Specific Binding Of 125I-insulin Research Articles

Insulin action in adipose tissue in type 1 diabetes

Background:Insulin action has been reported to be normal in type 1 diabetic patients. However, some studies have reported an insulin resistance state in these patients. The aim of this study was to investigate insulin resistance in a group of type 1 diabetic patients. We studied the insulin action in adipose tissue and analyzed the effects of duration of disease, body mass index (BMI), and glycosylated hemoglobin on insulin action at the receptor and postreceptor levels in adipocytes.Methods:Nine female type 1 diabetic patients with different durations of disease and eight nondiabetic female patients of comparable age and BMI were studied. 125I-insulin binding and U-[14C]-D-glucose transport was measured in a sample of subcutaneous gluteus adipose tissue obtained by open surgical biopsy from each subject.Results:The duration of disease was negatively correlated with both 125I-insulin binding capacity (r = −0.70, P < 0.05) and basal and maximum insulin-stimulated glucose transport (r = −0.87, P < 0.01, and r = −0.88, P < 0.01, respectively). Maximum specific 125I-insulin binding to the receptors in adipocytes was higher in the group of patients with a shorter duration of disease (P < 0.01). Basal and maximum insulin-stimulated glucose transport was significantly higher in the group with less than 5 years of disease (P < 0.01). No correlation was found between BMI and insulin action.Conclusion:Female type 1 diabetic patients have normal insulin action. There is a high glucose uptake in the early phase of the disease, although a longer duration of disease appears to be a contributing factor to a decrease in insulin action in these patients, and involving both receptor and postreceptor mechanisms.

Two separate receptors for insulin and insulinlike growth factors on arterial smooth muscle cells.

Insulin and insulinlike growth factors (IGF) are related polypeptides that have similar biological activities. Both factors produce metabolic effects as well as growth effects. Most cells have separate receptors for IGF and insulin. In the present study we have demonstrated specific IGF receptors in cultured smooth muscle cells of rat aorta. The properties and specificity of these receptors were compared with those of the insulin receptor in the same cell system. The specific binding of both 125I-IGF and 125I-insulin binding in the order of potency: insulin proinsulin IGF. The potency of IGF in displacing 125I-insulin was rapid and reversible. Maximal 125I-IGF binding occurred at 20 degrees C with a specific binding of 10%. At this temperature specific 125I-insulin binding was 1.3% and occurred in biphase. The pH optimum for 125I-IGF binding was between pH 7 and 8. Both receptors show a high degree of specificity. IGF, insulin and proinsulin competed for 125I-IGF binding in the order of potency: IGF proinsulin insulin. The potency of insulin in displacing 125I-IGF was about 2000 times lower than that of IGF itself. In addition, IGF, insulin and proinsulin competed for 125I-insulin was about 450 times lower than that of insulin. These results indicate two separate binding sites for insulin and IGF on arterial smooth muscle cells.

Diurnal variation in specific insulin binding to erythrocytes.

We studied the insulin binding to erythrocytes during a 24-h period in 11 normal subjects. Compared with 0800 and 1600 h the specific 125I-insulin binding decreased significantly at midnight. These changes were mainly due to alterations in insulin binding affinity rather than a decrease in receptor number. An inverse finding was obtained concerning the plasma insulin levels. In fact, the highest insulin concentrations were observed at midnight. The results suggest that diurnal variations in insulin receptor function occur in response to fluctuations of plasma insulin levels.

Insulin Binding to Erythrocytes in Children with Type I Diabetes Mellitus before, and after Insulin Treatment

125I-insulin binding to erythrocytes was studied in 24 children with type I diabetes mellitus before institution of insulin therapy, and in 10 normals. Mean specific 125I-insulin binding in the diabetics was significantly lower than in the controls (10.2 +/- 0.4% (SEM), as against 11.6 +/- 0.5%, P less than 0.05). In 8 diabetics the study was repeated after 3-6 weeks of therapy, at which time the insulin binding had increased to normal (12.1%, range 10.7-13.6). No correlation was found between insulin binding and blood glucose, plasma insulin or plasma beta-hydroxybutyrate. We conclude that insulin binding to the receptors is altered in the early phase of type I diabetes.

Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of 125I-insulin and of insulin-related factor-1 (125I-IGF-1) by plasma membranes of the rat liver and brain. The IC50 value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (104 ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC50 value in the case of 125I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 104 ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.

Receptor of Insulin-Like Growth Factor 1 in Tissues of the Mollusc Anodonta cygnea

To identify insulin-like receptors in the mollusc Anodonta cygnea, specific binding of 125I-insulin and 125I-IGF-1 by WGA-purified glycoprotein fractions of foot muscles and neural ganglia is studied. The binding sites for IGF-1 are detected for the first time in invertebrates, both in the muscles, and in the neural tissue of the mollusc. The level of 125I-IGF-1 binding in the muscle tissue was equal to 2.8 ± 0.1, in the neural tissue, to 4.0 ± 0.2% per 5 µg of protein. The equilibrium dissociation constant (Kd) was equal to 4.8 ± 0.3 and 4.3 ± 0.2 nM, respectively. The relative affinity of the binding sites to insulin did not exceed 1% of their affinity to IGF-1. Binding of 125I-insulin in the muscle tissue was not detected; the level of labeled insulin binding in the neural tissue was equal to 0.5% per 5 µg of protein. In the sarcolemmal fraction of the mollusc foot, IGF-1 and, to a lesser degree, insulin at a dose of 100 nM initiated phosphorylation of tyrosine in a protein with mol. mass of 70 kDa. The minor band of the phosphorylation was also detected in the zone of protein of 80 kDa. The conclusion is made about the existence in molluscan tissues of high-conserved receptors-tyrosine kinases identical by functional parameters to the mammalian receptor of IGF-1. From this, it is suggested that the peptides close by structure to vertebrate IGF-1 may be involved in physiological processes in A. cygnea. The problem of the nature of the insulin-binding sites in the molluscan neural tissue is discussed.

The Adipose Obese Gene Product, Leptin: Evidence of a Direct Inhibitory Role in Ovarian Function

Leptin, a recently-discovered hormonal product of the obese gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety and improves reproductive performance if injected into mice lacking circulating leptin. Therefore, we tested the hypothesis that leptin signals metabolic information to the reproductive system by directly affecting granulosa cell function. Doses of 10–300 ng/ml leptin had no effect (P > 0.10) on basal or insulin-induced numbers of granulosa cells cultured from small (1–5 mm) or large (≥8 mm) bovine follicles. Similarly, 30 and 300 ng/ml leptin had no effect (P > 0.10) on basal estradiol production. However, leptin, in a dose-dependent manner, inhibited (P < 0.05) insulin-induced progesterone and estradiol production by granulosa cells from small and large follicles. Leptin did not compete for specific 125I-insulin binding to granulosa cells. Furthermore, specific binding of 125I-leptin was demonstrable in granulosa cells. In conclusion, leptin, at physiological levels, can directly attenuate insulin-induced steroidogenesis of granulosa cells without affecting proliferation of this ovarian cell type. These results provide evidence to support the hypothesis that leptin can act as a metabolic signal to the reproductive system via direct action at the ovarian level.

The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function.

Leptin, a recently-discovered hormonal product of the obese gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety and improves reproductive performance if injected into mice lacking circulating leptin. Therefore, we tested the hypothesis that leptin signals metabolic information to the reproductive system by directly affecting granulosa cell function. Doses of 10-300 ng/ml leptin had no effect (P > 0.10) on basal or insulin-induced numbers of granulosa cells cultured from small (1-5 mm) or large (> or = 8 mm) bovine follicles. Similarly, 30 and 300 ng/ml leptin had no effect (P > 0.10) on basal estradiol production. However, leptin, in a dose-dependent manner, inhibited (P < 0.05) insulin-induced progesterone and estradiol production by granulosa cells from small and large follicles. Leptin did not compete for specific 125I-insulin binding to granulosa cells. Furthermore, specific binding of 125I-leptin was demonstrable in granulosa cells. In conclusion, leptin, at physiological levels, can directly attenuate insulin-induced steroidogenesis of granulosa cells without affecting proliferation of this ovarian cell type. These results provide evidence to support the hypothesis that leptin can act as a metabolic signal to the reproductive system via direct action at the ovarian level.

