The Rab3-interacting molecule RIM is expressed in pancreatic beta-cells and is implicated in insulin exocytosis

The putative Rab3 effector RIM (Rab3-interacting molecule) was detected by Northern blotting, RT-PCR and Western blotting in native pancreatic beta-cells as well as in the derived cell lines INS-1E and HIT-T15. RIM was localized on the plasma membrane of INS-1E cells and beta-cells. An involvement of RIM in insulin exocytosis was indicated by transfection experiments of INS-1E cells with the Rab3 binding domain of RIM. This domain enhanced glucose-stimulated secretion in intact cells and Ca(2+)-stimulated exocytosis in permeabilized cells. Co-expression of Rab3A reversed the effect of RIM on exocytosis. These results suggest an implication of RIM in the control of insulin secretion.     

L-亮氨酸对克隆的胰岛β细胞株INS-1E细胞分泌胰岛素的葡萄糖依赖性研究

目的:探讨L-亮氨酸对克隆的胰岛β细胞株INS-1E细胞分泌胰岛素的刺激作用及其葡萄糖依赖性。方法:INS-1E细胞经传代培养2 d后,在Krebs-Ringer缓冲液中37℃培养箱预培养30 min,再用含有不同浓度葡萄糖和不同浓度L-亮氨酸的改良Krebs-Ringer缓冲液培养60 min,然后留取上清液进行胰岛素测定。结果:L-亮氨酸在0.1~10 mmol.L-1范围不增加16.7mmol.L-1葡萄糖刺激的INS-1E细胞的胰岛素分泌,仅20 mmol.L-1的L-亮氨酸促进葡萄糖诱导的胰岛素分泌;10 mmol.L-1L-亮氨酸在1.1、3.3、6.7 mmol.L-1葡萄糖存在的情况下促进INS-1E细胞的胰岛素分泌,而在11.1、16.7、25 mmol.L-1葡萄糖存在的情况下无促进胰岛素分泌的作用。结论:本研究显示在无刺激胰岛素分泌的葡萄糖浓度条件下,10 mmol.L-1L-亮氨酸即显示了刺激INS-1E细胞分泌胰岛素的作用,在较高葡萄糖的条件下,10 mmol.L-1L-亮氨酸的作用减弱或消失。     

Mitochondria-derived glutamate at the interplay between branched-chain amino acid and glucose-induced insulin secretion

In pancreatic beta-cells, glutamate has been proposed to mediate insulin secretion as a glucose-derived factor, although it is also considered for its sole catabolic function. Hence, changes in cellular glutamate levels are a matter of debate. Here, we investigated the effects of glucose and the glutamate precursor glutamine on kinetics of glutamate levels together with insulin secretion in INS-1E beta-cells. Preincubation at low (1 mM) glucose resulted in reduced cellular glutamate levels, which were doubled by exposure to glutamine. In glutamine-deprived cells, 5 mM glucose restored glutamate concentrations. Incubation at 15 mM glucose increased cellular glutamate, along with stimulation of insulin secretion, following both glutamine-free and glutamine-rich preincubations. Nuclear magnetic resonance (NMR) spectroscopy of INS-1E cells exposed to 15 mM D-[1-(13)C]glucose revealed glutamate as the major glucose metabolic product. Branched-chain amino acids, such as leucine, reduced cellular glutamate levels at low and intermediate glucose. This study demonstrates that glucose stimulates glutamate generation, whereas branched-chain amino acids promote competitive glutamate expenditure.     

Oxidized but not acetylated low-density lipoprotein reduces preproinsulin mRNA expression and secretion of insulin from HIT-T15 cells

