125I-insulin Binding Research Articles

Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle

The distribution of glucose transporters and of insulin receptors on the surface membranes of skeletal muscle was studied, using isolated plasma membranes and transverse tubule preparations. (i) Plasma membranes from rabbit skeletal muscle were prepared according to Seiler and Fleischer (1982, J. Biol. Chem. 257,13862–13871), and transverse tubules from rabbit skeletal muscle were prepared according to Rosemblatt et al. (1981 , J. Biol. Chem. 256, 8140–8148) as modified by Hidalgo et al. (1983 , J. Biol. Chem. 258, 13937–13945). The membranes were identified by the abundance of nitrendipine receptors in the transverse tubules, and their relative absence from the plasma membranes. (ii) Plasma membranes and transverse tubules were also isolated from rat skeletal muscle, according to a novel procedure that isolates both fractions from the same common homogenate. (iii) Glucose transporters were detected by d-glucose protectable binding of the specific inhibitor [ 3H]cytochalasin B, and insulin receptors were detected by saturable binding of 125I-insulin. The concentration of glucose transporters was about threefold (rabbit) or fivefold (rat) higher in the transverse tubule membrane compared to the plasma membrane, whereas the insulin receptor concentration was about the same in both membranes. These results indicate that the glucose transporters on the surface of the muscle are preferentially segregated to the transverse tubules, and this poses interesting consequences on the functional response of glucose transport to insulin in skeletal muscle.

Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes. Inverse relationship to glycogen content.

Cultured rat hepatocytes were used to characterize the relationship between cellular glycogen content and the basal rate, as well as response to insulin of glycogen synthesis. Depending on the concentration of medium glucose, glycogen-depleted monolayers accumulated glycogen between 24 and 48 h of culture up to the fed in vivo level. Insulin at 100 nM stimulated glycogen deposition 20-fold at 1 mM and 1.5-fold at 50 mM glucose. The rate of further glycogen storage decreased with time and increasing glycogen content. In hepatocytes preincubated with 1-50 mM glucose during 24-48 h, short-term basal and insulin-dependent incorporation of 10 mM [14C]glucose into glycogen was inversely related to the actual cellular glycogen content. This was not due to different intracellular dilution of the label, since the specific radioactivity of UDP-glucose was similar in all groups. 125I-Insulin binding indicated that insulin receptors were also not involved in this phenomenon. An inverse relationship was also found between glycogen content and the stimulation of glycogen synthase I activity by insulin, whereas the basal activity of the enzyme was dissociated from the rate of incorporation of [14C]glucose. Basal net glycogen deposition at 10 mM glucose was also inversely related to cellular glycogen; however, no such relation was evident in the presence of insulin due to the overlapping inhibition of glycogenolysis. These studies suggest that the glycogen-mediated inhibition of the activation of glycogen synthase I is operative in the cultured hepatocyte and leads to an apparent inverse relationship between the actual glycogen content and basal as well as insulin-dependent glycogenesis.

Characterization of insulin-like growth factor binding to human granulosa cells obtained during in vitro fertilization.

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I. Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 +/- 0.30%/10(5) cells (SEM, n = 9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (alpha IR-3) inhibited 125I-IGF-I but not 125I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the alpha subunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays. IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.

Identification of insulin receptors on rat Sertoli cells

The binding of insulin to rat Sertoli cells was investigated to establish if effects of insulin on Sertoli cells can be mediated via insulin receptors. Sertoli cells were isolated from the testes of 3-week-old rats, and preincubated for 3 days in the absence of hormones. Binding of 125I-porcine insulin to the Sertoli cells was 75–80% specific and this binding was time- and pH-dependent and reversible. Scatchard analysis of the binding data resulted in curvilinear plots with a high affinity binding of K d = 1.8 × 10 −9 M. Porcine and bovine insulin competed equally well for 125I-porcine insulin binding. Porcine proinsulin was 10–50 times less potent, corresponding to its lower biological activity. Insulin-like growth factor-I (IGF-I) was 30–40 times less potent, indicating low affinity binding of IGF-I to the insulin receptor. Lutropin which was used as a control gave no competition with the 125I-insulin binding. Affinity labelling of Sertoli cell membrane proteins with 125I-insulin using the cross-linking agent disuccinimidylsuberate revealed binding of insulin to (a) protein(s) of M r > 300000 or M r = 130000 after electrophoresis under non-reducing or reducing conditions, respectively. Affinity labelling with 125I-insulin was largely prevented by unlabelled insulin. It is concluded that the protein of M r 130000 may represent the α-subunit of the insulin receptor. The presence of insulin receptors as well as IGF-I receptors on cultured rat Sertoli cells may suggest that insulin and IGF-I have specific functions in regulating the maturation and activities of Sertoli cells during the initiation and maintenance of spermatogenesis.

