Modulation of catecholamine release by endogenous adenosine in the rat adrenal medulla

Adenosine was shown to inhibit norepinephrine (NE) release from sympathetic nerve endings. The purpose of this study was to examine whether endogenous adenosine restrains NE and epinephrine release from the adrenal medulla. The effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl) xanthine (DPSPX), on epinephrine and NE release induced by intravenous administration of insulin in conscious rats were examined. Plasma catecholamines were measured by HPLC with an electrochemical detector. DPSPX significantly increased plasma catecholamine in both control rats and rats treated with insulin. The effect of DPSPX on plasma catecholamine was significantly greater in rats treated with insulin. Additional experiments were performed in adrenalectomized rats to investigate the contribution of the adrenal medulla to the effect of DPSPX on plasma catecholamine. The effect of DPSPX and insulin on epinephrine in adrenalectomized rats was significantly reduced compared with that of the controls. Finally, we tested whether endogenous adenosine restrains catecholamine secretion partially through inhibiting the renin-angiotensin system. The effect of DPSPX on plasma catecholamine in rats pretreated with captopril (an angiotensin-converting enzyme inhibitor) was reduced. These results demonstrate that under basal physiological conditions, endogenous adenosine tonically inhibits catecholamine secretion from the adrenal medulla, and this effect is augmented when the sympathetic system is stimulated. The effect of endogenous adenosine on catecholamine secretion from the adrenal medulla is achieved partially through the inhibitory effect of adenosine on the renin-angiotensin system.     

Uric acid may inhibit glucose-induced insulin secretion via binding to an essential arginine residue in rat pancreatic beta-cells

Uric acid (1a) suppresses basal insulin release in isolated rat pancreatic islets and inhibition of glucose-stimulated insulin secretion (GSIS) occurs right at hyperuricaemic levels (0.4 mM). Conversely, 1 mM guanidinium urate (2a) was completely ineffective, strongly suggesting that binding to an essential arginine residue triggers the inhibitory effect. A specific recognition of 1a molecule at the crucial beta-cell receptor is probably involved in the blocking glucose signal transduction.     

Inhibition of adipose S-100 protein release by insulin

The release of S-100 protein brought about in rat epididymal fat pads by 10 microM epinephrine was inhibited by about 50% in the presence of more than 8 nM insulin. The inhibitory effect of insulin was also observed in the release of S-100 protein induced by isoproterenol or adrenocorticotropin (ACTH), but not in the release induced by a high concentration (5 mM) of dibutyryl cyclic AMP. Since insulin suppressed (to about 50%) the increase in cyclic AMP content induced by epinephrine under the same conditions, it is suggested that the inhibitory mechanism is mediated by the cyclic AMP levels in adipocytes. The S-100 protein release induced by catecholamine was significantly decreased (to about 50%) in the fat pads obtained from insulin-injected rats. In contrast, in the fat pads obtained from diabetic or long-term starved rats, the S-100 protein release was greatly enhanced, showing several-fold higher levels of basal release in the absence of hormones, and S-100 protein contents in the epididymal adipose tissues of these rats were significantly lower than those of the control rats. These results suggest that the S-100 protein content in adipocytes is regulated by insulin as well as the lipolytic hormones.     

Effects of 2-deoxy-D-glucose, oligomycin and theophylline on in vitro glycerol metabolism in rat adipose tissue: response to insulin and epinephrine.

The effects of 2-deoxy-D-glucose (2DG), oligomycin and theophylline on the in vitro production and metabolism of glycerol and its response to insulin and epinephrine were studied in epididymal fat pads from fed rats. 2-DG failed to affect basal or epinephrine stimulated glycerol production but it decreased the uptake of 1-14 C-glycerol by the tissue and its conversion to glyceride-glycerol. Oligomycin also failed to affect the basal production of glycerol but it inhibited the effect of epinephrine on this parameter as well as the uptake and utilization of 1-14-C-glycerol. Theophylline enhanced the production of glycerol by the tissue and this effect was not further augmented by epinephrine. Theophyline also inhibited the uptake and utilization of 1-14C-glycerol; the most pronounced effect of theophylline was observed in the formation of 14C-fatty acids from 1-14C-glycerol in the presence of glucose. Insulin, but not epinephrine, decreased the inhibitory effect of theophylline on glycerol utilization. It is concluded that these compounds affect more intensely the ability of adipose tissue to metabolize glycerol than to release it through lipolysis. The pathway for glycerol utilization in adipose tissue appears to be more sensitive to changes in the availability of ATP than the mechanisms responsible for the release of glycerol from the tissue.     

