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.

Insulin receptors in isolated human adipocytes. Characterization by photoaffinity labeling and evidence for internalization and cellular processing.

We photolabeled and characterized insulin receptors in isolated adipocytes from normal human subjects and then studied the cellular fate of the labeled insulin-receptor complexes at physiologic temperatures. The biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)des-PheB1-insulin (NAPA-DP-insulin) was used to photoaffinity label the insulin receptors, and the specifically labeled cellular proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. At saturating concentrations, the binding of 125I-NAPA-DP-insulin to the isolated adipocytes at 16 degrees C was rapid (half-maximal in approximately 1 min and maximal in approximately 10 min) and approximately 25% of the specifically bound ligand was covalently linked to the cells by a 3-min exposure to long-wave (366 nm) ultraviolet light. Analysis of the photolabeled cellular proteins by PAGE in the absence of disulfide reductants revealed the specific labeling of a major protein band of Mr 330,000 and two less intense bands of Mr 295,000 and 260,000. Upon reduction of disulfide bonds with dithiothreitol, all three unreduced forms of the insulin receptor were converted into a major labeled Mr-125,000 band and a less intensely labeled Mr-90,000 band. The labeling of the Mr-125,000 receptor subunit was saturable and native porcine insulin effectively inhibited (half-maximal inhibition at 12 ng/ml) the photolabeling of this binding subunit by NAPA-DP insulin. When intact adipocytes photolabeled at 16 degrees C (a temperature that inhibits endocytosis) were immediately trypsinized, all of the labeled receptor bands were converted into small molecular weight tryptic fragments, indicating that at 16 degrees C all of the labeled insulin-receptor complexes remained on the cell surface. However, when the photolabeled cells were further incubated at 37 degrees C and then trypsinized, a proportion of the labeled receptors became trypsin insensitive, indicating that this fraction has been translocated to the cell interior and thus was inaccessible to the trypsin in the incubation medium. The intracellular translocation of the labeled receptors was observed within 2 min, became half-maximal by 10 min, and maximal by approximately 30 min of incubation at 37 degrees C. Cellular processing of the internalized insulin-receptor complexes also occurred, since incubation at 37 degrees C (but not 16 degrees C) resulted in the generation of a Mr-115,000 component from the labeled receptors. Inclusion of chloroquine, a drug with lysosomotropic properties, in the incubation media caused a time-dependent increase (maximal increase of 50% above control by 2 h at 37 degrees C) in the intracellular pool of labeled receptors. In contrast to these findings in human adipocytes, no appreciable internalization of insulin-receptor complexes and no chloroquine effect was observed in cultures human IM-9 lymphocytes during a 1-h incubation at 37 degrees C. We concluded that in isolated human adipocytes: (a) the subunit structure of insulin receptors is the same as that reported for several other tissues, (b) insulin-receptor complexes are rapidly internalized and processed at physiologic temperatures, and (c) the cellular processing of insulin-receptor complexes occurs at one or more chloroquine-sensitive intracellular site(s).

Photoaffinity labeling of insulin receptors in viable cultured human lymphocytes. Demonstration of receptor shedding and degradation.