Radioreceptor assay for insulin formulations.

A sensitive radioreceptor assay was developed for pharmaceutical insulin formulations with the use of rat liver membranes and 125I-insulin. The addition of unlabeled insulin or insulin analogues inhibited the binding of 125I-insulin to the receptors in a concentration-dependent way. Insulin concentrations between 3 x 10(-9) and 3 x 10(-8) M were used for a three-point assay which fulfilled pharmacopoeial statistical requirements for validity of the assay. The coefficient of variation for five assays was 4%. Potency estimates of insulin preparations obtained with the radioreceptor assay correlated well with corresponding estimates from in vivo assays. Both assays also gave similar potency readings for insulin preparations that were partly degraded during storage at elevated temperatures, but the radioreceptor method was more precise.

The effect of continuous subcutaneous insulin infusion on insulin binding to erythrocytes in diabetes mellitus

The effect of continuous subcutaneous insulin infusion (CSII) on glycaemic control and insulin binding to erythrocytes was studied in six diabetic patients. A marked improvement in blood glucose control during CSII was observed in these patients previously on conventional therapy. Specific 125I-insulin binding to erythrocytes of the diabetics before the institution of CSII was significantly lower than that of age-, weight- and sex-matched nondiabetic subjects, 6.5 +/- 0.1% vs. 9.8 +/- 0.3% (P less than 0.05). After 4-6 months of CSII, insulin binding was restored to normal levels. This normalization in insulin binding to erythrocytes of patients on CSII was due to a 35% increase in active binding sites, together with a small change in binding affinity.

Insulin Binding and its Action in Adipocytes of Hyperthyroid and Hypothyroid Rats

The studies of the effects of thyroid hormone on insulin secretion and glucose metabolism have been made, but the results have been controversial.In order to evaluate the effects of thyroid hormone at the cellular level, insulin binding and insulin-induced lipogenesis in isolated rat epididymal fat cells were studied in control, hyperthyroid and hypothyroid rats. Hyperthyroidism was induced by daily intraperitoneal injection of sodium L-thyroxine and hypothyroidism by a single injection of 131I. The adipocytes were isolated by treatment of collagenase as originally described by Gliemann (1967).The results were as follows: 1) The fasting serum insulin levels of hyperthyroid (21.6 ± 3.7 uU/ml) and hypothyroid groups (20.5 ± 7.0 uU/ml) were not different from the value of control (23.1 ± 11 uU/ml). The fasting blood glucose level of hypothyroid group (164.9 ± 12.0 mg/dl) was higher than the values of the controls (148.2 ± 13.2 mg/dl) or the hyperthyroid group (147.0 ± 12.5 mg/dl), (p<.005).2) The specific 125I-insulin binding of the hyperthyroid group was not different from the value of the controls, but the value of the hypothyroid group was higher than the value of the controls (p<.005).3) The insulin receptor concentration of the hypothyroid group (1.09 ± 0.02 ng/0.5×105 cells) was higher than the value of the controls (0.72 ± 0.01 ng/0.5×105 cells) or the hyperthyroid group (0.79 ± 0.02 ng/0.5×105 cells), (p<.05).4) The average affinities of the receptors in all groups showed an inverse correlation with the insulin concentration. The average affinity of the hypothyroid group was higher than the value of the control or the hyperthyroid group.5) Insulin-induced lipogenesis was reduced proportionately in all insulin concentrations in both the hyperthyroid and hypothyroid groups compared with the dose-response curve of the control group. The maximal amount of lipogenesis of the hyperthyroid and hypothyroid groups were 63.4% (p<.05) and 32.3% (p<.005) of the controls, respectively.These studies suggest that thyroid hormone may regulate the concentration of insulin receptors, and altered thyroid states reduce insulin-induced lipogenesis in the adipocyte at postreceptor levels.

Fasting and refeeding alter the insulin receptor tyrosine kinase in chicken liver but fail to affect brain insulin receptors.

