Insulinotropic effect of the non-steroidal compound STX in pancreatic β-cells

The non-steroidal compound STX modulates the hypothalamic control of core body temperature and energy homeostasis. The aim of this work was to study the potential effects of STX on pancreatic β-cell function. 1-10 nM STX produced an increase in glucose-induced insulin secretion in isolated islets from male mice, whereas it had no effect in islets from female mice. This insulinotropic effect of STX was abolished by the anti-estrogen ICI 182,780. STX increased intracellular calcium entry in both whole islets and isolated β-cells, and closed the K(ATP) channel, suggesting a direct effect on β-cells. When intraperitoneal glucose tolerance test was performed, a single dose of 100 μg/kg body weight STX improved glucose sensitivity in males, yet it had a slight effect on females. In agreement with the effect on isolated islets, 100 μg/kg dose of STX enhanced the plasma insulin increase in response to a glucose load, while it did not in females. Long-term treatment (100 μg/kg, 6 days) of male mice with STX did not alter body weight, fasting glucose, glucose sensitivity or islet insulin content. Ovariectomized females were insensitive to STX (100 μg/kg), after either an acute administration or a 6-day treatment. This long-term treatment was also ineffective in a mouse model of mild diabetes. Therefore, STX appears to have a gender-specific effect on blood glucose homeostasis, which is only manifested after an acute administration. The insulinotropic effect of STX in pancreatic β-cells is mediated by the closure of the K(ATP) channel and the increase in intracellular calcium concentration. The in vivo improvement in glucose tolerance appears to be mostly due to the enhancement of insulin secretion from β-cells.     

GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice

GABA has been proposed to inhibit insulin secretion through GABAB receptors (GABABRs) in pancreatic beta-cells. We investigated whether GABABRs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABAB1 subunit of the GABABR (GABAB(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABAB1 subunit in islet membranes and immunohistochemistry for insulin and GABAB1 were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABABR agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABAB(-/-) mice showed fasting and fed glucose levels similar to WT. GABAB(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABAB(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABAB(-/-) islets. In GABAB(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABAB(-/-) males. Results demonstrate that GABABRs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABABRs induces alterations in pancreatic histology, physiology, and insulin resistance.     

Resveratrol-induced inhibition of insulin secretion from rat pancreatic islets: evidence for pivotal role of metabolic disturbances

Resveratrol is a stilbene present in different plant species and exerting numerous beneficial effects, including prevention of diabetes and attenuation of some diabetic complications. Its inhibitory effect on insulin secretion was recently documented, but the exact mechanism underlying this action remains unknown. Experiments employing diazoxide and a high concentration of K(+) revealed that, in depolarized pancreatic islets incubated for 90 min with resveratrol (1, 10, and 100 microM), insulin secretion stimulated by glucose and leucine was impaired. The attenuation of the insulin secretory response to 6.7 mM glucose was not abrogated by blockade of intracellular estrogen receptors and was found to be accompanied by diminished islet glucose oxidation, enhanced lactate production, and reduced ATP levels. Glucose-induced hyperpolarization of the mitochondrial membrane was also reduced in the presence of resveratrol. Moreover, in depolarized islets incubated with 2.8 mM glucose, activation of protein kinase C or protein kinase A potentiated insulin release; however, under these conditions, resveratrol was ineffective. Further studies also revealed that, under conditions of blocked voltage-dependent calcium channels, the stilbene reduced insulin secretion induced by a combination of glucose with forskolin. These data demonstrate that resveratrol 1) inhibits the amplifying pathway of insulin secretion, 2) exerts an insulin-suppressive effect independently of its estrogenic/anti-estrogenic activity, 3) shifts islet glucose metabolism from mitochondrial oxidation to anaerobic,4) fails to abrogate insulin release promoted without metabolic events, and 5) does not suppress hormone secretion as a result of the direct inhibition of Ca(2+) influx through voltage-dependent calcium channels.     