Insulin Binding and Internalization in Hagfish Red Blood Cells

Binding of porcine 125I-insulin (0.15 nM) to hagfish red blood cells was time-dependent, reaching equilibrium after 1 hr at 10°. The specific 125I-Insulin bound to receptors from a T1/2 of 60 min in cells suspended in medium alone to 23 min in medium containing 8 μM nonradioactive insulin. Porcine insulin and desoctapeptide insulin competed for specific binding of 125I-insulin in a dose-dependent manner, whereas glucagon and somatostatin did not. For porcine insulin. Scatchard analysis produced a curvilinear plot, suggesting multiple affinity binding sites with high-affinity and low-affinity association constant (Ka) 0.2 × 109M-1 and 0.27 × 107M-1 respectively. A total of 2090 binding sites per hagfish red blood cell was calculated. Sixty-two percent of the bound 125I-insulin was found to be internalized into the hagfish red blood cells. Less degradation of 125I-insulin was observed by Sephadex G-50 chromatography compared to human red blood cells.

Insulin resistance in rats harboring growth hormone—secreting tumors: Decreased receptor number but increased kinase activity in liver

Growth hormone (GH) is a potent antagonist of insulin action, and this resistance occurs primarily at a post-binding step(s). To elucidate the underlying mechanisms, the effects of chronic GH excess on the structure and function of insulin receptors partially purified from the liver were examined in rats harboring GH-secreting tumors. Insulin resistance was established in this animal model of GH hypersecretion by a hyperinsulinemic euglycemic clamp. Specific binding of 125I-insulin and receptor number were reduced in tumor animals by 40% and 62%, respectively, reflecting downregulation of the insulin receptor by hyperinsulinemia in these animals. Receptors from tumor animals showed a 50% increase in β-subunit phosphorylation and in the kinase activity toward the synthetic polypeptide Glu4: Tyr1 when measured in vitro in the absence of insulin; however, the incremental stimulation by insulin (170 nmol/L) of the phosphorylation of either the β-subunit or Glu4:Tyr1 was not different between control and experimental animals. There was no difference between the two groups in Glu4:Tyr1 phosphorylation measured after immunodepletion of receptors by antibodies to the insulin receptor, indicating that the observed alteration in the kinase activity of tumor animals was intrinsic to the insulin receptor. Exposure to chronic GH excess did not alter insulin receptor structure, as evidenced by electrophoretic mobility under reducing and nonreducing conditions. The enhanced basal kinase activity of the receptor from tumor animals may reflect a more highly phosphorylated state of the receptor (and hence elevated enzyme activity) in these animals due to elevated serum insulin levels. These results demonstrate that the hepatic insulin resistance in rats chronically exposed to GH excess is not due to impaired insulin receptor kinase activity.

Characterization of Insulin Receptors in Chicken Kidneys: Effect of Nutritional Status

In chickens, the kidneys actively contribute to gluconeogenesis. A cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) is present in this tissue but is absent in liver. Cytosolic renal PEPCK is nutritionally and hormonally controlled which indicates a likely contribution of insulin in the control of this enzyme (and other renal functions). The present studies characterize renal insulin receptors in the chicken. The effects of the following nutritional conditions were examined: fed, 48 hr fasted, and 24 hr refed following a 48-hr fast. PEPCK activity was increased by the 48-hr fast and returned to normal after refeeding. Specific binding of 125I-insulin to renal membranes was time-, temperature-, and protein-dependent. Unlabeled insulin was more potent than IGF-1 in inhibiting 125I-insulin binding; the ratio of potencies for insulin and IGF-1, however, was dependent upon the nutritional state. Insulin binding was significantly higher (P < 0.05) following 48 hr fasting and lower (P < 0.05) following refeeding compared to ad libitum feeding. Receptor affinity was similar irrespective of the nutritional state. Solubilized and wheat germ agglutinin purified renal insulin receptors were devoid of ATPase activity in contrast to hepatic receptors. The sizes of α- and β-subunits of renal receptors were similar to those of hepatic receptors: 135 and 95 kDa, respectively. Insulin-stimulated autophosphorylation of the β-subunit was decreased, although not significantly, by prolonged fasting. Phosphorylation of artificial substrate: poly(Glu-Tyr) 4:1 was significantly decreased by the 48-hr fast at high insulin concentrations (10 and 100 nM). Kinase activities of renal insulin receptors from fed or refed chickens were very similar. In conclusion, typical insulin receptors are present in chicken kidneys. These receptors exhibit a regulation at the level of their number and kinase activity in a fashion similar to that found for hepatic receptors. The present results suggest a role for insulin in chicken renal function.