Insulin receptors from chicken liver and brain were studied following alterations in the nutritional state. Chickens were either fasted for 48 h, fasted for 48 h and then refed for 24 h, or fed a regular diet ad libitum. 125I-Porcine insulin binding was significantly elevated in liver membranes from the fasted animals and lowered in refed chickens when compared to preparations from ad libitum fed chickens. These changes in 125I-insulin binding were inversely related to the levels of plasma insulin and since receptor affinities for insulin were similar in each group, they probably represent alterations in receptor number. Apparent Mr of alpha subunits of the insulin receptors was unaffected by alterations in the nutritional states. The presence of ATPase-like activities that co-eluted with liver insulin receptors from wheat germ agglutinin lectin columns but not from pea lectin columns necessitated the use of both pea and wheat germ agglutinin for liver insulin receptor purification. The insulin receptors purified from both lectin columns were recognized by anti-insulin receptor antiserum and had similar affinities for insulin which were unaltered by the nutritional state. Insulin-stimulatable autophosphorylation of the beta subunit of the insulin receptor was lower in livers from fasted chickens and intermediate in refed chickens. Furthermore, basal and insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr) 4:1 was significantly less in the fasting state and intermediate in the refed state compared to the ad libitum fed state. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in all three states suggesting that the differences in insulin-induced phosphorylation are due to a change in maximal stimulation and not a change in insulin sensitivity. In contrast to the alterations seen with liver receptors, brain insulin receptors were unaffected by these alterations in nutritional state. These findings suggest that: liver insulin receptors are affected by altering the nutritional state; insulin binding to liver membranes is inversely related to plasma insulin levels; and tyrosine kinase is decreased both in fasted and refed animals suggesting an uncoupling of the normal interaction between alpha subunit and beta subunit in liver insulin receptors.

Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors.

Insulin-like growth factor (IGF) I (greater than or equal to 10(-10)M, insulin-like growth factor II (greater than or equal to 10(-9) M), insulin (greater than or equal to 10(-9) M, and epidermal growth factor (EGF, greater than or equal to 10(-11) M) caused rapid membrane ruffling in KB cells. The morphological change was observed within 1 min after the addition of these growth factors and was accompanied by microfilament reorganization, but not by microtubule reorganization. IGF-I, IGF-II, and insulin induced morphologically very similar or identical membrane ruffles with the order of potency IGF-I greater than IGF-II greater than insulin, whereas EGF-induced membrane ruffles were morphologically different. KB cells possessed EGF receptors, type I IGF receptors, and insulin receptors, but few or no type II IGF receptors. Monoclonal antibody against type I IGF receptors, which completely inhibited the binding of 125I-IGF-I to the cells but did not inhibit the binding of 125I-insulin, caused marked inhibition of IGF-I (10(-8) M)-stimulated membrane ruffling. IGF-II (10(-8) M)-stimulated membrane ruffling was partially inhibited in the presence of this antibody, but insulin (10(-7) M)-stimulated membrane ruffling was only slightly inhibited. In contrast, monoclonal antibody against insulin receptors blocked insulin (10(-7) M) stimulation, but not IGF-I (10(-8) M) stimulation, of membrane ruffling. Thus, this study provides evidence that IGF-I and insulin act mostly through their own (homologous) receptors and that IGF-II acts by cross-reacting with both type I IGF and insulin (heterologous) receptors in causing rapid alterations in cytoskeletal structure.

Diacylglycerol modulation of insulin receptor from cultured human mononuclear cells. Effects on binding and internalization.

Tumor-promoting phorbol esters alter binding of growth factors and hormones to their specific receptors. Action of diacylglycerols, endogenous phorbol ester analogues, on 125I-labeled insulin binding to its receptor from human cells was therefore investigated. A variety of 1,2-diacylglycerols and 1,3-diacylglycerols inhibited 125I-insulin binding to intact human monocyte-like (U-937) and lymphoblastoid (IM-9) cells in a dose-, time-, and temperature-dependent manner within 30 sec at 37 degrees C in a fashion analogous to that of the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate (TPA). Inhibition of insulin binding by diacylglycerols, analyzed by Scatchard plot, seems to be due to altered binding affinity of the insulin receptor. Diacylglycerol effects were reversible, were seen regardless of the order of addition of 125I-insulin and diacylglycerols, and were demonstrated only with occupied insulin receptors. Corresponding fatty acids or phospholipids did not affect specific insulin binding to the intact U-937 cells. Diacylglycerols also inhibited binding of 125I-insulin-like growth factor (IGF) I but not that of 125I-human growth hormone (HGH) to the human cells. The non-tumor-promoting phorbols (phorbol, 4-alpha-phorbol, phorbol-12,13-distearate) did not affect insulin binding to intact cells. Both diacylglycerols and TPA stimulated internalization of 125I-insulin by U-937 and IM-9 cells. The ability of diacylglycerol to mimic the effects of TPA on the insulin receptor supports the concept of diacylglycerols as endogenous phorbol diester analogues even though the sole role of protein kinase C in our system is doubtful.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-surface insulin receptor cycling and its implication in the glycogenic response in cultured foetal hepatocytes.

