Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.     

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.     

Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling

The kinetics of receptor internalization and recycling was directly determined in adipocytes by measuring 125I-insulin binding to total, intracellular, and cell-surface insulin receptors. In the absence of insulin 90% of all receptors were on the cell-surface and 10% were intracellular. Insulin (100 ng/ml) rapidly altered this distribution by translocating surface receptors to the cell-interior through a temperature and energy dependent process. Surface-derived receptors were seen within cells as early as 30 s and accumulated intracellularly at the rate of approximately 20,000/min (t 1/2 = 2.7 min). After 6 min the size of the intracellular receptor pool plateaued (for up to 2 h), with 30% of surface receptors residing within the cell. This plateau was due to the attainment of an equilibrium between receptor uptake and recycling, since removal of insulin (to stop receptor uptake) was followed by both a rapid depletion of intracellular receptors and a a concomitant and stoichiometric reappearance of receptors on the cell-surface. Receptors were efficiently recycled, with little or no net loss observed even after 4 h of insulin treatment; however, recycling could be partially inhibited (approximately 10%) by several agents (e.g. chloroquine and Tris). Tris treatment of adipocytes in the presence of insulin led to 50% loss of surface and total receptors at 2 and 4 h, respectively. Since chloroquine prevented the decrease in total receptors, but not the loss of surface receptors, it appears that Tris impairs recycling by diverting a portion of incoming receptors to a chloroquine-inhibitable degradative site. From these results we conclude that: 1) insulin triggers endocytotic uptake of insulin-receptor complexes; 2) internalized receptors are then rapidly reinserted into the plasma membrane, and the receptors can traverse this recycling pathway within 6 min; 3) prolonged recycling does not normally result in measurable receptor loss, but when receptors are prevented from recycling, they become trapped intracellularly and are shunted to a chloroquine-sensitive degradative pathway; and 4) chloroquine and Tris are only partially effective inhibitors of receptor recycling.     

Replacement of the human insulin receptor transmembrane and cytoplasmic domains by corresponding domains of the oncogene product v-ros leads to accelerated internalization, degradation, and down-regulation

Internalization, degradation, and insulin-induced down-regulation of insulin receptors were studied comparatively in transformed Chinese hamster ovary (CHO) cell lines, CHO.T and CHO.IR.ros, respectively expressing either the wild-type human insulin receptor (hIR) or a mutated hybrid receptor in which the transmembrane and cytoplasmic domains of hIR were replaced by corresponding domains of the transforming protein p68gag-ros (v-ros) of avian sarcoma virus UR2. At 37 degrees C, degradation of insulin receptors photoaffinity labeled on the cell surface (440 kDa) was most rapid for the hybrid hIR.ros (t1/2 1.0 +/- 0.1 h), intermediate for the wild-type hIR (t1/2 2.7 +/- 0.5 h), and slowest for the endogenous CHO insulin receptors (t1/2 3.7 +/- 0.7 h). Initial intracellular accumulation of the hIR.ros hybrid was also most rapid, reaching maximal amounts in 20 min following which the receptors disappeared rapidly from the intracellular compartment. In contrast, intracellular accumulation of the receptors in the CHO.T and CHO cells was slower, reaching maximal amounts in 60 min, and rapid disappearance of the receptors from the intracellular compartment did not occur. Chloroquine, a lysosomotropic agent, inhibited degradation of both the wild-type hIR and the chimeric hIR.ros and increased their intracellular accumulation. However, the chloroquine effect was much more marked for the hIR.ros receptors whose intracellular accumulation was increased by greater than 300% (in comparison with approximately 60% increase for the wild-type hIR), demonstrating marked intracellular degradation of the hybrid hIR.ros at chloroquine-sensitive sites. Insulin-induced down-regulation of the cell surface hIR.ros (52% loss in 3 h) was also more marked than the wild-type hIR (approximately 30% loss in 3 h). Thus, in the hybrid hIR.ros receptor, which was shown previously to exhibit insulin-stimulated autophosphorylation and kinase activity but not insulin-stimulated metabolic function, the capacity for internalization and down-regulation is not only preserved but is also markedly accelerated. These findings suggest that 1) the postreceptor coupling mechanisms mediating insulin-induced receptor internalization, degradation, and down-regulation are different from those mediating metabolic functions; and 2) v-ros may contain the structural information directing accelerated receptor catabolism.     

Absence of down-regulation of the insulin receptor by insulin. A possible mechanism of insulin resistance in the rat.

