Difference In Insulin Binding Research Articles

Reversible change in thiol redox status of the insulin receptor alpha-subunit in intact cells.

In this study, we used maleimidobutyrylbiocytin to examine possible alteration that may occur in the redox state of the insulin receptor (IR) sulfhydryl groups in response to reduced glutathione (GSH) or N-acetyl-L-cysteine (NAC). Short-term treatment of intact cells expressing large numbers of IR with GSH or NAC led to a rapid and reversible reduction of IR alpha-subunit disulfides, without affecting the receptor beta-subunit thiol reactivity. The overall integrity of the oligomeric structure of IR was maintained, indicating that neither class I nor class II disulfides were targeted by these agents. Similar findings were obtained in cells transfected with IR mutants lacking cysteine524, one of the class I disulfides that link the two IR alpha-subunits. Membrane-associated thiols did not participate in GSH- or NAC-mediated reduction of IR alpha-subunit disulfides. No difference in insulin binding was observed in GSH-treated cells; however, ligand-mediated increases in IR autophosphorylation, tyrosine phosphorylation of cellular substrates, and dual phosphorylation of the downstream target mitogen-activated protein kinase were inhibited at concentrations of GSH (10 mM or greater) that yielded a significant increase in IR alpha-subunit thiol reactivity. GSH did not affect IR signaling in the absence of insulin. Our results provide the first evidence that the IR alpha-subunit contains a select group of disulfides whose redox status can be rapidly altered by the reducing agents GSH and NAC.

Does dietary fat influence insulin action?

What is clear from the research thus far is that dietary fat intake does influence insulin action. However, whether the effect is good, bad, or indifferent is strongly related to the fatty acid profile of that dietary fat. The evidence has taken many forms, including in vitro evidence of differences in insulin binding and glucose transport in cells grown with different types of fat in the incubation medium, in vivo results in animals fed different fats, relationships demonstrated between the membrane structural lipid fatty acid profile and insulin resistance in humans, and finally epidemiological evidence linking particularly high saturated fat intake with hyperinsulinemia and increased risk of diabetes. This contrasts with the lack of relationship, or even possible protective effect, of polyunsaturated fats. In particular, habitual increased n-3 polyunsaturated dietary fat intake (as fish fats) would appear to be protective against the development of glucose intolerance. It is reassuring that the patterns of dietary fatty acids that appear beneficial for insulin action and energy balance are also the patterns that would seem appropriate in the fight against thrombosis and cardiovascular disease. Mechanisms, though, still need to be defined. However, there are strong indicators that defining the ways in which changes in the fatty acid profile of membrane structural lipids are achieved, and in turn influence relevant transport events, plus understanding the processes that control accumulation and availability of storage lipid in muscle may be fruitful avenues for future research. One of the problems of moving the knowledge gained from research at the cellular level through to the individual and on to populations is the need for more accommodating research designs. In vitro studies may provide in-depth insights into intricate mechanisms, but they do not give the "big picture" for practical recommendations. On the other hand, correlational studies tend to be fairly blunt instruments, requiring large numbers that are very often not feasible if a greater depth of understanding of the biological processes is to be incorporated. There may be benefit in turning to the clinical case study as a framework for a more comprehensive analysis of the links between dietary fats and insulin action. The real challenge is to keep the depth of analysis rigorous enough to be able to explain and accommodate individual variation (i.e., the diversity of both environmental and genetic backgrounds) while at the same time satisfying the cultural need to provide appropriate overall dietary guidelines. Finally, David Kritchevsky brought to our attention a delightful quote from Mark Twain: "There is something fascinating about science. One gets such a wholesale return of conjecture for such a trifling investment of fact." In the field of dietary fats and the Metabolic Syndrome, this quotation is, unfortunately, apt. Much more research is necessary to define how dietary fats really work to affect insulin action. Well designed, long-term studies in "free range" humans must be undertaken if dietary guidelines for the Metabolic Syndrome are to be based on anything more than a "trifling" amount of "fact."

