The inhibitory effect of cadmium on the secretory activity of the isolated perfused rat pancreas

Perfusion of the isolated rat pancreas with cadmium (Cd) (1 × 10^?3 and 5 × 10^?4m) inhibits the insulin secretory response to glucose (300 mg/100 ml), tolbutamide (40 mg/100 ml), and potassium ions (30 mEq/liter). Cadmium inhibition of pancreatic secretory activity is immediate in onset, and not reversed by simple washout of the organ with perfusion medium. Perfusion of the inhibited organ with a combination of glucose and theophylline results in partial reversal of the inhibition. It is suspected that Cd-induced inhibition of insulin secretion may be mediated through interference with calcium uptake by the pancreatic beta cell.     

U—73122对猪子宫平滑肌细胞和胰腺β细胞株RINm5F的电压依赖…

目的：研究Ｕ－７３１２２对细胞内钙离子浓度和电压依赖性钙通道的作用。方法 用Ｆｕａ－２荧光测定胞浆钙浓度和用穿孔膜片箝记录全细胞钙电流。结果：Ｕ－７３１２２呈剂量相关明显地降低ＲＩＮｍ５Ｆ细胞与子宫平滑肌细胞的去极化诱导的钙电流，并抑制ＫＣｌ诱导的与Ｂａｙ－Ｋ－８６４４诱导的胞浆钙浓度的增加。Ｕ－７３１２２的这种作用对子宫平滑肌细胞要比ＲＩＮｍ５Ｆ细胞强，而一种非磷脂酶Ｃ抑制剂Ｕ－３１２２类似物Ｕ     

Effects of bezafibrate on beta-cell function of rat pancreatic islets

Amphetamine increases extracellular dopamine and induces locomotor and stereotypical behaviors in rats. This study examined the effect of the dopamine D2/D3 receptor antagonist sulpiride (50 mg/kg s.c.) on the dopaminergic response to amphetamine (0.5, 2.0, or 8.0 mg/kg i.p.) in male Sprague-Dawley rats. Extracellular dopamine in the striatum was monitored using in vivo microdialysis and high performance liquid chromatography with electrochemical detection. Dopamine concentration curves were analyzed using non-linear regression and residual F-testing. Amphetamine enhanced extracellular dopamine in a dose-dependent manner. Sulpiride augmented the increase in dopamine induced by 0.5 and 2 mg/kg amphetamine by decreasing the rate of dopamine concentration fall off in the extracellular space (P<0.05). Sulpiride also potentiated the amount of dopamine increased by 8 mg/kg amphetamine, but did so by affecting the maximum concentration achieved (P<0.05), not the onset or offset rates. We conclude that the primary effect of a dopamine D2/D3 receptor antagonist is a potentiation of the effect of amphetamine on extracellular striatal dopamine levels, which may contribute to the enhanced stereotypic effects observed when paired with amphetamine.     

Effects of α-ketocarboxylic acids and 4-pentenoic acid on insulin secretion from the perfused rat pancreas

Pyruvic acid, α-ketobutyric acid, α-ketovaleric acid, α-ketooctanoic acid, α-ketononanoic acid, α-ketoisovaleric acid, α-ketoglutaric acid, α-ketoadipic acid, hydroxypyruvic acid, and 4-pentenoic acid did not induce significant insulin secretion from the perfused rat pancreas, β-phenyl-pyruvic acid and α-keto-β-methylvaleric acid induced only minimal insulin secretion from the perfused rat pancreas which was characterized by a monophasic secretory pattern. Both α-ketocaproic acid and α-ketoisocaproic acid were potent insulin secretagogues which induced a biphasic insulin secretory pattern comparable to the secretory pattern in response to glucose. The insulin secretory potency of this group of chemically related α-ketoacids is discussed.     

Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and beta-cell damage in rat pancreas.

