Cyclic AMP modulates insulin binding and induces post-receptor insulin resistance of glucose transport in isolated rat adipocytes

The effect of cAMP on insulin binding and insulin stimulation of glucose transport was investigated in isolated rat adipocytes. Preincubation for 30 min in medium containing 16 mmol/l glucose and either db-cAMP or bromo-cAMP in concentrations of 10(-4)-10(-3) M inhibited high affinity binding of insulin by 15 to 30% and glucose transport by 30 to 50%. Preincubation with IBMX (10(-4)-10(-3) M) reduced insulin binding by 25% and glucose transport by 70%. Closer analysis of these data indicated that preincubation with these compounds caused not only a decrease in insulin binding but also a post-receptor resistance. High intracellular cyclic AMP-levels seem therefore to induce insulin resistance at both receptor and post-receptor levels.     

Phosphorylation control by insulin in adipocytes is interfered with at a post-receptor step by phosphoinositol and glucosamine.

Inositol-phosphates, glucosamine and glucose-6-phosphate blocked the effects of insulin on target protein phosphorylation in adipocytes, but the unsubstituted or sulphated derivatives of inositol or of glucose, or N-acetyl-glucosamine were without effect. The insulin stimulated tyrosine phosphorylation of the insulin receptor was not affected. The sugar-phosphates inositol-phosphate and glucose-6-phosphate did not enter into the cells. They also blocked the insulin-like effects of a potential second messenger of insulin, a phospho-oligosaccharide (POS), which has previously been shown to mimic the effects of insulin on protein phosphorylation in intact cells.     

Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.     

Effect of lipid peroxidation on the insulin receptor in rat adipocytes

Lipid peroxidation is studied for its effect on insulin receptors in isolated rat adipocytes. The results suggest that addition of two peroxidants (3 mM cumene hydroperoxide and 0.2 mM Fe2+) leads to malondialdehyde accumulation and binding inhibition through insulin receptors quantity and affinity decrease.     

Heat tolerance in rats following administration of streptozotocin,glucose, insulin and glucose plus insulin

Rise in rectal temperature (T_re) and survival time was determined on exposure to 38°C in adult normoglycemic and diabetic (streptozotocin treated) rats and 1 h following glucose feeding or insulin administration or both, and in young rats with and without glucose feeding or insulin treatment. The heat tolerance of adult animals treated with streptozotocin and insulin plus glucose and of adult and young animals treated with glucose feeding or insulin was less than that of their respective normoglycemic controls. The rectal temperature on exposure to heat in the treated animals was significantly higher than that of controls in the adult, but not in young rats. Exposure to heat of the normoglycemic and glucose-fed animals resulted in a rise in blood glucose in the adults and a fall in the young. The already raised blood glucose level in the streptozotocin-treated animals rose further on exposure to heat. The rate of recovery of the blood glucose was not significantly altered by exposure of the animals to heat 60 min after administration of insulin or insulin plus glucose.     

Cold tolerance in rats following administration of streptozotocin,glucose, insulin and glucose plus insulin

Fall in rectal temperature (T_re) and survival time was determined on exposure to–20°C in adult normoglycemic and diabetic (streptozotocin treated) rats and 1 h following glucose feeding or insulin administration or both and on exposure to–10°C in young rats with and without glucose feeding. The susceptibility to frostbite was determined by exposure of the limbs to freezing mixture of–19°C or–23°C. The rate of fall of T_re was less and the survival time more in glucose and insulin plus glucose treated animals. On the other hand, the rate of fall of T_re was more and the survival time less, in dia betic and insulin-treated animals. The rectal temperature at which the animal died was the same in the control and the treated animals. The susceptibility to frost bite was more in insulin treated and diabetic animals and less in glucose-fed animals. Exposure to cold during the second h after glucose or glucose plus insulin injection did not alter the blood glucose from that obtained at room temperature. In insulin-treated animals the rate of rise of blood glucose during the second h was much higher at low temperature than at room temperature. The rise in blood glucose in diabetic animals was much higher than in normoglycemic animals exposed to cold.     

Measurements of glycolytic flux rate in brown adipocytes. Effects of insulin, noradrenaline and streptozotocin diabetes

Glycolytic flux was estimated in brown adipocytes by [3-3H]-glucose detritiation. Without insulin the process was slightly stimulated by noradrenaline or palmitate. Insulin stimulated glucose detritiation by 4-fold. Noradrenaline stimulated the process in the presence of insulin and synergism between these hormones was observed. Palmitate did not stimulate glucose detritiation in the presence of insulin suggesting that the effect of noradrenaline is not secondary to stimulation of lipolysis. With insulin, cells from streptozotocin-diabetic rats showed lower rates of glucose detritiation. Extracts from these cells also had lower maximum activities of phosphofructokinase.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.     

