Lysosomal and non-lysosomal pathways of intracellular insulin degradation in isolated rat hepatocytes

The amount of 125I-insulin associated with freshly isolated hepatocytes was increased 50% in the presence of 0.2 mM chloroquine (CQ) after 2 h of incubation. The degradation of insulin by the hepatocytes incubated with CQ was significantly diminished as compared with control cells. Hepatocytes incubated with 125I-insulin in the presence of CQ showed a slower rate of ligand dissociation than control cells. More TCA-precipitable and less TCA-soluble material appeared in the dissociation buffer of CQ-treated cells. However, CQ inhibited only 25-35% of intracellular insulin degradation. Non-lysosomal intracellular insulin degradation appears to be responsible for the remaining portion of the ligand degradation by isolated hepatocytes.     

Receptor-mediated degradation of insulin in isolated rat adipocytes. Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [TyrA14-125I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [125I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the 'insulin' peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Uptake and degradation of insulin and alpha 2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.     

Uptake and degradation of insulin and α2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways

The cell association and degradation of insulin and α₂-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 μM) reduced both the uptake of α₂-macroglobulin · trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2–3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of α₂-macroglobulin · trypsin markedly without affecting that of insulin. Leupeptin (100 μM) increased uptake and reduced degradation of α₂-macroglobulin · trypsin without affecting insulin. Dansylcadaverine (500 μM) almost abolished uptake and degradation of α₂-macroglobulin · trypsin but had little effect on insulin. Moreover, uptake and degradation of α₂-macroglobulin · trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.     

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.     

Two pathways for insulin metabolism in adipocytes

Using selected conditions, the appropriate collagenase, albumin and cell treatment, a preparation of isolated adipocytes was developed with no extracellular insulin degrading activity. Cell mediated insulin degradation rates were 0.68%±0.05%/100 000 cell/h using trichloracetic acid precipitability as a measure. Chloroquine (CQ) increased cell-associated radioactivity and decreased degradation while dansylcadaverine (DC), PCMBS and bacitracin (BAC) decreased degradation with no effect on binding. Extraction and chromatography of the cell-associated radioactivity showed 3 peaks, a large molecular weight peak, a small molecular weight peak and an insulin-sized peak. CQ, DC and BAC all decreased the small molecular weight peak while CQ and DC also increased the peak of large molecular weight radioactivity. Cell mediated insulin degradation in the presence of combinations of inhibitors suggested two pathways in adipocytes, one affected by inhibitors of the insulin degrading enzyme (IDE) (bacitracin and PCMBS) and the other altered by cell processing inhibitors (DC, CQ and phenylarsenoxide). Chloroquine altered the pattern of the insulin-sized cell-associated HPLC assayed degradation products, further supporting two pathways of degradation; one a chloroquine-sensitive and one a chloroquine-insensitive pathway.     

Recycling of photoaffinity-labeled insulin receptors in rat adipocytes. Dissociation of insulin-receptor complexes is not required for receptor recycling

We have used an iodinated, photoreactive analog of insulin, 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, to covalently label insulin receptors on the cell surface of isolated rat adipocytes. Following internalization of the labeled insulin-receptor complexes at 37 degrees C, we measured the rate and extent of recycling of these complexes using trypsin to distinguish receptors on the cell surface from those inside the cell. The return of internalized photoaffinity-labeled receptors to the cell surface was very rapid at 37 degrees C proceeding with an apparent t 1/2 of 6 min. About 95% of the labeled receptors present in the cell 20 min after the initiation of endocytosis returned to the cell surface by 40 min. Recycling was slower at 25 and 16 degrees C compared to 37 degrees C and essentially negligible at 12 degrees C or in the presence of energy depleters. Addition of excess unlabeled insulin had no effect on the recycling of photoaffinity-labeled insulin receptor complexes, whereas monensin, chloroquine, and Tris partially inhibited this process. These data indicate that dissociation of insulin from internalized receptors is not necessary for insulin receptor recycling. Furthermore, agents which have been shown to prevent vesicular acidification inhibit the recycling of insulin receptors by a mechanism other than prevention of ligand dissociation.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

Evidence for surface glycoprotein involvement in the intracellular bioactivity of insulin in rat adipocytes.