The aim of the present study was the evaluation of possible protective effects of quercetin (QE) against beta-cell damage in experimental streptozotocin (STZ)-induced diabetes in rats. STZ was injected intraperitoneally at a single dose of 50 mg kg(-1) for diabetes induction. QE (15 mg kg(-1) day, intraperitoneal (i.p.) injection) was injected for 3 days prior to STZ administration; these injections were continued to the end of the study (for 4 weeks). It has been believed that oxidative stress plays a role in the pathogenesis of diabetes mellitus (DM). In order to determine the changes of cellular antioxidant defense system, antioxidant enzymes such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase (CAT) activities were measured in pancreatic homogenates. Moreover we also measured serum nitric oxide (NO) and erythrocyte and pancreatic tissue malondialdehyde (MDA) levels, a marker of lipid peroxidation, if there is an imbalance between oxidant and antioxidant status. Pancreatic beta-cells were examined by immunohistochemical methods. STZ induced a significant increase lipid peroxidation, serum NO concentrations and decreased the antioxidant enzyme activity. Erythrocyte MDA, serum NO and pancreatic tissue MDA significantly increased (P < 0.05) and also the antioxidant levels significantly decreased (P < 0.05) in diabetic group. QE treatment significantly decreased the elevated MDA and NO (P < 0.05), and also increased the antioxidant enzyme activities (P < 0.05). QE treatment has shown protective effect possibly through decreasing lipid peroxidation, NO production and increasing antioxidant enzyme activity. Islet cells degeneration and weak insulin immunohistochemical staining was observed in STZ induced diabetic rats. Increased staining of insulin and preservation of islet cells were apparent in the QE-treated diabetic rats. These findings suggest that QE treatment has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic beta-cell integrity.     

Effect of diltiazem on insulin secretion. II. Experiments on perfused rat pancreas, anesthetized dogs and conscious rats.

Effect of diltiazem on insulin secretion was investigated in the perfused rat pancreas. Experiments were also carried out in anesthetized dogs and conscious rats with and without glucose loading. In the perfused rat pancreas, diltiazem reduced both glucose- and tolbutamide-induced insulin secretion and these effects of diltiazem were reversed with removal of the compound. Inhibition of the glucose-induced insulin secretion caused by diltiazem was counteracted by increasing the concentration of calcium ion. In experiments on intact animals, diltiazem at vasoactive doses produced no significant influence on the basal level of plasma insulin or glucose-induced insulin secretion. These data taken together with findings in previously reported work suggest that diltiazem reduces insulin secretion from pancreatic B-cells in vitro possibly by the calcium-antagonistic property, while the compound exhibits practically no inhibitory action on the insulin secretion in vivo.     

Microsomal enzyme activity in perfused rat liver

The activities of microsomal enzymes were observed during perfusion of livers isolated from both normal and phenobarbital treated rats. The hydroxylation of aniline and the O-demethylation of p-nitroanisol did not decrease substantially (by 10 per cent only) in the 9000 g fraction prepared from livers perfused for 4 hr.N-demethylation of aminopyrine was reduced by 60 per cent in the same experimental conditions. A similar decrease of N-demethylation was also observed when livers were isolated from animals treated with phenobarbital. The content of cytochrome P-450 in the microsomes was stable during a 4-hr perfusion of isolated liver. The decrease of N-demethylation of aminopyrine could be partly restored by the simultaneous infusion of nicotinic acid, which is a precursor of NAD and NADP biosynthesis.     

Effect of MgATP on pinacidil-induced displacement of glibenclamide from the sulphonylurea receptor in a pancreatic beta-cell line and rat cerebral cortex.

1. The effects of blockers and openers of K+ channels on binding of [3H]-glibenclamide to microsomes obtained from a pancreatic beta-cell line (HIT-T15) or rat cerebral cortex were examined. 2. The blockers quinine, chlorpromazine and thiopentone and the openers cromakalim [(+/- ) 6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1- pyrrolidyl)-2H-benzo[b]pyran-3-ol] and minoxidil sulphate did not significantly interact with the sulphonylurea receptor of HIT-cells both at phosphorylating (presence of MgATP) and dephosphorylating (absence of MgATP) conditions. 3. In the absence of MgATP, pinacidil (200-500 microM) did not significantly displace [3H]-glibenclamide binding to microsomes from HIT-cells. The displacement of [3H]-glibenclamide binding was strongly enhanced by MgATP and was due to a decrease in the number of high affinity binding sites for glibenclamide. 4. MgATP enhanced pinacidil-induced inhibition of [3H]-glibenclamide binding to microsomes from rat cerebral cortex. 5. The effect of MgATP on pinacidil-induced inhibition of [3H]-glibenclamide binding was maintained after solubilization of the membranes from HIT-cells or rat cerebral cortex. 6. It is concluded that the sulphonylurea receptor is regulated not only by sulphonylureas but also by the K+ channel openers, diazoxide and pinacidil, and by protein phosphorylation. The binding sites for sulphonylureas and these K+ channel openers are not identical, but appear to be located at a single protein or at tightly associated proteins.     

