Pituitary adenylate cyclase-activating polypeptide (PACAP): occurrence in rodent pancreas and effects on insulin and glucagon secretion in the mouse

Summary Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that occurs in several tissues, e.g., in the gut. We have studied PACAP-like immunoreactivity in the pancreas of rat and mouse, and the effects of PACAP-38 on basal and stimulated insulin and glucagon secretion in the mouse. Immunofluorescence staining demonstrated the presence of PACAP-like immunoreactivity in nerve fibers in both the rat and mouse pancreas. The nerve fibers were seen in the exocrine pancreas and surrounding the islets. Occasionally, the nerve fibers occurred within the islets. Most PACAP-positive nerve fibers innervated the intrapancreatic ganglia, although no nerve cell bodies contained PACAP-like immunoreactivity. In-vivo experiments in mice revealed that basal plasma glucagon levels were increased by PACAP-39 injected intravenously at dose levels exceeding 1.8 nmol/kg. Furthermore, PACAP-38 (7 nmol/kg) potentiated the plasma glucagon response to the cholinergic agonist carbachol (0.16 mol/kg). This potentiation was reduced to simple addition by pretreatment with a combined - and -adrenergic blockade by phentolamine (35 mol/kg) and propranolol (8.5 mol/kg). Moreover, PACAP-38 inhibited a carbachol-induced increase in the level of plasma insulin in the absence but not in the presence of adrenergic blockade. PACAP-38 increased basal plasma insulin levels and increased basal plasma glucose levels 6 min and 10 min, respectively, after injection of the peptide. We conclude that PACAP-like immunoreactivity exists in nerve fibers innervating the mouse and rat pancreas, particularly the intrapancreatic ganglia, and that PACAP-38 augments both basal and carbachol-stimulated glucagon secretion in the mouse.     

Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol.

Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO.     

Galanin-immunoreactive nerves in the mouse and rat pancreas

Summary Galanin-containing nerve fibers have previously been observed in the human, dog, and pig pancreas. Whether the mouse and rat pancreas also contain galanin nerve fibers has been a matter of debate. Therefore, we examined the distribution of galanin in the mouse and the rat pancreas. Further, the possible localization of galanin to adrenergic nerves was studied using sequential immunostaining for galanin and tyrosine hydroxylase (TH). In the mouse pancreas, numerous galanin-immunoreactive (GIR) nerve fibers occurred around blood vessels. They were less numerous in the exocrine parenchyma and in association with the islets. In contrast, in the rat pancreas, only a few GIR nerves were found. They were located around blood vessels and scattered in the exocrine parenchyma. Occasionally, GIR nerves were also observed in the islets. There was a dense distribution of TH-immunoreactive fibers in both the mouse and the rat pancreas. Sequential immunostaining revealed co-localization of galanin and TH immunoreactivity in nerve fibers in both the mouse and the rat pancreas. Following chemical sympathectomy using 6-hydroxydopamine (6-OHDA), not all GIR nerves disappeared. In the mouse pancreas a remaining population of galanin nerves was found around blood vessels, and occasionally in the islets. In the rat pancreas, a few GIR nerves were seen also after chemical sympathectomy. We conclude that intrapancreatic GIR nerves also occur in the mouse and the rat. These findings suggest that many of the GIR nerves are adrenergic but that non-adrenergic, possibly intrinsic or sensory GIR nerves exist as well in both the mouse and the rat pancreas.     

Lipopolysaccharides impair insulin gene expression in isolated islets of Langerhans via Toll-Like Receptor-4 and NF-κB signalling

Background

Type 2 diabetes is characterized by pancreatic β-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that the signalling cascade activated by lipopolysaccharides (LPS) binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. In this study, we tested the hypothesis that LPS alters insulin gene expression via TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in islets.

Methodology/Principal Findings

A 24-h exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of pancreas-duodenum homebox-1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Accordingly, LPS exposure also decreased glucose-induced insulin secretion. LPS repression of insulin, PDX-1 and MafA expression, as well as its inhibition of insulin secretion, were not observed in islets from TLR4-deficient mice. LPS inhibition of β-cell gene expression in rat islets was prevented by inhibition of the NF-κB pathway, but not the p38 mitogen-activated protein kinase (p38 MAPK) pathway.