Effect of glimepiride (HOE 490) on insulin receptors of skeletal muscles from genetically diabetic KK-Ay mouse

A new sulfonylurea, glimepiride (HOE 490), has been developed for the glycemic control in non-insulin-dependent diabetes mellitus. We examined the effect of glimepiride on glucose and insulin levels in KK-Ay mice, an animal model of non-insulin-dependent diabetes mellitus, which is characterized by hyperglycemia and hyperinsulinemia. Administration of glimepiride (0.5 mg/kg/day) for 8 weeks to KK-Ay mice resulted in decrease in glucose (297 ± 36 to 250 ± 51 mg/dl) and insulin (76 ± 14 to 41 ± 14 μU/ml) levels. To clarify the mechanism of the agent, we examined the effect of this new drug on insulin receptors in the skeletal muscles. There was no difference in insulin binding to the receptors from both glimepiride-treated and -untreated KK-Ay mice muscles. The insulin-stimulated autophosphorylation of insulin receptors from KK-Ay mice was decreased compared to that from normal mice (5 ± 1 vs. 39 ± 13% over basal). Glimepiride did not ameliorate impaired insulin-stimulated insulin receptor autophosphorylation. To determine the effect of glimepiride on post-insulin receptor signaling pathway, we measured 2-[ 3H]glycerol incorporation into diacylglycerol in the cultured rat fibroblast cell line overexpressing human insulin receptors. Glimepiride (100 μM) as well as insulin (10 nM) significantly stimulated diacylglycerol production. These results suggest that glimepiride has a potent extrapancreatic effect on glucose metabolism and may directly stimulate glucose transport activity through phospholipid signaling pathway, but not through insulin receptor kinase signaling pathway.

Postbinding insulin resistance around parturition in the isolated rat epitrochlearis muscle

To determine whether insulin resistance exists in maternal skeletal muscle during pregnancy and how it returns to normal during the postpartum period, 3-O-methyl[14C]-D-glucose transport and [125I]insulin-binding activities were measured in isolated rat epitrochlearis muscle. Maximally insulin-stimulated methylglucose transport activity was decreased on day 20 of pregnancy and on days 1 and 4 post partum; it returned to the nonpregnant level by day 9. The insulin-binding activity did not change significantly throughout pregnancy, increased on days 1 and 4 post partum, and returned to the nonpregnant level by day 9. There was no significant difference in insulin binding or insulin-stimulated methylglucose transport activity between lactating and nonlactating animals. These results suggest that insulin resistance caused by postbinding changes in epitrochlearis muscle develops during late pregnancy and continues at least until day 4 post partum. Lactation does not appear to have a significant effect on insulin resistance.

Postbinding insulin resistance around parturition in the isolated rat epitrochlearis muscle

To determine whether insulin resistance exists in maternal skeletal muscle during pregnancy and how it returns to normal during the postpartum period, 3-O-methyl[¹⁴C]-D-glucose transport and [¹²⁵I]insulin-binding activities were measured in isolated rat epitrochlearis muscle. Maximally insulin-stimulated methylglucose transport activity was decreased on day 20 of pregnancy and on days 1 and 4 post partum; it returned to the nonpregnant level by day 9. The insulin-binding activity did not change significantly throughout pregnancy, increased on days 1 and 4 post partum, and returned to the nonpregnant level by day 9. There was no significant difference in insulin binding or insulin-stimulated methylglucose transport activity between lactating and nonlactating animals. These results suggest that insulin resistance caused by postbinding changes in epitrochlearis muscle develops during late pregnancy and continues at least until day 4 post partum. Lactation does not appear to have a significant effect on insulin resistance.

Insulin stimulates inositol incorporation in hippocampus of lean but not obese Zucker rats

We have previously reported that intraventricular insulin is ineffective in decreasing the body weight of obese (fa/fa) Zucker rats. In the present study, we report that insulin at concentrations of 1 and 5 nM significantly stimulates incorporation of 3H-myoinositol into inositol phosphates (29±5% and 20±6% stimulation over control levels) and membrane inositol lipids (29±4% and 20±6% stimulation over control levels) within hippocampal slices from lean (Fa/Fa) Zucker rats (n=3 preparations). Smaller but significant stimulations were observed in the Fa/fa Zucker rat (n=5) hippocampus [10±3 and 13±4% stimulation over control levels (inositol phosphates) and 10±4 and 14±3% stimulation over control levels (inositol lipids) with 1 and 5 nM insulin]. Insulin had no effect on 3H-inositol incorporation into hippocampal slices from obese (fa/fa) Zucker rats (n=5). No difference in insulin binding to hippocampal membranes was observed among the three genotypes. These findings confirm the behavioral observation of insulin insensitivity within the fa/fa Zucker CNS and suggest that this insensitivity may be gene-dose-related.

