Ca2+-dependent exocytosis of L-glutamate by alphaTC6, clonal mouse pancreatic alpha-cells

Pancreatic islet cells express receptors and transporters for L-glutamate and are thus believed to use L-glutamate as an intercellular signaling molecule. However, the mechanism by which L-glutamate appears in the islets is unknown. In the present study, we investigated whether L-glutamate is secreted through exocytosis by alphaTC6 cells (clonal mouse pancreatic alpha-cells). An appreciable amount of L-glutamate was released from cultured cells after the addition of KCl or A23187 in the presence of Ca2+ and 10 mmol/l glucose in the medium. The KCl-induced glutamate release was significantly reduced when assayed in the absence of Ca2+ or when the cells were pretreated with EGTA-AM. The KCl-induced Ca2+-dependent glutamate release was inhibited approximately 40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 micromol/l. The degree of KCl-induced Ca2+-dependent glutamate release was correlated with an increase in intracellular [Ca2+], as monitored by fura-2 fluorescence. Botulinum neurotoxin type E inhibited 55% of the KCl-induced Ca2+-dependent glutamate release, followed by specific cleavage of 25 kDa synaptosomal-associated protein. Furthermore, bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, inhibited 40% of the KCl-induced Ca2+-dependent glutamate release. Immunoelectronmicroscopy with antibodies against synaptophysin, a marker for neuronal synaptic vesicles and endocrine synaptic-like microvesicles, revealed a large number of synaptophysin-positive clear vesicles in cells. Digitonin-permeabilized cells took up L-glutamate only in the presence of MgATP, which is sensitive to bafilomycin A1 or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (a proton conductor) but insensitive to either oligomycin or vanadate. From these results, it was concluded that alphaTC6 cells accumulate L-glutamate in the synaptophysin-containing vesicles in an ATP-dependent manner and secrete it through a Ca2+-dependent exocytic mechanism. The Ca2+-dependent glutamate release was also triggered when cells were transferred in the medium containing 1 mmol/l glucose, suggesting that low glucose treatment stimulates the release of glutamate. Our results are consistent with the idea that L-glutamate is secreted by alpha-cells through Ca2+-dependent regulated exocytosis.     

Expression of the vesicular inhibitory amino acid transporter in pancreatic islet cells: distribution of the transporter within rat islets

gamma-Aminobutyric acid (GABA) is stored in microvesicles in pancreatic islet cells. Because GAD65 and GAD67, which catalyze the formation of GABA, are cytoplasmic, the existence of an islet vesicular GABA transporter has been postulated. Here, we test the hypothesis that the putative transporter is the vesicular inhibitory amino acid transporter (VIAAT), a neuronal transmembrane transporter of GABA and glycine. We sequenced the human VIAAT gene and determined that the human and rat proteins share over 98% sequence identity. In vitro expression of VIAAT and immunoblotting of brain and islet lysates revealed two forms of the protein: an approximately 52-kDa and an approximately 57-kDa form. By immunoblotting and immunohistochemistry, we detected VIAAT in rat but not human islets. Immunohistochemical staining showed that in rat islets, the distribution of VIAAT expression parallels that of GAD67, with increased expression in the mantle. GABA, too, was found to be present in islet non-beta-cells. We conclude that VIAAT is expressed in rat islets and is more abundant in the mantle and that expression in human islets is very low or nil. The rat islet mantle differs from rat and human beta-cells in that it contains only GAD67 and relatively increased levels of VIAAT. Cells that express only GAD67 may require higher levels of VIAAT expression.     

Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion.

Ghrelin, a novel growth hormone-releasing peptide isolated from human and rat stomach, is a 28-amino acid peptide with a posttranslational acylation modification that is indispensable for stimulating growth hormone secretion by increasing intracellular Ca(2+) concentration. It also functions in the regulation of feeding behavior, energy metabolism, and gastric acid secretion and motility. Using two different antibodies against the NH(2)- and COOH-terminal regions of ghrelin, we studied its localization in human and rat pancreas by immunohistochemistry. Ghrelin-immunoreactive cells were identified at the periphery of pancreatic islets in both species. Ghrelin co-localized exclusively with glucagon in rat islets, indicating that it is produced in alpha-cells. We identified ghrelin and des-acyl ghrelin in the rat pancreas using reverse-phase high-performance liquid chromatography combined with two radioimmunoassays. We also detected mRNA encoding ghrelin and its receptor in the rat pancreatic islets. Ghrelin increased the cytosolic free Ca(2+) concentration in beta-cells and stimulated insulin secretion when it was added to isolated rat pancreatic islets. These findings indicate that ghrelin may regulate islet function in an endocrine and/or paracrine manner.     