A photosensitive derivative of radiolabeled insulin, SANAH-125I-insulin, was prepared by reacting N-succinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH) with 125I-insulin. Cultured IM-9 cells were incubated with SANAH-125I-insulin at 16 degrees C in the dark. They were then washed, photolyzed, solubilized, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Under disulfide reducing conditions, a single specific band of Mr 125,000 was obtained. The characteristics of the labeling of this band with SANAH-125I-insulin (specificity, time course, concentration effect) were the same as that of 125I-insulin interaction with the IM-9 cells and the labeling process did not affect cell viability. The solubilized photolabeled insulin receptor fraction was enriched by first adsorbing to agarose-bound wheat germ agglutinin and the material eluted with N-acetyl-D-glucosamine was then analyzed by SDS-PAGE and autoradiography. Under nonreducing conditions, a major receptor band of Mr 320 K and a minor band of 280 K were obtained. Upon disulfide bond reduction with increasing concentrations of dithiothreitol, a major band of Mr 125 K and two minor bands of Mr 210 K and 94 K were seen. When cells photolabeled at 16 degrees C were further incubated at 37 degrees C, there was a time-dependent loss of intact receptors into the incubation buffer. In contrast, no similar shedding of labeled receptors was observed from isolated rat adipocytes. Following shedding, the labeled IM-9 insulin receptors rapidly disappeared from the incubation buffer (half-time approximately 1.5 h). These results demonstrate the feasibility of photoaffinity labeling, characterizing, and following the fate of insulin receptor in viable cells. Thus receptor photoaffinity labeling should provide a suitable approach for studies of the biologic fate of insulin receptors in cells that are targets for insulin action.

Insulin-stimulated glucose transport and insulin internalization share a common postbinding step in adipocytes.

We recently demonstrated that chymotrypsin substrate analogues inhibit receptor-mediated insulin internalization in isolated rat adipocytes. In this study, the effect on glucose transport of inhibiting insulin internalization with these agents was examined. Glucose transport was assayed by measuring [3H]-2-deoxyglucose uptake, and internalized insulin was measured after rapidly dissociating surface-bound insulin with an acidic buffer. The chymotrypsin substrate analogue N-acetyl-Tyr ethyl ester inhibited insulin internalization by 85% while increasing surface-bound insulin by 80-110%. Under these conditions, ATP levels were minimally altered, and basal glucose transport was unchanged; however, insulin-stimulated glucose transport was decreased by 86%. The inhibition of insulin-stimulated glucose transport was not overcome by supramaximal concentrations (400 ng/ml) of insulin. When insulin internalization and insulin-stimulated glucose transport were measured in the presence of increasing concentrations of N-acetyl-Tyr ethyl ester (0.1-1 mM), a strong and highly significant correlation (r = .97, P less than .001) was found between inhibition of insulin internalization and inhibition of insulin-stimulated glucose uptake. Fragments of N-acetyl-Tyr ethyl ester that do not inhibit insulin internalization were also without effect on insulin-stimulated glucose transport. In addition to N-acetyl-Tyr ethyl ester, four other chymotrypsin substrate analogues that are effective inhibitors of insulin internalization also markedly inhibited insulin-stimulated glucose transport. These results indicate that insulin internalization and insulin-stimulated glucose transport share a common postbinding step in adipocytes and that this step is inhibitable by chymotrypsin substrate analogues.

Degradation of insulin receptors in rat adipocytes.

Insulin receptors on viable rat adipocytes were affinity-labeled using a biologically active and photosensitive analogue of insulin, 125I-B2(2-nitro, 4 azidophenylacetyl)-des-PheB1-insulin (125I-NAPA-DP-insulin). The radiolabeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of 125I-NAPA-DP-insulin (40 ng/ml) to rat adipocytes at 16 degrees C, followed by photolysis, resulted in the specific labeling of essentially one protein with an apparent molecular weight of 430-450,000 daltons. When this radiolabeled protein was treated with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a 125,000-dalton protein and to a lesser extent in a 90,000-dalton protein. In addition, there was a minimal amount of specific labeling of a 115,000-dalton protein. Under certain experimental conditions, the nonreduced form of the photoaffinity-labeled receptor appeared as a heterogeneous population of proteins having apparent molecular weights of 430,000, 350,000, and 270,000 daltons. Subsequent to photoaffinity labeling of insulin receptors at 16 degrees C, adipocytes were incubated at 37 degrees C for various periods of time to allow for internalization. This resulted in an initial rapid loss of radioactivity in the 430,000- and 125,000-dalton bands. At 60 min the amount of radioactivity in each of these bands was approximately 50% of that present before incubation at 37 degrees C and stayed constant for 120 min. A first-order plot of the decline in receptor-associated radioactivity was biphasic with the initial phase having a half-life of 1.4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein tyrosine phosphatase 1B interacts with the activated insulin receptor.

Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase of unknown function, although increasing evidence supports a role for this phosphatase in insulin action. We have investigated the interaction of PTP1B with the insulin receptor using a PTP1B glutathione S-transferase (GST) fusion protein with a point mutation in the enzyme's catalytic domain. This fusion protein is catalytically inactive, but the phosphatase's phosphotyrosine binding site is maintained. The activated insulin receptor was precipitated from purified receptor preparations and whole-cell lysates by the inactive PTP1B-GST, demonstrating a direct association between the insulin receptor and PTP1B. A p120 of unknown identity was also precipitated from whole-cell lysates by the PTP1B fusion protein, but IRS-1 (pp185) was not. A catalytically inactive [35S]PTP1B-fusion protein bound directly to immobilized insulin receptor kinase domains and was displaced in a concentration-dependent manner. Finally, tyrosine-phosphorylated PTP1B was precipitated from whole-cell lysates by an anti-insulin receptor antibody after insulin stimulation. The site of interaction between PTP1B and the insulin receptor was studied using phosphopeptides modeled after the receptor's kinase domain, the NPXY domain, and the COOH-terminal. Each phosphopeptide inhibited the PTP1B-GST:insulin receptor interaction. Study of mutant insulin receptors demonstrated that activation of the kinase domain is necessary for the PTP1B:insulin receptor interaction, but receptors with deletion of the NPXY domain or of the COOH-terminal can still bind to the PTP1B-GST. We conclude that PTP1B can associate directly with the activated insulin receptor at multiple different phosphotyrosine sites and that dephosphorylation by PTP1B may play a significant role in insulin receptor signal transduction.

The NPEY sequence is not necessary for endocytosis and processing of insulin-receptor complexes.

The tetrameric amino acid sequence AsnProXTyr (NPXY), where X represents any amino acid, is conserved in the intracytoplasmic domains of several membrane proteins and has been postulated to play a role in receptor-mediated endocytosis. The human insulin receptor (hIR) contains a single copy of the sequence AsnProGluTyr (NPEY) in its intracytoplasmic domain. To determine if this putative consensus sequence is necessary for endocytic functions of hIR, we constructed a mutant receptor, hIR delta NPEY, that lacks NPEY sequence, stably expressed this mutant receptor in Chinese hamster ovary cells, and then studied its endocytic functions. When compared to wild type hIR similarly expressed in Chinese hamster ovary cells, the hIR delta NPEY mutant exhibited: 1) normal subunit organization and insulin binding affinity; 2) essentially normal internalization of covalent photoaffinity labeled insulin-receptor complexes; and 3) normal internalization of receptor-bound [125I]insulin as well as normal degradation and release of the internalized insulin. Therefore, we conclude that the NPEY sequence in the juxtamembrane domain of hIR is not necessary for its endocytic function.

Insulin receptors on cultured hypothalamic cells: functional and structural differences from receptors on peripheral target cells.

We have studied the functional and structural characteristics of insulin receptors on cultured rat hypothalamic cells. The receptors on these cells are specific for insulin, but have a lower binding affinity than that measured in nonneuronal tissues. Neither acute (2-h) nor long term (24-h) exposure of the hypothalamic cells to high insulin concentrations resulted in receptor down-regulation. However, insulin is internalized in these cells and accumulated in the presence of the lysomotropic agent chloroquine. Acute exposure to insulin does not alter initial rate of 2-deoxyglucose transport in hypothalamic cells, but does cause a stimulation of aminoisobutyric acid uptake. Photoaffinity labeling of the receptors of the hypothalamic cells with a biologically active photosensitive insulin revealed a major specifically labeled band of 115K mol wt and a minor band of 40K mol wt under disulfide-reducing conditions compared to bands of 125K and 90K mol wt seen after labeling of the insulin receptors of adipocytes. The receptor proteins in hypothalamic cells under nonreducing conditions (420K, 370K, and 310K mol wt) were also smaller than those in adipocytes. Thus, the insulin receptors of cultured hypothalamic cells differ from insulin receptors on peripheral target tissues in both functional and structural aspects.

