Effect of insulin on glucagon binding to isolated rat epididymal adipocytes

Effect of insulin on glucagon binding to rat epididymal adipocytes was studied in vitro. [125I]iodoglucagon binding to isolated adipocytes was increased by preincubation of the cells with insulin. Maximal increase was observed with 7 X 10(-10) M insulin. In Scatchard analysis, [125I]iodoglucagon competition data generated one binding site with a single affinity for glucagon binding in the cells pretreated with buffer alone. Pretreatment of the cells with insulin increased the affinity without changes in the number of binding sites. [125I]iodoglucagon binding to isolated adipocytes was not affected by pretreatment of the cells with luteinizing hormone, follicle-stimulating hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon binding to adipocytes.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

Metabolic integrity and stability of isolated rat brown adipocytes

Using rats previously anesthetized with pentobarbital and with subtle modifications to existing procedures, rat brown adipocytes of good yield, viability, and stability were consistently obtained. The metabolic integrity of the cells, indicated by an 8–11 fold norepinephrine-dependent stimulation of respiration, was reproducible between cell preparations and maintained even after 3–5 hours of cell storage at room temperature. The ATP content of freshly isolated and stored cells per mg DNA (using a modified DNA assay) closely reflected the in vivo content determined from freeze clamped tissue.     

Adrenergic modulation of insulin and glucagon secretion from the isolated perfused rat pancreas

In order to observe the effect of the adrenergic system on pancreatic glucagon secretion in the isolated perfused rat pancreas, phenylephrine, an alpha-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, were added to the perfused solution. 1.2 microM phenylephrine suppressed glucagon secretion at 2.8 mM glucose, and it also decreased insulin secretion at 11.1 mM glucose. 240 nM isoproterenol enhanced glucagon secretion not only at 2.8 mM glucose, but also at 11.1 mM glucose, as well as insulin secretion at 11.1 mM. In order to study the role of intra-islet noradrenalin, phentolamine, an alpha-adrenergic antagonist, and propranolol, a beta-adrenergic antagonist, were infused with the perfused solution. 10 and 100 microM phentolamine caused an increase in insulin secretion, and 25 microM propranolol decreased insulin secretion, while they did not cause any change in glucagon secretion. From these results, it can be concluded that alpha-stimulation suppresses not only insulin but also glucagon secretion, while beta-stimulation stimulates glucagon secretion, as well as insulin secretion. Intra-islet catecholamine may have some effect on the B cell, whereas it seems to have no influence on the A cell.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Inhibitory effect of insulin on prostacyclin production by isolated rat adipocytes

Physiologic concentrations of insulin completely inhibited the norepinephrine-induced increment in the production of 6-keto-prostaglandin (PG) F_1α, the stable derivative of prostacyclin (PGI₂), by isolated rat adipocytes. The inhibition of PGI₂ production by insulin in isolated rat adipocytes supports the view that the elevated plasma level of 6-keto-PGF_1α in rats with non-ketotic diabetes mellitus and diabetic ketoacidosis is derived at least in part from production of PGI₂ by the adipocyte cell mass.     

Stimulation by prostaglandin E2 of glucagon and insulin release from isolated rat pancreas.

To ascertain whether prostaglandins (PG) may play a role in the secretion of glucagon and in an attempt to elucidate the conflicting observations on the effects of PG on insulin release, the isolated intact rat pancreas was perfused with solutions containing 1.1 x 10(-9) to 1.8 x 10(-5)m PGE2. In the presence of 5.6 mM glucose significant increments in portal venous effluent levels of glucagon and insulin were observed in response to minimal concentrations of 2.8 X 10(-8) and 1.4 X 10(-7) PGE2, respectively; a dose-response relationship was evident for both hormones at higher concentrations of PGE2. When administered over 60 seconds, 1.4 X 10(-6)M PGE2 resulted in a significant increase in glucagon levels within 24 seconds and in insulin within 48 seconds. Ten-minute perfusions of 1.4 X 10(-6)M PGE2 elicited biphasic release of both islet hormones; Phase I glucagon release preceded that of insulin. Both phases of the biphasic glucagon and insulin release which occurred in response to 15-minute perfusions of 10 mM arginine were augmented by PGE2. These observations indicate that PGE2 can evoke glucagon and insulin release at concentrations close to those observed by others in the extracts of rat pancreas. We conclude that PG may be involved in the regulation of secretion of glucagon and insulin and may mediate and/or modify the pancreatic islet hormone response to other secretagogues.     