Identification of Ia-bearing cells in rat, dog, pig, and human islets of Langerhans

Cells expressing Ia-like antigens (Ia) are believed to provide the primary recognition signals for allograft rejection. In order to clarify the localization of Ia within islets of Langerhans in animals most commonly used as transplantation models and in humans, we performed both light microscopic (LM) and electron microscopic (EM) immunocytochemical studies with the following panel of monoclonal antibodies directed against Ia: B1F6 (antidog, antirat, and antihuman Ia) and B2E8 (antidog and antihuman Ia), generated in our laboratory; OX6 (antirat Ia); and L243 (antihuman Ia). By LM, B1F6-labeled or B2E8-labeled dog islets and B1F6-labeled or OX6-labeled rat islets all exhibited Ia-specific localization on 0-15 spheroidal or stellate cells per islet. Ultrastructural identification of these cells by indirect immunoperoxidase labeling of unfixed isolated islets revealed Ia-specific cell surface binding mostly on sparsely distributed cells that exhibited ultrastructural characteristics typical of monocytes or macrophages; no specific correlation between cell shape and cell type was apparent by EM. The presence of a resident population of monocytes and macrophages in these islets was confirmed by cytochemical localization of nonspecific esterase activity. Islet endothelium was Ia-negative, as were endocrine cells. Studies of human islets with B1F6, B2E8, and L243 confirmed our preliminary findings that, although there exists a sparse population of Ia-positive leukocytes similar to those present in dog and rat islets, Ia is also expressed extensively and constitutively on the human islet vascular endothelium. This differential localization of Ia in rats and dogs compared with humans could have important implications for the development of successful strategies for the immunomodulation of human islet allografts. We have now shown by immunopurification of 125I-labeled splenocyte plasma membrane antigens, that our MoAbs B1F6 and B2E8 also recognize porcine Ia, and immunocytochemical studies with these MoAbs demonstrated that Ia is also localized on the vascular endothelium in pig islets. Hence, this species may prove to be uniquely suitable as a model in which the immune response functions of Ia-bearing islet endothelium can be explored.     

Identification and cloning of a beta-cell-specific zinc transporter, ZnT-8, localized into insulin secretory granules

SLC30A8, a novel member of the zinc transporter (ZnT) family, was identified by searching the human genomic and expressed sequence tag (EST) databases with the amino acid sequence of all known human ZnT. The protein (369 amino acids) predicted from this gene, ZnT-8, contains six transmembrane domains and a histidine-rich loop between transmembrane domains IV and V, like the other ZnT proteins. We demonstrated by RT-PCR on cDNA libraries and human tissue extracts that the ZnT-8 gene is solely transcribed in the pancreas, mainly in the islets of Langerhans. The gene, named SLC30A8, was cloned and sequenced. Confocal immunofluorescence analysis revealed that a ZnT-8-EGFP (enhanced green fluorescent protein) fusion product colocalized with insulin in the secretory pathway granules of the insulin-secreting INS-1 cells. Exposure of the ZnT-8-EGFP stably expressing HeLa cells to 75 micromol/l zinc caused an accumulation of zinc in intracellular vesicles compared with cells expressing EGFP alone. These results identified ZnT-8 as a ZnT specific to the pancreas and expressed in beta-cells. Because ZnT-8 facilitates the accumulation of zinc from the cytoplasm into intracellular vesicles, ZnT-8 may be a major component for providing zinc to insulin maturation and/or storage processes in insulin-secreting pancreatic beta-cells.     

Changes of A, B and D cells in Langerhans islets in pancreatic cancers of hamsters.

In order to clarify the effect of pancreatic hormones on the oncogenesis of pancreatic cancer, the kinetics of the B, A and D cells in the islets of Langerhans were studied in hamsters with pancreatic cancer induced by di-iso-propanol nitrosamine (DIPN). Tumors appeared histologically 8 weeks and duct adenocarcinomas became evident 12 weeks after the administration of DIPN. Although the area of the islets did not change in 8 to 16 weeks, the numbers of B cells was decreased 8 weeks after the administration of DIPN and of A and D cells was decreased at 16 weeks. The area occupied by B cells in proportion to the number of islet cells showed a significant decrease 8 weeks after the administration of DIPN. Since insulin has been reported to have a trophic effect on the exocrine pancreas, our findings suggest that pancreatic B cells start to decrease at the same time that pancreatic cancer begins to form. Thus, insulin appears to play an important role locally in the oncogenesis of pancreatic cancer in acinar cells.     