Lectins specific for D-mannose (concanavalin A), N-acetyl-D-glucosamine (wheat-germ agglutinin) or D-galactose (Ricinus communis agglutinin I) inhibited insulin binding and activated glucose transport in rat adipocytes [Cherqui, Caron, Capeau & Picard (1982) Mol. Cell. Endocrinol. 28, 627-643]. In the present investigation, the intracellular activities of insulin and lectins on lipogenesis and protein synthesis were studied under conditions where neither agent had an effect on membrane transport processes. (1) When glucose transport was rate-limiting (0.5 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 2.4-3-fold. (2) When passive diffusion of glucose was amplified (10 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 1.6-1.8-fold even in the presence of 50 microM-cytochalasin B, which completely blocked glucose transport. (3) Insulin (6 ng/ml), concanavalin A and wheat-germ agglutinin (40 micrograms/ml) stimulated the incorporation of L-[U-14C]leucine into fat-cell protein 1.5-fold but did not modify alpha-aminoisobutyric acid uptake or 14C-labelled protein degradation. (4) Peanut and soya-bean agglutinins (specific for O-glycosidically-linked oligosaccharides), known not to alter insulin binding, were ineffective. (5) Lectin effects were dose-dependent and were markedly inhibited by specific monosaccharides (50 mM). (6) Insulin and lectin maximal effects were not additive and were completely abolished by neuraminidase treatment of fat-cells (0.05 unit/ml). These data indicate involvement of surface sialylated glycoproteins of the complex N-linked type in the insulin stimulation of glucose and amino acid intracellular metabolic processes. They suggest, together with our previous results, that the transmission of the insulin signal for both membrane and intracellular effects occurs via glycosylated effector entities of, or closely linked to, the insulin-receptor complex.     

Metabolism of photoaffinity-labeled insulin receptors by adipocytes. Role of internalization, degradation, and recycling

Insulin receptors on isolated rat adipocytes were photoaffinity-labeled with a biologically active photo-derivative of insulin (iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin) in order to study the metabolism of surface receptors after binding insulin. Adipocytes were incubated with iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin (40 ng/ml) at 16 degrees C until specific binding reached equilibrium, subjected to photolysis, and then incubated at 37 degrees C to follow the metabolism of the covalent insulin-receptor complexes. Susceptibility of labeled insulin receptors to tryptic digestion was used to distinguish between receptors on the cell surface and those inside the cell. Following incubation of photoaffinity-labeled adipocytes at 37 degrees C, there was an initial rapid loss of insulin receptors from the cell surface. The internalization of insulin receptors occurred at a significantly faster rate than the loss of receptors from the cell, resulting in an accumulation of intracellular receptors. The proportion of surface-derived receptors inside the cell reached an apparent steady state after 30 min and represented about 20% of the labeled receptors originally on the cell surface. Chloroquine had no effect on the internalization of insulin receptors but inhibited their degradation. Cycloheximide inhibited both internalization and degradation of insulin receptors. After 60 min at 37 degrees C, the disappearance of insulin receptors from the cell surface slowed markedly and the overall loss of insulin receptors from the cell was minimal. If chloroquine was added at this time, there was a marked increase in the loss of receptors from the cell surface with a concomitant 2-fold increase in the intracellular pool of surface-derived receptors. From these observations, we conclude that 1) internalization is not rate-limiting in insulin receptor degradation, 2) chloroquine has no effect on the internalization of insulin receptors but inhibits the intracellular degradation of receptors, 3) cycloheximide interferes with both the internalization and degradation of insulin receptors, and 4) the plateau in the loss of labeled receptors from the cell surface after 60 min at 37 degrees C could be due to a new steady state balance between internalization and recycling of photoaffinity-labeled receptors.     

Sulphonylureas-induced increase in insulin binding and glucose metabolism by isolated rat adipocytes

The effects of oral hypoglycaemic drugs, SPC-703 (n-/p-toluenesulphonyl/-5-methyl-2-pirazoline-1-carbonami de) and tolbutamide on insulin binding and glucose metabolism by isolated adipocytes were studied. After 10 days of administration of both sulphonylurea derivatives, no differences were observed in insulin concentration between both experimental and the control groups of animals, despite a significant fall in blood glucose level. SPC-703 and tolbutamide in concentrations of 1 mM added in vitro to the suspension of adipocytes had no effect on insulin binding or on basal and insulin simulated glucose metabolism. Daily administration of 300 mg/kg body weight of SPC-703 or tolbutamide for 10 days resulted in 48% and 34% increase of specific binding of insulin by adipocytes, respectively. From the Scatchard plot analysis we noted that the increase of binding resulted from increased affinity of insulin receptors for hormone. Simultaneous increase in basal and insulin stimulated glucose metabolism by adipocytes, as measured by 14CO2 production and 14C incorporation into cellular lipids, was observed. The results indicate that hypoglycaemic action of sulphonylureas may be explained by increased affinity of insulin receptors and the stimulating action of these compounds on peripheral glucose metabolism.     

Catecholamine-induced insulin resistance of glucose transport in isolated rat adipocytes.

