Potential mechanism of the stimulatory action of insulin on insulin-like growth factor II binding to the isolated rat adipose cell. Apparent redistribution of receptors cycling between a large intracellular pool and the plasma membrane

Previous studies have proposed that insulin increases the binding of insulin-like growth factor II (IGF-II) in isolated rat adipose cells at 24 degrees C by increasing receptor affinity (Ka). This study re-examines these observations under conditions in which receptor-ligand internalization is blocked by 1 mM KCN. In the absence of KCN, adipose cells bind 0.71 amol of IGF-II/cell with low apparent affinity (0.030 nM-1), of which greater than 75% is not accessible to trypsin. In contrast, in the presence of KCN, IGF-II binding is decreased by 95% and its apparent affinity increased to 0.21 nM-1. Moreover, greater than 60% of the bound IGF-II now is sensitive to trypsin. In either the absence or presence of KCN, approximately 20% of the cell's total IGF-II receptors are present in the plasma membranes and approximately 80% in the low density microsomes. Insulin induces a 5-fold increase in cell surface IGF-II receptors without a change in affinity when IGF-II binding is measured in the presence of KCN. Similarly, insulin increases IGF-II receptor concentration in the plasma membranes and concomitantly decreases that in the low density microsomes. Receptor affinity in these two subcellular membrane fractions is not affected by incubation of intact cells with either insulin or KCN and is similar to that observed in intact cells in the presence of KCN. Addition of KCN prior to insulin abolishes all of these effects of insulin. These data suggest that (a) the effects of KCN reflect a selective blockade of endocytosis; (b) in the absence of KCN, IGF-II binds to receptors of constant affinity that cycle between the plasma membrane and an intracellular pool resulting in an accumulation of intracellular IGF-II; (c) insulin induces an increase in IGF-II binding by causing a steady state redistribution of receptors from this intracellular pool to the plasma membrane; and (d) this redistribution in the intact cell can only be detected using Scatchard analysis when recycling of the receptors is prevented by KCN.     

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.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters

Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane fraction and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with corresponding fractions from control cells. Intracellular vesicles containing insulin-responsive glucose transporters (GT) have been isolated by immunoadsorption from the microsomal fraction (Biber, J. W., and G. E. Lienhard. 1986. J. Biol. Chem. 261:16180-16184). All of the transferrin receptors in this fraction were localized in these vesicles; however, because the GT vesicles contain approximately 30-fold fewer transferrin receptors than GT, on the average only one vesicle in three contains a transferrin receptor. The binding of 125I-pentamannose 6-phosphate BSA to 3T3-L1 adipocytes at 4 degrees C was used to monitor surface insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptors. Exposure of cells to insulin at 37 degrees C for 5 min resulted in a 2.5-4.5-fold increase in surface receptors. There was a corresponding 20% decrease in the amount of IGF-II receptors in the microsomal membranes prepared from insulin-treated cells, as assayed by immunoblotting. Moreover, the IGF-II receptors and GT were located in the same intracellular vesicles, since antibodies to the carboxyterminal peptide of either protein immunoadsorbed vesicles containing 70-95% of both proteins initially present in the microsomal fraction. In conjunction with other studies, these results indicate that in 3T3-L1 adipocytes, three membrane proteins (the GT, the transferrin receptor, and the IGF-II receptor) respond similarly to insulin, by redistributing to the surface from intracellular compartment(s) in which they are colocalized.     

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.     