Expression of GLUT1 and GLUT2 glucose transporter isoforms in rat islets of Langerhans and their regulation by glucose.

Previous studies revealed that rat islets express the GLUT2-liver facilitative glucose transporter isoform, a glucose carrier with a low affinity for glucose but a high capacity for glucose transport. These studies indicated the presence of a second glucose transporter in rat islets; however, they did not indicate to which of the five known facilitative glucose transporters it corresponded. In this study, we isolated RNA from rat islets of Langerhans and confirmed the presence of GLUT2 mRNA. In addition, we present data indicating that the second isoform expressed in islets is the GLUT1-erythrocyte isoform. The effect of culturing islets in 5.5, 8.3, or 11.1 mM glucose on the levels of GLUT1 and GLUT2 mRNA also was examined. The levels of GLUT1 and GLUT2 mRNA were two- and threefold higher, respectively, in islets cultured for 24 h in 11.1 mM glucose compared with those incubated in the presence of 5.5 mM glucose. Therefore, the previously observed increase in GLUT2 mRNA levels in the islets of rats made hyperglycemic by chronic infusion of glucose can be mimicked in vitro, implying that glucose regulates GLUT2 mRNA expression.     

Automated large-scale isolation, in vitro function and xenotransplantation of porcine islets of Langerhans

To evaluate the potential of utilizing porcine islet tissue as an alternative to human islet tissue for transplantation, we developed a method for the isolation of large amounts of highly purified porcine islets, and assessed the in vitro and in vivo function of the isolated islets after 1, 4, and 7 days of culture. The pancreatic duct of the splenic lobe was cannulated and distended by injection of Hanks' balanced salt solution containing 1.5 mg/ml collagenase. The pancreas was then processed by a modification of the automated digestion-filtration method developed in this laboratory, and with purification accomplished by Euro-Ficoll gradients (dialyzed Ficoll in Eurocollins solution), consisting of two layers of 1.108 and 1.091 g/cm3 density, topped with a layer of HBSS. The postpurification yield was 5203 +/- 645 (mean +/- SEM) islets per gram of pancreas with a number of islet equivalents (IE) per gram pancreas (islet equivalence: 150-microns-sized islets) of 3551 +/- 305, and a volume of 6.27 +/- 1.7 mm3 islet tissue per gram of pancreas. The islet purity exceeded 90%. Overnight-cultured, perifused porcine islets released 53.1 +/- 8.2 pM insulin/200 IE at 3.3 mM glucose, and 114.9 +/- 25.4 pM insulin/200 IE at 16.7 mM glucose (P less than 0.001 vs. basal output). When theophylline was added, insulin secretion increased to 264.2 +/- 63.2 pM/200 IE (P less than 0.001 vs. basal secretion and P less than 0.005 vs. secretion at 16.7 mM glucose). After 4 days of culture, the islets still responded to secretagogues. The functional integrity of the isolated islets was confirmed by reversal of diabetes in aL3T4 antibody-treated C57B/B6 diabetic mice: normoglycemia was promptly restored by transplanting 1000 overnight- or 7-day-cultured (24 degrees C) islets under the kidney capsule. These results suggest that continued improvements of porcine islet isolation and culture could permit the use of porcine islets in immunoalteration and immunoisolation studies that may lead to eventual human transplantation.     

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.     

Ionic, electrical, and secretory effects of inhibitors of arachidonic acid metabolism in mouse pancreatic beta-cells.

Mouse islets were used to study the effects of inhibitors of cyclooxygenase and lipoxygenase pathways on insulin release, ionic fluxes, and beta-cell membrane potential. The cyclooxygenase inhibitors, Na-salicylate and Na-acetylsalicylate, potentiated glucose-induced insulin release, despite a decrease in Ca influx evidenced by inhibition of the Ca-dependent electrical activity in beta-cells and 45Ca efflux from islets perifused with a medium containing Ca. This paradox can probably be explained by a mobilization of intracellular Ca (acceleration of 45Ca efflux in the absence of Ca) with subsequent activation of K+ channels (acceleration of 86Rb efflux) and repolarization of the membrane. These effects of salicylate could not be ascribed to a change in intracellular pH because they were not mimicked by 2-Cl-benzoate, which has a similar pK as salicylate but increased insulin release by stimulating Ca influx in beta-cells. Among the other cyclooxygenase inhibitors tested, indomethacin caused a slight potentiation of insulin release accompanied by marginal increases in 45Ca efflux and electrical activity, whereas flurbiprofen and ibuprofen were ineffective. Among the lipoxygenase inhibitors, compound BW 755c reversibly decreased glucose-induced insulin release by inhibiting Ca influx in beta-cells, but nordihydroguaiaretic acid had no effect. Inhibitors of arachidonic acid metabolism have effects on ionic fluxes and beta-cell membrane potential, which may explain some of the changes in insulin release they produce.     