Immunocytochemical localization of alpha-protein kinase C in rat pancreatic beta-cells during glucose-induced insulin secretion

To investigate the role of protein kinase C (PKC) in the regulation of insulin secretion, we visualized changes in the intracellular localization of alpha-PKC in fixed beta-cells from both isolated rat pancreatic islets and the pancreas of awake unstressed rats during glucose-induced insulin secretion. Isolated, perifused rat islets were fixed in 4% paraformaldehyde, detergent permeabilized, and labeled with a mAb specific for alpha-PKC. The labeling was visualized by confocal immunofluorescent microscopy. In isolated rat pancreatic islets perifused with 2.75 mM glucose, alpha-PKC immunostaining was primarily cytoplasmic in distribution throughout the beta-cells. In islets stimulated with 20 mM glucose, there was a significant redistribution of alpha-PKC to the cell periphery. This glucose-induced redistribution was abolished when either mannoheptulose, an inhibitor of glucose metabolism, or nitrendipine, an inhibitor of calcium influx, were added to the perifusate. We also examined changes in the intracellular distribution of alpha-PKC in the beta-cells of awake, unstressed rats that were given an intravenous infusion of glucose. Immunocytochemical analysis of pancreatic sections from these rats demonstrated a glucose-induced translocation of alpha-PKC to the cell periphery of the beta-cells. These results demonstrate that the metabolism of glucose can induce the redistribution of alpha-PKC to the cell periphery of beta-cells, both in isolated islets and in the intact animal, and suggest that alpha-PKC plays a role in mediating glucose-induced insulin secretion.     

Effect of Dexamethasone on Insulin Secretion: Examination of Underlying Mechanisms

ObjectiveTo study the mechanism of increased insulin secretion in response to short-term administration of dexamethasone.MethodsMale Wistar rats were injected intraperitoneally with dexamethasone (dexamethasone; 200 mcg/kg body weight per day) or saline for 3 consecutive days. Insulin secretion in response to glucose, ionomycin, and KCl was quantified in islets isolated from the animals, and the amount of glucokinase was measured by Western blot.ResultsDexamethasone-treated animals had 1.18-fold higher fasting blood glucose concentration and 6.5-fold increase in fasting serum insulin concentration compared with findings from animals injected with saline. Compared with islets isolated from control rats, islets from dexamethasone-treated rats secreted more insulin at 60 minutes in response to 5.5 mM glucose (416.4 vs 115.6 fmoles/10 islets, P = .011) and in response to 16.6 mM glucose (985.5 vs 520.6 fmoles/10 islets, P = .014); no change in insulin secretion was observed at 10 minutes. Insulin secretion from islets of dexamethasone-treated rats and control rats was not differentially augmented in response to either ionomycin or potassium chloride. Glucokinase expression was not altered by treatment with dexamethasone.ConclusionsAugmentation of insulin secretion in response to glucose in the pancreatic islets from dexamethasone-treated rats is preserved in islets studied in vitro. The increase in glucose-stimulated insulin secretion appears to be mediated by steps upstream to β -cell membrane depolarization and the attended increase in intracellular calcium in the signaling pathway of insulin secretion. (Endocr Pract. 2010;16:763-769)     

5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways

AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic beta-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 microM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K(+) (K(ATP)) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5mM) AICAR did not affect K(ATP) activity or intracellular ([Ca(2+)](i)). AICAR also did not inhibit (86)Rb(+) efflux from islets isolated from Sur1(-/-) mice that lack K(ATP) channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K(ATP) channel-dependent and -independent pathways.     

Neurotensin is a regulator of insulin secretion in pancreatic beta-cells

Neurotensin (NT) is secreted from neurons and gastrointestinal endocrine cells. We previously reported that the three NT receptors (NTSRs) are expressed in pancreatic islets and beta cell lines on which we observed a protective effect of NT against cytotoxic agents. In this study, we explored the role of NT on insulin secretion in the endocrine pancreatic beta cells. We observed that NT stimulates insulin secretion at low glucose level and has a small inhibiting effect on stimulated insulin secretion from isolated islets or INS-1E cells. We studied the mechanisms by which NT elicited calcium concentration changes using fura-2 loaded islets or INS-1E cells. NT increases calcium influx through the opening of cationic channels. Similar calcium influxes were observed after treatment with NTSR selective ligands. NT-evoked calcium regulation involves PKC and the translocation of PKCα and PKC? to the plasma membrane. Part of NT effects appears to be also mediated by PKA but not via the Erk pathway. Taken together, these data provide evidence for an important endocrine role of NT in the regulation of the secretory function of beta cells.     

Cell swelling-induced signaling for insulin secretion bypasses steps involving G proteins and PLA2 and is N-ethylmaleimide insensitive.

