I-insulin Binding Research Articles

持続血糖モニター（CGM）による血糖変動の把握が有用であったインスリン抗体陽性の糖尿病の1例

A 76-year-old male was diagnosed to have diabetes mellitus at 57 years of age and oral hypoglycemic agents were started. Aspart, a human insulin analogue, was initiated because of a poor glycemic control in July 2009, at 74 years of age. He complained of dizziness and faintness caused by hypoglycemia in the early morning and afternoon. He was admitted to another hospital in August 2010; his plasma insulin levels were markedly elevated (11,000 μU/ml), and 125 I-insulin binding rate of anti-insulin antibodies was 89.5 %. He was transferred to this hospital in September 2010, and a Scatchard analysis of his anti-insulin antibodies showed low affinity and high capacity. The daily profiles of blood glucose were evaluated precisely by continuous glucose monitoring (CGM). CGM showed low blood glucose levels in the early morning and afternoon, when human insulin injections were given twice daily. In addition, insulin and diet therapies were adjusted to prevent hypoglycemia, and accordingly, blood glucose control was improved. Hence, CGM is therefore considered to be useful in the management of brittle diabetes cause by anti-insulin antibodies. (Less)

Glycogen synthase kinase‐3β: homologous regulation of cell surface insulin receptor level via controlling insulin receptor mRNA stability in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, 48 h-treatment with 20 mmol/L LiCl, 1 mmol/L valproic acid, 30 micromol/L SB216763, 30 micromol/L SB415286, or 100 nmol/L insulin, a condition that inhibits constitutive active glycogen synthase kinase-3 (GSK-3), decreased cell surface (125)I-insulin binding capacity by approximately 39%, without altering the K(d) value; LiCl, SB216763 or insulin decreased insulin receptor (IR) and IR precursor levels, attenuating insulin-induced Tyr-autophosphorylation of IR. LiCl increased inhibitory Ser9-phosphorylation of GSK-3beta at 6 h, decreasing (125)I-insulin binding at 24 h. SB216763-induced (125)I-insulin binding reduction (IC(50) = 3 micromol/L) was preceded by beta-catenin level increase by SB216763 (EC(50) = 11 micromol/L), a hallmark of GSK-3 inhibition. Insulin-induced rapid (> 1 min) Ser9-phosphorylation of GSK-3beta (Nemoto et al. 2006) was followed by approximately 48% decrease of IR level. LiCl did not stimulate endocytosis, nor proteolysis of IR. LiCl destabilized IR mRNA (t(1/2) = 9.3 vs. 6.5 h), decreasing IR mRNA level by approximately 47%, without altering IR gene transcription. Decreases of (125)I-insulin binding and IR level, as well as increased Ser9-phosphorylation of GSK-3beta were restored to the control levels by washing the test compound-treated cells. Thus, GSK-3beta regulates IR level via controlling IR mRNA stability.

Altered Hypothalamic Leptin, Insulin, and Melanocortin Binding Associated with Moderate-Fat Diet and Predisposition to Obesity

Rats with a genetic predisposition to develop diet-induced obesity (DIO) have a preexisting reduction in central leptin and insulin sensitivity. High-fat diets also reduce sensitivity to leptin, insulin, and melanocortin agonists. We postulated that such reduced sensitivities would be associated with decreased binding to the hypothalamic leptin, insulin, and melanocortin receptors in selectively bred DIO rats and in rats fed a high-energy (HE; 31% fat) diet for 7 wk. On HE diet, DIO rats gained 15% more weight and had 121% heavier fat pads and 70% higher leptin levels than low fat chow-fed DIO rats. Diet-resistant (DR) rats gained no more weight on HE diet but had 48% heavier fat pads and 70% higher leptin levels than chow-fed DR rats. Compared with DR rats, DIO (125)I-leptin binding was 41, 36, and 40% lower in the hypothalamic dorsomedial, arcuate, and dorsomedial portion of the ventromedial nuclei, respectively, and arcuate (125)I-insulin binding was 31% lower independent of diet. In contrast, hypothalamic melanocortin binding did not differ between DIO and DR rats. However, HE diet intake lowered lateral hypothalamic melanocortin-3 and melanocortin-4 receptor and hippocampal insulin binding of both DIO and DR rats and hypothalamic paraventricular nucleus melanocortin-4 receptor binding only in DR rats. Neither genotype nor diet affected substantia nigra or ventral tegmental area binding. These results corroborate our previous findings demonstrating a preexisting decrease in DIO hypothalamic leptin and insulin signaling and demonstrate that HE diet intake reduces hypothalamic melanocortin and hippocampal insulin binding.

