Glucose induces anion conductance and cytosol-to-membrane transposition of ICln in INS-1E rat insulinoma cells.

The metabolic coupling of insulin secretion by pancreatic beta cells is mediated by membrane depolarization due to increased glucose-driven ATP production and closure of K(ATP) channels. Alternative pathways may involve the activation of anion channels by cell swelling upon glucose uptake. In INS-1E insulinoma cells superfusion with an isotonic solution containing 20 mM glucose or a 30% hypotonic solution leads to the activation of a chloride conductance with biophysical and pharmacological properties of anion currents activated in many other cell types during regulatory volume decrease (RVD), i.e. outward rectification, inactivation at positive membrane potentials and block by anion channel inhibitors like NPPB, DIDS, 4-hydroxytamoxifen and extracellular ATP. The current is not inhibited by tolbutamide and remains activated for at least 10 min when reducing the extracellular glucose concentration from 20 mM to 5 mM, but inactivates back to control levels when cells are exposed to a 20% hypertonic extracellular solution containing 20 mM glucose. This chloride current can likewise be induced by 20 mM 3-Omethylglucose, which is taken up but not metabolized by the cells, suggesting that cellular sugar uptake is involved in current activation. Fluorescence resonance energy transfer (FRET) experiments show that chloride current activation by 20 mM glucose and glucose-induced cell swelling are accompanied by a significant, transient redistribution of the membrane associated fraction of ICln, a multifunctional 'connector hub' protein involved in cell volume regulation and generation of RVD currents.     

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.     

INS-1E细胞葡萄糖毒性模型的建立

目的:建立胰岛细胞系INS-1E细胞的葡萄糖毒性模型。方法:将INS-1E细胞分别在不同葡萄糖浓度(5.5 mmol/L、16.7mmol/L、25 mmol/L、30 mmol/L)的1640完全培养基中培养不同时间(48 h、72 h、96 h、120 h),分别在不同时间点取细胞进行细胞功能检测,实时荧光定量PCR法检测胰岛素m RNA的表达,ELISA检测葡萄糖刺激的胰岛素的分泌。结果:与对照组相比,高糖浓度(5.5 mmol/L、16.7 mmol/L、25 mmol/L、30 mmol/L)培养基中培养48 h后,INS-1E细胞的胰岛素合成和分泌的功能均增加(P均0.05),随着培养基中葡萄糖浓度的升高以及培养时间的延长,INS-1E细胞胰岛素合成及分泌的功能逐渐下降,当在葡萄糖浓度为30 mmol/L的培养基中培养120 h后,胰岛素m RNA合成及葡萄糖刺激的胰岛素分泌均显著降低(P均0.01)。结论:INS-1E细胞在30 m M的葡萄糖中培养120 h形成稳定的葡萄糖毒性模型。     

Pro-nucleotide inhibitors of adenylyl cyclases in intact cells

9-substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3'-phosphoryl derivatives of 2',5'-dideoxyadenosine (2',5'-dd-Ado) and beta-l-2',5'-dd-Ado, protected 5'-phosphoryl derivatives of beta-l-2',3'-dd-Ado, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl- or three S-acyl-2-thioethyl-substituents. These pro-nucleotides were tested for their capacity to block forskolin-induced increases in [(3)H]cAMP in OB1771 and F442A preadipocytes and human macrophages prelabeled with [(3)H]adenine. A striking selectivity for 2',5'-dd-Ado-3'-phosphoryl derivatives was observed. Cyclosaligenyl-derivatives (IC(50) approximately 2 microm) were much less potent than S-acyl-2-thioethyl-derivatives. Best studied of these was 2',5'-dd-Ado-3'-O-bis(S-pivaloyl-2-thioethyl)-phosphate, which blocked [(3)H]cAMP formation in preadipocytes (IC(50) approximately 30 nm) and suppressed opening of cAMP-dependent Cl(-) channels in cardiac myocytes (IC(50) approximately 800 nm). None of the pro-nucleotides inhibited adenylyl cyclase per se, whether isolated from rat brain or OB1771 cells. These compounds exhibit the hallmarks of prodrugs. Data suggest they are taken up, are deprotected, and are converted to a potent inhibitory form to inhibit adenylyl cyclase, but only by intact cells. The availability and characteristics of these prodrugs should make them useful for blocking cAMP-mediated pathways in intact cell systems, in biochemical, pharmacological, and potentially therapeutic contexts.     