Conclusions/Significance

Our findings demonstrate that LPS inhibit β-cell gene expression in a TLR4-dependent manner and via NF-κB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function.     

Studies on the acetylcholinesterase (ache)-positive and -negative autonomic axons supplying smooth muscle in the normal and 6-hydroxydopamine (6-OHDA)treated rat iris

Summary The adrenergic and cholinergic innervation to the rat iris has been studied at a light and electron microscopic level. Catecholamine fluorescence histochemistry showed adrenergic nerves to be present in both the dilatator and the constrictor pupillae regions. At a fine structural level the terminal innervation of the iris was studied and criteria for the differentiation between presumptive adrenergic and presumptive cholinergic axon terminals were examined. To aid this examination presumptive adrenergic axons were either labelled with the false adrenergic transmitter, 5-hydroxydopamine, or chemical sympathectomy performed using 6-hydroxydopamine. The value of using acetylcholinesterase staining as a marker for cholinergic nerve terminals was also studied.Results showed a mixed adrenergic/cholinergic innervation to the dilatator pupillae. In the constrictor pupillae an exclusively cholinergic innervation was found although adrenergic and cholinergic nerves were found supplying the blood vessels and at the dilatator-constrictor interface. These findings are discussed with regard to innervation-function relationships in the iris.     

EVIDENCE FOR DEGENERATION OF SYMPATHETIC NERVE TERMINALS CAUSED BY THE ORTHO- AND PARA-QUINONES OF 6-HYDROXYDOPAMINE

6-Hydroxydopamine and its corresponding ortho- and para-quinones were injected intraperitoneally into male Swiss-Webster mice. Measurements made 72 h after injection showed that all three compounds caused a decrease in the uptake of [³H]norepinephrine into slices of mouse heart tissue in vitro, a decrease in the endogenous content of heart norepine-phrine, and a disappearance of the adrenergic nerve plexus of the mouse iris as viewed by fluorescence histochemistry. These data suggest that both the ortho- and para-quinones of 6-hydroxydopamine are capable of producing a chemical sympathectomy similar to that caused by 6-hydroxydopamine.     

Combined effect of adenosine,alpha adrenergic and adenosine antagonists on serum insulin and insulin secretion from rat pancreatic islets

• 1.1. The effect of adenosine separately or in combination with alpha-1 adrenergic antagonist prazosin and alpha-2 adrenergic antagonist yohimbine as well as adenosine antagonists 8-phenyltheophylline and xanthine amine conjugate on glucose-induced insulin secretion from isolated rat pancreatic islets was studied.
• 2.2. Their in vivo effects on serum glucose and insulin levels were also investigated. Adenosine at 10 and 100 μM inhibited significantly, insulin secretion from the isolated islets whereas at 10 mM slightly increased the secretion of insulin.
• 3.3. Prazosin used at 100 μM inhibited insulin secretion. When it combined with adenosine (10 μM) it augmented the inhibitory effect of adenosine.
• 4.4. In vivo prazosin (21 mg/kg bodywt) caused a hyperglycaemia which was accompanied by hypoinsulinaemia.
• 5.5. Concurrent administration of this drug with adenosine neither affect the hyperglycaemic nor the hypoinsulinaemic effects of adenosine.
• 6.6. On the other hand, yohimbine (100 μM) has no effect neither separately nor in combination with adenosine (10 μM) in modulating the inhibitory effect of adenosine on insulin secretion.
• 7.7. When Yohimbine administered at 19.5 mg/kg body wt it did not alter serum glucose but it markedly increased the serum insulin level. Its combined administration with adenosine reduced the hyperglycaemic effect of adenosine with a remarkable increase in serum insulin.
• 8.8. Both adenosine-antagonists were ineffective in alteration of insulin secretion.
• 9.9. However, combination of 8-phenyltheophylline with adenosine (10 μM) totally blocked the inhibitory effect of adenosine on insulin secretion while xanthine amine conjugate failed to prevent this effect of adenosine.
• 10.10. These results indicate that the inhibitory effect of adenosine on insulin secretion is neither mediated via alpha-1 nor alpha-2 adrenoceptors. It might be via activation of specific adenosine receptors on rat islets which are sensitive to blockade by 8-phenyltheophylline.