The effects of protease inhibitors on basal and insulin-stimulated lipid metabolism, insulin binding, and signaling

The objective of our research was to investigate the effects of the protease inhibitors ritonavir, saquinavir, and indinavir on triglyceride synthesis, lipolysis, insulin binding, and signaling in differentiating 3T3 L1 pre-adipocytes. Saquinavir, ritonavir, and indinavir all stimulated triglyceride (TG) synthesis. Additionally, all concentrations of protease inhibitors employed (i.e., 0.1 micro M to 10 micro M) significantly decreased insulin-stimulated TG synthesis. No effects of any of the protease inhibitors were observed either on basal lipolysis or after stimulation of lipolysis with 100 nM noradrenaline. Specific (125)I-insulin binding was observed to be decreased by exposure to all the protease inhibitors throughout the period of adipocyte phenotype development. This was mediated by indinavir through a receptor decrease and had no effect on receptor affinity. During differentiation with ritonavir (i.e., 1-11 days post addition of differentiating cocktail), insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation was ascertained (day 11) and found to be decreased in the ritonavir exposed cells when compared with control cells. The results reported herein demonstrate protease inhibitor effects on basal TG synthesis while exhibiting decreased insulin-stimulated TG synthesis at physiological concentrations of protease inhibitors. These effects may be subsequent to decreased insulin binding and/or IRS-1 tyrosine phosphorylation.

Distinct effects of ketone bodies on down-regulation of cell surface insulin receptor and insulin receptor substrate-1 phosphorylation in adrenal chromaffin cells.

Treatment (>/=24 h) of cultured bovine adrenal chromaffin cells with ketoacidosis-related concentrations (>/=3 mM) of acetoacetate (but not beta-hydroxybutyrate, acetone, and acidic medium) caused a time- and concentration-dependent reduction of cell surface (125)I-insulin binding by ~38%, with no change in the K(d) value. The reduction of (125)I-insulin binding returned to control nontreated level at 24 h after the washout of acetoacetate-treated cells. Acetoacetate did not increase the internalization rate of cell surface insulin receptor (IR), as measured in the presence of brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network. Acetoacetate (10 mM for 24 h) lowered cellular levels of the immunoreactive IR precursor molecule (approximately 190 kDa) and IR by 22 and 28%, respectively. Acetoacetate decreased IR mRNA levels by approximately 23% as early as 6 h, producing their maximum plateau reduction at 12 and 24 h. The half-life of IR mRNA was shortened by acetoacetate from 13.6 to 9.5 h. Immunoprecipitation followed by immunoblot analysis revealed that insulin-induced (100 nM for 10 min) tyrosine-phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated by 56% in acetoacetate-treated cells, with no change in IRS-1 level. These results suggest that chronic treatment with acetoacetate selectively down-regulated the density of cell surface functional IR via lowering IR mRNA levels and IR synthesis, thereby retarding insulin-induced activation of IRS-1.

Down-regulation of cell surface insulin receptor and insulin receptor substrate-1 phosphorylation by inhibitor of 90-kDa heat-shock protein family: endoplasmic reticulum retention of monomeric insulin receptor precursor with calnexin in adrenal chromaffin cells.