We examined the effect of oxidized low-density lipoprotein (oxLDL) on the insulin secretion in the culture of HIT-T15 cell line, an islet beta-cell line derived from a hamster pancreatic tumor. In order to check the uptake of modified LDL by HIT-T15 cells, we prepared DiI-labeled native LDL (nLDL), acetylated LDL (AcLDL), and oxLDL. After the addition of each LDL into the cultures of HIT-T15 cells, fluorescence microscopic study was done. It was suggested that AcLDL and oxLDL were taken up by HIT-T15 cells, as well as nLDL. mRNA expression of the LDL receptor, CD36, and SR-B1 was detected in HIT-T15 by RT-PCR. The medium insulin level was measured in the culture of HIT-T15 cells with each LDL. oxLDL significantly reduced the insulin secretion stimulated by various concentrations of glucose, the intracellular content of insulin, and the expression of preproinsulin mRNA compared to the control cultures without LDL addition. In contrast, nLDL and AcLDL had no effect on the insulin secretion, the intracellular insulin level, or the expression of preproinsulin mRNA. MTT assay findings (reflecting cell numbers) were not different between cultures with and without LDLs. These results indicated that oxLDL disturbed the insulin metabolism of HIT-T15 cells.     

Cytosolic and mitochondrial malic enzyme isoforms differentially control insulin secretion

In islet beta-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP(+)-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD(+)-preferred (ME2) expressed at approximately 50%, and the NADP(+)-specific (ME3) at approximately 10% compared with ME1. Selective reduction (89 +/- 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mM:41 +/- 6%, p < 0.01) and amino acid (4 mM glutamine +/- 10 mM leucine: 39 +/- 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 +/- 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 +/- 4% (p < 0.01). Modeling of the metabolism of [U-(13)C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.     

Increased methylation of endogenous 20-kDa protein in HIT beta-cell during insulin secretion

Enzymatic methylation of endogenous proteins in clonal pancreatic beta-cell, HIT-T15, was investigated. When cell extract incubated with S-adenosyl-L-[methyl-3H]methionine was subjected to SDS-PAGE followed by fluorography, endogenous 20-kDa protein was highly [methyl-3H]-labeled. The increase of methylation was correlated with insulin secretion, when the cells were treated with secretagogue; at 5.5mM glucose, insulin secretion increased by 2.5-fold, while the 20-kDa methylation to about 3.2-fold. In the case of forskolin, another secretagogue, at 0.1mM, the methylation increased by approximately 4.5-fold. This increase of 20-kDa methylation was inhibited when the cells were treated with 3mM EGTA to inhibit insulin secretion by depleting extracellular calcium ion, indicating intercausal relation between methylation and insulin secretion. The [methyl-3H]-labeled amino acids were identified by thin layer chromatography as N(G)-methylated arginines. While arginyl residues in Gly-Arg-Gly sequence are known to be posttranslationally methylated, a synthetic nonapeptide, GGRGRGRGG, competed with the 20-kDa methylation; at 1 and 10 micro M nonapeptides, 62% and 78% of 20-kDa methylation were inhibited, respectively. Furthermore, Western immunoblot analysis of HIT cell extract against GGRGRGRGG antibodies strongly immunoreacted with the 20-kDa protein. These results suggested that methylation of the endogenous 20-kDa protein might play some role in insulin secretion.     

Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane.     

2-Ketoisocaproate transport in insulin-secreting cells

The transport of the nutrient secretagogue 2-ketoisocaproate (KIC) was studied in isolated rat pancreatic islets and in the HIT-T15 insulinoma cell line using an oil-filtration technique. In both islets and HIT-T15 cells, KIC uptake was a slow process, not reaching equilibrium within 10 min KIC transport was not dependent upon Na⁺ in the medium, was not inhibited by -cyano-4-hydroxy-cinnamate nor by 2-amino-2-norborane carboxylic acid (BCH) and did not appear to be electrogenic. Evidence was obtained to suggest that KIC uptake occurred via passive diffusion into the cell of the undissociated acid species. This possibility was supported by the apparent unsaturability of KIC uptake in HIT-T15 cells. Addition of 10–30 mM KIC to dispersed islets cells or HIT-T15 cells produced a rapid intracellular acidification. In islets, the rate of transport of 10 mM KIC was comparable with oxidation rate of the keto-acid suggesting that uptake could be rate-limiting factor for KIC oxidation and thus stimulated insulin release. However, in HIT-T15 cells, the rate of uptake of KIC greatly exceeded the oxidation rate. The low rate of KIC oxidation could explain the poor secretory response of HIT-T15 cells to KIC     

Adrenomedullin inhibits insulin exocytosis via pertussis toxin-sensitive G protein-coupled mechanism

