Possible effect of bromocriptine on the insulin receptor

The administration of 0.00011 mg/g weight/day of bromocriptine (CB154) for 7 days to Wistar rats, improved the peripheral glucose uptake without significant changes in plasma insulin level, during the intravenous glucose tolerance test (0.33 g/kg). The mode of the bromocriptine action on binding of 125I insulin to erythrocyte insulin receptors has been evaluated. The total number of sites was greater with bromocriptine (513.1 +/- 124.1 pM/1,CB154 815.6 +/- 107.9 pM/l) (p less than 0.01). The high affinity/low capacity compound of insulin receptor, in CB154 rats (51.8 +/- 16.8 pM/l) was higher than in normal rats (18.3 +/- 8.9 pM/l) (p less than 0.005). Additional studies indicated that CB154 had no effect on the rate of association and dissociation of 125I insulin from rats erythrocyte insulin receptors. The degradation of insulin or the erythrocyte receptor sites do not change, after the treatment with CB154.     

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.     

Bacitracin enhances intracellular accumulation of insulin in rat adipocytes

Bacitracin (1 mg/ml) markedly increased (approx. 75%) the cell-associated specifically bound 125I-labelled insulin without altering the affinity of the binding sites. Bacitracin also exerted a modest inhibitory effect on the degradation of insulin in the incubation medium determined as radioactivity not precipitated by trichloroacetic acid (from 9.6 to 4.8%). The effect on insulin binding was about 5-times as sensitive as the effect on degradation. The increased binding was due to intracellular accumulation of radioactivity which could not be removed by treating the cells with trypsin. This increase was not seen when the internalization process was reduced by ATP-depletion or low temperature. Since the trypsin-sensitive fraction of cell-associated radioactivity was apparently not altered, it is suggested that bacitracin, in addition to its well-known inhibition of extracellular degradation, also inhibits the intracellular degradation of insulin.     

Varying brain insulin concentrations differentially regulate the fetal brain insulin receptor

We investigated the downregulating effect of varying states (physiologic and pharmacologic) of systemic and intracranial hyperinsulinism on the 28 to 30 day fetal rabbit brain insulin receptor. Alloxan-induced maternal diabetes (n = 5) produced mild fetal hyperinsulinemia (D) (plasma insulin concentrations = 59.80 +/- 8.10 microU/ml, control = 26.25 +/- 3.70; p less than 0.01), whereas systemic administration (IMI) of 1.0 U (n = 4) and 2.0 U (n = 4) of insulin to the fetus resulted in moderate (103.13 +/- 34.63 microU/ml) and severe (288.3 +/- 51 microU/ml) fetal hyperinsulinemia respectively. All three states of systemic hyperinsulinemia neither altered the fetal brain insulin content nor the brain insulin receptor number and affinity. 0.01 U (n = 4) of intracranial insulin administration (ICI) increased the brain insulin content four-fold (p less than 0.01) but did not alter the brain insulin receptor number or affinity. 0.1 (n = 5) and 2.0 U (n = 7) of intracranial insulin increased the brain insulin content to supraphysiologic concentrations (p less than 0.01) and decreased the fetal brain insulin receptor number (p less than 0.01), the affinity remaining constant. We conclude that 1) regardless of the ability of insulin to cross the blood brain barrier, the downregulation of the brain insulin receptor is insulin dose-dependent and 2) the downregulation of the fetal brain insulin receptor is not a physiologic but a pharmacologic effect of insulin.     

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.     

Up-regulation of insulin receptors with dexamethasone in cultured human urinary bladder carcinoma cells

Cultured human urinary bladder carcinoma cells ( JTC -32) were used to investigate the regulation of insulin receptors by dexamethasone. When the cells were preincubated with dexamethasone at 37 degrees C, insulin binding sites increased up to 24 h. A large increase in insulin binding sites took place for 14 h of preincubation with dexamethasone. At lower concentrations of dexamethasone (less than 1 nM), no significant increase in insulin binding sites was observed, but the maximal increase was observed at more than 10 nM. Scatchard plots showed that dexamethasone increased the number of high affinity insulin binding sites (2.8 fold) without any change in the apparent equilibrium constant in JTC -32 cells. In addition, this steroid hormone also increased the number of low affinity insulin binding sites (1.6 fold) with a small change in the apparent equilibrium constant. Although insulin and dexamethasone did not affect the number of cells or the amount of cellular proteins per dish, dexamethasone plus insulin slightly increased them.     