Epoxyeicosatrienoic acids stimulate glucagon and insulin release from isolated rat pancreatic islets

Metapyrone and eicosatetraynoic acid but not indomethacin are effective inhibitors of the secretory response of isolated rat pancreatic islets to arginine and glucose. Epoxyeicosatrienoic acids, products of the cytochrome P-450-NADPH dependent arachidonic acid epoxygenase activity, are potent and selective mediators for the in vitro release of either insulin or glucagon from preparations of isolated rat pancreatic islets.     

Phosphorylation of carnitine palmitoyltransferase and activation by glucagon in isolated rat hepatocytes

Effects of glucagon and forskolin on the phosphorylation and changes of activity of carnitine palmitoyltransferase (CPT) have been studied in isolated rat hepatocytes using anti-CPT immunoglobulin. When the activity was determined in lysed hepatocytes after glucagon or forskolin treatment, it was found to be stimulated 30-80% mainly through increased affinity for palmitoyl-CoA. By SDS electrophoresis of the immunoprecipitates, CPT subunit (Mr 69000) was noted to be phosphorylated 4-5-fold with glucagon (1.2 X 10(-7) M) and forskolin (0.1 mM) over control. These results indicate that hepatic ketogenesis is regulated with glucagon by phosphorylation of CPT through cAMP-dependent protein kinase.     

Adrenalectomy-induced alterations in glucagon binding and lipolysis in isolated rat adipocytes.

Adipocytes from adrenalectomized rats nearly lost their lipolytic response to glucagon concomitant with a 90% decrease in the number of glucagon receptors per cell. Quantitative analysis of the relation between amount of cell-bound glucagon and hormone-stimulated lipolysis revealed that the ability of the remaining 10% of glucagon receptors to induce lipolysis was not impaired. Binding of the beta-adrenergic antagonist [3H]dihydroalprenolol and maximal lipolysis induced by (-)-isoproterenol, (Bu)2cAMP, 3-isobutyl-1-methylxanthine, and adenosine deaminase were reduced only 10 to 20% after adrenalectomy. Furthermore, glucagon-stimulated cAMP production was greatly decreased in adrenalectomized animals, but isoproterenol-stimulated cAMP production was not. Hydrocortisone replacement in adrenalectomized rats only partially prevented the loss of glucagon receptors and glucagon effects on both cAMP production and lipolysis. These findings suggest that lipolytic cascade distal to hormone receptors was not greatly impaired in adipocytes after adrenalectomy and that the unresponsiveness of these cells to glucagon was mostly due to a marked reduction in the number of glucagon receptors.     

Sulphonylureas-induced increase in insulin binding and glucose metabolism by isolated rat adipocytes

The effects of oral hypoglycaemic drugs, SPC-703 (n-/p-toluenesulphonyl/-5-methyl-2-pirazoline-1-carbonami de) and tolbutamide on insulin binding and glucose metabolism by isolated adipocytes were studied. After 10 days of administration of both sulphonylurea derivatives, no differences were observed in insulin concentration between both experimental and the control groups of animals, despite a significant fall in blood glucose level. SPC-703 and tolbutamide in concentrations of 1 mM added in vitro to the suspension of adipocytes had no effect on insulin binding or on basal and insulin simulated glucose metabolism. Daily administration of 300 mg/kg body weight of SPC-703 or tolbutamide for 10 days resulted in 48% and 34% increase of specific binding of insulin by adipocytes, respectively. From the Scatchard plot analysis we noted that the increase of binding resulted from increased affinity of insulin receptors for hormone. Simultaneous increase in basal and insulin stimulated glucose metabolism by adipocytes, as measured by 14CO2 production and 14C incorporation into cellular lipids, was observed. The results indicate that hypoglycaemic action of sulphonylureas may be explained by increased affinity of insulin receptors and the stimulating action of these compounds on peripheral glucose metabolism.     