     

Adrenergic control of pancreatic glucagon secretion

In order to characterize the adrenergic control of pancreatic A cell, the effect on the glucagon secretion of three sympathomimetic substances (epinephrine, isoproterenol, phenylephrine) and two adrenergic blockers (propranolol and phentolamine) have been separately examined by the isolated perfused rat pancreas. The study was performed in basal state and during glucagon hypersecretion induced by arginine or glucopenia. Epinephrine and isoproterenol infusion determined a prompt an sustained glucagon release both in the basal state and during glucagon hypersecretion. The effect of phenylephrine infusion was slight. In the presence of propranolol, glucagon secretion induced by metabolic stimulus was significantly depressed. The glucagon secretion in the same experimental conditions was insignificantly enhanced by phentolamine. Finally propranolol infusion reverse the glucagon secretion induced by phenylephrine. In conclusion the pancreatic glucagon secretion in our model of study is clearly induced by B adrenergic receptor stimulation.     

Effect of functional adrenalectomy on glucagon secretion and circulating catecholamines during insulin hypoglycemia in the dog

The present study was carried out to determine whether an increase in the pancreatic immunoreactive glucagon (IRG) secretion during the acute phase of insulin-induced hypoglycemia depends on circulating catecholamines of adrenal origin. Hypoglycemia was induced by a bolus insulin injection (0.15 IU/kg, i.v.) in dogs anesthetized with sodium pentobarbital (35 mg/kg, i.v.). Plasma aortic epinephrine (E) and norepinephrine (NE) concentrations increased significantly 30 min after the injection of insulin. At this time point, a functional adrenalectomy (diversion of bilateral adrenal venous blood from the systemic circulation) was performed for 5 min. The increased aortic E and NE concentrations significantly decreased reaching, within 5 min, a level below the corresponding preinjection control value. The basal output of pancreatic IRG (6.58 +/- 1.12 ng/min, n = 6) significantly increased (24.93 +/- 2.77 ng/min, p less than 0.05, n = 6) 30 min after insulin injection. During the functional adrenalectomy, the increased pancreatic IRG output diminished rapidly, within 5 min, to approximately 50% (11.73 +/- 3.19 ng/min, p less than 0.05, n = 6) of the value observed 30 min after insulin administration. In the other group of dogs receiving sham adrenalectomy, the increased aortic E and NE concentrations and pancreatic IRG output following insulin injection remained elevated above the levels observed immediately before the sham adrenalectomy. The net decrease in IRG output during the adrenalectomy was significant (p less than 0.05) compared with the corresponding net IRG output observed in the sham group.(ABSTRACT TRUNCATED AT 250 WORDS)     

The determination of alpha-adrenergic receptor concentration on rat pancreatic islet cells

The selective a2 adrenergic antagonist yohimbine has been shown to prevent the noradrenaline induced inhibition of insulin secretion from isolated rat islets of Langerhans, Binding studies utilizing [³H]yohimbine showed specific binding to dispersed rat islet cells with a K_d of 2.9 nM and receptor concentration of 645 fmols/mg protein. The use of chloroquine to inhibit receptor recycling did not affect binding of the ligand. Binding studies and secretion data are consistent with the suggestion that adrenergic receptors of the ₂ sub-type may play a dominant role in the regulation of insulin secretion.     

Metabolic signals produced by purine ribonucleosides stimulate proinsulin biosynthesis and insulin secretion.

Inosine, guanosine and adenosine strongly stimulated proinsulin biosynthesis and insulin secretion in isolated mouse pancreatic islets. None of the purine ribonucleosides stimulated insulin secretion in rat islets, although as reported [jain & Logothetopoulos (1977) Endocrinilogy 100, 923-927] inosine and guanosine, but no adenosine, were potent stimulants of proinsulin biosynthesis in this species. The purine bases had no effect in either species. D-Ribose, which enhanced proinsulin biosynthesis at 0.3 and 0.6 mM but not at 5mM in rat pancreatic islets [jain & Logothetopoulos (1977) Endocrinology 100, 923-927], produced no secretory signals in rat islets and was without any effect on proinsulin biosynthesis and insulin secretion in mouse islets. The rates of oxidation of 14C-labelled purine ribonucleosides and D-ribose in islets of the two species correlated well with their effectiveness as inducers of insulin secretion and proinsulin biosynthesis. Specific inhibitors of purine ribonucleoside phosphorylase, adenosine deaminiase and of purine ribonucleoside transport suppressed the stimulatory effects of nucleosides in pancreatic islets without altering the effect of D-glucose. The same inhibitors also markedly diminished the oxidation rats of the labelled purine ribonucleosides. The experiments clearly indicate that porinsulin biosynthesis and insulin secretion are modulated through metabolic signals and not through interactions of intact substrate molecules with cell receptors.     