Thiopental and ether anaesthesia decrease the prostaglandin receptor density of the rat liver.

Male Sprague-Dawley rats were killed either by decapitation or during anaesthesia with thiopental or diethylether by aortectomy. Livers were removed and liver plasma membranes were prepared using standard techniques. Direct binding experiments with 3H-PGE1 and 3H-iloprost revealed heterogeneity of the binding sites (high and low affinity binding sites), whereas 3H-PGE2 demonstrated only high affinity binding to the liver. The highest binding capacity for all radioligands was found for livers after decapitation. Livers obtained during anaesthesia showed a significantly (p less than 0.05 to p less than 0.001) lower binding capacity and binding affinity for 3H-PGE1, 3H-PGE2 and 3H-iloprost. The reduction in binding activity was more pronounced in livers obtained during inhalation than thiopental anaesthesia. Specific binding amounted to 82.1 +/- 7% for 3H-PGE2, 75.3 +/- 9% for 3H-PGE1 and 78.9 +/- 8% for iloprost in livers obtained after decapitation. In livers obtained during anaesthesia specific prostaglandin binding was significantly (p less than 0.01) decreased, again being more pronounced during inhalation than thiopental anaesthesia. These results suggest that some anaesthetics interfere with prostaglandin receptors of the liver.     

Hormone-induced conformational changes in the hepatic insulin receptor

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.     

Erythrocyte insulin receptor characteristics and erythrocyte membrane lipid composition in healthy men

The cell membrane plays an important role in the mechanism of insulin action. To test whether erythrocyte insulin receptor characteristics are related to the erythrocyte membrane lipid composition, 11 healthy volunteers were studied. The relationship between insulin binding to erythrocytes, the number of receptors per cell and the affinity of receptors to insulin on the one hand and total phospholipid fatty acid (FA) composition and cholesterol/phospholipid molar ratio in the erythrocyte membrane on the other hand were evaluated. 1. We found a significant negative correlation between specific insulin binding and the proportion of n-6 essential FA in erythrocyte membrane phospholipids, especially linoleic acid (r = -0.82, p less than 0.01) and arachidonic acid (r = -0.73, p less than 0.05). On the other hand, a significant positive correlation between insulin binding and the proportion of nonessential FA (r = +0.65, p less than 0.05) was seen. Number of receptors per cell and the affinity of receptors were not significantly related to phospholipid FA composition. 2. There was no significant correlation between insulin receptor characteristics and the cholesterol/phospholipid molar ratio in the erythrocyte membrane. The data presented support the hypothesis that the FA pattern of membrane total phospholipids may modify the properties of insulin receptors.     

Insulin receptors in human mononuclear leucocytes. II. Binding and degradation of insulin in obese subjects.

The binding of insulin to the receptors on circulating mononuclear cells of obese subjects is significantly decreased when compared to the binding in normal subjects. This fenomenon is due to the reduction of the number of insulin receptors rather than reduction in affinity. The insulin degradation is also reduced, but a very strong correlation, similar to that demonstrated in normal subjects exist between insulin binding to its receptors and insulin degradation.     