Different effects of tolbutamide and diazoxide in alpha, beta-, and delta-cells within intact islets of Langerhans

Interaction between the different types of cells within the islet of Langerhans is vital for adequate control of insulin release. Once insulin secretion becomes defective, as in type 2 diabetes, the most useful drugs to increase insulin release are sulfonylureas. It is well-known that sulfonylureas block K(ATP) channels, which results in depolarization of the membrane that provokes calcium influx and increases intracellular calcium concentration ([Ca2+]i), which thereby triggers insulin secretion. The sulfonamide diazoxide produces the opposite effect: it activates K(ATP) channels, resulting in a decreased insulin secretion. Despite such evidence, little is known about the effect of sulfonylureas and sulfonamides in non-beta-cells of the islet of Langerhans. In this article, we describe the effects of tolbutamide and diazoxide on [Ca2+]i in alpha-, beta-, and delta-cells within intact islets of Langerhans. Tolbutamide elicits an increase in [Ca2+li in beta- and delta-cells, regardless of glucose concentrations. Remarkably, tolbutamide is without effect in alpha-cells. When diazoxide is applied, glucose-induced [Ca2+]i oscillations in beta- and delta-cells are abolished, whereas [Ca2+]i oscillations in alpha-cells remain unaltered. Furthermore, the existence of sulfonylurea receptors is demonstrated in beta-cells but not in alpha-cells by using binding of glybenclamide-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) combined with immunostaining for insulin and glucagon.     

Regulation of amino acid arginine transport by lipopolysaccharide and nitric oxide in intestinal epithelial IEC-6 cells

As a precursor for nitric oxide (NO) synthesis and an immune-enhancing nutrient, amino acid L-arginine plays a critical role in maintaining intestine mucosal integrity and immune functions in sepsis. However, the relationship between intestinal arginine transport and NO synthesis in sepsis remains unclear. In the present study, we investigated the effects of lipopolysaccharide (LPS) and NO on the arginine transport in cultured rat intestinal epithelial IEC-6 cell. Near-confluent IEC-6 cells were incubated with LPS (0-50 μg/ml) in serum-free Dulbecco’s modified Eagles’s medium, in the presence and absence of the NO donor sodium nitroprusside (SNP, 0–500 μmol/L) and the inducible nitric oxide synthase (iNOS) inhibitor N-ω-nitro-L-arginine (NNA, 0–1000 μmol/L) for various periods of time (0-48 hours). Arginine transport activity, arginine transporter CAT1 mRNA and protein levels were measured with transport assay, Northern blot analysis, and Western blot analysis, respectfully. LPS increased arginine transport activity in a time- and dose-dependent fashion. Prolonged incubation of LPS (24 hours, 25 μg/ml) resulted in a 3-fold increase of arginine transport activity (control: 28 ±5; LPS: 92 ±20 pmol/mg/ min, P < 0.05), with the System y⁺ as the predominant arginine transport system, and a 2-fold increase of System y⁺ CAT1 mRNA and transporter protein levels (P < 0.05). LPS increased the arginine transport System y⁺ maximal velocity (V_max, control: 1484 ±180; LPS: 2800 ±230 pmol/mg/min, P<0.05) without affecting the transport affinity (K_m, control: 76 ±8; LPS: 84 ±14 μmol/L, p = NS). The LPSinduced arginine transport activity was blocked by sodium nitroprusside (SNP) (control: 25 ±6; LPS: 97 ±26*; SNP: 22 ±0.4⁺; LPS+SNP: 33 ±10.3⁺ pmole/mg/min, *P < 0.01 and ⁺p = NS, compared with control). In contrary, the LPS-induced arginine transport activity was further augmented by NNA (control: 18 ±3.2; LPS: 59 ±2.7*; NNA: 26.3 ±5.8; LPS + NNA: 127 ±18⁺ pmol/mg/min; *P < 0.01 compared with control and ⁺P < 0.01 compared with control or LPS). LPS-stimulates arginine transport activity in IEC-6 cells via a mechanism that involves increase of transport System y⁺ mRNA levels and transporter protein levels. The LPS-stimulated arginine transport activity is regulated by the availability of nitric oxide. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 14–18, 2005 (oral presentation). This work was supported in part by The Society for Surgery of Alimentary Tract Career Development Award (M.P.) and National Institute of Diabetes and Digestive and Kidney Disease Grant DK-62165 (M.P.).     

