Effect of hyperglycemia and hyperinsulinemia on rat red blood cell insulin receptors and catecholamines: relationship with cellular ageing

Insulin receptor activity and its relationship with catecholamines in rat young, middle aged and old red blood cells were investigated in short term (4-6) weeks and long term (6-8 months) hyperglycemia and hyperinsulinemia. Loss of insulin receptor activity is linear with cellular ageing and norepinephrine and epinephrine levels increase with age together with levels of glycosylated hemoglobin in control animals and this correlation is altered in hyperglycemia and hyperinsulinemia. These results suggest that loss of insulin receptor in cellular ageing is probably part of a more generalised alteration which is possibly brought about by glycosylation.     

Modulation of insulin receptors and catecholamines in rat brain in hyperglycemia and hyperinsulinemia

Insulin receptor activity and its relationship with catecholamines and serotonin were investigated in rat whole brain membranes, synaptosomes and choroid plexus in alloxan induced short term and long term hyperglycemia and hyperinsulinemia. Insulin receptor activity was measured by [125I]insulin binding and catecholamines by high performance liquid chromatography with electrochemical detection. While choroid plexus insulin receptors modulate along with norepinephrine, dopamine and serotonin with the changes in insulin and/or plasma glucose levels, insulin receptor activity in synaptosomes and total membranes is not affected to a great extent except in long term hyperglycemia.     

Neonatal STZ model of type II diabetes mellitus in the Fischer 344 rat: characteristics and assessment of the status of the hepatic adrenergic receptors

The Fischer 344 rat was found to be extremely sensitive to the diabetogenic effects of neonatally injected streptozotocin (STZ): injection of 40-100 mg/kg STZ at 1.5 days postnatal produced in the adult graded levels of hyperglycemia in males but not the females. The optimal dose in the 1.5 day old male was 80 mg/kg: it produced hyperglycemia without affecting growth or thyroid status in the adult. The neonatally STZ-injected adult rat displayed characteristics consistent with type II diabetes: mild hyperglycemia accentuated by fasting or consumption of a high fat diet; little change in insulin levels; slight elevation in glucagon levels; no alterations in ketones. Using radioligand binding techniques to isolated rat liver plasma membranes, compared to the control state, the type II diabetic state was found to have: no effect on either alpha(2)- or beta-adrenergic receptor binding; a decrease in the major dominant alpha(1)-adrenergic receptor, reflecting a decrease in receptor numbers but not their affinity; an increase in the plasma membrane calcium transport system, potentially depleting intracellular calcium stores essential for producing an alpha(1)-adrenergic receptor response. Since the alpha(1)-adrenergic receptor-calcium effector system is critical for the actions of catecholamines in the rat, these results suggest that the liver in the type II diabetic state may be refractory to the actions of catecholamines. We propose that the diabetes-evoked decrease in the dominant adrenergic receptor-effector system through which catecholamines act may be the cellular expression of defective glucocounterregulation in the diabetic state.     

Rat brain insulin degrading enzyme in insulin and thyroid hormones imbalances

Rat brain insulin degrading enzyme activity and its relationship with insulin receptor were investigated in experimental hyperglycemia, hyperinsulinemia, hypothyroidism and hyperthyroidism. Insulin degrading enzyme activity was assessed in synaptosomes and high speed cytosol using [125I]insulin. Levels of insulin degrading enzyme were changed in high speed cytosol in insulin and thyroid hormone imbalances. These results suggest that insulin degrading enzyme in brain is predominantly active in cytosol and is subject to regulation by insulin and thyroid hormones. Probably it plays some role in long term effects of insulin in brain.     

Modulation of insulin receptors and catecholamines in rat brain in hyperthyroidism and hypothyroidism

Insulin receptor activity and its relationship with catecholamines and serotonin were investigated in rat brain using Triton X-100 extracts from total membranes, synaptosomes and choroid plexus in experimental hypothyroidism and hyperthyroidism. Insulin receptor activity was assessed by binding to [125I]insulin and catecholamines by high performance liquid chromatography. In choroid plexus thyronines effects are well pronounced and there is modulation vis a vis plasma hormone concentrations. Triiodothyronine levels increase in brain in all experimental groups. This suggests that rat may serve as a useful model for thyronine homeostasis in brain and there may be involvement of very complex regulatory mechanisms in glucose tolerance.     