The insulin-receptor cycle was investigated in cultured foetal rat hepatocytes by determining the variations in insulin-binding sites at the cell surface after short exposure to the hormone. Binding of 125I-insulin was measured at 4 degrees C after dissociation of prebound native insulin. Two protocols were used: exchange binding assay and binding after acid treatment; both gave the same results. Cell-surface 125I-insulin-receptor binding decreased sharply (by 40%) during the first 5 min of 10 nM-insulin exposure (t1/2 = 2 min) and remained practically constant thereafter; subsequent removal of the hormone restored the initial binding within 10 min. This fall-rise sequence corresponded to variations in the number of insulin receptors at the cell surface, with no detectable change in receptor affinity. The reversible translocation of insulin receptors from the cell surface to a compartment not accessible to insulin at 4 degrees C was hormone-concentration- and temperature-dependent. SDS/polyacrylamide-gel electrophoresis after cross-linking of bound 125I-insulin to cell-surface proteins with disuccinimidyl suberate showed that these variations were not associated with changes in Mr of binding components, in particular for the major labelled band of Mr 130,000. The insulin-receptor cycle could be repeated after intermittent exposure to insulin. Continuous or intermittent exposure to the hormone gave a similar glycogenic response, contrary to the partial effect of a unique short (5-20 min) exposure. A relationship could be established between the repetitive character of the rapid insulin-receptor cycle and the maximal expression of the biological effect in cultured foetal hepatocytes.

Glucose starvation alters insulin but not IGF-I binding to Chinese hamster ovary (CHO) cells

The pattern of cellular protein glycosylation can be altered in CHO cells by glucose starvation. When wild type CHO cells are deprived of glucose, 125I-insulin binding increases from a B F of 0.033 ± 0.004 to 0.063 ± 0.011, due to an increase in receptor affinity. The already elevated insulin binding to mutant B4-2-1 CHO cells, whose genetic defect causes abnormal glycosylation mimicking the pattern seen in the glucose starved normal cells, is not affected by glucose starvation. In neither cell line is 125I-IGF-I binding affected by glucose starvation. These data support the hypothesis that abnormal glycosylation can alter insulin binding to its receptor. Furthermore, there is a striking difference in the susceptibility of IGF-I and insulin receptors to alterations in glycosylation.

Insulin resistance in uremia: In vitro model in the rat liver using human serum to study mechanisms

We have previously demonstrated in a rat model of chronic uremia that the liver is resistant to insulin. To further investigate the mechanism(s) of insulin resistance in uremia, primary cultures of normal rat hepatocytes were incubated with varying dilutions ( 1 10 to 1 10,000 ) of sera from undialyzed end stage uremic and normal humans for 20 hours. We then studied insulin action, binding, and postbinding events. Dilutions of uremic sera as low as 1 1,000 rendered the hepatocytes resistant to maximal concentrations of insulin with regard to [ 14C]acetate incorporation into lipids. The dose response curve for insulin-stimulated [ 14C]aminoisobutyric acid uptake demonstrated a shift to the right in hepatocytes incubated with uremic sera when compared with those incubated with normal sera. The 125I-insulin binding sites and affinity, 125I-insulin internalization and degradation, insulin receptor structure, autophosphorylation of the insulin receptor, and its tyrosine-specific kinase activity were normal in the hepatocytes rendered resistant to insulin by uremic sera. However, these cells failed to generate the chemical mediator or second messenger of insulin action, as assessed by its ability to stimulate pyruvate dehydrogenase (PDH) in liver mitochondria from normal rats. We concluded that uremic sera renders normal rat hepatocytes resistant to insulin. Insulin resistance is a postinsulin receptor kinase defect possibly due to lack of the generation of the chemical mediator of insulin action. This in vitro cell model may be useful to further define the mechanism(s) and the serum factor(s) responsible for insulin resistance in uremia in the absence of complicating hormonal and substrate changes that occur in vivo.