Insulin-induced redistribution of the insulin-like growth factor II/mannose 6-phosphate receptor in intact rat liver

The ability of acute insulin treatment to elicit a redistribution of the liver insulin-like growth factor-II/ mannose 6-phosphate (IGF-II/M6P) receptor has been studied in rats, using cell fractionation. Injection of insulin (0.4-50 microg) led to a time- and dose-dependent decrease in IGF-II binding activity in Golgi-endosomal (GE) fractions, along with an increase in activity in the plasma membrane (PM) fraction; only receptor number was affected. Quantitative subfractionation of the microsomal fraction on sucrose density gradients showed that IGF-II binding activity distributed similarly to galactosyltransferase (a Golgi marker), at slightly higher densities than in vivo internalized (125)I-insulin, and at lower densities than 5' nucleotidase and alkaline phosphodiesterase (two plasma membrane markers). Insulin treatment led to a slight time-dependent and reversible shift of IGF-II binding activity toward higher densities. Subfractionation of the GE fraction on Percoll gradients showed that IGF-II binding activity was broadly distributed, with about 60% at low densities coinciding with galactosyltransferase and early internalized (125)I-insulin and with 40% at high densities in the region of late internalized (125)I-insulin. Insulin treatment caused a time-dependent and reversible shift of the distribution of IGF-II binding activity toward low densities. On SDS-PAGE, the size of the affinity-labeled IGF-II/M6P receptor was comparable in GE and PM fractions (about 255 kDa), but on Western blots receptor size was slightly lower in the latter (245 kDa) than in the former (255 kDa). Insulin treatment did not affect the size, but modified the abundance of the IGF-II/M6P receptor in a manner similar to that of IGF-II binding. In vivo chloroquine treatment fully suppressed the changes in IGF-II binding activity in liver GE and PM fractions observed in insulin-treated rats. We conclude that insulin elicits a time-dependent and reversible redistribution of liver IGF-II receptors from Golgi elements and endosomes to the plasma membrane, presumably via early endosomes.     

The type II insulin-like growth factor receptor is internalized and recycles in the absence of ligand

Recent studies have demonstrated that ligand-bound insulin-like growth factor (IGF)-II receptors on the adipocyte cell surface are rapidly internalized into an intracellular membrane fraction prior to recycling to the plasma membrane (Oka, Y., Rozek, L. M., and Czech, M. P. (1985) J. Biol. Chem. 260, 9435-9442). In order to evaluate whether these subcellular movements of IGF-II receptors in fat cells require their binding to ligand, cell surface IGF-II receptors of insulin-treated fat cells were iodinated with Na125I and lactoperoxidase at 15 degrees C. IGF-II receptors were then localized by immunoadsorption from solubilized cell surface plasma membranes and intracellular low density microsomes derived from labeled cells. When fat cells were homogenized immediately after iodination, most of the labeled IGF-II receptors were associated with the plasma membrane fraction. However, when iodinated fat cells were incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors progressively decreased in the plasma membrane fraction and concomitantly increased in the low density microsome fraction with a half-time of about 5 min. The rate of increase of radiolabeled IGF-II receptors appearing in the low density microsomes of labeled fat cells incubated with insulin was not changed by the addition of a saturating concentration of IGF-II. These results indicate that cell surface IGF-II receptors are rapidly internalized and recycled even in the absence of ligand binding in insulin-treated adipocytes.     

Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity

The effects of insulin therapy in streptozotocin diabetic rats on the glucose transport response to insulin in adipose cells have been examined. At sequential intervals during subcutaneous insulin infusion, isolated cells were prepared and incubated with or without insulin, and 3-O-methylglucose transport was measured. Insulin treatment not only reversed the insulin-resistant glucose transport associated with diabetes, but resulted in a progressive hyperresponsiveness, peaking with a 3-fold overshoot at 7-8 days (12.1 +/- 0.3 versus 3.4 +/- 0.1 fmol/cell/min, mean +/- S.E.) and remaining elevated for more than 3 weeks. During the peak overshoot, glucose transporters in subcellular membrane fractions were assessed by cytochalasin B binding. Insulin therapy restored glucose transporter concentration in the plasma membranes of insulin-stimulated cells from a 40% depleted level previously reported in the diabetic state to approximately 35% greater than control (38 +/- 4 versus 28 +/- 2 pmol/mg of membrane protein). Glucose transporter concentration in the low-density microsomes from basal cells was also restored from an approximately 45% depleted level back to normal (50 +/- 4 versus 50 +/- 6 pmol/mg of membrane protein), whereas total intracellular glucose transporters were further increased due to an approximately 2-fold increase in low-density microsomal membrane protein. However, these increases remained markedly less than the enhancement of insulin-stimulated glucose transport activity in the intact cell. Thus, insulin treatment of diabetic rats produces a marked and sustained hyperresponsive insulin-stimulated glucose transport activity in the adipose cell with little more than a restoration to the non-diabetic control level of glucose transporter translocation. Because this enhanced glucose transport activity occurs through an increase in Vmax, insulin therapy appears to be associated with a marked increase in glucose transporter intrinsic activity.     