Uncoupling protein-2 attenuates glucose-stimulated insulin secretion in INS-1E insulinoma cells by lowering mitochondrial reactive oxygen species

Glucose-stimulated insulin secretion (GSIS) by pancreatic β cells is regulated by mitochondrial uncoupling protein-2 (UCP2), but opposing phenotypes, GSIS improvement and impairment, have been reported for different Ucp2-ablated mouse models. By measuring mitochondrial bioenergetics in attached INS-1E insulinoma cells with and without UCP2, we show that UCP2 contributes to proton leak and attenuates glucose-induced rises in both respiratory activity and the coupling efficiency of oxidative phosphorylation. Strikingly, the GSIS improvement seen upon UCP2 knockdown in INS-1E cells is annulled completely by the cell-permeative antioxidant MnTMPyP. Consistent with this observation, UCP2 lowers mitochondrial reactive oxygen species at high glucose levels. We conclude that UCP2 plays both regulatory and protective roles in β cells by acutely lowering GSIS and chronically preventing oxidative stress. Our findings thus provide a mechanistic explanation for the apparently discrepant findings in the field.     

Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms

During angiogenesis, endothelial cells (ECs) degrade their surrounding extracellular matrix (ECM) to facilitate invasion. How interactions between ECs and other cells within their microenvironment facilitate this process is only partially understood. We have utilized a tractable 3D co-culture model to investigate the proteolytic mechanisms by which pre-committed or more highly committed mesenchymal cells stimulate capillary formation. On their own, ECs invade their surrounding matrix, but do not form capillaries. However, in the presence of either mesenchymal stem cells (MSCs) or fibroblasts, ECs form polarized, tubular structures that are intimately associated with mesenchymal cells. Further, ECs up-regulate gene expression of several extracellular proteases upon co-culture with either mesenchymal cell type. The administration of both broad spectrum and specific protease inhibitors demonstrated that MSC-stimulated capillary formation relied solely on membrane-type matrix metalloproteinases (MT-MMPs) while fibroblast-mediated sprouting proceeded independent of MMP inhibition unless the plasminogen activator/plasmin axis was inhibited in concert. While other studies have established a role for the ECM itself in dictating proteolysis and matrix degradation during capillary morphogenesis, the present study illustrates that heterotypic cellular interactions within the microenvironment can direct the proteolytic mechanisms required for capillary formation.     

Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells

Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane electrical activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidation and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addition, citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy production and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.     

Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion

Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic beta-cells (INS-1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS-1E mitochondria [Affourtit and Brand (2006) Biochem. J. 393, 151-159]. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazol-sensitive, oligomycin-resistant respiration) was nearly four times higher in INS-1E cells than in myoblasts. This relatively high leak activity was decreased more than 30% upon knock-down of UCP2 (uncoupling protein-2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20% of INS-1E respiration. UCP2 knock-down enhanced GSIS (glucose-stimulated insulin secretion), consistent with a role for UCP2 in beta-cell physiology. We propose that the high mitochondrial proton leak in beta-cells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion.     

Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential

Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (ΔΨ_m) during GSIS are lacking. Using high resolution respirometry and parallel ΔΨ_m monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and ΔΨ_m during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the “toxic” effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, ΔΨ_m jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10–20 mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.     

Glucose increases extracellular [Ca2+] in rat insulinoma (INS-1E) pseudoislets as measured with Ca2+-sensitive microelectrodes

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors.     