Alterations in the Hepatic Insulin Receptor Kinase in Genetic and Acquired Obesity in Rats*

Obesity is associated with insulin resistance and type II diabetes mellitus. In the present study, we have characterized hepatic insulin receptor function in two animal models of obesity: the Zucker fatty rat (ZFR), a model of genetic obesity with severe hyperinsulinemia, and the Sprague-Dawley rat with dietary obesity, a model of acquired obesity. Zucker fatty rats were also treated with streptozotocin (STZ) in an effort to examine the effects of relative insulin deficiency and hyperglycemia in the setting of obesity. Using wheat germ agglutinin-purified insulin receptor extracted from liver, no significant difference in insulin binding was identified in either model of obesity. beta-Subunit autophosphorylation was significantly decreased in both obese models relative to that in controls (72% in the obese ZFR and 49% in the overfed Sprague-Dawley model). Kinase activity, as measured by phosphorylation of the 1142-1153 synthetic peptide, was also decreased in both models of obesity by 22% and 64%, respectively. In the Zucker rat, STZ treatment led to an 80% increase in receptor concentration and a further 70% increase in beta-subunit autophosphorylation per receptor, whereas tyrosine kinase activity toward substrate was not altered. Since kinase activity is closely linked to autophosphorylation, we determined the fraction of autophosphorylated (activated) receptors vs. non-phosphorylated (inactive) receptors by using antiphosphotyrosine antibody to precipitate receptors bound with [125I]insulin. There was no significant difference in the percentage of activated insulin receptors in the dietary obese, ZFR, or STZ-treated Zucker rat vs. that in the controls. In all models, the percentage of activated receptors ranged from 32-46% of the total receptor pool. These data suggest that in genetic and acquired obesity, autophosphorylation of the beta-subunit is reduced and is a limiting factor in insulin receptor activation. A similar fraction of all receptors appears to undergo some level of autophosphorylation; however, full autophosphorylation and, thus, activation of the receptor do not occur, and this results in a decrease in kinase activity. This block in autophosphorylation may account for significant reductions in insulin receptor kinase function in obesity.

Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man.

Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.

Effect of short-term consumption of a high fat diet on glucose tolerance and insulin sensitivity in the rat.

The mechanism by which the consumption of high fat, low carbohydrate diets impair glucose tolerance and decrease insulin sensitivity is poorly understood. In an attempt to clarify this question, intravenous glucose tolerance and insulin action in the liver and skeletal muscle were examined in rats after two weeks feeding of either a high fat (HF: 66% energy as fat) or a low fat (LF: 12% energy as fat) diet. Both diets had a P/S ratio (ratio of polyunsaturated to saturated fat in the diet) of 1.3. The high fat diet resulted in mild impairment of intravenous glucose tolerance. Postprandial glucose levels were elevated in the presence of a sustained insulin response. In vitro insulin-stimulated glucose utilisation was decreased significantly in soleus muscle of HF rats, as indicated by decreased [14C]glucose incorporation into muscle glycogen. In contrast, muscle lipogenesis from glucose was not affected by dietary composition. There was no difference in insulin binding to soleus muscle of HF and LF rats, indicating a dissociation between insulin receptor binding and post-receptor metabolic events. Dietary composition did not influence the incorporation of increasing [14C]glucose loads into muscle glycogen or lipid in vivo. However, the HF diet was associated with reduced incorporation of [14C]glucose into lipids and glycogen in the liver and, to a smaller extent, reduced incorporation into adipose tissue lipids in vivo. These results suggest that the mechanism by which HF diets impaired glucose tolerance was mainly hepatic in origin. Decreased glucose uptake, secondary to reduced glucokinase activity, may result in a reduction in glucose utilisation in the liver.

Characterization of insulin binding to slices of slow and fast twitch skeletal muscles in the rabbit.