The insulin-like effect of growth hormone on insulin-like growth factor II receptors is opposed by cyclic AMP. Evidence for a common post-receptor pathway for growth hormone and insulin action.

The counter-regulatory effects of beta-adrenergic stimulation and cyclic AMP on the insulin-like action of growth hormone (GH) on the subcellular distribution of insulin-like growth factor II (IGF-II) receptors were studied in fat cells from hypophysectomized (Hx) and sham-operated rats. For comparison, the effect of insulin on this process was also studied. Basal IGF-II binding was increased by approx. 2-fold in cells from Hx as compared with sham-operated animals. The stimulatory effect of insulin was decreased in Hx cells, mainly due to a basal redistribution but also to a reduced total number of receptors. GH exerted an acute insulin-like effect in cells from Hx rats and stimulated the translocation of IGF-II receptors from an intracellular pool to the plasma membrane. beta-Adrenergic stimulation with isoprenaline or addition of the non-metabolizable cyclic AMP-analogue N6-monobutyryl cyclic AMP induced a cellular resistance to both GH and insulin and also reduced the responsiveness to these hormones. Adenosine exerted a modulatory effect on both hormones. Binding of 125I-labelled GH to its receptors was not significantly changed by any of these factors. It is concluded that: (1) beta-adrenergic stimulation and cyclic AMP induce a cellular GH resistance at a level distal to the GH-binding site, and (2) the insulin-like effect of GH shares a common pathway with insulin which occurs at the post-binding level.     

Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C

Phosphorylation of hormone receptors by protein kinase C (PKC) may be involved in the regulation of receptor recycling. We have studied the recycling and the phosphorylation state of the insulin growth factor (IGF) II/mannose 6-phosphate (Man-6-P) receptor in microvascular endothelial cells from rat adipose tissue. Scatchard analysis showed these cells have over 2 x 10(6) receptors/cell with an affinity constant of 1 x 10(9) M-1. In the presence of phorbol myristate acetate (PMA), an activator of PKC and analog of diacylglycerol, IGF-II receptor number increased in the plasma membrane by 60% without changes in the binding affinity. This increase in cell surface receptor number was confirmed by affinity cross-linking and 125I-surface labeling studies, occurred with a half-time of 20 min, and was reversible upon withdrawal of PMA. The redistribution of IGF-II/Man-6-P receptors was not due to an inhibition of internalization which was in fact stimulated by PMA. The effect of PMA on IGF-II receptor recycling correlated with its stimulation of PKC activity. Furthermore, after down-regulation of cellular PKC levels by preincubation with PMA, PMA was unable to activate residual PKC activity in the membranous pool or increase IGF-II receptor number at the cell surface. The phosphorylation state of the IGF-II/Man-6-P receptor was determined by 32P labeling of intact cells and immunoprecipitation with anti-receptor antibodies. In the basal state, the receptor was phosphorylated only on serine residues which was increased by 75% after treatment with PMA. In contrast, IGF-II decreased receptor phosphorylation and plasma membrane binding in a parallel and dose-dependent manner. Thus, PKC-stimulated serine phosphorylation of IGF-II/Man-6-P receptor may promote the translocation of the receptor to the cell surface, whereas IGF-II-stimulated dephosphorylation of the receptor may lead to a decrease in the number of cell surface receptors. These data suggest a role for PKC-mediated serine phosphorylation in the regulation of intracellular trafficking of receptors in endothelial cells.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific d-glucose-inhibitable [³H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4–5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7·10⁶/cell). Insulin does not influence the K_d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15·10³ mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Role of protein kinase C in the regulation of glucose transport in the rat adipose cell. Translocation of glucose transporters without stimulation of glucose transport activity