Hexose transport after glucose refeeding of glucose-starved human fibroblasts: 1. The effects of tunicamycin and cycloheximide 2. Insulin binding and action

Hexose transport in glucose-starved human fibroblasts was readily reversed by glucose refeeding. This hexose transport reversal was not inhibited by tunicamycin (1.5 μg/ml) but was blocked by cycloheximide (20 μg/ml). The ability of insulin (100 mU/ml) to stimulate hexose transport was returned by glucose refeeding and this was not affected by tunicamycin. Cycloheximide which blocked the glucose refeeding effect on hexose transport, decreased the ability of insulin to stimulate hexose transport. Specific 125I-insulin binding was increased by glucose refeeding of glucose-starved cells and this change in binding was inhibited by tunicamycin and cycloheximide. Thus, it appears that under the conditions employed in human fibroblasts, the ability of insulin to stimulate hexose transport is differentially regulated more by factors affecting basal hexose transport than by those affecting changes in insulin binding.

The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level.

Sodium vanadate has several insulin-like effects. To determine whether vanadate acts via the insulin receptor, I investigated the effect of vanadate on glucose transport (2-deoxyglucose uptake) in adipocytes that had been treated to decrease the number of insulin receptors. Trypsin (100 micrograms/ml) caused greater than 95% loss of 125I-insulin binding and rendered glucose transport resistant to both insulin and an anti-insulin-receptor antibody. However, vanadate caused an 8-fold increase in the transport rate [EC50 (concn. giving 50% of maximum effect) 0.2 mM] in both control and trypsin-treated cells, demonstrating that the insulin receptor does not have to be intact for vanadate to stimulate glucose transport. Insulin receptors were depleted by treatment of adipocytes with insulin (100 ng/ml) in the presence of Tris (which blocks receptor recycling). A 2 h treatment caused 60% loss of receptors, and a shift to the right in the dose-response curve for insulin stimulation of glucose transport (EC50 0.3 ng of insulin/ml in controls, 1.2 ng/ml in treated cells). The response to vanadate was again unaffected. Treatment with insulin for 4 h caused a 67% decrease in insulin binding and, in addition to the rightward shift in the insulin dose-response curve, a decrease in basal and maximal transport rates (which cannot be explained by decreased insulin receptor number). The EC50 of vanadate was again equal in control and treated cells, but glucose transport in the presence of a maximally effective concentration of vanadate (1 mM) was decreased. I conclude that the effect of vanadate on glucose transport is independent of the insulin receptor. Induction of a post-receptor defect (which may be a decrease in the total number of cellular glucose transporters) by prolonged exposure to insulin decreases the potency of a maximally effective concentration of vanadate. The findings demonstrate that vanadate stimulates glucose transport by an effect at a level distal to the insulin receptor.

Insulin-receptor development in normal and diabetic pregnancies. Role of membrane fluidity.

In the hyperinsulinemic offspring of the diabetic mother, both significant macrosomia and postnatal hypoglycemia are thought to be due to increased insulin sensitivity. The purpose of this study is to characterize changes in insulin-receptor development in fetal offspring of an experimental model of diabetic pregnancy. Two groups of Sprague-Dawley female rats were studied after timed mating. Both groups received injections of either vehicle (controls) or streptozocin (diabetic), 40 mg/100 g body wt, on day 7 of pregnancy and were killed at either 17, 20, or 21 days of gestation. Maternal and fetal blood were assayed for glucose and insulin, and fetal liver membranes were prepared for 125I-labeled insulin binding, lipid composition, and fluorescence polarization studies with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Maternal and pooled fetal glucose levels were elevated in streptozocin-treated rats; however, pooled fetal insulin values were not elevated in the offspring of diabetic animals compared with controls (33 +/- 1 vs. 50 +/- 5 microU/ml). 125I-insulin binding was greater in fetal offspring of diabetic (FD) rat membranes at each gestational age studied [P less than .001 by analysis of variance (ANOVA)] due to significantly greater numbers of both high- and low-affinity receptors. The highest insulin-binding capacity was seen on membranes obtained from FD rats at day 21 (9.92 M X L-1 X 100 micrograms membranes-1 vs. 6.38 M X L-1 X 100 micrograms protein-1 in fetal control (FC) rats. At each gestational age, membranes from FDs had lower values for fluorescence polarization (using the probe DPH) than did gestational-age--matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of 125I-insulin binding sites in the rat hypothalamus by quantitative autoradiography

In vitro autoradiography and computer video densitometry were used to localize and quantify binding of 125I-insulin in the hypothalamus of the rat brain. Highest specific binding was found in the arcuate. dorsomedial, suprachiasmatic, paraventricular and periventricular regions. Significantly lower binding was present in the ventromedial nucleus and median eminence. The results are consistent with the hypothesis that insulin modulates the neural regulation of feeding by acting at sites in the hypothalamus.