BACKGROUND: This study was undertaken to examine putative mechanisms of calcium independent signal transduction pathway of cell swelling-induced insulin secretion. METHODS: The role of phospholipase A(2), G proteins, and soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) in insulin secretion induced by 30% hypotonic medium was studied using isolated rat pancreatic islets. RESULTS: In contrast to glucose stimulation, osmotically induced insulin secretion from pancreatic islets was not inhibited by 10 micromol/l bromoenol lactone, an iPLA(2) (Ca(2+) independent phospholipase) inhibitor. Similarly, preincubation of islets for 20 hours with 25 microg/ml mycophenolic acid to inhibit GTP synthesis fully abolished glucose-induced insulin secretion but was without effect on hypotonicity stimulated insulin release. Glucose-induced insulin secretion was prevented by preincubation with 20 nmol/l tetanus toxin (TeTx), a metalloprotease inactivating soluble SNARE. Cell swelling-induced insulin secretion was inhibited by TeTx in the presence of calcium ions but not in calcium depleted medium. The presence of N-ethylmaleimide (NEM, 5 mmol/l, another inhibitor of SNARE proteins) in the medium resulted in high basal insulin secretion and lacking response to glucose stimulation. In contrast, high basal insulin secretion from NEM treated islets further increased after hypotonic stimulation. CONCLUSION: G proteins and iPLA(2) - putative mediators of Ca(2+) independent signaling pathway participate in glucose but not in hypotonicity-induced insulin secretion. Hypotonicity-induced insulin secretion is sensitive to clostridial neurotoxin TeTx but is resistant to NEM.     

Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.     

Glucose stimulates Ca2+ influx and insulin secretion in 2-week-old beta-cells lacking ATP-sensitive K+ channels

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) and (b) a K(ATP) channel-independent amplification of secretion without further increase of [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus K(ATP) channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca(2+)](c) and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca(2+)](c) and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca(2+)](c) and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca(2+)](c) followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca(2+)](c) was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca(2+)](c) and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This K(ATP) channel-independent first phase [Ca(2+)](c) rise was attributed to a Na(+)-, Cl(-)-, and Na(+)-pump-independent depolarization of beta-cells, leading to Ca(2+) influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca(2+)](c) in beta-cells without functional K(ATP) channels.     

Selective potentiation of 5-methyl-2-(1-methylcyclohexyl)-4-oxazoleacetic acid (AD-4610) on glucose-induced insulin secretion

In the perfused pancreas from normal SD rats, AD-4610 (0.01-0.1 mM) potentiated biphasic insulin secretion induced by 7.5 mM of glucose. The concentration-response curve of insulin secretion to glucose was shifted leftwards with AD-4610 (0.1 mM) without altering either the threshold concentration of glucose to induce insulin secretion or the maximal insulin response to glucose, indicating increased sensitivity of the pancreatic B-cells to glucose. On the other hand, AD-4610 was 10-fold less effective in altering insulin secretion induced by arginine and glyceraldehyde. The effect of AD-4610 on insulin secretion and glucose metabolism was compared with that of tolbutamide in vivo. AD-4610 (100 mg/kg) potentiated insulin secretion induced by an intravenous glucose load, and also accelerated glucose metabolism without altering basal insulin secretion in normal rats. On the other hand, tolbutamide (20 mg/kg) increased basal insulin secretion, but slightly decreased glucose-induced insulin secretion. In yellow KK mice with hyperglycemia, AD-4610 (10-100 mg/kg) had a dose-dependent hypoglycemic action, but tolbutamide did not. Thus, AD-4610 stimulated insulin secretion in a glucose-dependent fashion and enhanced glucose metabolism in vivo. These results suggest that AD-4610 selectively potentiates glucose-induced insulin secretion by increasing the sensitivity of pancreatic B-cells to glucose and may be useful for treating human NIDDM through a different mechanism than that of tolbutamide.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.     

The apj receptor is expressed in pancreatic islets and its ligand, apelin, inhibits insulin secretion in mice

Apelin is the endogenous ligand of the G-protein coupled apj receptor. Apelin is expressed in the brain, the hypothalamus and the stomach and was recently shown also to be an adipokine secreted from the adipocytes. Although apelin has been suggested to be involved in the regulation of food intake, it is not known whether the peptide affects islet function and glucose homeostasis. We show here that the apj receptor is expressed in pancreatic islets and that intravenous administration of full-length apelin-36 (2 nmol/kg) inhibits the rapid insulin response to intravenous glucose (1 g/kg) by 35% in C57BL/6J mice. Thus, the acute (1-5 min) insulin response to intravenous glucose was 682+/-23 pmol/l after glucose alone (n=17) and 445+/-58 pmol/l after glucose plus apelin-36 (n=18; P=0.017). This was associated with impaired glucose elimination (the 5-20 min glucose elimination was 2.9+/-0.1%/min after glucose alone versus 2.3+/-0.2%/min after glucose plus apelin-36, P=0.008). Apelin (2 nmol/kg) also inhibited the insulin response to intravenous glucose in obese insulin resistant high-fat fed C57BL/6J mice (P=0.041). After 60 min incubation of isolated islets from normal mice, insulin secretion in the presence of 16.7 mmol/l glucose was inhibited by apelin-36 at 1 mumol/l, whereas apelin-36 did not significantly affect insulin secretion at 2.8 or 8.3 mmol/l glucose or after stimulation of insulin secretion by KCl. Islet glucose oxidation at 16.7 mmol/l was not affected by apelin-36. We conclude that the apj receptor is expressed in pancreatic islets and that apelin-36 inhibits glucose-stimulated insulin secretion both in vivo and in vitro. This may suggest that the islet beta-cells are targets for apelin-36.     