Insulin resistance occurs in rat adipocytes during pregnancy and lactation despite increased or normal insulin binding respectively; this suggests that a post-receptor defect exists. The possibility has been examined that, although insulin binding occurs normally, internalization of insulin or its receptor may be impaired in these states. Insulin produced a dose-dependent reduction in the number of insulin receptors on adipocytes from virgin rats maintained in culture medium, probably due to internalization of the hormone-receptor complex. In contrast, adipocytes from pregnant and lactating rats did not exhibit this 'down-regulation' phenomenon. Down regulation was, however, apparent in all groups when the experiments were performed in Tris buffer (where receptor recycling is inhibited), suggesting that in pregnant and lactating rats insulin receptors are rapidly recycled back to the plasma membrane, whereas in virgin rats this recycling process is less effective. Internalization of insulin was also determined by using 125I-labelled insulin. Adipocytes from pregnant and lactating rats appeared to internalize similar amounts of insulin to virgin rats. In the presence of the lysosomal inhibitor chloroquine, adipocytes from pregnant rats internalized more insulin than virgin or lactating rats. These results suggest that adipocytes from pregnant and lactating rats internalize insulin and its receptor normally, whereas intracellular processing of the insulin receptor may differ from that in virgin rats. In addition the rate of lysosomal degradation of insulin may be altered in adipocytes from pregnant rats.     

Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process

The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.     

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblast insulin receptors. These cells bind and internalize insulin normally. Biochemical assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 degrees C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 degrees C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The total number of complexes reached a maximum by 5 min and decreased rapidly thereafter with a t 1/2 of approximately 10 min. There was a distinct delay in the appearance, rate of rise, and peak of intracellular free and degraded insulin. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored, based on the ability of dissociated insulin to rebind to receptor upon neutralization of acidic intracellular vesicles with monensin. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.     

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.     

Mechanism of Free Fatty Acid Effects on Hepatocyte Insulin Receptor Binding and Processing

We determined whether the paimitate effects on hepatocyte insulin receptor binding and post-receptor trafficking were mediated by accelerated mitochondrial (β-oxidation or accumulation of intracellular fatty acyl-CoA derivatives and possibly protein acyiation. Preincubation of hepatocytes with moderate concentrations of paimitate (0.5 mM) resulted in a 23% decline in cell-surface binding and proportional decreases in receptor-mediated insulin internalization and degradation. Brief pretreatment of hepatocytes with the carnitine palmityltransferase-I inhibitor, methyl paimoxirate (MP), prevented 70% of the paimitate effects. At higher paimitate concentrations (2.0 mM), cell-surface binding was reduced by 34%, whereas internalization of the receptor complex was reduced by 78%. These effects were only partially prevented by MP pretreatment. Receptor-mediated insulin degradation increased by 34% and was uninfluenced by MP pretreatment. Octanoate, which is rapidly shunted into mitochondrial oxidation, produced a dose-dependent reduction in insulin binding, with proportional decreases in internalization and degradation. Similarly preincubation with 2.0 mM oleate, which, unlike palmitate, is not known to produce protein acylation, resulted in proportional decreases in insulin receptor binding and receptor-mediated internalization and degradation. High concentrations of octanoate or oleate (2.0 mM) did not reproduce the additive postreceptor effects of palmitate. We conclude that the receptor and post-receptor effects of moderate palmitate concentrations are closely linked to accelerated fatty acid oxidation. The post-receptor effects observed at higher concentrations involve other mechanisms, possibly relating to intracellular levels of palmityl-CoA derivatives.     

Diversity in the effects of extracellular ATP and adenosine on the cellular processing and physiologic actions of insulin in rat adipocytes

ATP or adenosine (1 mM) added to extracellular buffer abolished both chloroquine- and monensin-dependent accumulation of [125I]iodoinsulin in isolated rat adipocytes. The effects of ATP were not secondary to its conversion to adenosine and were mimicked by beta, gamma-methyleneadenosine 5'-triphosphate. ATP, but not adenosine, partially inhibited the binding of insulin to the cellular receptor. Neither ATP nor adenosine had any significant effect on both internalization of cell-bound insulin and externalization of the internalized hormone. The degradation of cell-bound insulin was reduced to a considerable extent by both 0.1 mM chloroquine and 5 mM ATP, to a lesser degree by 1 mM ATP, and not significantly by 1 or 5 mM adenosine. Physiologically, (a) 1 mM ATP had a strong, while 1 mM adenosine had a mild inhibitory effect on the insulin-stimulated glucose transport without affecting its basal activity, (b) both ATP and adenosine moderately stimulated basal as well as insulin-stimulated glycogen synthase, and (c) ATP, but not adenosine, transiently stimulated basal cAMP phosphodiesterase without affecting the insulin-stimulated enzyme. Phosphodiesterase in cells that had been exposed to ATP for 30 min was refractory to ATP added afresh, but not to insulin. These data suggest that (a) extracellular ATP may block the degradative pathway of insulin processing, (b) adenosine might render the ordinarily irreversible intracellular traffic of insulin reversible or modulate a pathway which is yet to be identified, (c) the previously reported effect of ATP on glycogen synthase may not involve phosphorylation, (d) ATP stimulates cAMP phosphodiesterase by a mechanism which is distinct from that of insulin, and (e) the degradative pathway of insulin processing may not be involved in the physiologic actions of the hormone on glycogen synthase and phosphodiesterase.     