Insulin binding was studied in rabbit semimembranosus proprius and psoas major muscles composed of slow-twitch oxidative (SO) and fast-twitch glycolytic (FG) fibers, respectively. For this purpose, we developed a technique using cryostat microtome muscle slices. Degradation of 125(I)-insulin during the incubation period was prevented by the addition of 1 mM bacitracin in the buffer. Specific binding to muscle slices plateaued by the 24 hrs. of incubation at 4 degrees C. It increased as a function of the amount of muscle, with a maximum binding occurring at about 5 mg of muscle slices. Triton X-100 has been shown to increase specific binding from a critical concentration of 10(-4) M with a maximum effect occurring at 3.3 10(-4) M. Under this condition, the binding was specific since displacement studies showed no inhibition of 125(I)-insulin binding by GH, HCG, ACTH and glucagon, whereas half maximal inhibition was achieved using 5 10(-10) M insulin, 3 10(-9) M IGF1 and 2 10(-8) M proinsulin. The analysis of the binding data yielded curvilinear Scatchard plots. The number of high affinity insulin receptors was higher in the SO muscle than in the FG muscle (4.3 +/- 0.7 vs 0.7 +/- 0.2 fmol/mg fresh muscle; P less than 0.001) with similar high affinity dissociation constants (Kd = 1.5 10(-10) M). Analogous results were obtained using muscle microsomal fractions. The differences in insulin binding might be related to the more intense metabolism of SO fibres which contract more often than FG fibres in vivo.

Insulin receptors in normal pregnant women and women with gestational diabetes.

In a serial study of insulin receptor binding to monocytes from normal pregnant women, a significant increase in insulin binding in mid pregnancy followed by a significant decrease in late pregnancy at tracer insulin concentration was found. No changes in the insulin concentration necessary to reduce tracer binding by 50% (ID50) were observed. At delivery, binding to isolated adipocytes was significantly lower in normal pregnant women than in non-pregnant normal controls while no difference in ID50 was observed. No differences in insulin binding at tracer insulin concentration to monocytes and adipocytes between normal weight women with gestational diabetes and healthy non-diabetic pregnant controls were found, but the ID50 was significantly lower in women with gestational diabetes diagnosed in late pregnancy than in pregnant controls at the same weeks of gestation.

Etiology and pathophysiology of gestational diabetes mellitus.

In pregnancy, several physiologic changes take place, the sum of which tends to reset the glucose homeostasis in the direction of diabetes. About 1-2% of all pregnant women develop an abnormal glucose tolerance in pregnancy, but most often glucose tolerance returns to normal postpartum. This condition is called gestational diabetes mellitus (GDM). The possibility that glucose tolerance deteriorates in pregnancy because of diabetes-like changes in the secretory function of the endocrine pancreas has been investigated in healthy controls and in normal-weight gestational diabetic subjects. The insulin responses to oral glucose and mixed meals are equally large in these two groups, but the insulin response per unit of glycemic stimulus is significantly lower in the gestational diabetic subjects than in the controls. Diabetes-like changes in glucagon secretion are not observed in either group. Insulin degradation is unaffected by human pregnancy and the proinsulin share of the total plasma insulin immunoreactivity does not increase in pregnancy. Insulin receptor binding to monocytes from normal pregnant women is increased in midpregnancy but is significantly decreased in late pregnancy. No difference in insulin binding (at tracer insulin concentration) to monocytes from healthy pregnant controls and gestational diabetic subjects is found. The insulin concentration necessary to reduce tracer insulin binding by 50% (ID50) is lower in the gestational diabetic subjects diagnosed in late pregnancy than in the pregnant controls. Together, these findings indicate that the number of insulin receptors on monocytes is decreased in GDM at this stage of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Human erythrocyte insulin receptors in normal male and female subjects.

Human red blood cells (RBC) have been shown to have highly specific insulin receptors. We have studied the binding characteristics of insulin to these receptors in erythrocytes from normal male and female subjects on their usual diet and physical activity. There were no significant differences in insulin binding in erythrocytes from females between the two phases of the menstrual cycle. However, the receptor concentration was higher in the 2nd half of the cycle accompanied by a reduction in affinity. Binding curves of 125-I-insulin to RBC from males were higher than females in either phases of the menstrual cycle, primarily due to an increase in receptor concentration when compared to females in the follicular phase and mediated by an increased affinity at low receptor occupancy when compared to females in the luteal phase. We speculated that the differences in the binding characteristics of 125I-insulin to RBC insulin receptors are mediated by differences in the levels of sex hormones.