Effects of the novel mitochondrial protein mimitin in insulin-secreting cells

Mimitin, a novel mitochondrial protein, has been shown to act as a molecular chaperone for the mitochondrial complex I and to regulate ATP synthesis. During Type 1 diabetes development, pro-inflammatory cytokines induce mitochondrial damage in pancreatic β-cells, inhibit ATP synthesis and reduce glucose-induced insulin secretion. Mimitin was expressed in rat pancreatic islets including β-cells and decreased by cytokines. In the ob/ob mouse, a model of insulin resistance and obesity, mimitin expression was down-regulated in liver and brain, up-regulated in heart and kidney, but not affected in islets. To further analyse the impact of mimitin on β-cell function, two β-cell lines, one with a low (INS1E) and another with a higher (MIN6) mimitin expression were studied. Mimitin overexpression protected INS1E cells against cytokine-induced caspase 3 activation, mitochondrial membrane potential reduction and ATP production inhibition, independently from the NF-κB (nuclear factor κB)-iNOS (inducible NO synthase) pathway. Mimitin overexpression increased basal and glucose-induced insulin secretion and prevented cytokine-mediated suppression of insulin secretion. Mimitin knockdown in MIN6 cells had opposite effects to those observed after overexpression. Thus mimitin has the capacity to modulate pancreatic islet function and to reduce cytokine toxicity.     

Evidence for multiple adrenoceptor sites in rainbow trout vasculature

The importance of neuronal and lumenal vascular adrenoceptors in the regulation of vascular reactivity was examined in rainbow trout (Oncorhynchus mykiss), in vivo and in vitro. In vivo, ganglionic blockade with hexamethonium or -adrenoceptor blockade, with either phentolamine or prazosin, produced similar (7 mmHg) decreases in dorsal aortic blood pressure. The drop in dorsal aortic pressure produced by phentolamine or prazosin was due to reduced systemic vascular resistance. Neither the -adrenoceptor antagonist, phenoxybenzamine nor chemical sympathectomy with 6-hydroxy-dopamine affected dorsal aortic pressure. However, after chemical sympathectomy, phenoxybenzamine lowered dorsal aortic pressure to levels similar to that produced by either phentolamine or prazosin. Plasma epinephrine and norepinephrine concentrations increased four- and twofold, respectively, in sympathectomized fish. Sympathectomy also produced a leftward shift in the epinephrine dose/response curve of the in vitro perfused splanchnic vasculature, placing the effective catecholamine concentration well within the in vivo plasma levels. These results indicate that following chemical sympathectomy arterial blood pressure is stabilized by circulating catecholamines through the combined effect of increased plasma catecholamine concentrations and increased sensitivity of vascular adrenoceptors. Phenoxybenzamine is incapable of blocking neuronal vascular adrenoceptors but is a potent antagonist of the up-regulated adrenoceptors, suggesting that the latter are localized on the lumenal side of the vessel.Abbreviations 6OH-DA 6-hydroxy dopamine - EC ₅₀ half-maximal response - EDTA ethylenediaminetetra-acetate - PE polyethylene - PBS phosphate-buffered saline - P _da dorsal aortic pressure - USP United States Pharmacopeia     

Transgenic mice expressing Shb adaptor protein under the control of rat insulin promoter exhibit altered viability of pancreatic islet cells.