Treatment (>/=6 h) of cultured bovine adrenal chromaffin cells with geldanamycin (GA) or herbimycin A (HA), an inhibitor of the 90-kDa heat-shock protein (Hsp90) family, decreased cell surface (125)I-insulin binding. The effect of GA was concentration (EC(50) = 84 nM)- and time (t(1/2) = 8.5 h)-dependent; GA (1 microM for 24 h) lowered the B(max) value of (125)I-insulin binding by 80%, without changing the K(d) value. Western blot analysis showed that GA (>/=3 h) lowered insulin receptor (IR) level by 83% (t(1/2) = 7.4 h; EC(50) = 74 nM), while raising IR precursor level by 100% (t(1/2) = 7.9 h; EC(50) = 300 nM). Pulse-label followed by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that monomeric IR precursor (~190 kDa) developed into the homodimeric IR precursor (approximately 380 kDa) and the mature alpha(2)beta(2) IR (~410 kDa) in nontreated cells, but not in GA-treated cells; in GA-treated cells, the homodimerization-incompetent form of monomeric IR precursor was degraded via endoplasmic reticulum (ER)-associated protein degradation. Immunoprecipitation followed by immunoblot analysis showed that IR precursor was associated with calnexin (CNX) to a greater extent in GA-treated cells, compared with nontreated cells. GA had no effect on IR mRNA levels and internalization rate of cell surface IRs. In GA-treated cells, insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated by 77%, with no change in IRS-1 level. Thus, inhibition of the Hsp90 family by GA or HA interrupts homodimerization of monomeric IR precursor in the ER and increases retention of monomeric IR precursor with CNX; this event retards cell surface expression of IR and attenuates insulin-induced activation of IRS-1.

High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes.

The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.

Cholesterol Depletion Disrupts Caveolae and Insulin Receptor Signaling for Metabolic Control via Insulin Receptor Substrate-1, but Not for Mitogen-activated Protein Kinase Control

Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Strålfors, P. (1999) FASEB. J. 13, 1961-1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with beta-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by (125)I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.

Insulin Receptor Antibodies Inhibit Insulin Uptake by the Liver

BackgroundInsulin receptor antibodies can induce severe hypoglycemia or insulin resistance in rare autoimmune syndromes. In vitro properties of these antibodies occasionally explain the clinical features of the syndrome, but direct...

Up-regulation of cell surface insulin receptor by protein kinase C-alpha in adrenal chromaffin cells: involvement of transcriptional and translational events.

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-alpha (PKC-alpha) and novel PKC-epsilon (but not atypical PKC-zeta) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-alpha. In the present study, chronic (>/=12 h) treatment of chromaffin cells with PDBu raised cell surface (125)I-insulin binding without altering the K(D) value; it developed in a concentration (EC(50) = 1.9 nM)-and time (t(1/2) = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC(50) = 6.4 nM) also increased (125)I-insulin binding by 97 or 88%, whereas the biologically inactive 4alpha-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on (125)I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of (125)I-insulin binding. Western blot analysis, using antibody against the beta-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC(50) = 2.3 nM) increased levels of insulin receptor precursor (approximately 190 kDa; t(1/2) = 7.1 h) and insulin receptor beta-subunit (t(1/2) = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by approximately 35% as soon as 3 h, producing its monophasic peak approximately 76% increases at 24 h. All of these increasing effects of PDBu and TMX on (125)I-insulin binding and insulin receptor beta-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-alpha up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Effects of dietary restriction on insulin resistance in obese mice.