Preliminary investigations of bony fish--tench (Tinca tinca L.)--erythrocyte insulin receptors.

Specific insulin receptors were identified on tench (Tinca tinca L.) erythrocytes. The samples of blood from 3-4 fish per experiment were pooled in summer. Erythrocytes were washed twice with saline solution, then centrifuged and resuspended in the assay buffer. Incubations were carried out in 0.5 ml samples containing 10(9) cells, with addition of 0.03 nmol/l of 125I-pork insulin in the absence or presence of unlabelled pork or salmon insulin (0.05-2000 nmol/l) for 24 h at 4 degrees C. Calculated from seven experiments specific binding of 125I-insulin was 6.2 +/- 0.2% (mean +/- SD). Scatchard analysis of the binding data from insulin radioreceptor assay showed two different binding sites with high (HAIR) and low (LAIR) affinity. KD values were 0.82 +/- 0.17 nmol/l and 2.05 +/- 1.62 mumol/l, and maximal binding capacity (Bmax) 24.9 +/- 1.6 fmol/10(9) cells and 17.5 +/- 12.2 pmol/10(9) cells, respectively (N = 4). If compared with pork insulin, six to seven times more of salmon insulin was required to displace 50% of iodine labelled hormone. Thus, insulin receptors of tench erythrocytes are similar to those described in other species of fish and other lower vertebrates.

Altered pattern of insulin receptor isotypes in skeletal muscle membranes of type 2 (non-insulin-dependent) diabetic subjects.

The human insulin receptor exists in two isoforms (HIR-A alpha-subunit 719 amino acids and HIR-B alpha-subunit 731 amino acids) which are generated by alternative splicing of a small exon and display distinct patterns of tissue-specific expression. Using the polymerase chain reaction we have recently shown that skeletal muscle of non-diabetic individuals contains predominantly mRNA encoding HIR-A while in skeletal muscle derived from subjects with Type 2 (non-insulin-dependent) diabetes mellitus similar amounts of each mRNA are expressed. We used a polyclonal antibody which discriminates between HIR-A and HIR-B to assess the isoform expression at the protein level. The antibody showed clearly distinct displacement of insulin binding in skeletal muscle membranes of non-diabetic subjects compared to Type 2 diabetic subjects (displacement of specific 125I-insulin binding: 13 non-diabetic subjects 70.0% +/- 14.34, 12 Type 2 diabetic subjects 32.6% +/- 17.45). A control antibody which does not discriminate between both isoforms showed similar displacement of 125I-insulin in membranes of non-diabetic and Type 2 diabetic subjects. These data suggest that the altered expression of receptor isotype mRNA in the skeletal muscle of Type 2 diabetic subjects leads to an altered receptor isoform pattern in the plasma membrane. While skeletal muscle membranes of non-diabetic subjects contain predominantly HIR-A, membranes of Type 2 diabetic subjects show an increased level of HIR-B in addition to HIR-A.

Down regulation of masked and unmasked insulin receptors in the liver of transgenic mice expressing bovine growth hormone gene

The interaction of insulin with its receptor was studied in microsomes from livers of transgenic mice expressing the bovine growth hormone gene with mouse metallothionein-1 promoter (MT/bGH) and in their normal (non-transgenic) littermates. Specific binding of 125I-insulin was detected in hepatic microsomes from normal and transgenic mice with an apparent Kd of 8 and 200 nM, for high and low affinity sites, respectively. The transgenic MT/bGH mice had a marked hyperinsulinism without significant elevation of plasma glucose levels. Under identical conditions of preparation and incubation, microsomes from the transgenic male and female mice bound 39% and 34% less insulin than those from their litter mates. Scatchard's analysis indicates that this decrease in binding is due to a decrease in the number of receptor sites. In contrast to the marked decrease in insulin binding to unmarked receptors, the levels of masked (also called cryptic) insulin receptors were similar (or slightly increased) in transgenic mice microsomes as compared to those of their normal litter mates.