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.

Insulin-stimulated glucose transport in cultured fibroblasts from normal and noninsulin-dependent (type II) diabetic human subjects.

We have studied the effects of insulin on glucose transport in cultured fibroblasts obtained from six normal subjects and six patients with type II or noninsulin-dependent diabetes mellitus. Initial rates of glucose transport were determined by measurement of 2-deoxy-D-glucose uptake. In both normal and diabetic fibroblasts, insulin stimulated the uptake of 2-deoxy-D-glucose in a dose-dependent manner. The maximal level of insulin stimulation of 2-deoxy-D-glucose uptake (approximately 35% over basal uptake values) and the half-maximally effective insulin concentration (approximately 1 nM) were essentially the same for both the normal and diabetic fibroblasts. Furthermore, the absolute basal and maximally insulin-stimulated 2-deoxy-D-glucose uptake values were also the same. These results demonstrate that cultured fibroblasts from patients with noninsulin-dependent diabetes mellitus have normal basal and insulin-stimulated glucose transport capacities. Thus, the postreceptor defects in the glucose transport system previously demonstrated in vivo and in freshly isolated adipocytes from type II diabetic patients are most likely due to in vivo environmental factors rather than to an intrinsic (genetic) cellular defect(s).

Profile of coronary artery risk factors in Ethiopian diabetic patients.

OBJECTIVES: To assess the coronary artery risk factors in Ethiopian diabetic patients. DESIGN: A cross sectional study done on a representative sample of diabetic patients to evaluate the coronary risk factors. SETTING: Hospital-based study. SUBJECTS: A total of 302 diabetic patients randomly selected from the 3000 regularly attending diabetic patients in the Tikur Anbessa Hospital Diabetes Centre of whom 161(53.3%) were males, 141(46.7%) were females, 140(46.4%) were Type 1 and 162(53.6%) were Type 2. OUTCOME MEASURES: Assessment of the coronary risk factors in diabetic patients. RESULTS: There were 20(6.6%) smokers and no ex-smokers. All smokers were males of whom five (25%) were Type 1 and 15(75%) were Type 2. Hypertension and obesity were found in 64(21.2%) and 69(22.8%) patients respectively. Sixty one (20.2%) patients and 43(14.2%) patients had hypercholesterolaemia and hypertriglyceridaemia respectively. CONCLUSION: Our diabetic patients share the risk factors for developing coronary artery disease like any other diabetic patient in the developed countries but at a lower level.

Retinopathy in patients of Tikur Anbessa Hospital diabetic clinic.

A total of 302 diabetic patients were selected from regular attendants of the Tikur Anbessa Hospital (TAH) diabetic clinic to determine the prevalence of retinopathy from December 1994 to March 1995. The mean age was 41.4 +/- 14.4 years (range 14-85). There were 160 males (53%) and 142 females (47%). One hundred forty (46%) were type 1 and 162 (53.6%) were type 2. The mean duration of diabetes was 9.4 +/- 5.4 years and the mean Hemoglobin Alc (HbAlc) was 10.4 + 2.2%. On the day of the examination the mean fasting blood glucose (FBG) and random blood glucose (RBG) were 195.5 +/- 79.9 mg/dl and 273.1 +/- 114.5 mg/dl respectively. The mean serum total cholesterol, triglycerides, LDL, VLDL and GDL were 166.5 +/- 45.5 mg/dl, 129.9 +/- 92.4 mg/dl, 94.5 +/- 36.4 mg/dl, 24.4 +/- 15.1 mg/dl and 44.3 +/- 11.5 mg/dl respectively. The overall prevalence of retinopathy was 37.8% out of which 108 patients (36.1%) had background retinopathy and 5 patients (1.7%) had proliferative retinopathy. The retina could not be visualized in three patients because of dense cataract. Retinopathy correlated positively with age, duration of diabetes and blood pressure respectively, however no significant correlation was seen with mean total HgAlc and serum lipids. Prevalence of retinopathy was comparable in type 1 and type 2 (p > 0.05). The prevalence of retinopathy in our patients relative to the duration of diabetes mellitus is high. Therefore, improving facilities for the diagnosis and treatment of retinopathy is recommended.