Islet cyclic AMP levels are not lowered during alpha 2-adrenergic inhibition of insulin release

Epinephrine-induced changes in insulin release and cyclic AMP levels were measured simultaneously in isolated rat islets. Forskolin was used to enhance islet cyclic AMP levels. Forskolin (30 microM) stimulated adenylate cyclase activity 10-fold in islet homogenates and raised cyclic AMP levels 5-fold in intact islets (both at low and high glucose). Insulin release was enhanced by forskolin only at high glucose. Epinephrine (0.1 microM) inhibited glucose- and forskolin-induced insulin release to basal rates. At the same time epinephrine potentiated forskolin-elevated cyclic AMP levels. In contrast epinephrine attenuated forskolin-stimulated adenylate cyclase activity in islet homogenates. At low glucose, both alpha 2- and beta-adrenergic blockade counteracted the epinephrine potentiation, each by 50%. At high glucose the effect was mainly beta-adrenergic in nature. The actions of epinephrine in the presence of a beta-blocker were mimicked by the alpha 2-agonist clonidine. Despite the variations in cyclic AMP levels stimulated insulin release was always inhibited by activation of alpha 2-receptors. Finally, insulin release stimulated by exogenous cyclic AMP was abolished by epinephrine. These results suggest that epinephrine inhibits insulin release at a step distal to the generation of cyclic AMP.     

Interactions of mononuclear cells of peripheral blood with islet cells in vitro

Peripheral blood mononuclear cells from Type I diabetic patients health donors or Wistar rats were cultured with rat islet cells for 18 h. Then TNF content of the medium and basal and stimulated insulin release were determined. Mononuclear cells from both healthy donors and diabetic patients could inhibit the insulin release with no correlation to TNF content. Addition of lipopolysaccharide resulted in a 5-7 times increase of TNF content of the medium followed by a more pronounced inhibition of insulin release. Rat mononuclear cells inhibited the beta-cell function almost completely and initially produced large amounts of TNF. The data indicate that inhibition of insulin release by blood mononuclear cells in vitro does not reflect anti beta-cell specific cellular immunity, involves cytokines and, probably depends upon the initial properties of the cells.     

Beta-cell function improved by supplementing basal insulin secretion in mild diabetes.

Insulin supplements, predominantly as a constant basal fish insulin infusion, were given to patients with mild diabetes to reduce the overnight fasting glucose level to normal. The basal plasma human insulin levels were reduced to subnormal levels by the infusion, and the insulin response to intravenous glucose was enhanced. The beta-cell in diabetes seems to be in a vicious circle in which an impaired insulin response to glucose produces hyperglycaemia, which stresses beta-cell function, making it more inefficient. A constant basal insulin supplement to induce basal normoglycaemia may benefit beta-cell function in diabetes.     

Adenosine and the regulation of insulin secretion by isolated rat islets of Langerhans.

The effect of adenosine in insulin secretion and adenylate cyclase activity of rat islets of Langerhans was investigated. Adenosine inhibited insulin secretion stimulated by glucose, glucagon, prostaglandin E2, tolbutamine and theophylline. Adenosine decreased basal adenylate cyclase activity of the islets as well as that stimulated by glucagon prostaglandin E2 and GTP, although fluoride-stimulated activity was not affected. Neither insulin secretion nor adenylate cyclase activity of the islets was affected by adenine, AMP or ADP. The inhibitory effect of adenosine on adenylate cyclase activity was not altered by either phenoxybenzamine (alpha-adrenergic blocker) or propranolol (beta-adrenergic blocker), suggesting that the effect is not mediated through the adrenergic receptors of the islet cells. These results suggest that the intracellular concentration of adenosine in the beta-cell may play a role in regulating insulin secretion and that this effect may be mediated via alterations in the activity of adenylate cyclase in the beta-cell.     

Effects of cytochalasins B and D on alloxan inhibition of insulin release

In isolated rat islets, cytochalasin-B, which potentiates glucose-induced insulin release and inhibits hexose transport, provided immediate protection from the inhibitory effect of alloxan on glucose-induced insulin release. Cytochalasin-D, which also potentiates glucose-induced insulin release, but exerts no detectable effect on hexose transport, provided no protection from the action of alloxan. These results indicate 1) the protective action of cytochalasin-B against the effect of alloxan is mediated on the beta-cell membrane in proximity to the hexose transport site, and 2) the ability of cytochalasin-B and D to potentiate glucose-induced insulin release appears unrelated to an interaction with the hexose transport site.     