Binding and biological effects of insulin, insulin analogues and insulin-like growth factors in rat aortic smooth muscle cells. Comparison of maximal growth promoting activities

Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of 125I-insulin was 0.9 +/- 0.2% of total 125I-insulin added, and the IC50-value was estimated to 8.9 pmol/l. The insulin analogue B10 Asp tended to be more potent than insulin in displacing 125I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of 125I-insulin-like growth factor-I after 4 h incubation at 10 degrees C was five times higher than the specific binding of insulin (4.4 +/- 0.4% of total 125I-insulin-like growth factor-I added), and the IC50-value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing 125I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for 3H-thymidine incorporation into DNA: insulin-like growth factor-I greater than B10 Asp greater than insulin-like growth factor-II greater than insulin greater than or equal to B28 Asp greater than B9 Asp/B27 Glu greater than proinsulin. The maximal effect of insulin-like growth factor-I on 3H-thymidine incorporation was 71 +/- 16% higher than the maximal effect of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose regulation of insulin receptor affinity in primary cultured adipocytes.

Treatment of primary cultured adipocytes with 20 mM glucose resulted in a progressive increase in specific 125I-insulin binding that began almost immediately (no lag period) and culminated in a 60% increase by 24 h. This effect was dose-dependent (glucose ED50 of 4.6 mM) and mediated by an increase in insulin receptor affinity. Moreover, it appears that glucose modulates insulin receptor affinity through de novo protein synthesis rather than through covalent modification of receptors, since cycloheximide selectively inhibited the glucose-induced increase in insulin binding capacity (ED50 of 360 ng/ml) and restored receptor affinity to control values. Importantly, insulin sensitivity of the glucose transport system was increased by glucose treatment (63%) to an extent comparable with the enhancement in receptor affinity, thus indicating a functional coupling between insulin binding and insulin action. When the long term effects of insulin were assessed (24 h), we found that insulin treatment reduced 125I-insulin binding by greater than 60% by down-regulating the number of cell surface receptors in a dose-dependent manner (insulin ED50 of 7.4 ng/ml). On the basis of these studies, we conclude that 1) insulin binding is subject to dual regulation (glucose controls insulin action by enhancing receptor affinity, whereas insulin controls the number of cell surface receptors); and 2) glucose appears to modulate insulin receptor affinity through the rapid biosynthesis of an affinity regulatory protein.

Regulation of hexose transport in respiration deficient hamster lung fibroblasts

The transport of [3H]2-deoxy-D-glucose (2DG) and [3H]3-O-methyl-D-glucose (3-OMG) was elevated in a respiration deficient (NADH coenzyme Q [Co Q] reductase deficient) Chinese hamster lung fibroblast cell line (G14). This sugar transport increase was related to an increased Vmax for 2DG transport, 26.9 +/- 4.2 nmoles 2DG/mg protein/30 sec in the G14 cell line vs 9.5 +/- 0.6 nmoles 2DG/mg protein/30 sec in the parental V79 cell line. No differences were observed in their respective Km values for 2DG transport (3.9 +/- .6 vs. 3.0 +/- .13 mM). Factors which increase sugar transport (e.g., glucose deprivation, serum or insulin exposure) or decrease sugar transport (e.g., serum deprivation) in the parental V79 cell line had little effect on sugar transport in the G14 respiration deficient cell lines. Amino acid transport, specific 125I-insulin binding to cells, and insulin-stimulated DNA synthesis, however, were similar in both cell lines. Exposure of both cell lines to varying concentrations of cycloheximide (0.1-50 micrograms/ml) for 4 h resulted in differential effects on 2DG transport. In the parental cell line (V79) low cycloheximide concentrations resulted in decreased 2DG transport, while higher concentrations (greater than or equal to 1 microgram/ml) resulted in elevated 2DG transport. In the G14 cell line, 2DG transport decreased at all concentrations of cycloheximide (up to 50 micrograms/ml). The data indicate that the G14 mutant has been significantly and specifically affected in the expression of sugar transport activity and in the regulatory controls affecting sugar transport activity.