Direct effects of adrenomedullin on insulin secretion from pancreatic beta-cells were investigated using a differentiated insulin-secreting cell line INS-1. Adrenomedullin (1-100 pM) inhibited insulin secretion at both basal (3 mM) and high (15 mM) glucose concentrations, although this inhibitory effect was not observed at higher concentrations of adrenomedullin. The inhibition of glucose-induced insulin secretion by adrenomedullin was restored with 12-h pretreatment with 1 microg/ml pertussis toxin (PTX), suggesting that this effect could be mediated by PTX-sensitive G proteins. Cellular glucose metabolism evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colorimetric assay was not affected by adrenomedullin at concentrations that inhibited insulin secretion. Moreover, electrophysiological studies revealed that 10 pM adrenomedullin had no effect on membrane potential, voltage-gated calcium currents, or cytosolic calcium concentration induced by 15 mM glucose. Finally, insulin release induced by cAMP-raising agents, such as forskolin plus 3-isobutyl-1-methylxanthine or the calcium ionophore ionomycin, was significantly inhibited by 10 and 100 pM adrenomedullin. In conclusion, adrenomedullin at picomolar concentrations directly inhibited insulin secretion from beta-cells. This effect is likely due to the inhibition of insulin exocytosis through the activation of PTX-sensitive G proteins.     

Inhibition of fatty acid translocase cluster determinant 36 (CD36), stimulated by hyperglycemia, prevents glucotoxicity in INS-1 cells

The purpose of the present study was to determine whether exposure of pancreatic islets to glucotoxic conditions changes fatty acid translocase cluster determinant 36 (CD36) and examine the role of CD36 on the induction of glucotoxicity. We measured the changes of CD36 and insulin secretion in high glucose (30 mM) exposed INS-1 cells and CD36 suppressed INS-1 cells by transfection of CD36 siRNA. The intracellular peroxide level of INS-1 cells increased in the high glucose media compared to normal glucose (5.6mM) media. The mRNA levels of insulin and PDX-1, as well as glucose stimulated insulin secretion (GSIS) were decreased in INS-1 cells exposed to high glucose media compared to normal glucose media, while CD36 and palmitate uptake were significantly elevated with exposure to high glucose media for 12h. The inhibition of CD36 reversed the decreased GSIS and intracellular peroxide level in INS-1 cells. These results suggest that high glucose may exacerbate glucotoxicity via increasing fatty acid influx by elevation of CD36 expression, and that CD36 may be a possible target molecule for preventing glucotoxicity in pancreatic beta-cells.     

The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic beta-cells: action of synaptotagmin at low micromolar calcium.

The Ca2+- and phospholipid-binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca2+-affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 microM cytosolic free Ca2+. The well-characterized synaptotagmin isoforms I and II are present in pancreatic beta-cell lines RINm5F, INS-1 and HIT-T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin-containing secretory granules whereas only minor amounts were found on synaptic vesicle-like microvesicles. Antibodies or Fab-fragments directed against the Ca2+-dependent phospholipid binding site of the first C2 domain of synaptotagmin I or II inhibited Ca2+-stimulated, but not GTPgammaS-induced exocytosis from streptolysin-O-permeabilized INS-1 and HIT-T15 cells. Transient expression of wild-type synaptotagmin II did not alter exocytosis in HIT-T15 cells. However, mutations in the Ca2+-dependent phospholipid binding site of the first C2 domain (Delta180-183, D231S) again inhibited only Ca2+-, but not GTPgammaS-evoked exocytosis. In contrast, mutations in the IP4-binding sites of the second C2 domain (Delta325-341; K327,328, 332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Delta180-183/K327,328,332Q) induced greater inhibition than mutant Delta180-183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.     

Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.

alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.     

Intracellular depletion of insulin by oleate is due to an inhibited synthesis and not to an increased secretion

In the polyclonal rat pancreatic beta-cell line INS-1, immunoreactive insulin (IRI, insulin and its precursors) and C-peptide (surrogate marker for mature insulin) were quantified after a 1-h incubation at 16.7 mM glucose with or without oleate. Oleate caused a 20% decrease (P 相似文献   

Dehydroepiandrosterone inhibits intracellular calcium release in beta-cells by a plasma membrane-dependent mechanism

Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.     