In many cases, development of insulin resistance has been linked to obesity and may contribute to mechanism of aging. The role of diet, irrespective of degree of obesity, in modulating insulin resistance and development of age degeneration disease remains uncertain. Lowered blood glucose levels are commonly associated with diet restriction (DR), which is an intervention shown to successfully retard aging and age associated disease. The effects of DR on blood glucose and insulin resistance were measured in yellow obese (A(vy)/A), lean black (a/a) mice and in another common inbred strain (B6C3F1) (at three different ages). The yellow obese mice become diabetic as a result of an insulin receptor defect which is not clearly understood. Insulin responses and radioinsulin binding were assayed in yellow obese and lean black mice fed either ad libitum (AL) or DR diets (YAL, BAL, YDR and YAL, respectively) at four different circadian intervals. The B6C3F1 controls were fed either AL (CAL) or DR (CDR) and measures were made at six circadian stages and three different ages. Within 23 days, DR produced a significant loss in body weight and a time-dependent 22-55% reduction in basal blood glucose levels in the yellow obese mice. Additionally, exogenous insulin produced circadian stage dependent (at the time of food intake) reductions in blood glucose in the YDR animals that were not present in YAL animals. (125)I-Insulin binding in liver was increased nearly 2-fold in YDR and BDR mice during the time of day that animals were active and eating. (125)I-Insulin binding was two-fold-higher in CDR mice at 4, 12 and >24 months of age. Binding decreased as a function of age in both the CAL and CDR animals. However, even in the >24 month group the CDR animals were found to have levels of binding that were as high as those found in younger CAL liver. The mechanism of action appears to be through resolution of insulin resistance by modulating an insulin receptor defect.

Prenatal Glucocorticoid Induced Fetal and Postnatal Growth Restriction is Mediated by Obese Gene (OB) or Leptin Receptors † 1559

Prenatal glucocorticoid (GC) therapy is an accepted practice in the prevention of respiratory distress syndrome in the neonate. Concerns regarding its use in early gestation and its side effects upon fetal and postnatal growth and metabolism persist. The recent cloning of the obese gene and obese gene receptors (OB-R) has shed some light upon mechanisms by which altered metabolism (anorexic/catabolic) and diminution in growth occurs. To determine if the ob gene (leptin) receptors mediate the growth reducing effects of GC, we intraperitoneally administered 2 doses of 0.3 mg/kg/day dexamethasone (GC) or vehicle (V) beginning d7, d9, d11, d13, d15, d17, and d19 (term ≈21d; n=7/age/group), and examined the binding of125 I-leptin (10-9M), in the presence and absence of 0.6 X 10-6M unlabeled leptin, to the d9, d11, d13, d15, d17, d19, and d21 frozen saggital embryonic liver sections. In addition, postnatal livers were studied on d15 and d30 (n=7/age/group) subsequent to GC or V administration on d19 gestation. Specific 125I-leptin binding was assessed by quantitation of the autoradiographs and phoshoimages by microdensitometry(Molecular Analyst Microdensitometry Program-1.4.1, NIH). GC reduced body weight at all ages (p < 0.001), and increased hepatic 125I-leptin binding at all ages with a maximal 3-fold increase noted at postnatal d15 (p< 0.005). This is in contrast to a 2-fold decline in hepatic125 I-insulin binding (p < 0.01) at postnatal d15, which served as a known control. We conclude that prenatal GC 1] increases hepatic leptin receptors from mid- to late gestation, 2] has a persistent enhancing effect upon postnatal hepatic leptin receptors. We speculate that prenatal GC increases the biological action of leptin peripherally, thereby affecting the metabolic mileu and the growth potential of the fetus and the newborn.

Insulin Receptor Downregulation in Isolated Hepatocytes of River Lamprey (Lampetra fluviatilis)

Insulin receptor downregulation in the isolated hepatocytes of lamprey (Lampetra fluviatilis) was studied at the ambient temperature for this species. Preincubation of hepatocytes with 10-9-10-8M unlabeled insulin decreased insulin binding capacity to 43, 37, and 34% at 4, 15, and 25°, respectively. Preincubation of hepatocytes in the presence of 10-10M unlabeled insulin had no effect on the 125 I-insulin binding. The maximal decrease in the 125 I-insulin binding was reached after 10 min of preincubation and was then maintained at a constant level for 2 hr. Competitive binding assays demonstrated that preincubation with unlabeled insulin resulted in a 45% decline in the number of binding sites. In rat adipocytes and frog hepatocytes used for the comparison, preincubation with insulin caused a 65% decrease in cell-surface receptors, while intracellular (internalized receptors) increased proportionally. In contrast, in the lamprey hepatocytes both cell surface and intracellular receptors decreased. We conclude that insulin receptors of the lamprey hepatocytes could be downregulated at the ambient temperature for the species and at the physiological concentrations of insulin (10-9-10-8M).