Modulation of noradrenaline-induced vasoconstriction in isolated perfused mesenteric arterial beds from obese Zucker rats in the presence and absence of insulin

The genetically obese Zucker rat (fa/fa) is an insulin-resistant animal model with early-onset severe hyperinsulinemia that eventually develops mild hypertension. Thus, it represents a model in which the effect of hyperinsulinemia - insulin resistance associated with hypertension on vascular reactivity can be examined. The purpose of this study was to investigate the contribution of endogenous nitric oxide (NO) and prostaglandins to reactivity to noradrenaline (NA) in the presence and absence of insulin in mesenteric arterial beds (MAB) from 25-week-old obese Zucker rats and their lean, gender-matched littermates. In the absence of insulin, bolus injection of NA (0.9-90 nmol) produced a dose-dependent increase in perfusion pressure in MAB from both lean and obese rats. Although there was no significant difference in NA pD2 (-log ED50) values, the maximum response of MAB from obese rats to NA was slightly but significantly reduced compared with that of MAB from lean rats. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 300 microM) enhanced and indomethacin (20 microM) inhibited pressor responses to NA in MAB from both obese and lean rats. Perfusion with insulin (200 mU/L, a level similar to that in obese rats in vivo) potentiated only the responses of the obese MAB to the two lowest doses of NA tested (0.9 and 3 nmol). In the presence of L-NMMA, insulin further potentiated the NA response in MAB from obese rats. Indomethacin, the prostaglandin H2/thromboxane A2 receptor antagonist SQ 29548 (0.3 microM), and the nonselective endothelin-1 (ET-1) receptor antagonist bosentan (3 microM) all abolished insulin potentiation of the NA response in obese MAB. These data suggest that concurrent release of NO and vasoconstrictor cyclooxygenase product(s) in MAB from both obese and lean Zucker rats normally regulates NA-induced vasoconstrictor responses. Furthermore, insulin increases the release of contracting cyclooxygenase product(s) and enhances reactivity to low doses of NA in MAB from obese rats. The effects of insulin may be partially mediated by ET-1 via ET receptors and are buffered to some extent by concomitant NO release. This altered action of insulin may play a role in hypertension in this hyperinsulinemic - insulin-resistant model.     

Binding and action of glucagon in isolated adipocytes from cortisol-treated rats

Evidence for pre-receptor, receptor and post-receptor glucagon defects was investigated in adipocytes from cortisol-treated rats. A decrease in glucagon binding due to a decreased number of receptors was observed. No changes in receptor affinity were detected. Both, the lipolytic response of glucagon and the ability of glucagon to increase basal and theophylline-stimulated cAMP accumulation remained unaltered. Moreover, a hyperglucagonemia accompanied by an increase in glucagon degradation in the serum of cortisol-treated rats was observed. Such alterations could represent a new mechanism by which glucocorticoids exert their biological actions.     

Microcalorimetric measurements of heat production in isolated rat brown adipocytes.

Heat production, oxygen consumption, and lipolysis in isolated interscapular brown adipocytes from the rat were investigated. Epinephrine, norepinephrine, and isoproterenol increased heat production in a concentration-dependent manner, showing, about 6-, 4-, and 5-fold higher effects than controls, respectively. The concentration of isoproterenol for threshold heat production and glycerol release were 10(-10) M and 10(-9) M, respectively. The fact that 10(-9) M isoproterenol increased heat production by about 3-fold while glycerol release had no effect at all indicates that calorimetry is more appropriate for investigation of brown adipocytes. At least the method is more sensitive than that of measuring glycerol release.     