Temperature and benzyl alcohol as probes of the antilipolytic mechanism of insulin action in adipocytes.

The temperature dependence of cAMP accumulation and glycerol release in response to epinephrine and insulin in adipocytes is examined. (1) Glycerol release in the presence of epinephrine demonstrated linear Arrhenius kinetics to 41 degrees C, and above 45 degrees C glycerol release was progressively inhibited. (2) In contrast, incubation of the cells with both epinephrine and insulin resulted in glycerol release rates that were relatively temperature insensitive. (3) Calculation of the efficacy of insulin to inhibit epinephrine-stimulated glycerol release as a function of temperature yielded a biphasic response, with a distinct optimum around 41 degrees C, in a similar manner to the effects of insulin on hexose transport activation determined previously. (4) A saturating dose of insulin (40 ng/ml) was found to have no significant effect on epinephrine-stimulated intracellular cAMP over the temperature range studied. (5) Addition of benzyl alcohol (to 40 mM) resulted in substantial inhibition of basal, epinephrine stimulated, and insulin inhibited glycerol release, without affecting the magnitude of insulin inhibition. We conclude from these studies that (a) insulin inhibition of glycerol release can not be mediated directly by intracellular cAMP modulation, (b) as in the case of hexose transport activation, the signalling mechanism by the occupied insulin receptor appears to be relatively independent of the membrane lipid environment.     

The effect of massive small bowel resection (MSBR) and small intestinal bypass (JIB) in the rat on the enteroinsular axis

The effect of massive small bowel resection (MSBR) and jejuno-ileal bypass (JIB) on the enteroinsular axis in rats was compared. Glucose levels after an oral glucose tolerance test were determined in MSBR, JIB and control animals. The response of the beta-cell mass to glucose and gastric inhibitory polypeptide (GIP) was established in the same animals using the isolated perfused pancreas model. Immunocytochemical and morphological studies were performed to monitor the adaptive changes seen in the intestine of these animals. The glucose response to the oral glucose load was blunted in both test groups with the fasting GIP levels in the JIB group being elevated and the MSBR group being reduced. The response of the isolated perfused pancreas to GIP showed a marked (70%) reduction of insulin release in the JIB rats and a slight but non-significant reduction in the MSBR rats. In both groups the insulin response to glucose alone appeared normal. The area of the pancreatic islets and the percentage of the total area consisting of the four islet cell types (B, A, D, PP) were unchanged. In the intestine the GIP cells were markedly reduced in number in the jejunum of the functional intestine of the JIB rats and the jejunum from the MSBR rats. The GIP cells in the jejunum of the bypass loop did not differ from the control jejunum. The results indicate that the high basal GIP levels seen in the JIB rats were the result of GIP secreted from the blind loop. This study also confirmed the decreased sensitivity of the beta-cells to GIP after JIB while indicating that MSBR has little if any effect on the response of the beta-cell to GIP. These data presented further evidence that the high basal GIP levels were causally related to the decreased insulin response in the JIB rats.     

Dynamin is functionally coupled to insulin granule exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion.     

Chronic administration of dehydroepiandrosterone reduces pancreatic beta-cell hyperplasia and hyperinsulinemia in genetically obese Zucker rats

The Zucker obese (fa/fa) rat is a model of hypertrophic/hyperplastic obesity. These rats develop marked hyperinsulinemia, insulin resistance, and pancreatic beta-cell hyperplasia. In the present study, chronic (22 weeks) administration of the 17-ketosteroid, dehydroepiandrosterone (DHEA), to obese Zucker rats significantly decreased body weight, and retroperitoneal and parametrial fat pad weights. In addition, beta-cell hyperplasia was reduced as well as pancreatic insulin content. DHEA treatment of lean Zucker rats also reduced body weight, fat depot weight, pancreatic islet diameter, and pancreatic insulin content. These data indicate that DHEA treatment appears to inhibit insulin synthesis and beta-cell proliferation. Whether this is due to a direct effect on the pancreas or due to improvement of peripheral insulin sensitivity remains to be elucidated.     