Human retinoblastoma Y79 cells contain both insulin-specific mRNA and insulin-binding sites

We previously reported the presence of insulin-like immunoreactivity in cells from the human retinoblastoma Y79 cell line. In the present study, in situ DNA hybridization techniques were applied, using a human insulin cDNA probe to investigate whether the insulin-like activity is due to local synthesis of insulin. Our results suggest that Y79 cells contain mRNA for the synthesis of insulin or a homologous peptide. In addition, 125I-insulin binding autoradiographic studies show that these cells also contain specific insulin-binding sites. It is suggested that insulin may play an autocrine and/or paracrine role in the maintenance and metabolism of the Y79 retinoblastoma cells.

Human Placental Insulin Binding in Normal and Well-Controlled Diabetic Patients

Previous studies of insulin binding to placentas of both insulin-dependent and untreated gestational diabetic patients have described placentas from diabetics to contain fewer insulin receptors than placentas from nondiabetic gravidas. However, these studies were done using membrane fractions prepared from the placentas and at a time when adequacy of antepartum glycemic control in the diabetic patients was not routinely evaluated by self blood sugar measurement or hemoglobin A1 assay. The current study compares specific 125I-insulin binding in vitro to intact placental villi from 15 normal patients with insulin binding to intact villi obtained from 15 insulin-dependent diabetic mothers whose fasting and postprandial blood sugars and hemoglobin A1 levels were maintained in a range normal for term pregnancy. We demonstrate that insulin binding to intact placental villi is the same in this group of diabetic patients as in the nondiabetic patients.

Effects of dietary fiber and protein on rat erythrocyte insulin-receptor interaction

The metabolic effects of cellulose on insulin receptor interaction (IRI) was studied at 10 and 20 percent protein levels in male weanling Sprague Dawley rats. One hundred and four (104) rats were divided into 8 groups of 13 each and randomly put on various dietary treatments as already reported elsewhere [Fed. Proceed. 1985; 44(5):6367]. Animals for radioreceptor studies (n=5 per group) were fed for 8 weeks. Blood samples were obtained from the rats via cardiac puncture under nembutal anesthesia. Erythrocyte IRI and serum insulin levels (IN) were measured by a standardized radioreceptor assay (10) and radioimmunoassay respectively. Fiber and protein intake had significant effect on IRI (p<0.01) and IN (p<0.05). Specific percent 125 I-insulin binding (SIB) was higher in the 20% protein group at all fiber levels while in the fiber free groups higher SIB was observed in the 10% protein group (p<0.01). At both protein levels, maximum SIB was observed at the 5% fiber level. While SIB in the absence of fiber was observed to be strictly a function of receptor site concentration (Ro) binding in the presence of fiber was a function of both Ro and empty site affinity (Ke). Fiber intake appears to interfere with the negative cooperative nature of the receptor sites by slowing down the rate of changing affinity from a high Ke to the limiting K f (filled site affinity) state as receptor occupancy [Y=Long [B]/Ro] increases. While no significant effect of calorie was observed on e and f, caloric intake (Cal) showed a positive correlation (r=0.8, p<0.001) with IN and a negative (r=0.32, p<0.02) correlation with Ro. It is concluded that: (I) diet can modulate erythrocyte IRI in growing animals, (II) the metabolic effects of dietary fiber depend on the combination with other nutrients, (III) adequate protein nutrition is essential for plasma membrane function at the cell receptor level in growing animals and (IV) the increase in IRI by dietary fiber is a function of both e, Ro and the sustenance of the average affinity of these homogeneous class of receptor sites in the high affinity states.