The possible role of protein kinase C in the regulation of glucose transport in the rat adipose cell has been examined. Both insulin and phorbol 12-myristate 13-acetate (PMA) stimulate 3-O-methylglucose transport in the intact cell ein association with the subcellular redistribution of glucose transporters from the low density microsomes to the plasma membranes, as assessed by cytochalasin B binding. In addition, the actions of insulin and PMA on glucose transport activity and glucose transporter redistribution are additive. Furthermore, PMA accelerates insulin's stimulation of glucose transport activity, reducing the t1/2 from 3.2 +/- 0.4 to 2.1 +/- 0.2 min (mean +/- S.E.). However, the effect of PMA on glucose transport activity is approximately 10% of that for insulin whereas its effect on glucose transporter redistribution is approximately 50% of the insulin response. Immunoblots of the GLUT1 and GLUT4 glucose transporter isoforms in subcellular membrane fractions also demonstrate that the translocations of GLUT1 in response to PMA and insulin are of similar magnitude whereas the translocation of GLUT4 in response to insulin is markedly greater than that in response to PMA. Thus, glucose transport activity in the intact cell with PMA and insulin correlates more closely with the appearance of GLUT4 in the plasma membrane than cytochalasin B-assayable glucose transporters. Although these data do not clarify the potential role of protein kinase C in the mechanism of insulin action, they do suggest that the mechanisms through which insulin and PMA stimulate glucose transport are distinct but interactive.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific D-glucose-inhibitable [3H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4-5 fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7 X 10(6)/cell). Insulin does not influence the Kd of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15 X 10(3) mol of glucose/min per mol of transporters at 37 degrees C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Hybrid receptors formed by insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) have low insulin and high IGF-1 affinity irrespective of the IR splice variant

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.     

Exercise modulates the insulin-induced translocation of glucose transporters in rat skeletal muscle

Insulin and acute exercise (45 min of treadmill run) increased glucose uptake into perfused rat hindlimbs 5-fold and 3.2-fold, respectively. Following exercise, insulin treatment resulted in a further increase in glucose uptake. The subcellular distribution of the muscle glucose transporters GLUT-1 and GLUT-4 was determined in plasma membranes and intracellular membranes. Neither exercise nor exercise----insulin treatment altered the distribution of GLUT-1 transporters in these membrane fractions. In contrast, exercise, insulin and exercise----insulin treatment caused comparable increases in GLUT-4 transporters in the plasma membrane. The results suggest that exercise might limit insulin-induced GLUT-4 recruitment and that following exercise, insulin may alter the intrinsic activity of plasma membrane glucose transporters.     

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

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

Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.     

Regulation of mannose 6-phosphate/insulin-like growth factor II receptor distribution by activators and inhibitors of protein kinase C