Effect of neonatal streptozotocin and thyrotropin-releasing hormone treatments on insulin secretion in adult rats

Neonatal STZ (nSTZ) treatment results in damage of pancreatic B-cells and in parallel depletion of insulin and TRH in the rat pancreas. The injury of B-cells is followed by spontaneous regeneration but dysregulation of the insulin response to glucose persists for the rest of life. Similar disturbance in insulin secretion was observed in mice with targeted TRH gene disruption. The aim of present study was to determine the role of the absence of pancreatic TRH during the perinatal period in the nSTZ model of impaired insulin secretion. Neonatal rats were injected with STZ (90 microg/g BW i.p.) and the effect of exogenous TRH (10 ng/g BW/day s.c. during the first week of life) on in vitro functions of pancreatic islets was studied at the age 12-14 weeks. RT-PCR was used for determination of prepro-TRH mRNA in isolated islets. Plasma was assayed for glucose and insulin, and isolated islets were used for determination of insulin release in vitro. The expression of prepro-TRH mRNA was only partially reduced in the islets of adult nSTZ rats when compared to controls. nSTZ rats had normal levels of plasma glucose and insulin but the islets of nSTZ rats failed to response by increased insulin secretion to stimulation with 16.7 mmol/l glucose or 50 mmol/l KCl. Perinatal TRH treatment enhanced basal insulin secretion in vitro in nSTZ animals of both sexes and partially restored the insulin response to glucose stimulation in nSTZ females.     

Saturated fatty acids inhibit hepatic insulin action by modulating insulin receptor expression and post-receptor signalling

Free fatty acids (FFAs) are proposed to play a pathogenic role in both peripheral and hepatic insulin resistance. We have examined the effect of saturated FFA on insulin signalling (100 nM) in two hepatocyte cell lines. Fao hepatoma cells were treated with physiological concentrations of sodium palmitate (0.25 mM) (16:0) for 0.25-48 h. Palmitate decreased insulin receptor (IR) protein and mRNA expression in a dose- and time-dependent manner (35% decrease at 12 h). Palmitate also reduced insulin-stimulated IR and IRS-2 tyrosine phosphorylation, IRS-2-associated PI 3-kinase activity, and phosphorylation of Akt, p70 S6 kinase, GSK-3 and FOXO1A. Palmitate also inhibited insulin action in hepatocytes derived from wild-type IR (+/+) mice, but was ineffective in IR-deficient (-/-) cells. The effects of palmitate were reversed by triacsin C, an inhibitor of fatty acyl CoA synthases, indicating that palmitoyl CoA ester formation is critical. Neither the non-metabolized bromopalmitate alone nor the medium chain fatty acid octanoate (8:0) produced similar effects. However, the CPT-1 inhibitor (+/-)-etomoxir and bromopalmitate (in molar excess) reversed the effects of palmitate. Thus, the inhibition of insulin signalling by palmitate in hepatoma cells is dependent upon oxidation of fatty acyl-CoA species and requires intact insulin receptor expression.     

Effect of insulin on glucose metabolism in adipocytes from virgin and late-pregnant rats.

Under basal conditions (zero insulin), paraovarian adipocytes from 19-day-pregnant rats exhibited the same rates of [U-14C]glucose conversion into CO2 and total lipids as did those from age-matched virgin rats. The dose-response curves for insulin stimulation of glucose metabolism were similar in both groups: maximal response (+100% over basal values) and high sensitivity (half-maximal effect at 0.05 nM-insulin). The present results suggest that the insulin resistance in vivo that occurs during late pregnancy may involve circulating factors lost in vitro.     

Effect of aging on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipocytes.

The effect of aging on the insulin binding parameters and on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipose tissue has been investigated. Using solubilized receptors from adipocyte plasma membranes, no significant differences were identified in either affinity or receptor number in adult vs old rats. Time courses for in vitro receptor phosphorylation revealed that both the initial rate of autophosphorylation and the maximal 32P incorporation were decreased by 40% in old (24-month) animals as compared to adult (3-month) control rats. The tyrosine phosphatase activity associated with the adipocyte plasma membranes does not account for the decreased kinase activity found in old rats. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in both groups of rats. However, the kinase activity showed a decreased responsiveness to the hormone in the old rats. Double reciprocal plot analysis of receptor phosphorylation revealed that the Km for ATP was not modified. In contrast, the insulin-stimulated Vmax value was decreased by two-fold in 24-month-old rats. The decrease in Vmax does not appear to be related to an increased basal phosphorylation level on Ser/Thr residues of the C terminus of the receptor beta-subunit. Thus, we conclude that the reduced insulin receptor kinase activity in adipose tissue from old rats is due, at least in part, to a defect of the intrinsic kinase activity of the insulin receptor.     

Comparison of anti-apoptotic signalling by the insulin receptor and IGF-I receptor in preadipocytes and adipocytes

We compared the effectiveness of insulin receptor (IR) and type I insulin-like growth factor (IGF) receptor (IGFR) cytoplasmic domains in mediating anti-apoptotic effects in 3T3-L1 preadipocytes and adipocytes. We used TrkC/IR and TrkC/IGFR chimeras, stably expressed in these cells and stimulated with neurotrophin-3 (NT-3), so as to avoid interference from endogenous receptors. After 24-h serum deprivation, 10% of preadipocytes and 2% of adipocytes appeared apoptotic as determined by fluorescence-activated cell sorter (FACS) analysis of cells stained with propidium iodide (PI) and Annexin V. When NT-3 was added, the two chimeras inhibited apoptosis to the same extent by 80% in preadipocytes and 50% in adipocytes. Mutation of juxtamembrane tyrosines (IR Y960F, IGFR Y950F) abrogated these anti-apoptotic effects. Qualitatively similar results were obtained by determination of viable rather than apoptotic cells. We conclude that IR and IGFR have equal potential to inhibit apoptosis in cell backgrounds, which are normally responsive to either IGF-I or insulin.     