The effect of glucocorticoid on 125I-insulin binding to human erythrocytes Possible postreceptor modulation of receptor binding

To evaluate the role of insulin receptors in the pathogenesis of insulin resistance observed in glucocorticoid excess, we measured 125I-insulin binding to circulating erythrocytes in 7 patients with Cushing's syndrome and 7 patients with adrenal insufficiency. Insulin receptor binding was higher in Cushing's syndrome and was lower in adrenal insufficiency, compared to normal subjects. Insulin binding decreased after transsphenoidal surgery in 2 patients with Cushing's syndrome. In addition, glucocorticoid treatment in 6 patients with adrenal insufficiency resulted in the increase of insulin binding. The biological significance of this phenomenon must await further investigation, but it does suggest that insulin resistance in glucocorticoid excess should be interpreted as an alteration of cellular mechanisms of insulin at a step distal to the insulin receptor. Increased insulin binding to the receptor is probably modulated by postreceptor events.

1239 DETECTION OF GESTATIONAL DIABETES IN THE FIRST TRIMESTER: ABNORMALITIES IN CELL MEMBRANE STRUCTURE AND FUNCTION

Detection of gestational diabetes (GD) is currently limited to the last trimester, whereas the associated letal abnormalities occur early in pregnancy due to disturbances in maternal metabolism. He have studied 39 pregnant subjects with varying degrees of carbohydrate tolerance at intervals during pregnancy. Heparinized blood samples (10-15cc) were obtained from all subjects after an overnight fast, for studies of [125]I-insulin binding, membrane phospholipid (PL) and membrane fluidity determinations using fluorescence polarization (FP). The data suggest that a detect in insulin binding and insulin action is present in women with GD, associated with disturbances in both membrane structure and biophysical behavior. This detect, similar to that seen in obese. Type II diabetic subjects is detectable betore 12 weeks gestation. Early detection ot GD, permitting the institution of appropriate measures of metabolic control in these subjects, should result in marked reduction of perinatal morbidity.

Insulin degrading system on isolated rat hepatocytes; enzymatic and immunologic characteristics of insulin-degrading activities

Insulin degradation by isolated rat hepatocytes was investigated. Using the preincubation method, the extracellular insulin-degrading activity was removed from the medium over 120 min at 15 degrees C and 60 min at 37 degrees C. The degradation of insulin was assayed by the ability of binding to specific receptors on isolated rat hepatocytes (rebound method) and the precipitability with trichloroacetic acid (TCA method). The degrading activities measured by the rebound method showed twice those by the TCA method, however, a positive correlation with high coefficient (r = 0.87, P less than 0.001) was demonstrated between both methods. The insulin-degrading activity by hepatocytes depended on time, temperature and cell concentrations, and the optimum pH was 7.0. As a result of kinetic analysis of insulin binding and degradation, the degradation velocity of 125I-insulin was inhibited by 50% at native insulin concentration of 7 X 10(-8) M, whereas the half-maximum inhibition of 125I-insulin binding was demonstrated at that of 4 X 10(-9) M. The Km for insulin degradation was 170 nM. In order to characterize the enzymatic properties of insulin-degrading activity by isolated hepatocytes, the effects of various compounds and anti-insulin-degrading enzyme (IDE) rabbit serum on insulin degradation were examined. N-ethylmaleimide and anti-IDE serum significantly inhibited the insulin-degrading activity, while dithiothreitol stimulated it, whereas chloroquine and NH4Cl had little effect on insulin-degrading activity. Finally, the immunoenzymatic labelling of hepatocytes by anti-IDE serum showed the presence of cell surface IDE on isolated hepatocytes. These results suggest that most of insulin-degrading activity by isolated rat hepatocytes is identical to pig muscle IDE. Therefore, it would seem that IDE plays an important role in insulin metabolism by isolated rat hepatocytes rather than lysosomes.

Reticulocytes and insulin binding to erythrocytes.

The relationship between erythrocyte insulin receptors and the reticulocytes were studied in a group with a varying degree of reticulocytosis. The maximal I-insulin binding to erythrocytes varied between 6% and 37% and it showed a highly significant positive correlation with the reticulocyte count (r=0.85). The highest binding was found in a patient with hereditary spherocytosis and very active hematopoiesis (312 x 10(9) reticulocytes per litre). The results suggest that insulin receptors are predominantly localized to the young erythrocytes, and that further studies are needed to characterize the dependence of the receptor on erythrocyte maturation.