Chloroquine stimulation of insulin binding to IM-9 lymphocytes: Evidence for action at a nonlysosomal site

Chloroquine has been shown to both increase insulin binding and decrease intracellular insulin degradation in several target cells. However, whether this increased binding is a consequence of decreased degradation is unclear. Accordingly, we studied the effects of chloroquine on insulin binding to IM-9 cultured lymphocytes, a cell type that does not degrade insulin in the cell interior. In these cells, chloroquine enhanced insulin binding in a dose dependent manner over the concentration range of 100 μM to 1 mM; at 1 mM binding was increased by 70%. Scatchard analysis indicated that chloroquine acted to increase receptor affinity. These studies indicate, therefore, that chloroquine can enhance the binding of insulin to its receptor via a mechanism that is independent of effects on intracellular insulin degradation.     

Chloroquine allows the secretion of internalized 125I-epidermal growth factor from fibroblasts

Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse-chase paradigm in which cell surface receptors were labelled with 125I-EGF at 4 degrees C, it was found that when 100 microM chloroquine was present in the 37 degrees C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor-mediated endocytosis and subcellular distribution of 125I-EGF-receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I-EGF released from the cells could rebind to surface receptors and be re-internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I-EGF whose rebinding was inhibited. Thus, the 125I-EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I-EGF and mechanisms of its preferential rebinding are discussed.     

Internalization of transforming growth factor-beta and its receptor in BALB/c 3T3 fibroblasts

The fate of 125I-labeled transforming growth factor-beta (125I-TGF beta) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I-TGF beta to cellular receptors at 4 degrees C is pH-sensitive, being markedly decreased at pH less than 6. Most (approximately 90%) of the 125I-TGF beta bound to cells at 4 degrees C can be removed by a brief treatment with acidic medium but is converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Cell-bound 125I-TGF beta is degraded at 37 degrees C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I-TGF beta from the cells. Cells allowed to accumulate 125I-TGF beta intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X-100; this treatment caused the cross-linking of internalized 125I-TGF beta with the 280-kilodalton TGF beta receptor component. Under conditions in which sustained binding and degradation of saturating 125I-TGF beta concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGF beta binding the TGF beta:receptor complex becomes rapidly internalized and that TGF beta is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGF beta receptors recycled after internalization or supplied by a large intracellular pool.     

Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.     

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.     

Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half- life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR- gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome- like elements. Extracting ATR-gold complexes with Triton X after a 30- min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.     

Insulin increases the cell surface concentration of alpha 2-macroglobulin receptors in 3T3-L1 adipocytes. Altered transit of the receptor among intracellular endocytic compartments

The present study shows that insulin causes an increase in the binding of alpha 2-macroglobulin (alpha 2M) to 3T3-L1 adipocytes. Scatchard analysis of the binding at 4 degrees C indicated an approximate 2-fold increase in the number of alpha 2M binding sites, with no change in the apparent affinity of the receptor. In addition, a 2-3-fold increase in the binding of monoclonal antibody 2C6, which recognizes a component of the alpha 2M receptor, was found in cells treated at 37 degrees C with insulin and then KCN to inhibit receptor endocytosis. An increased cellular accumulation of alpha 2M was also observed in response to insulin. Interestingly, the increase in the rate of accumulation of alpha 2M was significantly smaller than the increase in the number of alpha 2M receptors on the cell surface, suggesting that the rate of ligand internalization or subsequent processing is altered in response to insulin. Ultrastructural analysis of the internalization pathway of the alpha 2M receptor was performed using colloidal gold-coupled 2C6 monoclonal antibody. Control cells incubated for 20 min at 37 degrees C with the gold-conjugated antibody displayed 40% of cellular gold particles on the cell surface and 60% within intracellular structures. In insulin-treated cells this proportion was reversed, with 64% of the particles being found on the cell surface, and only 36% within intracellular structures. Significant differences in the distribution of gold particles among intracellular structures were detected between control and insulin-treated cells. Whereas in control cells, 18% of the total cellular gold particles internalized into tubulovesicles and multivesicular bodies, in insulin-treated cells only 3% of the gold particles were found within these structures. These data indicate that the movement of this receptor between endocytic compartments is altered in response to insulin, and suggest that the effect of insulin to increase the cell surface concentration of alpha 2M receptors and the accumulation of alpha 2M is due, at least in part, to alterations in the endocytic portion of the receptor recycling pathway.     