Regulation of human ovarian insulin receptors in vivo

Insulin participates in regulating ovarian function in normal and in pathological states. This effect of insulin may be mediated by ovarian insulin receptors. We have previously characterized human ovarian insulin receptors and began to examine their regulation in vitro. The present study examines regulation of human ovarian insulin receptors in vivo. Stromal ovarian tissue was obtained from 21 women during an indicated surgical procedure. Ten women were premenopausal and 11 were postmenopausal. Specific 125I-insulin binding to stromal ovarian fragments ranged from 2.5% to 7.3%/mg protein. 125I-insulin binding to stromal fragments correlated positively with 125I-insulin binding to circulating leucocytes (r = .57; P < .01). When postmenopausal and premenopausal women were analyzed separately, this relationship persisted in postmenopausal women (r = .70; P < .05), but not in premenopausal women. 125I-insulin binding to stromal overian fragments correlated negatively with age (r = −.63; P = .005). 125I-insulin binding to stromal ovarian fragments tended to correlate negatively with plasma insulin levels in postmenopausal women (r = −.67; P = .06), but not in premenopausal women. Plasma insulin levels correlated negatively with serum SHBG (r = − .62; P = .003). The percent free testosterone levels correlated positively with plasma insulin levels in premenopausal women (r = .95; P = .0001), but not in postmenopausal women. Our findings demonstrate that (1) ovarian 125I-insulin binding correlates well with 125I-insulin binding to circulating white cells, suggesting that insulin is an important regulator of ovarian insulin receptors, particularly after the menopause; (2) the lack of correlation between 125I-insulin binding to circulating white cells and to ovarian stromal fragments from premenopausal women suggests that factors other than insulin participate in insulin-receptor regulation in premenopausal ovaries; (3) the number of ovarian insulin receptors decreases with age; and (4) insulin inhibits SHBG production in premenopausal women, thereby increasing the biologic availability of testosterone. These findings are important in understanding the nature of ovarian dysfunction in such diverse entities as diabetes mellitus, syndromes of insulin resistance, obesity, and polycystic ovarian syndrome.

Liver Insulin Receptor Tyrosine Kinase Activity in a Rat Model of Type II Diabetes Mellitus and Obesity

Spontaneous hypertensive-corpulent rats (SHR/N-corpulent), homozygous for the corpulent gene (cp/cp), are obese, hyperinsulinemic and exhibit abnormal glucose tolerance and thus represent a model for type II diabetes and obesity. In view of their overall insulin resistance, we examined liver insulin receptor binding and tyrosine kinase activity from corpulent rats and lean littermates fed purified diets containing 54% sucrose or starch for about 12 wk. Specific 125I-insulin binding to crude liver membranes from female corpulent rats fed either starch or sucrose was reduced to approximately 50% of that seen in lean rats (14 vs. 7%). Affinity of insulin receptors was similar in all groups, suggesting that hyperinsulinemic corpulent rats possess fewer hepatic insulin receptors than do lean rats. Using similar numbers of wheat germ agglutinin-agarose (WGA)-purified insulin receptors with similar affinities for insulin, it was found that basal and insulin-stimulated phosphorylation of the synthetic tyrosine-specific kinase substrate poly(Glu, Tyr)4:1 was similar in lean and obese rats fed sucrose or starch. It is suggested that the contribution of the liver to the insulin resistance in obese SHR/N-cp rats probably lies distal to the insulin receptor tyrosine kinase.

Effects of fetal hypoinsulinemia on fetal hepatic insulin binding in the rat

Fetal hepatic insulin binding was studied in term fetal rats born to control mothers, mothers fasted for 48 h and mothers made hyperinsulinemic by the chronic, exogenous administration of insulin for 5 days prior to term. Maternal hyperinsulinemia was associated with fetal hypoglycemia and an approx. 70% reduction in fetal plasma insulin. Fetuses from these mothers exhibited an increase in hepatic insulin binding as indicated by a significant change in Scatchard analyses. No significant effect on fetal hepatic insulin binding by Scatchard analysis was seen with maternal fasting, despite a modest decrease in fetal plasma insulin. However, analysis of all animals showed that high-affinity fetal hepatic insulin binding and specific 125I-insulin binding were inversely correlated with fetal plasma insulin concentration. These results indicate that fetal rat liver, similar to adult rat liver, responds to a decrease in circulating insulin to below normal concentrations with an increase in insulin receptor binding.