BACKGROUND: The Src-homology 2 domain-containing adaptor protein Shb was recently cloned as a serum-inducible gene in the insulin-producing beta-TC1 cell line. Subsequent studies have revealed an involvement of Shb for apoptosis in NIH3T3 fibroblasts and differentiation in the neuronal PC12 cells. To assess a role of Shb for beta-cell function, transgenic mice utilizing the rat insulin promoter to drive expression of Shb were generated. MATERIALS AND METHODS: A gene construct allowing the Shb cDNA to be expressed from the rat insulin 2 promoter was microinjected into fertilized mouse oocytes and implanted into pseudopregnant mice. Mice containing a low copy number of this transgene were bred and used for further experimentation. Shb expression was determined by Western blot analysis. The insulin-positive area of whole pancreas, insulin secretion of isolated islets and islet cell apoptosis, glucose tolerance tests, and in vivo sensitivity to multiple injections of the beta-cell toxin streptozotocin were determined in control CBA and Shb-transgenic mice. RESULTS: Western blot analysis revealed elevated islet content of the Shb protein. Shb-transgenic mice displayed enhanced glucose-disappearance rates in response to an intravenous glucose injection. The relative pancreatic beta-cell area neonatally and at 6 months of age were increased in the Shb-transgenic mice. Islets isolated from Shb-transgenic mice showed enhanced insulin secretion in response to glucose and increased insulin and DNA content. Apoptosis was increased in islets isolated from Shb-transgenic mice compared with control islets both under basal conditions and after incubation with IL-1 beta + IFN-gamma. Rat insulinoma RINm5F cells overexpressing Shb displayed decreased viability during culture in 0.1% serum and after exposure to a cytotoxic dose of nicotinamide. Shb-transgenic mice injected with multiple doses of streptozotocin showed increased blood glucose values compared with the corresponding controls, suggesting increased in vivo susceptibility to this toxin. CONCLUSION: The results suggest that Shb has dual effects on beta-cell growth: whereas Shb increases beta-cell formation during late embryonal stages, Shb also enhances beta-cell death under certain stressful conditions and may thus contribute to beta-cell destruction in type 1 diabetes.     

Factors influencing insulin and glucagon secretion in lean and genetically obese mice.

The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.     

Transgenic overexpression of intraislet ghrelin does not affect insulin secretion or glucose metabolism in vivo

Whereas ghrelin is produced primarily in the stomach, a small amount of it is produced in pancreatic islets. Although exogenous administration of ghrelin suppresses insulin secretion in vitro or in vivo, the role of intraislet ghrelin in the regulation of insulin secretion in vivo remains unclear. To understand the physiological role of intraislet ghrelin in insulin secretion and glucose metabolism, we developed a transgenic (Tg) mouse model, rat insulin II promoter ghrelin-internal ribosomal entry site-ghrelin O-acyl transferase (RIP-GG) Tg mice, in which mouse ghrelin cDNA and ghrelin O-acyltransferase are overexpressed under the control of the rat insulin II promoter. Although pancreatic desacyl ghrelin levels were elevated in RIP-GG Tg mice, pancreatic ghrelin levels were not altered in animals on a standard diet. However, when Tg mice were fed a medium-chain triglyceride-rich diet (MCTD), pancreatic ghrelin levels were elevated to ～16 times that seen in control animals. It seems likely that the gastric ghrelin cells possess specific machinery to provide the octanoyl acid necessary for ghrelin acylation but that this machinery is absent from pancreatic β-cells. Despite the overexpression of ghrelin, plasma ghrelin levels in the portal veins of RIP-GG Tg mice were unchanged from control levels. Glucose tolerance, insulin secretion, and islet architecture in RIP-GG Tg mice were not significantly different even when the mice were fed a MCTD. These results indicate that intraislet ghrelin does not play a major role in the regulation of insulin secretion in vivo.     