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels

Understanding mechanisms by which glibenclamide stimulates insulin release is important, particularly given recent promising treatment by glibenclamide of permanent neonatal diabetic subjects. Antidiabetic sulfonylureas are thought to stimulate insulin secretion solely by inhibiting their high-affinity ATP-sensitive potassium (K(ATP)) channel receptors at the plasma membrane of beta-cells. This normally occurs during glucose stimulation, where ATP inhibition of plasmalemmal K(ATP) channels leads to voltage activation of L-type calcium channels for rapidly switching on and off calcium influx, governing the duration of insulin secretion. However, growing evidence indicates that sulfonylureas, including glibenclamide, have additional K(ATP) channel receptors within beta-cells at insulin granules. We tested nonpermeabilized beta-cells in mouse islets for glibenclamide-stimulated insulin secretion mediated by granule-localized K(ATP) channels by using conditions that bypass glibenclamide action on plasmalemmal K(ATP) channels. High-potassium stimulation evoked a sustained rise in beta-cell calcium level but a transient rise in insulin secretion. With continued high-potassium depolarization, addition of glibenclamide dramatically enhanced insulin secretion without affecting calcium. These findings support the hypothesis that glibenclamide, or an increased ATP/ADP ratio, stimulates insulin secretion in part by binding at granule-localized K(ATP) channels that functionally contribute to sustained second-phase insulin secretion.     

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

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

铃蟾肽对四氧嘧啶引起的大鼠糖尿病的预防作用

本工作从三个不同的层次对铃蟾肽防止胰岛 B 细胞损伤的作用进行了研究:(1)在整体水平,预先注射铃蟾肽(50μg/kg,iv)可明显抑制单独给予四氧嘧啶(200mg/kg,s.c.)引起的大鼠血糖升高和血浆胰岛素水平下降的趋势。(2)在离体胰腺灌流实验发现,在四氧嘧啶之前预灌流铃蟾肽(10~(-2)mmol/L)可使胰腺对高糖刺激产生反应性分泌;而仅以四氧嘧啶灌流时,胰腺对高糖刺激无反应。(3)在离体胰岛水平,初步研究了在四氧嘧啶引起胰岛 B 细胞功能改变时,铃蟾肽对胰岛内胰岛素、胰高血糖素和生长抑素分泌的影响。结果表明,铃蟾肽可防止四氧嘧啶引起的胰岛素和生长抑素分泌的抑制及胰高血糖素分泌的增加趋势。     

Leptin fully suppresses acetylcholine-induced insulin secretion and is reversed by tolbutamide in isolated perfused chicken pancreas.

So far, there has been no evidence for any direct pancreatic effect of leptin in the chicken. The present study was aimed at detecting chicken leptin receptor (cOb-R) expression in isolated chicken islets of Langerhans and to examine the direct effect of leptin on insulin secretion after stimulation by acetylcholine (1 micro M) + glucose (14 mM) from isolated perfused chicken pancreas. We will show that i) full length cOb-R mRNA was expressed in isolated pancreatic islets of chickens, ii) recombinant chicken leptin (10 nM) or diazoxide (100 micro M) rapidly (within 2 min) and significantly suppressed insulin secretion induced by acetylcholine stimulation without any change in volume outflow rate, iii) tolbutamide (100 micro M) introduced 10 min after leptin and perfused for 10 min fully reversed the suppressive effect of leptin on pre-established acetylcholine-induced insulin release. In conclusion, we found that leptin has a profound inhibitory influence upon insulin secretion in perfused chicken pancreas. The results suggest that leptin inhibits insulin secretion by acting before or at the level of K ATP channels in chicken pancreatic beta-cells. Further studies are warranted to clarify the specific inhibitory mechanism.     

Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for 1 wk in high glucose

Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic beta-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key beta-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by approximately 50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in (45)Ca(2+) influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca(2+) concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5-G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an approximately 50% reduction in the maximal glucose stimulation of insulin secretion.