Role of mitochondrial phosphate carrier in metabolism-secretion coupling in rat insulinoma cell line INS-1

In pancreatic β-cells, glucose-induced mitochondrial ATP production plays an important role in insulin secretion. The mitochondrial phosphate carrier PiC is a member of the SLC25 (solute carrier family 25) family and transports Pi from the cytosol into the mitochondrial matrix. Since intramitochondrial Pi is an essential substrate for mitochondrial ATP production by complex V (ATP synthase) and affects the activity of the respiratory chain, Pi transport via PiC may be a rate-limiting step for ATP production. We evaluated the role of PiC in metabolism-secretion coupling in pancreatic β-cells using INS-1 cells manipulated to reduce PiC expression by siRNA (small interfering RNA). Consequent reduction of the PiC protein level decreased glucose (10 mM)-stimulated insulin secretion, the ATP:ADP ratio in the presence of 10 mM glucose and elevation of intracellular calcium concentration in response to 10 mM glucose without affecting the mitochondrial membrane potential (Δψm) in INS-1 cells. In experiments using the mitochondrial fraction of INS-1 cells in the presence of 1 mM succinate, PiC down-regulation decreased ATP production at various Pi concentrations ranging from 0.001 to 10 mM, but did not affect Δψm at 3 mM Pi. In conclusion, the Pi supply to mitochondria via PiC plays a critical role in ATP production and metabolism-secretion coupling in INS-1 cells.     

Glucolipotoxicity in INS-1E cells is counteracted by carnitine palmitoyltransferase 1 over-expression

Effects of non-esterified fatty acids (FAs) are accentuated when applied together with elevated glucose through preferential use of glucose as fuel, which leads to decreased oxidation of FAs. We examined how over-expression of the mitochondrial FA transporter carnitine palmitoyltransferase 1 (CPT1) affects glucose-stimulated insulin secretion (GSIS), apoptosis and ER stress in INS-1E cells cultured in the presence of elevated levels of glucose and palmitate. INS-1E cells were infected with Tet-ON regulated adenovirus containing CPT1 and cultured for 48 h in the presence of 0.5 mM palmitate and 20 mM glucose. Over-expressing CPT1 lowered basal insulin secretion in a dose-dependent manner thereby improving GSIS from INS-1E cells. Also, apoptosis was alleviated and ER-stress markers p-eIF2α and CHOP were decreased in cells over-expressing CPT1. We conclude that regulated over-expression of CPT1 is beneficial for glucolipotoxic beta-cells.     

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.     

Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.     

SSTR2 mediates the somatostatin-induced increase in intracellular Ca(2+) concentration and insulin secretion in the presence of arginine vasopressin in clonal beta-cell HIT-T15

The effects of somatostatin (SRIF) are mediated through the seven transmembrane receptor family that signals via Gi/Go. To date, five distinct SRIF receptors have been characterized and designated SSTR1-5. We have characterized the SRIF receptor that mediates the increase in [Ca(2+)](i) and insulin secretion in HIT-T15 cells (Simian virus 40-transformed Syrian hamster islets) using high affinity, subtype selective agonists for SSTR1 (L-797,591), SSTR2 (L-779,976), SSTR3 (L-796,778), SSTR4 (L-803,087), SSTR5 (L-817,818) and PRL-2903, a specific SSTR2 antagonist. In the presence of arginine vasopressin (AVP), SRIF increased [Ca(2+)](i) and insulin secretion. Treatment with the SSTR2 agonist L-779,976 resulted in similar responses to SRIF. In addition, L-779,976 increased both [Ca(2+)](i) and insulin secretion in a dose-dependent manner. Treatment with L-779,976 alone did not alter [Ca(2+)](i) or basal insulin secretion. In the presence of AVP, all other SRIF receptor agonists failed to increase [Ca(2+)](i) and insulin secretion. The effects of SRIF and L-779,976 were abolished by the SSTR2 antagonist PRL-2903. Our results suggest that the mechanism underlying SRIF-induced insulin secretion in HIT-T15 cells be mediated through the SSTR2.