Evaluation of factors responsible for the inability of insulin to antagonize lipolysis due to high concentrations of catecholamines

Previous studies using rat adipocytes have shown that the ability of insulin to antagonize lipolysis induced by physiological concentrations of catecholamines is diminished at high concentrations of these hormones. Since such high concentrations of catecholamines cause an accumulation of free fatty acids, a decrease in cellular ATP level and a ‘short lived’ increase in cAMP (that is many fold higher than required to activate lipolysis maximally), we studied which of these modulates the antilipolytic activity of insulin. We found that inhibition of adenylate cyclase by virazole (2 mM), which lowers the initial cyclic AMP burst by about 70%, enables insulin to antagonize lipolysis at high isoproterenol concentrations. In contrast, reduction of cellular ATP level by 40% and 70%, using cyanide ion, or increasing free fatty acids in the medium to a level that suppresses the effects of insulin on glucose metabolism, failed to compromise the antilipolytic activity of the hormone. These data indicate that the inability of insulin to antagonize lipolysis induced by high isoproterenol concentrations is the direct consequence of the initial, larger burst of cyclic AMP.     

Loss of growth hormone-dependent characteristics of rat hepatocytes in culture

Summary The liver of rodents is sexually differentiated, i.e. the female liver differs from the male liver. This differentiation is largely controlled by the pattern of growth hormone (GH) secretion. We have attempted to maintain GH-dependent differentiation of cultured rat hepatocytes. We examined the level of alcohol dehydrogenase (ADH) activity, which responds to GH and is higher in female than in male liver, and the estrogen receptor, which is dependent on GH but is present in equal amounts in males and females. ADH activity was maintained in cells from male rats, but fell by 40% in cells from females in medium supplemented with insulin and dexamethasone. The estrogen receptor content of female cells fell dramatically to undetectable levels within 2 d of culture. Extensive supplementation of the medium failed to prevent the decrease in ADH activity in female cells; similarly, the addition of female sex steroids; rat serum; pituitary extracts; rat, human, or bovine GH; or ovine prolactin failed to maintain the enzyme activity. Insulin, dexamethasone, thyroid hormone plus GH or prolactin, or the combination of all five hormones also failed to prevent the loss of estrogen receptors. Short-term cultures of rat hepatocytes, although retaining the liver-specific expression of ADH at the male level, lose GH-dependent expression of the estrogen receptor and stimulation of ADH activity. Supported by grants AA 00081 and AA 06434 from the National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD.     

Hormonal control of the adenyl cyclase activity of adipose cell membranes prepared from badger, rabbit, fox and rat adipose tissues]

1. We have shown differences in hormonal regulation of adenylate cyclase activity in fat cell ghosts prepared from rat, rabbit, fox and badger adipose tissue, under the influence of catecholamines, ACTH and insulin. a) In the rat, catecholamines induced a large stimulation (+315%) of adenylate cyclase. b) In the rabbit, ACTH was the most effective hormone. c) In the fox and the badger, only catecholamines could stimulate adenylate cyclase. d) In both rat and rabbit, insulin did not reduce spontaneous enzymatic activity. Moreover, the activation of adenylate cyclase by ACTH in the rabbit was not altered by insulin, while in the rat, this hormone slightly decreased epinephrine stimulation. 2. Hormonal regulation of adenylate cyclase correlated with the lipolytic response.     

Tradition of basic and applied pigment cell research in Marseilles.

Melanogenesis, thyroid hormones and catecholamines synthesis share the same amino acid, free L-tyrosine for melanogenesis and catecholamines synthesis, L-tyrosine residues for thyroid hormones synthesis. The author describes basic and applied research on Melanogenesis and thyroid hormones metabolism in Biochemistry laboratory, School of Medicine, Marseilles since his arrival in 1962. He gives a short review of his participation in these two areas during his career in Marseilles, Yaoundé and Boston.     

Proteolytic processing of the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation

This report describes the use of an antibody directed against the carboxyl terminus of the insulin receptor beta subunit to assess the fate of the insulin receptor protein over the time course of insulin-induced receptor down-regulation. The insulin receptor beta subunit is lost from the cellular membranes of insulin-treated 3T3-C2 fibroblasts with a time course superimposable with the insulin-induced loss of cellular insulin binding activity. Concomitant with the time-dependent loss of the intact beta subunit from the membranes, a 61,000-Da fragment of the insulin receptor beta subunit accumulates in the cytosol of the cells in a time-dependent manner. The insulin-induced loss of the intact beta subunit from the cellular membranes is inhibited by cycloheximide. Chloroquine and the thiol protease inhibitors leupeptin and E-64 inhibit the insulin-induced loss of the intact beta subunit from the membranes and induce an accumulation of the intact subunit in the membranes. However, in the presence of leupeptin, E-64, or chloroquine, the insulin-induced loss of insulin binding activity occurs normally. These data indicate that down-regulation results in the loss of the intact beta subunit from the cellular membranes with the production of a fragment of the beta subunit in the cytosol. The protease responsible for the generation of the fragment is a thiol protease which requires acidic conditions. Since the insulin-induced proteolysis of the beta subunit can be totally inhibited under conditions where the insulin-induced loss of insulin binding activity proceeds normally, the proteolysis of the beta subunit is a process which is separate and distinguishable from the insulin-induced loss of insulin binding activity.     

Circadian oscillations of thyroid hormones, insulin and glucagon in the blood of laboratory rats in the course of the year

The concentration of thyroid hormones and insulin in the serum that of glucagon in the plasma and glucose in the blood was determined at 3-hour intervals, in the course of one day, at different times of the year, in adult male rats (Wistar strain) of a conventional breed kept under standard conditions with a 12:12 h light:dark regimen. The lowest thyroid hormone and glucagon concentrations and the highest insulin and blood glucose levels were found in the winter. In various seasons, circadian oscillations of the thyroid hormones culminated in the dark part of the day and that of glucagon in the light part, with the exception of the autumn. Circadian oscillation of insulin levels culminated at different times of day during the year. The pronounced changes found in the examined hormones in the laboratory rat at various times of the year are evidently the outcome of adaptation to changes in external environmental conditions during phylogenesis. In the polarity of changes in these indicators between the winter and the summer or the spring, the laboratory rat bears the closest resemblance to wild mammals.     

Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) regulation of insulin receptor function: phosphorylation of insulin receptor with A kinase decreases the insulin binding activity.

The effect of phosphorylation of insulin receptor with adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) on its insulin binding activity was investigated by using insulin receptors prepared from rat liver in vitro. A 95 KDa protein was phosphorylated by stimulation of insulin receptor kinase. This protein was also phosphorylated by A kinase. Analysis of phosphoamino acid showed that tyrosine residue(s) was phosphorylated by activation of insulin receptor kinase, whereas phosphoserine and phosphothreonine were dominantly generated by activation of A kinase. [125I] Iodoinsulin binding activity was decreased by prior phosphorylation of the receptor with A kinase. Scatchard analysis showed that the affinity for insulin was decreased by the phosphorylation with A kinase. Although the maximal activity of insulin receptor kinase was not affected by phosphorylation with A kinase, the insulin concentration which induced half maximal activity (ED50) of the receptor kinase was increased by the phosphorylation with A kinase. These results suggested that counter regulatory hormones whose actions are mediated by the generation of adenosine 3',5'-cyclic monophosphate regulate the insulin binding to the alpha subunit through phosphorylation of the beta subunit of insulin receptor.     

Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vitro.

Catecholamine treatment of isolated rat adipocytes decreases insulin binding and inhibits insulin stimulation of the glucose-transport system. There is increasing evidence that the insulin signal is transmitted after insulin is bound to the receptor via a tyrosine kinase, which is an intrinsic part of the receptor. To find whether the receptor kinase is modified by catecholamines, we solubilized and partially purified the insulin receptor of isoprenaline-treated adipocytes and studied the effect of insulin on its kinase activity. (1) Insulin increased the tyrosine autophosphorylation of the insulin receptor kinase from catecholamine-treated cells only 4-fold, compared with a 12-fold stimulation in control cells. (2) The rate of insulin-stimulated 32P incorporation into the receptor of isoprenaline-treated cells at non-saturating [32P]ATP concentrations (5 muM) was decreased to 5-8% of the values for receptor from control cells. (3) 125I-insulin binding to the partially purified receptor from catecholamine-treated cells was also markedly decreased. The insulin receptor from catecholamine treated cells bound 25-50% of the amount of insulin bound by the receptor from control cells at insulin concentrations of 10 pM-0.1 muM. Part of the impaired insulin-responsiveness of the receptor kinase of catecholamine-treated cells is therefore explained by impaired binding properties; however, an additional inhibition of the kinase activity of the insulin receptor from catecholamine-treated cells is evident. (4) This inhibition of kinase activity decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis revealed that the Km for ATP of the receptor kinase from isoprenaline-treated cells was increased to approx. 100 muM, compared with approx. 25 muM for receptor of control cells. (5) We conclude from the data that catecholamine treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of the mechanism leading to insulin-resistance in these cells.     