Intestinal motility responses to insulin and glucagon in streptozotocin diabetic rats

Myoelectrical and mechanical activities were chronically recorded by use of nichrome electrodes and miniaturized strain-gage transducers sutured on the serosa of the antrum, the duodenum, and the jejunum. In a first experiment (n = 6 rats) the early (0-6 h) and late (greater than 4 days) effects of streptozotocin (65 mg/kg i.v.) was recorded. In addition, the effect of insulin (1-5 IU/kg) and glucagon (6-200 micrograms/kg) administered intravenously were studied separately each in groups of seven normal and streptozotocin-induced diabetic-fed and fasted rats. The results indicated that within the 30 min following streptozotocin administration there was a significant stimulation of the duodenal and jejunal motility lasting 46 +/- 8 min. When diabetes was established as shown by the basal blood glucose level obtained in those rats (2.30 +/- 0.84 g/L), a progressive decrease of the frequency of the migrating myoelectric complex was observed along with a disorganization of the regular spiking activity phases without disturbing the basal electrical rhythm. Comparing with the basal level, a significant increase in the gastrointestinal motility indexes (MI) appeared both in fasted (p less than 0.01) and fed (p less than 0.05) normal animals, 13.1 +/- 1.6 min after an i.v. injection of 1 IU/kg insulin. Motor effects of glucagon were related to the dose. When used at 25 microgram/kg a disorganization of the spiking activity was observed with a stimulation of the contractile activity in the jejunum. At higher dosages, i.e., 100 micrograms/kg, it induced an immediate and significant decrease of motility at any level tested and lasting up to 20 +/- 7 min. The motility responses to both hormones were lower in diabetic than in normal rats.     

Autoantibodies to the insulin receptor activate glycogen synthase in rat adipocytes

Summary Autoantibodies to the insulin receptor mimic the effects of insulin on glycogen synthase and phosphorylase. The interaction of antibodies with adipocyte cell surface insulin receptors seems sufficient to promote stable changes in the activities of these intracellular enzymes, suggesting that internalization or processing of insulin is not important in the generation of these biological responses.     

In vitro effects of glucocorticoid on glucose transport in rat adipocytes: evidence of a post-receptor coupling defect in insulin action

The in vitro effect of glucocorticoid on insulin binding and glucose transport was studied with rat adipocytes. Isolated rat adipocytes were incubated with or without 0.70 microgram/ml (1.9 mumol) of hydrocortisone in TCM 199 medium at 37 degrees C, 5% CO2/95% air (v/v), pH 7.4, for 2, 4, and 8 h, and then fat cell insulin binding and insulin-stimulated 3-O-methylglucose transport were measured. Hydrocortisone did not affect insulin binding in terms of affinity or receptor number. Glucose transport in the absence of insulin was significantly decreased at the incubation time of 2 h and continued to decrease up to 8 h of incubation with hydrocortisone. Decreased insulin sensitivity of glucose transport (i.e., a right-ward shift of the dose response curve) was also demonstrated after 2 h incubation with hydrocortisone, and the ED50 of insulin was maximally increased at 4 h of incubation (0.53 ng/ml for treated vs. 0.22 ng/ml for control cells). Maximal insulin responsiveness was also significantly decreased in treated cells after 8 h incubation with hydrocortisone. When percent maximum glucose transport was expressed relative to receptor-bound insulin, the ED50 values of treated and control cells were 10.5 and 7.2 pg of bound insulin, per 2 X 10(5) cells, respectively. Thus, it was evident that glucocorticoid induced a post-receptor coupling defect in the signal transmission of insulin-receptor complex.     

Separate intracellular pathways for insulin receptor recycling and insulin degradation in isolated rat adipocytes

To gain insight into the sequence of events that follow endocytotic uptake of insulin receptor complexes, we have examined the interrelationship between the degradative pathway of the insulin ligand and the recycling pathway of the insulin receptor. Tris(hydroxymethyl)aminomethane and other nonamphoteric amines were found to selectively impair insulin receptor recycling while leaving the insulin-degradative pathway intact. In contrast, low concentrations of the lysosomotropic agent chloroquine markedly inhibited intracellular insulin degradation but had little or no affect on the recycling of internalized receptors. Thus, we conclude: (1) that insulin dissociates from its receptor after endocytotic uptake and both receptor and ligand follow a separate intracellular pathway; and (2) that receptor recycling and insulin degradation can be selectively inhibited by Tris and chloroquine, respectively, highlighting the potential usefulness of these agents as intracellular probes in the study of receptor-ligand metabolism.     

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.