Internalization of the neonatal brain insulin receptor

Using 10-15 day neonatal rabbit brain cells, we studied the internalization (n = 6) and intracellular degradation (n = 8) of specifically bound 125I-insulin. In addition we investigated the association between the internalization of the specifically bound 125I-insulin and the metabolic effects of insulin such as glucose (n = 13) and amino-acid (leucine) uptake (n = 6). Phenylarsine oxide (10 microM), an agent that inhibits the internalization of the insulin receptor (n = 6) decreased the specifically bound 125I-insulin in the intact and trypsin-resistant (inside) part of the brain cells by 50% (p less than 0.05). On the other hand chloroquine (100 microM), a lysosomotropic agent that interferes with the intracellular degradation of the insulin receptor (n = 8) increased two-fold the 125I-insulin specifically bound to the intact and trypsin resistant part of the cells (p less than 0.05). Both these agents did not alter the time-dependent basal glucose uptake by the brain cells. Glucose alone regulated its own uptake (n = 4) whereas 1 X 10(-6) M insulin did not augment the glucose uptake (n = 11+13) above basal. Similarly leucine regulated the leucine uptake (n = 4) but insulin did not alter this basal uptake by the brain cells (n = 6). In summary we observed no associated glucose or leucine uptake along with the presence of internalization and intracellular degradation of specifically bound 125I-insulin in the brain cells.     

The erythrocyte insulin receptor response to insulin induced hypoglycaemia

The response of the erythrocyte insulin receptor to a prolonged intravenous infusion of insulin has been measured in normal individuals during hypoglycaemia and when hypoglycaemia was prevented by the concurrent infusion of glucose. When euglycaemia was maintained, mean (+/- S.D.) specific insulin binding following the 5 hour insulin infusion was unchanged (6.9 +/- 2.1 to 6.65 +/- 2.2% bound per 2.25 X 10(9) erythrocytes). In the presence of mild hypoglycaemia, mean (+/- SD) specific insulin binding rose from 6.6 +/- 2.3 to 7.6 +/- 2.5% bound per 2.25 X 10(9) erythrocytes (P less than 0.01), after 5 hours. This increase was due to increased receptor affinity. It was not correlated with the increase in the concentration of any individual counter-regulatory hormone. Initial insulin receptor binding correlated strongly with the subsequent decline in plasma glucose concentration (r = 0.9527; P less than 0.01). Thus, acute hyperinsulinaemia, when associated with hypoglycaemia, does not result in downregulation of insulin receptors on erythrocytes but rather results in increased receptor binding. Consequently, the insulin receptor may not play an active role in protecting the individual against acute hypoglycaemia.     

Reciprocal changes of insulin and glucagon receptors in primary cultured hepatocytes

The specific [125I]insulin binding to primary cultured hepatocytes was significantly greater than that to freshly isolated hepatocytes. Low affinity insulin binding sites in cultured cells were 6-fold greater in number than those of freshly isolated cells without a significant change in high affinity sites. However, both sensitivity (insulin concentration for half maximum stimulation) and responsiveness (% of increase above the basal level) to insulin for the stimulation of ODC activity were similar for isolated and cultured cells indicating an important role of high affinity sites in the insulin action. On the other hand, the specific [125I]glucagon binding to cultured cells was significantly decreased. Low affinity glucagon binding sites in cultured cells decreased by about 50% in cultured cells without a significant change in high affinity sites. Both sensitivity and responsiveness to glucagon for the stimulation of ketogenesis from palmitate also decreased as compared with those of isolated cells, indicating an important role of low affinity sites in the glucagon action. These results indicate that insulin and glucagon receptors were reciprocally changed in cultured cells, as compared with isolated cells.     

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.     

Erythrocyte insulin receptor and glucose tolerance test in children treated with prednisolone

Insulin receptors of erythrocytes and oral glucose tolerance test (O-GTT) were investigated in sixteen children treated with prednisolone for various diseases. Ten patients (Group 1) received low doses of prednisolone (0.2-0.5 mg/kg body weight/day) and six patients (Group 2) received higher doses of prednisolone (1.5-2.0 mg/kg body weight/day). Compared to the values for controls, the sums of blood glucose (sigma BS) at O-GTT in both group 1 and group 2 patients were significantly elevated. (422 +/- 75 mg/dl, p less than 0.01 Group 1; 419 +/- 39 mg/dl, p less than 0.01 Group 2; 338 +/- 41 mg/dl controls) Significant differences were not observed in the sums of insulin concentration at O-GTT, fasting blood concentration and basal insulin levels among these two groups and the controls. There was a significant increase in the maximum insulin binding in group 2 (9.13 +/- 0.68% in group 2, 7.97 +/- 1.06% in controls, p less than 0.05), but not in group 1 (8.59 +/- 1.82%). There is no significant difference in binding affinity or the number of receptors between any of these two patients' groups and the controls. When patients in group 1 and group 2 were combined, sigma IRI levels were significantly elevated in the patients (p less than 0.05). These results suggested that prednisolone treatment with a smaller dosage as well as with the higher dosage resulted in a carbohydrate intolerance, the main cause of which is located in a postreceptor step (or steps) of insulin action.     