Insulin inhibition of tyrosine aminotransferase induction by dexamethasone is species-related

1. 1. Dexamethasone induces TAT in the embryonic chick liver, but this effect is not blocked by prior administration of insulin at a dose sufficient to increase the hepatic protein/DNA ratio. 2. 2. Similarly, insulin does not block dexamethasone induction of TAT in chick embryo hepatocytes in vitro. 3. 3. In contrast, insulin reduces TAT induction by dexamethasone in fetal rat hepatocytes by 35–40%. 4. 4. No significant differences in insulin binding were noted between chick and rat hepatocytes.

Influence of ambient glucose and insulin concentrations on adipocyte insulin binding

To elucidate factors of importance for insulin binding, fat cells from humans and rats were incubated under various experimental conditions for different periods of time. Human adipocytes incubated for 24 hours in the absence of insulin showed no significant difference in insulin binding compared with cells from freshly excised tissue. After 48 hours, however, an increased rate of binding (average 54%; P < 0.05) was obtained. The addition of insulin (2000 μU/ml) to the culture medium resulted in a decrease in insulin binding (average 33%; P < 0.05) compared with cells maintained in the absence of insulin. There was no apparent difference in receptor affinity, indicating that the altered binding was due to a change in receptor number. In the absence of insulin, elevating the glucose concentration of the medium from 0.8 mM to 22.4 mM did not significantly influence insulin binding. Rat adipocytes showed similar but more rapid changes. Thus, incubation for 24 hours without insulin caused an increase in insulin binding (average 37%; P < 0.05). This up-regulation was seen even in a high glucose concentration (28 mM) but was completely prevented by the presence of insulin in the medium. Furthermore, when rat adipocytes were incubated with insulin in the presence of a high glucose concentration (28 mM) there was a significant further decrease in insulin binding compared with that of parallel incubations performed in 5.6 mM glucose. Thus, even in the absence of TRIS buffer, insulin-dependent regulation of the number of binding sites is shown for both human and rat adipose tissue in vitro. Although this perturbation could be directly due to hormone-receptor interaction at the membrane level, the finding in rat adipocytes that the ambient glucose concentration can modulate this effect suggests the importance of post-receptor events.

The role of cell age in the difference in insulin binding between adult and cord erythrocytes.

Erythrocytes (RBCs) from cord blood are known to bind more insulin than those of adults. We have investigated the possibility that this difference is due to the younger cell age of the neonatal cells. Samples from 13 normal full term neonates and 12 adults were fractionated into 5 fractions of defined density by dextran gradient centrifugation. Fractions of comparable density, a variable known to correlate well with age, were then compared for insulin binding. Plasma insulin and glucose levels were the same in the two study groups. The density distribution curve was shifted to the left for the cord samples, indicating a younger cell age. The difference was most marked in the lightest fraction, which contained 4.66 + 0.46% (mean +/- SE) adult cells vs. 15.26 +/- 1.27% cord cells (P less than 0.01). Insulin binding was identical when fractions of equal density were compared in the two groups. The only exception was the lightest (youngest) fraction (density, less than 1.092), in which the cord cells displayed a considerably higher percentage of specific binding; (19.98 +/- 1.57 vs. 13.32 +/- 0.92 P less than 0.005). This difference was shown to be due to a high percentage of very light cells in cord samples, compared to adult samples, in this fraction which, unlike the others, did not have a lower limit of density. When a lower density cutoff was introduced (1.089 g/ml), the remaining cells of this fraction (density, 1.089-1.092) displayed quite similar percentages of specific binding (13.07 +/- 1.63 vs. 11.86 +/- 1.27). Cells below this density were virtually absent in the adult, comprising less than 0.3% of all adult cells compared to 7.2% of the cord cells. These results indicate that the difference in insulin binding between adult and cord cells is due to a younger RBC age distribution in cord blood. This difference is most marked in the youngest cell fractions. Age fractionation of RBCs by density gradient centrifugation appears to be a promising method of assessing RBC insulin receptors in situations in which substantial changes in cell age are seen.