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.     

Effects of neuromedin B on insulin and glucagon release from the isolated perfused rat pancreas

The effect of neuromedin B (NMB) on insulin and glucagon release was studied in isolated perfused rat pancreas. Infusion of NMB (10 nM, 100 nM and 1 microM) did not affect the insulin release under the perusate conditions of 5.5 mM glucose plus 10 mM arginine and 11 mM glucose plus 10 mM arginine, although 10 nM NMB tended to slightly suppress it under the perfusate condition of 5.5 mM glucose alone. The degree of stimulation of insulin release provoked by the addition of 5.5 mM glucose to the perfusate was not affected by the presence of 10 nM NMB. The glucagon release was slightly stimulated by the infusion of 100 nM and 1 microM NMB but not by 10 nM NMB under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The effect of C-terminal decapeptide of gastrin releasing peptide (GRP-10) was also examined and similar results were obtained; 10 nM and 100 nM GRP-10 did not affect insulin release and 100 nM GRP-10 stimulated glucagon release under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The present results concerning glucagon release are consistent with the previous results obtained with isolated perfused canine and porcine pancreas. However, the results regarding insulin release are not. Species differences in insulin release are also evident with other neuropeptides such as substance P and the mechanism of such differences remains for be clarified.     

Modulation of insulin and glucagon secretion from the perfused rat pancreas by the neurohypophysial hormones and by desamino-D-arginine vasopressin (DDAVP)

Marked stimulation of glucagon release and modest stimulation of insulin release were observed during in situ perfusion of the rat pancreas with AVP or OT. Glucagon release in response to AVP or OT (200 pg/ml) gradually increased over a 45 min perfusion period reaching maxima of 500% and 300% of the pre-stimulatory levels, respectively. Insulin release transiently increased by 100%. In each case release rates returned to control values immediately after withdrawal of the peptides. Total glucagon release was concentration dependent and linear from 20 pg to 20 ng AVP or OT/ml (r greater than .97). Pancreatic response to DDAVP perfused at 20 ng/ml was virtually indistinguishable from that induced by AVP at 200 pg/ml. This demonstration of a glucagonotrophic action of the neurohypophysial hormones in the in situ perfused rat pancreas confirms earlier studies using isolated islets and bolus IV injection.     

Stimulation by prostaglandin E2 of glucagon and insulin release from isolated rat pancreas

To ascertain whether prostaglandins (PG) may play a role in the secretion of glucagon and in an attempt to elucidate the conflicting observations on the effects of PG on insulin release, the isolated intact rat pancreas was perfused with solutions containing 1.1 × 10⁻⁹ to 1.8 × 10⁻⁵M PGE₂. In the presence of 5.6 mM glucose significant increments in portal venous effluent levels of glucagon and insulin were observed in response to minimal concentrations of 2.8 × 10⁻⁸ and 1.4 × 10⁻⁷M PGE₂, respectively; a dose-response relationship was evident for both hormones at higher concentrations of PGE₂. When administered over 60 seconds, 1.4 × 10⁻⁶M PGE₂ resulted in a significant increase in glucagon levels within 24 seconds and in insulin within 48 seconds. Ten-minute perfusions of 1.4 × 10⁻⁶M PGE₂ elicited biphasic release of both islet hormones; Phase I glucagon release preceded that of insulin. Both phases of the biphasic glucagon and insulin release which occurred in response to 15-minute perfusions of 10 mM arginine were augmented by PGE₂. These observations indicate that PGE₂ can evoke glucagon and insulin release at concentrations close to those observed by others in the extracts of rat pancreas. We conclude that PG may be involved in the regulation of secretion of glucagon and insulin and may mediate and/or modify the pancreatic islet hormone response to other secretagogues.