The tumor-promotor phorbol dibutyrate (PDBt) increases the binding of a neoglycoprotein containing mannose 6-phosphate (Man6P) and of insulin-like growth factor II (IGF-II) to the Man6P/IGF-II receptor at the cell surface. This effect is dependent on time and concentration and is also seen with synthetic 1-oleoyl-2-acetyl-sn-glycerol, but not with 4 alpha-phorbol, an inactive tumor-promoter. The increase is due to a 3-4-fold increase in the number of cell-surface, receptors, accompanied by a 1.6-fold increase in ligand-binding affinity. The internalization rate of the Man6P/IGF-II receptor is not affected by PDBt, suggesting that the redistribution of these receptors to the cell surface is due to an accelerated externalization rate. The redistribution of Man6P/IGF-II receptors did not impair the sorting of newly synthesized Man6P-containing ligands while uptake of these ligands is 2-4-fold increased. Inactivation or down regulation of protein kinase C decreased the binding of the Man6P-containing neoglycoprotein to 65% of controls. Incubation of cells with Man6P, IGF-I, IGF-II or epidermal growth factor induces a rapid redistribution of Man6P/IGF-II receptors to the plasma membrane [Braulke, T., Tippmer, S., Neher, E. & von Figura, K. (1989) EMBO J. 8, 681-686]. Incubation with PDBt prevented the effect of growth factors but not that of Man6P on receptor redistribution. Inactivation of protein kinase C did not affect the Man6P/IGF-II receptor redistribution induced by Man6P and growth factors. These data suggest that Man6P, growth factors and activation of protein kinase C by phorbol esters and diacylglycerols modulate Man6P/IGF-II receptor cell-surface binding by at least two independent mechanisms, receptor redistribution as well as an increase of binding affinity, which might be involved in regulation of endocytosis of ligands.     

Endocytosis of insulin-like growth factor II by a mini-receptor based on repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) plays an important role in controlling the extracellular level of the insulin-like growth factor II (IGF-II) by mediating its binding at the cell surface and delivery to lysosomes. Loss of the receptor is associated with an accumulation of IGF-II, which can cause perinatal lethality if it is systemic, or local proliferation and tumorgenesis if it is spatially restricted. The extracytoplasmic domain of the receptor consists of 15 homologous repeats, of which repeat 11 carries the IGF-II-binding site of the multifunctional receptor. To investigate whether repeat 11 is sufficient to mediate binding and internalization of IGF-II, a construct consisting of repeat 11 fused to the transmembrane and cytoplasmic domain of the M6P/IGF-II receptor was transfected into mouse embryonic fibroblasts. The construct was expressed as a stable membrane protein which binds IGF-II with a 10-fold lower affinity as observed for the M6P/IGF-II receptor and is found at the cell surface and in endosomes. It mediates the internalization of IGF-II and its delivery to lysosomes, suggesting that it can function as a IGF-II mini-receptor controlling the extracellular IGF-II level.     

Proposed mechanism for increased insulin-mediated glucose transport in adipose cells from young, obese Zucker rats. Large intracellular pool of glucose transporters

The mechanism for hyperresponsive insulin-mediated glucose transport in adipose cells from 30-day-old obese Zucker rats was examined. Glucose transport was assayed by measuring 3-O-methylglucose transport, and the concentration of glucose transporters was estimated by measuring specific D-glucose-inhibitable cytochalasin B binding. Insulin increased glucose transport activity by approximately 17 fmol/cell/min in cells from obese rats compared to 3 fmol/cell/min in lean littermates. Insulin increased the concentration of glucose transporters in the plasma membrane fraction by about 15 pmol/mg of membrane protein in both groups. The insulin-mediated decrease in the concentration of transporters in the low-density microsomal fraction was 30 pmol/mg of membrane protein for the obese rats compared to 15 pmol/mg of membrane protein for the lean controls. An estimated number of glucose transporters was calculated using membrane protein and enzyme recoveries for each group. Insulin increased the number of transporters in the plasma membrane by 3 X 10(6) sites/cell for the obese rats and only 0.6 X 10(6) sites/cell for the lean controls. In addition, insulin decreased the number of transporters/cell in the intracellular membrane pool by approximately 4 X 10(6) sites/cell for the obese rats and 0.9 X 10(6) sites/cells for the lean rats. The total number of transporters/cell was about 7 X 10(6) sites/cell for the obese animals and 1.6 X 10(6) sites/cell for the lean controls. In the basal state, more than 80% of these transporters were located in the intracellular pool for both the lean and obese rats. Thus, the marked hyperresponsive insulin-mediated glucose transport observed in adipose cells from 30-day-old obese Zucker rats may be the consequence of a marked increase in the number of glucose transporters in the intracellular pool.