Effect of pioglitazone in combination with insulin therapy on glycaemic control, insulin dose requirement and lipid profile in patients with type 2 diabetes previously poorly controlled with combination therapy.

AIM: The aim of this randomized placebo-controlled study was to evaluate the safety and efficacy of pioglitazone administered alone or in combination with metformin in reducing insulin dosage requirements for improved glycaemic control in patients with type 2 diabetes previously poorly controlled with combination therapy. METHODS: In this multicentre, double-blind study, 222 patients with haemoglobin A1c (HbA(1c))>8.0% at screening treated with combination therapy initially were given titrated insulin therapy (to fasting plasma glucose <140 mg/dl) and then were randomly assigned to 20-week treatment with pioglitazone or placebo in combination with insulin, with or without concurrent metformin therapy. More than 98% of patients were taking metformin prior to and during the study. RESULTS: Pioglitazone significantly reduced (p < 0.05) insulin dose requirements 2 weeks after treatment initiation. At study end relative to baseline, pioglitazone reduced daily insulin dosages by 12.0 units (p < 0.001), a 21.5% (12.0/55.8 units at baseline) group mean average reduction. Relative to placebo, pioglitazone reduced daily insulin dosages by 12.7 units [95% confidence interval [CI]: -17.5, -8.0], while improving mean HbA(1c) levels [adjusted mean HbA(1c) change: pioglitazone, -1.6% vs. placebo, -1.4% (not statistically different)]. Pioglitazone also significantly increased high-density lipoprotein cholesterol levels [adjusted mean difference: +4.5 (95% CI: 2.6-6.5) mg/dl], decreased triglyceride levels [-43.9 (-69.2, -18.6) mg/dl], shifted low-density lipoprotein (LDL) particle concentrations from small [pattern B, -13.6% (-17.7%, -9.5%)] to large [pattern A, +15.1% (10.8%, 19.5%)] and increased mean LDL particle size [+3.8 (2.6, 4.9) A]. More pioglitazone-treated patients experienced oedema (9.0 vs. 4.5%) and weight gain (9.1 vs. 2.7%) than placebo patients. CONCLUSIONS: Pioglitazone in combination with insulin therapy improved glycaemic control, reduced insulin dose requirements and improved lipid profiles in patients with type 2 diabetes previously poorly controlled with combination therapy.

Internalized insulin-receptor complexes are unidirectionally translocated to chloroquine-sensitive degradative sites. Dependence on metabolic energy.

Insulin receptors on the surface of isolated rat adipocytes were photoaffinity labeled at 12 degrees C with the iodinated photoreactive insulin analogue, 125I-B2 (2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, and the pathways in the intracellular processing of the labeled receptors were studied at 37 degrees C. During 37 degrees C incubations, the labeled 440-kDa insulin receptors were continuously internalized (as assessed by trypsin inaccessibility) and degraded such that up to 50% of the initially labeled receptors were lost by 120 min. Metabolic poisons (0.125-0.75 mM 2,4-dinitrophenol (DNP) and 1-10 mM NaF), which led to dose-dependent depletion of adipocyte ATP pools, inhibited receptor loss, and caused up to 3-fold increase in intracellular receptor accumulation. This effect was due to inhibition of intracellular receptor degradation, and there was no apparent effect of the metabolic poisons on initial internalization of the receptors. Following maximal intracellular accumulation of labeled insulin receptors in the presence of NaF or DNP, removal of these agents resulted in a subsequent, time-dependent degradation of the accumulated receptors. However, when the lysosomotropic agent, chloroquine (0.2 mM), was added immediately following removal of the metabolic poisons, further degradation of the intracellularly accumulated receptors was prevented, suggesting that the chloroquine-sensitive degradation of insulin receptors occurs distal to the site of inhibition by NaF or DNP. To confirm this, maximal intracellular accumulation of labeled receptors was first allowed to occur in the presence of chloroquine and the cells were then washed and reincubated in chloroquine-free media in the absence or presence of NaF or DNP. Under these conditions, degradation of the intracellularly accumulated receptors continued to occur, and NaF or DNP failed to block the degradation. In summary, these results indicate that the loss of cell surface insulin receptors in adipocytes involves: 1) initial internalization of the receptors to a nondegradative intracellular compartment by a process that is relatively insensitive to ATP depletion, followed by 2) a highly energy-dependent unidirectional translocation of the receptors from this compartment to chloroquine-sensitive site(s) of degradation.