β-adrenergic insulin release and adrenergic innervation of mouse pancreatic islets

Summary An investigation of the stereospecificity of -adrenergic insulin release, its relation to -adrenergic blockade and the adrenergic innervation of the pancreatic islets was performed in the mouse. It was observed that in vivo -adrenergic stimulation of insulin release by isopropylnoradrenaline was stereospecific for the L-stereoisomer and selectively blocked by the L-isomer of the -adrenergic antagonist L-propranolol. D-propranolol had no effect. Pretreatment of mice with a dose of D-isopropylnoradrenaline devoid of insulin releasing activity, slightly increased the subsequent insulin response to a halfmaximal dose of L-isopropylnoradrenaline. Basal insulin secretion was blocked by L-propranolol (-adrenergic blockade) and increased by phentolamine (-adrenergic blockade). A -blocked insulin response to L-isopropyl-noradrenaline could be overcome by -adrenergic blockade depending on the dose of the -agonist, suggesting a close association between the adrenergic receptors.The adrenergic innervation of the islet cells was studied by electron microscopic autoradiography after injection of ³H-L-noradrenaline. It was observed that labelled adrenergic nerve terminals were associated with both A₁(D-), A₂ and B-cells. The nerves were mainly distributed in the periphery of the islets either as single axons or as bundles. The majority of the terminals were associated with A₂-cells, the most frequent cell type in the islet periphery. However, in all islets examined terminals were found close to B-cells. Adrenergic terminals often caused indentations in the contour of an islet cell and were separated from the islet cell membrane only by a narrow intercellular space, about 20 nm in width.It is concluded that the islet cells of the mouse are equipped with the morphological substrate for direct adrenergic regulation. Further it is suggested that the B-cell is supplied with L-stereospecific -adrenergic receptors and that the - and -adrenergic receptors are at least partially interrelated.     

Acute exposure of beta-cells to troglitazone decreases insulin hypersecretion via activating AMPK

Background

It has been recognized that insulin hypersecretion can lead to the development of insulin resistance and type 2 diabetes mellitus. There is substantial evidence demonstrating that thiazolidinediones are able to delay and prevent the progression of pancreatic β-cell dysfunction. However, the mechanism underlying the protective effect of thiazolidinediones on β-cell function remains elusive.

Methods

We synchronously detected the effects of troglitazone on insulin secretion and AMP-activated protein kinase (AMPK) activity under various conditions in isolated rat islets and MIN6 cells.

Results

Long-term exposure to high glucose stimulated insulin hypersecretion and inhibited AMPK activity in rat islets. Troglitazone-suppressed insulin hypersecretion was closely related to the activation of AMPK. This action was most prominent at the moderate concentration of glucose. Glucose-stimulated insulin secretion was decreased by long-term troglitazone treatment, but significantly increased after the drug withdrawal. Compound C, an AMPK inhibitor, reversed troglitazone-suppressed insulin secretion in MIN6 cells and rat islets. Knockdown of AMPKα2 showed a similar result. In MIN6 cells, troglitazone blocked high glucose-closed ATP-sensitive K⁺ (K_ATP) channel and decreased membrane potential, along with increased voltage-dependent potassium channel currents. Troglitazone suppressed intracellular Ca^2 + response to high glucose, which was abolished by treatment with compound C.

Conclusion

Our results suggest that troglitazone provides β-cell “a rest” through activating AMPK and inhibiting insulin hypersecretion, and thus restores its response to glucose.

General significance

These data support that AMPK activation may be an important mechanism for thiazolidinediones preserving β-cell function.     

Glucocorticoid-induced changes in insulin secretion related to the metabolism and ultrastructure of pancreatic islets

The effect of adrenalectomy and dexamethasone-treatment on insulin secretion was studied and related to the changes observed in the glucose oxidation, calcium uptake, cAMP and insulin content, as well as the ultrastructure of pancreatic rat islets. It was found that adrenalectomy was followed by a decreased glucose-induced insulin secretion, glucose oxidation, calcium uptake, cAMP and insulin content without any remarkable change observed at the ultrastructural level. Conversely, adrenalectomized-rats supplemented with dexamethasone showed an increased glucose-induced insulin secretion, glucose oxidation, calcium uptake and cAMP content but a diminished islet insulin content. At the ultrastructural level, a clear picture of increased secretory activity was found, with diminished number of mature B granules and greater number of pale granules, while rough endoplasmic reticulum and Golgi complex frequently appeared hypertrophic. These changes were only observed in the B cells. On account of our results, we might suggest that insulin secretion is partially controlled by glucocorticoid circulating levels throughout their effect on pancreatic islet metabolism.     

GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion

GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12 to 16 week-old mice strongly (2.5-fold) increased basal (1 mmol/l) and moderately (40%) potentiated glucose (20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Down-regulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and β-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type-2 diabetes.