Lipase Inhibition and Hormonal Status,Body Composition and Gastrointestinal Processing of a Liquid High-Fat Mixed Meal in Moderately Obese Subjects

DRENT, MADELEINE L, CORRIE POPP-SNIJDERS, HERMAN J ADER, JAN BMJ JANSEN AND EDUARD A VAN DER VEEN. Lipase inhibition and hormonal status, body composition and gastrointestinal processing of a liquid high-fat mixed meal in moderately obese subjects. Obes Res. The effect of Orlistat, a lipase inhibitor used in the treatment of obesity was studied on gastrointestinal transit time, on body composition and on hormones known to be influenced by the degree of hydrolysis of nutritional triglycerides or by reduced nutrient intake and absorption. After a placebo run-in period 14 patients were randomized to a 12-week treatment period on Orlistat 360 mg per day (mean body weight 93.1 ± 9.8 kg) or placebo (mean body weight 90.7 ± 10.5 kg). At randomization and after 12 weeks body weight, body composition, thyroid hormones, catecholamines, insulin-like growth factor I (IGF-I) and IGF-binding protein 3 were measured. During 4 hours after consumption of a liquid fat-rich mixed meal containing study medication, 15 g lactulose and 25 g xylose, blood levels of glucose, insulin, c-peptide, glucagon, triglycerides, free fatty acids, cholecys-tokinin, pancreatic polypeptide and xylose and expiration air levels of hydrogen were measured. Weight loss was 4.2 ± 3.5 kg in the Orlistat group versus 3.0 ± 1.9 kg in the placebo group. Fat mass decreased to an equal degree, whereas lean body mass remained stable. No differences were found for thyroid hormones, catecholamines, IGF-I and IGFBP-3 levels. By comparing the areas under the curve (AUC) and the peak levels at randomization (acute effects) of insulin and c-peptide a tendency was found to be increased in the Orlistat group, whereas those of xylose were increased significantly, suggesting faster gastric empyting after Orlistat. No differences were found in the other parameters. By comparing the changes in responses (longer term effects) no significant differences were found. In conclusion, the presence in the gut of undigested and unabsorbed fat does not seem to have a relevant influence on hormonal status and body composition in a small group of moderately obese patients.     

Effects of dysthyroidism on central catecholaminergic neurons

After 15 years of research, it is clear that alterations in thyroidal status affect catecholaminergic neurons in the developing as well as in the adult brain. Experiments on fetal catecholaminergic brain areas grafted into the anterior eye chamber of adult thyroidectomized rat have shown the thyroid hormone dependency of the morphological differentiation of catecholaminergic neurons originating from the substantia nigra and the locus coeruleus. Furthermore, thyroid hormones also affect the metabolism of catecholaminergic neurons. Neonatal hypothyroidism induced either by ¹³¹I or by an antithyroid drug decreases the concentration of dopamine, noradrenaline and the activity of tyrosine hydroxylase at least in whole brain studies. Treatments with l-thyroxine of neonatally thyroidectomized rats reverse these neurochemical changes in a both time and dose dependent manner. These presynaptic modifications are associated with a decrease in the number of catecholaminergic receptors in different brain areas. On the opposite, experimental neonatal hyperthyroidism induced by daily administration of l-triiodothyronine increases the synthesis as well as the utilization of catecholamines. These changes are also associated with an alteration of catecholaminergic receptors. Despite numerous studies, there is, so far, no clear conclusion on the effects of neonatal dysthyroidism on the development of each catecholaminergic group. However, from these studies, it appears that the intensity of neonatal dysthyroidism greatly varies, depending of the monoamine and the brain area studied. The utilization of fetal brain cell cultures growing in a chemically defined medium has permitted to demonstrate the direct effect of thyroid hormones on fetal brain cells and the morphological effects of triiodothyronine on the size and the neurite length and arborization of fetal hypothalamic dopaminergic neurons.In the adult brain, hypothyroidism induced by surgical thyroidectomy, decreases the rate of catecholamines synthesis, decreases the number of alpha noradrenergic receptors and has no effect on striatal dopaminergic receptors. In contrast, hyperthyroidism increases the rate of catecholamines synthesis and induced an hypersensitivity of noradrenergic receptors. The intensity of the effects of dysthyroidism seems to be dependent on the monoamine and the brain area studied.In conclusion, it can be proposed that in the neonate thyroid hormones act on CA neuron activity mostly through a morphogenetic effect whereas in the adulthood they directly affect CA metabolism.     