Metabolic studies of adipose tissue in acute uremia

The effect of acute uremia on lipoprotein lipase (LPL) activity in rat adipose tissue and on the response of the isolated adipocytes to insulin was assessed. LPL activity in adipose tissue and in adipocytes of the uremic rats was decreased compared with values in sham-operated controls. Also, the adipocytes from uremic rats released significantly less than control amounts of LPL. In contrast, glucose oxidation by adipocytes isolated from uremic rats was not different from controls, and there was no difference in insulin binding or in insulin-stimulated glucose oxidation in the two groups. Triglyceride injected into the uremic rats was cleared at about half the control rate. Thus, the specific reduction in LPL activity in adipose tissue may be responsible, at least in part, for the defective removal of triglyceride. However, it is unlikely that the reduced LPL is due to a generalized toxic effect of uremia on adipose tissue since no significant alteration in insulin binding and glucose oxidation was found.

Regenerating Rat Liver: Insulin and Glucagon Serum Levels and Receptor Binding*

Serum insulin and glucagon levels and liver plasma membrane receptor binding were studied in rats after partial hepatectomy. To clarify whether the surgical stress and decreased food intake that accompanies partial hepatectomy influenced these parameters, sham-operated rats were also studied. When sham-operated rats were compared to nonoperated controls, there was a 30% fall in insulin levels and a significant rise in the number of insulin receptors. In contrast, glucagon levels and glucagon receptor binding were unchanged. When partially hepatectomized rats were compared to sham-operated rats, there was no significant change in either insulin levels or the number of plasma membrane insulin receptors. Insulin degradative activity, however, was decreased in liver plasma membranes from partially hepatectomized animals, causing an apparent increase in [125I]iodoinsulin binding to this organelle. Bacitracin, an inhibitor of insulin degradation, abolished this difference in insulin binding. Glucagon levels rose by 65% after partial hepatectomy, whereas the number of glucagon receptors decreased significantly. The studies demonstrate, therefore, that after partial hepatectomy, serum insulin levels and insulin receptor binding in liver are altered, and these alterations are due to surgical stress and decreased food intake. Glucagon levels and glucagon receptor binding are also altered after partial hepatectomy, but these alterations are due to liver regeneration per se.

The insulin receptor in myotonic dystrophy.

Insulin binding was studied using circulating monocytes obtained from seven patients with myotonic dystrophy and seven control subjects. During an oral glucose tolerance test the 2 h plasma glucose value was significantly higher in patients with myotonic dystrophy compared to controls, and 5 out of the 7 patients demonstrated hyperinsulinemia. This combination of abnormal glucose tolerance and hyperinsulinemia suggests insulin resistance. There was no difference in insulin binding to monocyte receptors between the two groups. These results suggest that no abnormality exists in the insulin receptor in myotonic dystrophy.

THE INSULIN RECEPTOR IN NORMAL AND OBESE PERSONS

Using [125I]insulin at 172 pmol/1 (1 ng/ml) the binding of insulin to mononuclear leucocytes isolated from peripheral blood was studied. Our present study comprised 21 healthy subjects (22-33 years old, 90-110% of ideal weight) and a comparable group of 22 obese subjects (20-37 years old, minimum 150% of ideal weight). A significant difference in insulin binding was found between the two groups, the mean specific insulin binding fraction in normals being 1.92 +/- 0.58 (s) X 10(-2) and that for the obese 1.19 +/- 0.41 (s) X 10(-2) (P less than 0.01). No correlation was found between body weight and the number of insulin receptors in the obese subjects. However, the number of insulin receptors was negatively correlated to fat cell size (P less than 0.05). Insulin receptors in subjects were also negatively correlated to fasting plasma insulin (P less than 0.05). Insulin receptors were studied in 11 obese subjects before and after 10 days of fasting. A significant increase in the number of insulin receptors was observed with a simultaneous decrease in plasma insulin to normal values. The results indicate that obesity complicated by hyperinsulinism is associated with a decrease in the number of insulin receptors compared with the normal. This finding may in part explain the decreased insulin sensitivity of the hyperinsulinaemic obese.