The effects of pre-incubation with isoprenaline and noradrenaline on insulin binding and insulin stimulation of D-glucose transport in isolated rat adipocytes are reported. (1) Pre-incubation of the cells with isoprenaline (0.1-10 microM) in Krebs-Ringer-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] buffer (30 min, 37 degrees C) at D-glucose concentrations of 16 mM, in which normal ATP levels were maintained, caused a rightward-shift in sensitivity of D-glucose transport to insulin stimulation by 50% and a decrease in maximal responsiveness by 30% (2) [A14-125I]insulin binding was reduced significantly by 35% at insulin concentrations less than 100 mu-units/ml and Scatchard analysis showed that this consisted mainly of a decrease in high-affinity binding. (3) Pre-incubation with catecholamines under the same conditions but at low glucose concentrations (0-5 mM) caused a fall in intracellular ATP levels of 65 and 45% respectively. (4) The fall in ATP additionally lowered insulin binding by 50% at all insulin concentrations and a parallel shift of the binding curves in the Scatchard plot showed that this was due to a decrease in the number of receptors. (5) At low and high ATP concentrations the insulin stimulation of D-glucose transport was inhibited to a similar extent. (6) Pre-incubation with catecholamines thus inhibited insulin stimulation of D-glucose transport in rat adipocytes mainly by a decrease in high-affinity binding of insulin, which was not mediated by low ATP levels. This mechanism may play a role in the pathogenesis of catecholamine-induced insulin resistance in vivo.     

Effect of lipid peroxidation on the insulin receptor in rat adipocytes

Lipid peroxidation is studied for its effect on insulin receptors in isolated rat adipocytes. The results suggest that addition of two peroxidants (3 mM cumene hydroperoxide and 0.2 mM Fe2+) leads to malondialdehyde accumulation and binding inhibition through insulin receptors quantity and affinity decrease.     

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)     

Autoantibodies to the insulin receptor activate glycogen synthase in rat adipocytes

Summary Autoantibodies to the insulin receptor mimic the effects of insulin on glycogen synthase and phosphorylase. The interaction of antibodies with adipocyte cell surface insulin receptors seems sufficient to promote stable changes in the activities of these intracellular enzymes, suggesting that internalization or processing of insulin is not important in the generation of these biological responses.     

Modulation of GLUT4 and GLUT1 recycling by insulin in rat adipocytes: kinetic analysis based on the involvement of multiple intracellular compartments

The trafficking kinetics of GLUT4 and GLUT1 in rat epididymal adipocytes were analyzed by a four-compartment model based upon steady-state pool sizes of three intracellular fractions and one plasma membrane fraction separated and assessed under both basal and insulin-stimulated states. The steady-state compartment sizes provided relative values of the kinetic coefficients characterizing the rate of each process in the loop. Absolute values of these coefficients were obtained by matching the simulated half-times to those observed experimentally and reported in the literature for both basal and insulin-stimulated states. Our analysis revealed that insulin modulates the GLUT4 trafficking at multiple steps in the rat adipocyte, not only reducing the endocytotic rate constant 3-4-fold and increasing the exocytotic rate 8-24-fold but also increasing the two rate coefficients coupling the three intracellular compartments 2-6-fold each. Furthermore, GLUT1 was completely segregated from GLUT4 in two of the three intracellular compartments, and its steady-state distribution is consistent with a four-compartment model of GLUT1 recycling involving an insulin sensitive endocytosis step in common with the GLUT4 system, but with all other processes being insensitive to insulin.     

Inhibitory effect of insulin on prostacyclin production by isolated rat adipocytes

Physiologic concentrations of insulin completely inhibited the norepinephrine-induced increment in the production of 6-keto-prostaglandin (PG) F_1α, the stable derivative of prostacyclin (PGI₂), by isolated rat adipocytes. The inhibition of PGI₂ production by insulin in isolated rat adipocytes supports the view that the elevated plasma level of 6-keto-PGF_1α in rats with non-ketotic diabetes mellitus and diabetic ketoacidosis is derived at least in part from production of PGI₂ by the adipocyte cell mass.     

Cyclic AMP modulates insulin binding and induces post-receptor insulin resistance of glucose transport in isolated rat adipocytes

The effect of cAMP on insulin binding and insulin stimulation of glucose transport was investigated in isolated rat adipocytes. Preincubation for 30 min in medium containing 16 mmol/l glucose and either db-cAMP or bromo-cAMP in concentrations of 10(-4)-10(-3) M inhibited high affinity binding of insulin by 15 to 30% and glucose transport by 30 to 50%. Preincubation with IBMX (10(-4)-10(-3) M) reduced insulin binding by 25% and glucose transport by 70%. Closer analysis of these data indicated that preincubation with these compounds caused not only a decrease in insulin binding but also a post-receptor resistance. High intracellular cyclic AMP-levels seem therefore to induce insulin resistance at both receptor and post-receptor levels.     

The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation

The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.