Studies of biosynthesis of insulin by isolated perfused rat pancreas.

Incorporation studies with 3H-leucine have demonstrated that proinsulin is formed as a precursor of insulin in the perfused pancreas. A dependency of the insulin synthesis on the glucose concentration was revealed, based on a direct stimulation of the proinsulin synthesis. The conversion of proinsulin into insulin was not accelerated by glucose stimulation. The conversion also occurred in the absence of glucose.     

Structural requirements for the cytoprotective actions of mono-unsaturated fatty acids in the pancreatic beta-cell line, BRIN-BD11

BACKGROUND AND PURPOSE: Exposure of pancreatic beta-cells to long-chain free fatty acids leads to differential responses according to the chain length and degree of unsaturation. In particular, long-chain saturated molecules such as palmitate (C16:0) cause apoptosis, whereas equivalent mono-unsaturated species (for example, palmitoleate (C16:1)) are not overtly toxic. Moreover, mono-unsaturates exert a powerful cytoprotective response against a range of proapoptotic stimuli. However, the structural requirements that determine cytoprotection have not been determined and form the basis of the present study. EXPERIMENTAL APPROACH: BRIN-BD11 and INS-1 beta-cells were exposed either to the saturated fatty acid palmitate, or to serum withdrawal, to mediate cytotoxicity. The protective effects of a wide range of mono-unsaturated fatty acid derivatives were tested in cytotoxicity assays. Effector caspase activity was also measured and correlated with viability. KEY RESULTS: The cytotoxic actions of palmitate were inhibited dose-dependently by long-chain mono-unsaturated fatty acids with a defined potency order C18:1>C16:1>C14:1. The configuration of the double bond was also important with cis forms being more potent than trans forms. Alkylated mono-unsaturated fatty-acid derivates were also cytoprotective, although their efficacy declined as the alkyl chain length increased. Cytoprotection was achieved rapidly on addition of mono-unsaturates and correlated with a rapid and dramatic inhibition of caspase-3/7 activity in palmitate-treated cells. CONCLUSIONS AND IMPLICATIONS: The data reveal the structural requirements that dictate the cytoprotective actions of mono-unsaturated fatty acids in pancreatic beta-cells. Metabolic activation is not required and the data point at the potential involvement of a fatty acid receptor in mediating cytoprotection.     

Effects of sodium tungstate on insulin and glucagon secretion in the perfused rat pancreas

Both the direct effect of sodium tungstate on insulin and glucagon secretion in the perfused rat pancreas, and the insulin response to glucose and arginine in pancreases isolated from tungstate-pretreated rats were studied. Infusion of tungstate stimulated insulin output in a dose-dependent manner. The insulinotropic effect of tungstate was observed at normal (5.5 mM), and moderately high (9 mM) glucose concentrations, but not at a low glucose concentration (3.2 mM). Tungstate-induced insulin output was blocked by diazoxide, somatostatin, and amylin, suggesting several targets for tungstate at the B-cell secretory machinery. Glucagon release was not modified by tungstate. Pancreases from chronically tungstate-treated rats showed an enhanced response to glucose but not to arginine. Our results indicate that the reported reduction of glycemia caused by tungstate administration is, at least in part, due to its direct insulinotropic activity. Furthermore, chronic tungstate treatment may prime the B-cell, leading to over-response to a glucose stimulus.     