Effects of metalloendoprotease inhibitors on insulin binding, internalization and processing in adipocytes.

The effects of metalloendoprotease inhibitors on insulin binding, internalization, and processing were studied in isolated rat adipocytes. The metalloendoprotease inhibitor phosphoramidon caused a marked (threefold) increase in intracellular insulin accumulation without affecting surface binding. The dipeptide metalloendoprotease substrate analogues benzyloxycarbonyl-Gly-Phe-NH2 and benzyloxycarbonyl-Gly-Leu-NH2 caused similar large increases in intracellular insulin but also caused a doubling of cell surface bound insulin. The effect on surface binding was due to increased insulin receptor affinity as demonstrated by Scatchard analysis and the benzyloxycarbonyl-Gly-Phe NH2 induced inhibition of the dissociation of prebound insulin from the cell surface. These results suggest a role for endogenous metalloendoprotease-like enzymes in insulin processing by rat adipocytes.

Insulin signal transduction by a mutant human insulin receptor lacking the NPEY sequence. Evidence for an alternate mitogenic signaling pathway that is independent of Shc phosphorylation.

The cytoplasmic juxtamembrane domain of the human insulin receptor (hIR) contains a single copy of the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) (residues 969-972 in the exon 11-containing B-isoform), which exists in the context of NPXY. In this study, we examined the role of NPEY972 in mediating insulin signal transduction and cellular biological effects. Transfected Chinese hamster ovary cell lines expressing either the wild-type hIR-B isoform (hIR.WT) or a mutant receptor lacking the NPEY972 sequence (hIRDeltaNPEY) and control Chinese hamster ovary.Neo cells were used to comparatively analyze the following insulin effects: in vivo receptor tyrosine autophosphorylation and kinase activity, signal transduction to downstream signaling molecules, and stimulation of glycogen and DNA synthesis. The results showed that in comparison to hIR.WT, the hIRDeltaNPEY mutant demonstrated the following: (a) normal insulin-mediated receptor tyrosine phosphorylation, but approximately 50% reduction in phosphorylation of p185-(insulin receptor substrate-1) and binding of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (b) an enhanced stimulation of PI 3-kinase enzymatic activity, (c) a complete inability to phosphorylate Shc, (d) minimal impairment of insulin sensitivity for glycogen synthesis, and (e) an augmented response to insulin-stimulated DNA synthesis via a high capacity, low sensitivity pathway. These results demonstrate the following: 1) the NPEY972 sequence is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate-1 and p85 or for mediating insulin's metabolic and mitogenic effects, 2) the NPEY972 sequence is necessary for Shc phosphorylation, and 3) the absence of Shc phosphorylation releases the constraints on maximal insulin-stimulated mitogenic response, thus indicating that alternate signaling pathway(s) exist for this insulin action. This alternate pathway appears to be associated with enhanced activation of PI 3-kinase and is of high capacity and low sensitivity.

Adipocyte insulin receptor. Generation of a cryptic domain of the alpha-subunit during internalization of hormone-receptor complexes.

The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.