Glucose and insulin stimulate heparin-releasable lipoprotein lipase activity in mouse islets and INS-1 cells. A potential link between insulin resistance and beta-cell dysfunction

Lipoprotein lipase (LpL) provides tissues with triglyceride-derived fatty acids. Fatty acids affect beta-cell function, and LpL overexpression decreases insulin secretion in cell lines, but whether LpL is regulated in beta-cells is unknown. To test the hypothesis that glucose and insulin regulate LpL activity in beta-cells, we studied pancreatic islets and INS-1 cells. Acute exposure of beta-cells to physiological concentrations of glucose stimulated both total cellular LpL activity and heparin-releasable LpL activity. Glucose had no effect on total LpL protein mass but instead promoted the appearance of LpL protein in a heparin-releasable fraction, suggesting that glucose stimulates the translocation of LpL from intracellular to extracellular sites in beta-cells. The induction of heparin-releasable LpL activity was unaffected by treatment with diazoxide, an inhibitor of insulin exocytosis that does not alter glucose metabolism but was blocked by conditions that inhibit glucose metabolism. In vitro hyperinsulinemia had no effect on LpL activity in the presence of low concentrations of glucose but increased LpL activity in the presence of 20 mm glucose. Using dual-laser confocal microscopy, we detected intracellular LpL in vesicles distinct from those containing insulin. LpL was also detected at the cell surface and was displaced from this site by heparin in dispersed islets and INS-1 cells. These results show that glucose metabolism controls the trafficking of LpL activity in beta-cells independent of insulin secretion. They suggest that hyperglycemia and hyperinsulinemia associated with insulin resistance may contribute to progressive beta-cell dysfunction by increasing LpL-mediated delivery of lipid to islets.     

Two-dimensional gel proteome reference map of INS-1E cells

The insulin-producing INS-1E rat cell line is widely used as a model for studying β-cells. It is a well-characterized cell line, mainly used in diabetes research. We established a 2-DE reference map for INS-1E cells. Using MALDI-TOF/TOF-MS/MS, we identified 546 spots. These included various proteins with an important role in β-cell physiology and with known roles as crucial proteins for diabetes development. We believe that the availability of this reference map will enhance our knowledge of β-cell physiology.     

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.     

Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms

Neurotensin, bradykinin and somatostatin inhibited in a time- and concentration-dependent manner prostaglandin E1- or forskolin-stimulated cAMP production in neuroblastoma N1E115 cells. Cell treatment with 1 microgram/ml pertussis toxin for 6 hours reversed the inhibition elicited by peptides after short incubation periods (less than or equal to 1 min) but, in contrast, had no effect after longer incubation periods (greater than or equal to 3 min). Fluoroaluminate also inhibited prostaglandin E1-stimulated cAMP production in N1E115 cells, and this effect was not reversed by pertussis toxin. The 6 hour treatment with pertussis toxin was shown to be sufficient to ADP ribosylate virtually all of the 41 kD protein substrate corresponding to the alpha subunit of Gi. Protein kinase C activation with phorbol ester did not inhibit basal or stimulated cAMP production. Our data point to the existence of both pertussis toxin sensitive and insensitive mechanisms of neuropeptide-mediated inhibition of cAMP formation in N1E115 cells. The toxin insensitive response is not mediated by protein kinase C. The possibility is discussed that it results from the activation of a pertussis toxin insensitive G protein.     

Palmitate-induced changes in protein expression of insulin secreting INS-1E cells

Elevated blood levels of glucose and lipids in individuals with type 2 diabetes mellitus have been observed to cause impairment of insulin secretion from pancreatic β-cells. Chronic exposure to either of the circulating fatty acid oleate or palmitate has different effects on the β-cell. Whereas palmitate causes functional impairment of the β-cell and apoptosis, oleate has only minor negative effects on β-cell function and mass. The aim of the present study was to delineate mechanisms by which the fatty acids affect the β-cell differently. In particular, the aim was to identify β-cell proteins exclusively regulated by palmitate. INS-1E cells were cultured for 24 h in medium supplemented with palmitate or oleate. After culture, cells were lysed and subjected to two-dimensional gel electrophoresis. Proteins specifically regulated by palmitate were excised from the gel and identified by peptide mass fingerprinting using MALDI-TOF MS. Proteins exclusively regulated by palmitate were classified into proteins of carbohydrate or protein metabolism and Ca²⁺ or mRNA binding proteins. The specific palmitate-induced down-regulation of enzymes of glycolysis, proteins of protein turnover and anti-apoptotic protein may contribute to explain the different effects exerted by palmitate and oleate on β-cell function and mass.