Iodoacetamide-induced sensitization of the pancreatic β-cells to glucose stimulation

At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose concentrations, iodoacetamide exerted both an initial stimulatory and a subsequent inhibitory action. When islets were perifused with 1mm-iodoacetamide and 17mm-glucose the inhibitory action predominated after about 15min of transient stimulation. With decreasing concentrations of iodoacetamide the stimulatory phase was gradually prolonged, and with 0.003-0.1mm-iodoacetamide stimulation only was observed for 75min. Prolonged stimulation was also noted after a short pulse of iodoacetamide. Similar responses to 0.1mm-iodoacetamide were observed with islets from normal mice. With islets from ob/ob-mice the effect of 0.1mm-iodoacetamide was reproduced with 0.1mm-iodoacetate, whereas 0.1mm-acetamide had no apparent effect. Iodoacetamide increased the V(max.) of glucose-stimulated insulin release without altering the apparent K(m) for glucose. Leucine, glibenclamide or theophylline could not replace glucose in this synergistic action with iodoacetamide. Iodoacetamide rather inhibited the insulin-releasing action of theophylline. Iodoacetamide-induced potentiation of the glucose-stimulated insulin release was rapidly and reversibly inhibited by mannoheptulose, adrenaline, or calcium deficiency. The potentiating effect on insulin release was not paralleled by effects on glucose oxidation or on islet fructose 1,6-diphosphate. However, the inhibitory action of iodoacetamide might be explained by inhibition of glycolysis as evidenced by an inhibition of glucose oxidation and a rise of fructose 1,6-diphosphate. The results support our previous hypothesis that thiol reagents can stimulate insulin release by acting on relatively superficial thiol groups in the beta-cell plasma membrane. Glycolysis seems to be necessary in order for iodoacetamide to stimulate in this way.     

Pertussis toxin enhances the beta-adrenergic and blocks the alpha 2-adrenergic regulation of renin secretion in renal cortical slices

The adrenergic regulation of renin secretion was studied in renal cortical slices from control and pertussis toxin-treated rats. Pertussis toxin was used to study the role of adenylate cyclase in the control of renin release. It was observed that isoproterenol and epinephrine stimulated renin secretion and that clonidine decreased both basal and isoproterenol-stimulated renin secretion in the control group. Pertussis toxin: a) increased significantly basal renin secretion, b) displaced to the left the concentration-response curve for isoproterenol and epinephrine and magnified the response to epinephrine and c) abolished the inhibitory effect of clonidine on renin secretion. This work confirms our previous results obtained in vivo and suggests a direct effect of pertussis toxin on the cells that secrete renin.     

Ethanol decreases basal insulin secretion from HIT-T15 cells

Various epidemiological studies suggest that alcohol intake is one of the risk factors leading to type II or non-insulin-dependent diabetes mellitus (NIDDM), but the effect of alcohol on beta-cell function remains unexplored. To study the mechanism of the diabetogenic action of ethanol, we investigated the effect of ethanol on beta-cell functions using a single clonal beta-cell line, HIT-T15 cells. When HIT cells were treated with ethanol, the metabolic activity judged by MTT assay was inhibited in dose- and time dependent manners, but cytotoxicity was not observed. Ethanol also inhibited basal insulin secretion by 30% compared to the untreated control. However, glucose-stimulated insulin secretion was not impaired by ethanol although the basal insulin secretion was inhibited. These results imply that ethanol exert beta-cells to overwork in order to compensate inhibition of the basal secretion. This finding may at least in part explain the diabetogenic action of ethanol.     

Genistein, a plant-derived isoflavone, counteracts the antilipolytic action of insulin in isolated rat adipocytes

Genistein is a phytoestrogen exerting numerous biological effects. Its direct influence on adipocyte metabolism and leptin secretion was previously demonstrated. This study aimed to determine whether genistein antagonizes the antilipolytic action of insulin in rat adipocytes. Freshly isolated adipose cells were incubated for 90 min with epinephrine, epinephrine with insulin and epinephrine with a specific inhibitor of protein kinase A (H-89) at different concentrations of genistein (0, 6.25, 12.5, 25, 50 and 100 μM). Genistein failed to affect epinephrine-induced glycerol release, however, the inhibitory action of insulin on epinephrine-induced lipolysis was significantly abrogated in cells exposed to the phytoestrogen (12.5–100 μM). The increase in insulin concentration did not suppress the genistein effect. Its inhibitory influence on the antilipolytic action of insulin was accompanied by a substantial rise in cAMP in adipocytes. This rise appeared despite the presence of 10 nM insulin in the incubation medium. Further experiments, in which insulin was replaced by H-89, revealed that the antilipolytic action of protein kinase A inhibitor on epinephrine-induced lipolysis was not affected by genistein. This means that genistein counteracted the antilipolytic action of insulin due to the increase in cAMP levels and activation of protein kinase A in adipocytes. The observed attenuation of the inhibitory effect of insulin on triglyceride breakdown evoked by genistein was not related to its estrogenic activities, as evidenced in experiments employing the intracellular estrogen receptor blocker, ICI 182,780. Moreover, it was found that genistein-induced impairment of the antilipolytic action of insulin was not accompanied by changes in the proportion between fatty acids and glycerol released from adipocytes. The ability of genistein to counteract the antilipolytic action of insulin may contribute to the decreased triglyceride accumulation in adipose tissue.     