Mechanisms altered beta-adrenergic responsiveness in the hyperthyroid and hypothyroid turkey erythrocyte

Studies on the relationship between thyroid hormone and the beta-adrenergic catecholamines have been carried out in the turkey erythrocyte. Conditions of thyroid hormone excess and deficiency were examined with respect to their effects on the beta receptor itself, as well as to their effects on associated biochemical and physiological indices of beta receptor function, including agonist stimulated adenylate cyclase activity, cellular cyclic AMP generation, and catecholamine-induced stimulation of potassium ion influx. Erythrocytes obtained from hypothyroid turkeys showed a marked (approximately 50%) reduction in beta receptor number without any change in receptor affinity for agonists or antagonists. Catecholamine-sensitive adenylate cyclase activity and cellular cyclic AMP levels were similarly reduced. The sensitivity of these cells to agonist-stimulated potassium influx was significantly decreased, but maximal agonist-stimulated transport rate was unchanged. Analysis of the quantitative relationship between beta receptor number, agonist concentration, and level of catecholamine-stimulated potassium influx indicates that, at any given absolute level of receptor occupancy, the level of agonist-stimulated potassium influx is identical in hypothyroid and normal erythrocytes, and that the diminished physiological sensitivity of the hypothyroid cell is attributable in its entirety to a reduction in beta receptor number per se. The results obtained in the hyperthyroid turkey erythrocyte were strikingly different. Here, beta receptor number, binding affinity for agonists and antagonists, catecholamine-sensitive adenylate cyclase activity, and maximal cyclic AMP levels were all unchanged. In contrast, maximal agonist-stimulated potassium ion transport was markedly reduced, while the concentration of isoproterenol required for half-maximal stimulation was only slightly increased. Analysis of the relationship between beta receptor number, agonist concentration, and catecholamine-stimulated potassium influx rate indicates that, at all absolute levels of beta receptor occupancy, the stimulation of monovalent cation influx is markedly blunted in the hyperthyroid cell. In contrast to the findings in the hypothyroid cell, where decreased physiologic sensitivity to catecholamines is directly attributable to a reduction in beta receptor number, the primary abnormality responsible for diminished catecholamine responsiveness in the hyperthyroid cell would appear to be located at a point "distal" to the beta receptor itself.     

Unidirectional actions of insulin and Ca2+-dependent hormones on adipocyte pyruvate dehydrogenase

Norepinephrine and epinephrine, in the presence of the beta-adrenergic antagonist propranolol (10(-5) M), stimulated adipocyte pyruvate dehydrogenase at low concentrations but inhibited the enzyme at higher concentrations. The alpha-adrenergic agonist, phenylephrine, rapidly stimulated pyruvate dehydrogenase activity in a dose-dependent manner with maximal stimulation observed at 10(-6) M. The stimulation of pyruvate dehydrogenase by phenylephrine was mediated via alpha 1-receptors. Inhibition of pyruvate dehydrogenase by catecholamines was mediated via beta-adrenergic receptors, since the beta-agonist, isoproterenol, and dibutyryl cAMP produced similar effects. Like insulin, alpha-adrenergic agonists increased the active form of pyruvate dehydrogenase without changing the total enzyme activity and cellular ATP concentration. The effects induced by maximally effective concentrations of insulin and alpha-adrenergic agonists were nonadditive. The ability of phenylephrine and methoxamine to stimulate pyruvate dehydrogenase and phosphorylase and to inhibit glycogen synthase was not affected by the removal of extracellular Ca2+. Similarly, the stimulation of pyruvate dehydrogenase and glycogen synthase by insulin was also observed under the same conditions. However, when intracellular adipocyte Ca2+ was depleted by incubating cells in a Ca2+-free buffer containing 1 mM ethylene glycol bis(beta-amino-ethyl ether)-N,N,N' -tetraacetic acid, the actions of alpha-adrenergic agonists, but not insulin, on pyruvate dehydrogenase were completely abolished. Vasopressin and angiotensin II also stimulated pyruvate dehydrogenase in a dose-dependent manner with enhancement of glucose oxidation and lipogenesis. Our results demonstrate that the Ca2+ -dependent hormones stimulate pyruvate dehydrogenase and lipogenesis in isolated rat adipocytes, and the action is dependent upon intracellular, but not extracellular, Ca2+.