Development of insulin and epidermal growth factor receptors during the differentiation of rat preadipocytes in primary culture

An homogeneous cell population isolated from the inguinal tissue of 3-day-old rats is able to proliferate in primary culture. In the presence of a physiological concentration of insulin (1.5 nM) it converts into cells exhibiting the morphology and the biochemical characteristics of adipocytes. Insulin and epidermal growth factor (EGF) receptors were studied during both the exponential growth and the adipose conversion phases of these cells. Binding experiments with 125I-labelled peptides were performed directly in the culture dishes. The number of high affinity insulin binding sites increased, during the entire culture period studied, reaching 18 days after plating the value of 10,600 x 2360. Control cells (cultured in the presence of anti-insulin antibody) exhibited an increase of the concentration of insulin binding sites from no more than 500 sites/cell to 6880 +/- 1710 sites/cell between dat 0 and 9 (corresponding to the exponential growth phase); this increase was followed by a rapid reduction in insulin receptors during the stationary phase. The density of EGF binding sites increased between day 0 and 4 (one cell cycle), whether the cells were maintained or not with insulin, and plateaued thereafter. Mature adipocytes freshly isolated from the inguinal tissue of 3-day-old rats had no detectable EGF binding sites, but their content in high affinity binding sites for insulin was similar to that of cells after complete adipocyte conversion in primary culture.     

Association of specific immune response to pork and beef insulin with certain HLA-DR antigens in type 1 diabetes.

To test the association of HLA-DR antigens with high-responder and low-responder status to either beef or pork insulin, insulin antibodies in diabetic sera were separated into those with average low and those with average high affinity and their insulin-binding capacities for each insulin determined. Significantly less binding of pork insulin by the high affinity antibodies occurred in the group of patients with DR3 antigens compared with those with DR4 antigens (p less than 0.01) and DR3/4 antigens (p less than 0.01). The difference in the binding capacity of beef insulin by the high affinity antibodies between the groups with DR3 and DR4 antigens was less pronounced but still significant. The high-responder status of DR3/4 antigens to pork insulin suggests that the gene or genes associated with HLA-DR4, and responsible for a high response to pork insulin, are dominant to genes associated with HLA-DR3 and a low response. If extended to human insulin and different HLA-DR and HLA-B antigen patterns, these finding should help in the therapeutic selection of the appropriate insulin and thus reduce the induction of an anti-insulin response in patients with diabetes.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

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.     

Effect of exercise on insulin binding and glucose transport in adipocytes of normal humans

Acute exercise increases insulin binding to its receptors on blood cells. Whether the enhanced insulin binding explains the exercise-induced increase in glucose uptake is unclear, since insulin binding and glucose uptake have not been measured simultaneously in a target tissue of insulin. In this study, we determined insulin binding and the rate of glucose transport in adipocytes obtained by needle biopsy from 10 healthy men before and after 3 h of cycle-ergometric exercise. During the exercise, plasma glucose (P less than 0.01) and insulin (P less than 0.001) fell and serum free fatty acid level rose 4.3-fold (P less than 0.001). 125I-insulin binding to adipocytes remained unchanged during exercise. The rate of basal glucose transport clearance fell from 28.1 +/- 5.7 fl.cell-1.s-1 to 22.9 +/- 5.6 fl.cell-1.s-1 (P less than 0.005), and the insulin-stimulated increase in glucose transport rate rose from 196 +/- 26 to 279 +/- 33% (P less than 0.025) during the exercise. Thus, in the adipocytes during exercise, the basal glucose transport rate and the responsiveness of glucose transport to insulin changed in the absence of alterations in insulin binding. These data indicate that the exercise-induced changes in insulin binding show tissue specificity and do not always parallel alterations in glucose transport.