Mechanisms of action of cyclosporin A on islet alpha- and beta-cells. Effects on cAMP- and calcium-dependent pathways.

The involvement of cAMP- and calcium-dependent pathways on the inhibitory effect of CsA (0.5 micrograms/ml) on insulin and glucagon release was studied in collagenase-isolated islets. CsA suppressed by 50% the release of insulin in pertussis toxin treated islets stimulated by 20 mM D-glucose. CsA blocked glucagon and insulin release induced by 0.2 mM IBMX (80% and 50% respectively). Similarly it inhibited glucagon and insulin release induced by 1 microM A23187 (53% and 40% respectively). CsA also abolished 0.1 microM glucagon-induced insulin release and 10 ng/ml VIP-induced glucagon release (70% and 38% respectively). The glucagon response to 2 mM D-glucose and to 10 mM arginine was decreased 25% and 45% respectively by CsA. The inhibitory effect of 0.1 microM somatostatin on insulin release was significantly abolished by CsA (p less than 0.001 vs control). On the other hand 1 microM forskolin induced insulin and glucagon release was not modified by CsA. Rats treated with CsA (10 mg/kg body wt) during 10 days showed hyperglycaemia, hypoglucagonemia and higher contents of pancreatic glucagon. It is concluded that CsA affects alpha- and beta-cell function, in vivo and in vitro, acting through calcium and cAMP-dependent pathways. This latter pathway involves the Ca(2+)-calmodulin dependent phosphodiesterase and the regulatory proteins Gs and Gi.     

Prolactin, progesterone, and dexamethasone coordinately and adversely regulate glucokinase and cAMP/PDE cascades in MIN6 beta-cells

Islet cells undergo major changes in structure and function to meet the demand for increased insulin secretion during pregnancy, but the nature of the hormonal interactions and signaling events is incompletely understood. Here, we used the glucose-responsive MIN6 beta-cell line treated with prolactin (PRL), progesterone (PRG), and dexamethasone (DEX, a synthetic glucocorticoid), all elevated during late pregnancy, to study their effects on mechanisms of insulin secretion. DEX alone or combined with PRL and PRG inhibited insulin secretion in response to 16 mM glucose-stimulating concentrations. However, in the basal state (3 mM glucose), the insulin levels in response to DEX treatment were unchanged, and the three hormones together maintained higher insulin release. There were no changes of protein levels of GLUT2 or glucokinase (GK), but PRL or PRG treatment increased GK activity, whereas DEX had an inhibitory effect on GK activity. alpha-Ketoisocaproate (alpha-KIC)-stimulated insulin secretion was also reduced by DEX alone or combined with PRL and PRG, suggesting that DEX may inhibit distal steps in the insulin-exocytotic process. PRL treatment increased the concentration of intracellular cAMP in response to 16 mM glucose, suggesting a role for cAMP in potentiation of insulin secretion, whereas DEX alone or combined with PRL and PRG reduced cAMP levels by increasing phosphodiesterase (PDE) activity. These data provide evidence that PRL and to a lesser extent PRG, which increase in early pregnancy, enhance basal and glucose-stimulated insulin secretion in part by increasing GK activity and amplifying cAMP levels. Glucocorticoid, which increases throughout gestation, counteracts only glucose-stimulated insulin secretion under high glucose concentrations by dominantly inhibiting GK activity and increasing PDE activity to reduce cAMP levels. These adaptations in the beta-cell may play an important role in maintaining the basal hyperinsulinemia of pregnancy while limiting the capacity of PRL and PRG to promote glucose-stimulated insulin secretion during late gestation.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.