associated 125I-insulin resides in the cell interior reflecting a dynamic equilibrium between the rate of insulin endocytosis and the rate at which internalized insulin is processed and extruded from cells. Since this percentage decreased to 15% at 16 degrees C, it appears that internalization is more temperative-sensitive than the intracellular processing of insulin. When 125I-insulin was preloaded into the cell interior, it was found that internalized insulin was rapidly released to the medium at 37 degrees C (t 1/2 = 6.5 min) and consisted of both degraded products and intact insulin (as assessed by trichloroacetic acid precipitability and column chromatography). Since 75% of internalized insulin was ultimately degraded, and 25% was released intact, this indicates that degradation is the predominant pathway. To determine when incoming insulin enters a degradative compartment, cells were continually exposed to 125I-insulin and the composition of insulin in the cell interior over time was assessed. After 2 min all endocytosed insulin was intact, between 2-3 min degradation products began accumulating intracellularly, and by 15 min equilibrium was reached with 20% of internalized insulin consisting of degraded products. Degraded insulin was then released from the cell interior within 4-5 min after endocytotic uptake, since this was the earliest time chloroquine was found to inhibit the release of degradation products. Moreover, the final release of degraded insulin was not inhibitable by the energy depleter dinitrophenol. Thus, within the degradative pathway, insulin enters lysosomes by 2.5-3 min and is released to the medium by simple diffusion after an additional 1.5-2 min.

Dual pathways for the intracellular processing of insulin. Relationship between retroendocytosis of intact hormone and the recycling of insulin receptors.

Adipocytes process insulin through either of two pathways: a retroendocytotic pathway that culminates in the release of intact insulin, and a degradative pathway that terminates in the intracellular catabolism and release of degraded ligand. Mechanistically, these pathways were found to differ in several ways. First, temporal differences were found in the rate at which intact and degraded products were extruded. After 125I-insulin was preloaded into the cell interior, intact ligand was completely released during the first 10 min (t 1/2 = 2 min), whereas degraded insulin was released at a much slower rate over 1 h (t 1/2 greater than 8 min). Secondly, it was found that chloroquine profoundly inhibited the insulin degradative pathway, resulting in the intracellular accumulation of intact ligand and a reduction in the release of degraded products. In contrast, however, chloroquine was without effect on the retroendocytotic processing of insulin. Based on the known actions of chloroquine, it appears that retroendocytosis of insulin does not involve vesicular acidification or dissociation of the insulin-receptor complex and that insulin is most likely carried to the cell exterior in the same vesicles (either receptor-bound or free) as those mediating recycling receptors. Interestingly, accumulation of undergraded insulin within chloroquine-treated cells did not result in the release of additional intact ligand, suggesting that once insulin enters the degradative compartment it is committed to catabolism and cannot exit the cell through the retroendocytotic pathway. A third difference was revealed by the finding that extracellular unlabeled insulin (100 ng/ml) markedly accelerated the rate at which preloaded 125I-insulin was released from adipocytes (t 1/2 of 3 min versus 7 min in controls cells). Analysis of the composition of the released products revealed that extracellular insulin rapidly augmented (over 10 min) in a dose-dependent manner (5-200 ng/ml) the amount of insulin released intact (from 25 to 38% of preloaded counts; insulin ED50 = 10 ng/ml). Although extracellular insulin had no effect on the early extrusion of degraded insulin, the release of catabolized products was reduced at later times. The interpretation of these results is that the rate or amount of incoming insulin-receptor complexes can effect a sorting process (prior to bifurcation) such that a proportion of insulin is shunted from the slower degradative pathway to the more rapid retroendocytotic pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin binding and degradation in isolated hepatocytes from streptozotocin injected rats

Isolated hepatocytes from streptozotocin injected rats bound the same amount of [ 125I]monoiodoinsulin as hepatocytes from control rats. Scatchard analysis confirmed that insulin receptor number and affinity were the same for both groups. Relatively more cell-associated radioactivity was located intracellularly in hepatocytes from streptozotocin injected rats. Pretreatment with chloroquine resulted in a smaller increase in intracellular [ 125I]monoiodo-insulin in cells isolated from streptozotocin injected rats than for control cells. These results suggest that intracellular insulin processing occurs more slowly in hepatocytes isolated from streptozotocin injected rats than from control rats.

Selective effects of inhibitors of hormone processing on insulin action in isolated hepatocytes.

Several compounds known to alter receptor-mediated endocytosis were tested for their effect on insulin action in isolated rat hepatocytes. Dansylcadaverine blocked the inhibitory effect of insulin on hepatic protein degradation, but was without effect on the inhibition produced by high concentrations of amino acids. Dansylcadaverine also was without effect on the stimulatory action of insulin on glucose incorporation into glycogen, but totally blocked the insulin stimulation of alanine uptake. No inhibition of basal or (Bu)2cAMP-stimulated alanine uptake was observed with dansylcadaverine. These results indicate that dansylcadaverine has selective effects on some but not all of insulin's actions. Methylamine also blocked insulin stimulation of alanine transport, although at higher doses, methylamine decreased basal and (Bu)2cAMP-stimulated uptake due to cellular toxicity. Chloroquine, an inhibitor of intracellular insulin processing, had no effect on insulin-stimulated alanine transport. To examine the site of action of dansylcadaverine on insulin processing, the binding (cell-associated radioactivity) and degradation of [125I]iodoinsulin by hepatocytes were examined in the presence and absence of dansylcadaverine. Dansylcadaverine delayed the time required to achieve maximal insulin binding and produced a dose-dependent decrease in cell-mediated insulin degradation. Acid dissociation and proteolytic digestion methods were used to differentiate between [125I]iodoinsulin bound to the cell surface vs. that which had been internalized. Dansylcadaverine did not block internalization as measured by either of these criteria, but did block the increase in cell-associated radioactivity produced by chloroquine, suggesting that dansylcadaverine blocks insulin processing after internalization but before the chloroquine-sensitive step. These data show that some of the hepatic actions of insulin, namely inhibition of protein degradation and stimulation of alanine transport, are blocked by certain inhibitors of cellular hormone processing, suggesting that these actions of insulin may require postreceptor events involving insulin metabolism.

Metabolic effects of bacitracin in isolated rat hepatocytes.

Bacitracin is a proteolytic inhibitor which interacts with the intracellular processing of insulin. Its effects on pyruvate, fatty acid and amino acid metabolism were examined in rat hepatocyte suspensions. Bacitracin (0.25-1.0 mM) increased the oxidation of [1-14C]pyruvate by 50-70% and presumably therefore increased the flux through pyruvate dehydrogenase. This was found both in the presence of extracellular Ca2+ and in its absence, but not in the presence of 2 mM-2-chloropropionate, which inhibits pyruvate dehydrogenase kinase. Insulin did not further stimulate [1-14C]pyruvate oxidation in the presence of 1 mM-bacitracin. Bacitracin decreased 14CO2 formation from [2-14C]pyruvate (20-40%) and [U-14C]palmitate (30-70%), suggesting a decreased flux through the tricarboxylic acid cycle. Fatty acid oxidation before acetyl-CoA formation was also decreased. Bacitracin decreased the incorporation of label from [3H]leucine into protein in the absence of insulin, but not in its presence. Bacitracin is commonly used in studies on insulin action. Our results suggest that in such studies the effects noted may be related not only to an interaction of bacitracin with the intracellular processing of insulin but also to direct metabolic effects of bacitracin independent of insulin.

Internalization and intracellular processing of insulin and insulin receptors in adipocytes

Internalization and intracellular processing of insulin and insulin receptors in adipocytes