Interrelationship among insulin,glucagon and somatostatin secretory responses to exendin-4 in the perfused rat pancreas

We have investigated the effect of exendin-4 on insulin, glucagon and somatostatin output in the perfused rat pancreas. At 9 mM glucose, exendin-4 potentiated the insulin and somatostatin responses to arginine and reduced the glucagon response to this amino acid. Thus, this reduction might be thought to be paracrine-mediated through the concomitant increase in insulin and somatostatin concentrations. At 3.2 mM glucose, exendin-4 did not affect insulin secretion, reduced glucagon release and stimulated somatostatin output. Furthermore, exendin-4 reduced glucagon secretion as induced by a glucose decline (from 11 to 3.2 mM) without affecting insulin or somatostatin responses. In summary, exendin-4 stimulated insulin and somatostatin secretion and reduced glucagon release. The glucagonostatic effect of exendin-4 was observed under conditions in which insulin and somatostatin were not affected, thus indicating that exendin-4, per se, inhibits A-cell secretion. Indeed, an additional glucagonostatic effect of exendin-4, mediated by its stimulation of insulin and/or somatostatin secretion, cannot be ruled out.     

G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell

It is increasingly clear that some of the effects of both free and derivatised long chain fatty acids in pancreatic beta-cells are mediated by a group of G-protein coupled receptors. Some of these display close structural homology while others are more divergent. This Commentary reviews the expression and functional roles of three such molecules, GPR40, GPR119 and GPR120. GPR40 is the best characterised of this group and appears to mediate the acute stimulatory effects of long chain fatty acids on insulin secretion. GPR40 has also been proposed as a potential mediator of fatty acid toxicity but this is more controversial. GPR119 is also involved in stimulation of insulin secretion and responds primarily to ethanolamide derivatives of long chain fatty acids and also to some lysophospholipids rather than to free fatty acids. It may represent a useful target for the development of new insulin secretagogues aimed to enhance insulin release in patients with type 2 diabetes. GPR120 is the most enigmatic of the lipid-responsive cell-surface receptors and its function remains to be established. It has been proposed to play a cytoprotective role in certain other cell types but it is unclear whether it fulfils a similar function in beta-cells.     

G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell

It is increasingly clear that some of the effects of both free and derivatised long chain fatty acids in pancreatic beta-cells are mediated by a group of G-protein coupled receptors. Some of these display close structural homology while others are more divergent. This Commentary reviews the expression and functional roles of three such molecules, GPR40, GPR119 and GPR120. GPR40 is the best characterised of this group and appears to mediate the acute stimulatory effects of long chain fatty acids on insulin secretion. GPR40 has also been proposed as a potential mediator of fatty acid toxicity but this is more controversial. GPR119 is also involved in stimulation of insulin secretion and responds primarily to ethanolamide derivatives of long chain fatty acids and also to some lysophospholipids rather than to free fatty acids. It may represent a useful target for the development of new insulin secretagogues aimed to enhance insulin release in patients with type 2 diabetes. GPR120 is the most enigmatic of the lipid-responsive cell-surface receptors and its function remains to be established. It has been proposed to play a cytoprotective role in certain other cell types but it is unclear whether it fulfils a similar function in beta-cells.     

Diltiazem inhibition of glucose-induced insulin secretion from the isolated perfused rat pancreas

The effect of the calcium channel blocker diltiazem on stimulated insulin release from the isolated perfused rat pancreas was studied. Rats were treated for seven days with three dosages of diltiazem. Both phases of glucose-stimulated insulin secretion from the isolated perfused rat pancreas were diminished dose dependently.     

ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function

This review focuses on the emerging evidence that reactive oxygen species (ROS) derived from glucose metabolism, such as H₂O₂, act as metabolic signaling molecules for glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Particular emphasis is placed on the potential inhibitory role of endogenous antioxidants, which rise in response to oxidative stress, in glucose-triggered ROS and GSIS. We propose that cellular adaptive response to oxidative stress challenge, such as nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant induction, plays paradoxical roles in pancreatic beta-cell function. On the one hand, induction of antioxidant enzymes protects beta-cells from oxidative damage and possible cell death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two premises are potentially relevant to impairment of beta-cells occurring in the late and early stage of Type 2 diabetes, respectively. In addition, we summarized our recent findings that persistent oxidative stress due to absence of uncoupling protein 2 activates cellular adaptive response which is associated with impaired pancreatic beta-cell function.     