Relationship between the affinity and proteolysis of the insulin receptor. Evidence that higher affinity receptors are preferentially degraded.

125I-Insulin binding to rat liver plasma membranes initiated two processes that occurred with similar time courses: an increase of receptor affinity for hormone and degradation of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000. Inhibitors of serine proteinases prevented alpha subunit degradation without affecting the affinity change. This shows that the change of affinity is not produced by receptor proteolysis and that the intact alpha subunit of the insulin receptor can exist as a higher or lower affinity species. Hormone binding was much more rapid than receptor proteolysis and the initial rate of alpha subunit degradation was independent of the concentration of occupied lower affinity receptors. Only persistent hormone binding and the accumulation of higher affinity insulin-receptor complexes led to significant receptor proteolysis. As the incubation time between 125I-insulin and membranes increased, the rate at which hormone dissociated from Mr 135,000 complexes diminished, whereas hormone dissociated from Mr 120,000 complexes slowly after brief or extended incubations. These observations suggest that 125I-insulin binds to membranes to form low affinity complexes that are not substrates for proteolysis. A slow conformational change produces higher affinity hormone-receptor complexes that are selectively degraded. Thus, the conversion between states of affinity may play a role in the regulation of receptor proteolysis and, consequently, insulin action in cells.

Insulin binding to the blood-brain barrier in the streptozotocin diabetic rat.

125I-Insulin binding to isolated brain microvessels from control, streptozotocin diabetic, and insulin-treated diabetic rats was measured. The binding was highest in the control (21.1 +/- 1.8%/mg capillary protein) and lowest in the diabetic (14.8 +/- 1.9%, p less than 0.01) animals. Administration of 2 U of protamine zinc insulin per day increased the maximum binding in the diabetic rats to 17.2 +/- 2.1%. Scatchard analyses of the binding showed that the major difference between the diabetic and the control animals was a decrease in the number of both high- and low-affinity sites in the diabetic animals. To test whether the failure of up-regulation in the hypoinsulinemic diabetic animal was related to an inherent defect in the endothelial cell or resulted from the diabetic milieu, cultured brain endothelial cells were tested for their capacity to up- and down-regulate their insulin receptors in vitro. In response to 100 ng/ml insulin for 12 h, these cells down-regulated their insulin receptors. When the insulin was removed, the insulin receptors returned to control levels. These studies showed that in vitro brain capillary endothelial cells have the capacity to increase their insulin receptors in response to a low-insulin environment, whereas in vivo the microvessels decrease their insulin receptors in response to diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

Sephadex (G-50 fine grade)-gel chromatography and trichloroacetic acid (TCA) precipitation were used to investigate the effects of chloroquine and bacitracin on the nature of cell-associated radioactivity in studies on the binding and degradation of 125I-insulin in cultured rat hepatocytes. Sephadex peak I, eluted with the void volume, increased with hepatocyte incubation time and comprised 6% of total cell-bound radioactivity at 120 min. However, all radioactivity in this peak was due to unspecific binding. Peak II, corresponding to intact insulin, represented 95% of specifically cell-associated label at 5 min and decreased to 77% at 120 min. Peak III, containing the final low-Mr degradation products, increased with incubation time (22% of specifically bound label at 120 min). The TCA-precipitable and TCA-soluble fractions of hepatocytes extracted with 0.1% SDS were within 4-7% of the proportions of radioactivity in peaks II and III respectively. Scatchard plots based on insulin-binding data from Sephadex chromatography or TCA precipitation were identical. Dissociation studies revealed that at least 75% of the intact insulin associated with the hepatocytes was bound to receptors at the cell surface. Bacitracin increased the proportion of cell-associated intact hormone and decreased that of ligand degraded when analysed by either Sephadex chromatography or TCA precipitation. The proportion of surface-bound to internalized intact hormone remained unaltered, indicating that bacitracin acted predominantly at the cell surface. In the presence of chloroquine, which dramatically increased the contribution of peak I to specific binding, 'intact' insulin was substantially overestimated when determined as the TCA-precipitable fraction. In addition, all peak I material and 50% of cell-associated label in peak II was trapped intracellularly, thereby pointing to the lysosomal or prelysosomal site of action of this drug.