异丙酚对大鼠不同脑区兴奋性和抑制性氨基酸类神经递质释放的影响

目的 评价异丙酚对大鼠皮质区、丘脑区、海马区和纹状体区兴奋性和抑制性氨基酸类神经递质释放的影响,探讨异丙酚麻醉的中枢机制.方法 Wistar大鼠12只,雌雄各半,体重300～350 g,麻醉后参照Paxinos和Waston定位图谱按照不同脑区坐标预埋微透析探针套管,恢复4 d后,随机分为2组(n=6):对照组和异丙酚组,分别尾静脉输注生理盐水6 ml/kg和异丙酚120 mg·kg-1·h-1,1 h后收集微透析液,采用高效液相色谱-电化学法测定微透析液中氨基酸类神经递质(谷氨酸、天冬氨酸、γ-氨基丁酸和甘氨酸)浓度.结果 与对照组比较,异丙酚组各脑区谷氨酸、天冬氨酸水平降低,γ-氨基丁酸、甘氨酸水平升高(P<0.05或0.01).结论 异丙酚抑制大鼠不同脑区(皮质区、丘脑区、海马区和纹状体区)兴奋性氨基酸类神经递质释放而增强抑制性氨基酸类神经递质释放,可能是其麻醉的中枢机制之一.     

cDNA and deduced amino acid sequence of mouse matrix gla protein: one of five glutamic acid residues potentially modified to gla is not conserved in the mouse sequence.

A cDNA library was constructed using the mouse osteoblastic cell line MC3T3-E1 treated with 1 alpha,25-dihydroxyvitamin D3, based on the finding that the treatment increased ninefold the expression of 0.7 kb matrix gla protein (MGP) mRNA. cDNA clones encoding mouse MGP were isolated from the library. The nucleotide sequence showed an open reading frame of 312 nucleotides encoding 104 amino acids. Murine MGP shared 84-89% amino acid sequence homology with bovine, rat, and human MGP. However, there are five glutamic acid residues potentially modified to gamma-carboxyglutamic acid (gla) in those species; in murine MGP, lysine replaced glutamic acid 37. Also, an extra tyrosine was added at the carboxyl terminus. The significance of the substitution is discussed in relation to the gamma-carboxylation sites in MGP protein.     

Effect of dynorphin on insulin and somatostatin secretion, calcium uptake, and c-AMP levels in isolated rat islets of Langerhans

Dynorphin-[1-13], at concentrations of 5.8 X 10(-12) to 5.8 X 10(-9) M, stimulated insulin secretion from isolated islets of Langerhans of the rat, in medium containing 6 mM glucose. Higher concentrations of dynorphin had no significant effect on secretion. Dynorphin (5.8 X 10(-9) M) was unable to initiate insulin release from islets in the presence of 2 mM glucose, or to increase insulin secretion further in the presence of 20 mM glucose or 6 and 12 mM glyceraldehyde. Dynorphin-induced insulin secretion from islets was blocked by verapamil (5 microM) or by chlorpropamide (72 microM), but not by a mu opiate receptor antagonist, naloxone (0.11 microM), or by ICI 154129, a specific antagonist for the delta receptor (0.25 microM). Dynorphin had no effect on islet somatostatin secretion, under conditions in which insulin secretion was greatly stimulated. Glucose (20 mM) and glyceraldehyde (6 and 12 mM) significantly increased both insulin and somatostatin secretion. Dynorphin (5.8 X 10(-9) M) increased 45Ca2+ uptake into islets, and also increased intracellular islet c-AMP levels. These changes persisted when higher concentrations of dynorphin were used. These results suggest that (1) dynorphin is a very potent stimulus for insulin secretion; (2) dynorphin does not affect somatostatin secretion in static incubations of islets, in the same way as does glucose and glyceraldehyde; (3) dynorphin's effects may involve increased calcium ion movement and can be blocked by verapamil; (4) dynorphin can also increase islet c-AMP, and could thereby modulate the responsiveness of other secretagogues; (5) the actions of dynorphin on insulin secretion are not mediated by delta or mu opiate receptors in islets.