Uncoupling between the insulin-receptor cycle and the cellular degradation of the hormone in cultured foetal hepatocytes. Effect of drugs and temperature that inhibit insulin degradation.

Sequential changes in the numbers of cell-surface receptors induced by a transitory exposure to insulin in cultured 18-day foetal-rat hepatocytes were investigated in the presence of drugs and at a temperature of 22 degrees C, which inhibit cellular insulin degradation. Chloroquine (70 microM) and monensin (3 microM) did not greatly change the initial rate of internalization of cell-surface receptor sites after exposure to 10 nM-insulin, but led to a steady state after 20 min, which represented 40% of the initial binding, compared with 5 min and 60% in the absence of the drug. Moreover, these drugs strongly decreased the proportion of receptor sites recovered at the cell surface after subsequent removal of the hormone. They were ineffective when insulin was not present. The removal of monensin together with the hormone allowed partial restoration of cell-surface receptor sites and degradation of cell-associated insulin to start again at the initial speed, indicating a reversible effect of the drug. During this phase, the drug concentration-dependence for the two effects showed that receptor recycling was restored with concentrations of monensin not as low as for insulin degradation. The effect of vinblastine (50-100 microM) was similar to that of chloroquine and monensin, whereas no modification in the internalization and recovery processes was observed in the presence of bacitracin concentrations (1-3 mM) that inhibit insulin degradation by 70%. A temperature of 22 degrees C did not prevent the receptor internalization, but had a slowing effect on the recycling process, which appeared to vary in experiments where insulin degradation remained inhibited. The present study shows that the process of insulin degradation mediated by receptor endocytosis is not a prerequisite for insulin-receptor recycling in cultured foetal hepatocytes.     

Insulin induces chloroquine-sensitive recycling of insulin-like growth factor II receptors but not of glucose transporters in rat adipocytes

Incubation of insulin-treated rat adipocytes with chloroquine, in a time- and concentration-dependent manner, was observed to inhibit the insulin-stimulated increase in insulin-like growth factor II (IGF-II) binding activity, whereas no significant change in IGF-II binding was observed in the absence of insulin. The incremental increase of insulin-stimulated IGF-II binding was inhibited 50% by 0.2 mM chloroquine within 15 min and was nearly completely abolished by 60 min. Interestingly, IGF-II binding was never observed to decrease below the binding value in cells without insulin treatment even when incubation was extended to 180 min. Scatchard analysis of IGF-II binding as well as the specific binding of an anti-IGF-II receptor antibody demonstrated that the loss of IGF-II binding in the insulin-stimulated chloroquine-treated adipocytes was due to a decrease in the number of cell-surface IGF-II receptors, whereas the total number of cellular IGF-II receptors was unaltered. The effect of chloroquine was observed to be reversible, temperature-dependent, and sensitive to the metabolic poison KCN. Furthermore, NH4Cl was also observed to inhibit insulin-stimulated increase in IGF-II binding. In contrast, chloroquine or NH4Cl did not inhibit the basal or insulin-stimulated glucose transport activity. Photoaffinity labeling of the glucose transporter with [3H]cytochalasin B also demonstrated that the basal and insulin-stimulated subcellular distribution of the glucose transporters was unaltered by chloroquine treatment. These results suggest that 1) insulin induces a constitutive, acidotropic agent-sensitive recycling of IGF-II receptor and 2) the glucose transporter and IGF-II receptor do not share the same insulin-regulated intracellular trafficking pathways.     

Effects of vesicle-adipocyte interaction on regulation of insulin receptor recycling

The effect of interacting isolated rat adipocytes with small, unilammelar vesicles on insulin receptor internalization and processing was studied. Treatment of freshly isolated cells with vesicles containing phosphatidylcholine and phosphatidylserine followed by incubation in 35 mM Tris-containing buffer considerably reduced the chloroquine-induced increase in cell-associated 125I-insulin and significantly inhibited the time and insulin dependent loss of surface insulin receptors. The internal receptor pool, as measured by insulin binding to detergent solubilized adipocytes, was relatively smaller in vesicle-treated cells. Concomitant with a slower rate of receptor internalization, insulin-sensitive hexose uptake also demonstrated significantly slower kinetics of decreased response with time. These results support the conclusion that pretreatment of fat cells with phospholipid vesicles inhibits normal insulin receptor cycling.