Effect of galanin on endocrine secretions from the isolated perfused rat stomach and pancreas

Nerves containing galanin immunoreactivity have been shown to be present in the stomach and pancreas. The present experiments were designed to test the effect of galanin on the release of gastric somatostatin-like immunoreactivity (SLI) and immunoreactive gastrin (IR-G), pancreatic SLI and immunoreactive insulin (IR-I) from the isolated perfused rat stomach and pancreas respectively. Galanin (2 X 10(-10)-5 X 10(-8) M) inhibited gastric SLI and IR-G release dose dependently. At a concentration of 10(-8) M, galanin also suppressed IR-I release stimulated by gastric inhibitory polypeptide (2 X 10(-10) M) in the presence of 8.9 X 10(-3) M glucose. Pancreatic SLI release under this condition was not altered. Thus the present study suggests that galanin might be involved in the neural regulation of gastric and pancreatic endocrine secretions.     

Oleic acid and glucose regulate glucagon-like peptide 1 receptor expression in a rat pancreatic ductal cell line

The glucagon-like peptide 1 receptor (GLP1R) plays a critical role in glucose metabolism and has become an important target for a growing class of drugs designed to treat type 2 diabetes. In vitro studies were designed to investigate the effect of the GLP1R agonist, exenatide (Ex4), in “on-target” RIN-5mF (islet) cells as well as in “off-target” AR42J (acinar) and DSL-6A/C1 (ductal) cells in a diabetic environment. Ex4 increased islet cell proliferation but did not affect acinar cells or ductal cells at relevant concentrations. A high caloric, high fat diet is a risk factor for impaired glucose tolerance and type-2 diabetes. An in vitro Oleic acid (OA) model was used to investigate the effect of Ex4 in a high calorie, high fat environment. At 0.1 and 0.4 mM, OA mildly decreased the proliferation of all pancreatic cell types. Ex4 did not potentiate the inhibitory effect of OA on cell proliferation. Akt phosphorylation in response to Ex4 was diminished in OA-treated ductal cells. GLP1R protein detected by western blot was time and concentration dependently decreased after glucose stimulation in OA-treated ductal cells. In ductal cells, OA treatment altered the intracellular localization of GLP1R and its co-localization with early endosome and recycling endosomes. Chloroquine (lysosomal inhibitor), N-acetyl-l-cysteine (reactive oxygen species scavenger) and wortmannin (a phosphatidylinositol-3-kinase inhibitor), fully or partially, rescued GLP1R protein in OA-pretreated, glucose-stimulated ductal cells. The impact of altered regulation on phenotype/function is presently unknown. However, these data suggest that GLP1R regulation in ductal cells can be altered by a high fat, high calorie environment.     

胰岛β细胞葡萄糖毒性分子机制的研究进展

慢性高糖可损伤胰岛β细胞,这被称为“葡萄糖毒性”。胰岛β细胞葡萄糖毒性涉及多种机制包括氧化应激、内质网应激、线粒体功能失调、炎症、糖基化终末产物形成、低氧状态与低氧诱导因子激活、胰岛β细胞分化表型丧失,等等。目前认为,氧化应激、内质网应激和胰岛β细胞分化表型的丧失在胰岛β细胞葡萄糖毒性中的作用较为明确;其中氧化应激在糖尿病胰岛β细胞葡萄糖毒性中发挥中心性作用。     

Effects of verapamil, ouabain, and ethacrynic acid on calcium retention in perfused rat kidneys.

A Sprague-Dawley rat kidney perfusion technique was used in situ to study the effect of verapamil, ouabain, and ethacrynic acid on renal calcium retention. The technique involves perfusion of the kidneys via the abdominal aorta and then through the left and right renal arteries and dorsal aorta. Verapamil (1 mM) in Krebs-improved Ringer solution increased calcium retention in the kidneys by approximately 117% compared with controls. With Na-free Krebs-improved Ringer solution, calcium retention increased by only 92%. However, in Krebs-improved Ringer solutions containing 59 and 122 mequivalents of Na, calcium retention in the kidney increased by 46 and 43%, respectively, compared with controls. With Krebs-improved Ringer solution containing 15 mM ouabain, calcium retention in the kidney decreased by 29.5%, whereas with 15 mM ouabain plus 1 mM ethacrynic acid in the perfusate, the effect on calcium retention in the kidney was insignificant compared with controls. These results suggest that two sodium-dependent calcium-transporting systems exist at the peritubular side of the kidney tubules: (1) a Na(+)-Ca+2 countertransport system sensitive to verapamil and (2) a Na(+)-Ca+2 cotransport system sensitive to the intracellular concentration of sodium.