Beta-cell PDE3B regulates Ca2+-stimulated exocytosis of insulin

cAMP signaling is important for the regulation of insulin secretion in pancreatic beta-cells. The level of intracellular cAMP is controlled through its production by adenylyl cyclases and its breakdown by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE3B is involved in the regulation of nutrient-stimulated insulin secretion. Here, aiming at getting deeper functional insights, we have examined the role of PDE3B in the two phases of insulin secretion as well as its localization in the beta-cell. Depolarization-induced insulin secretion was assessed and in models where PDE3B was overexpressed [islets from transgenic RIP-PDE3B/7 mice and adenovirally (AdPDE3B) infected INS-1 (832/13) cells], the first phase of insulin secretion, occurring in response to stimulation with high K(+) for 5 min, was significantly reduced ( approximately 25% compared to controls). In contrast, in islets from PDE3B(-/-) mice the response to high K(+) was increased. Further, stimulation of isolated beta-cells from RIP-PDE3B/7 islets, using successive trains of voltage-clamped depolarizations, resulted in reduced Ca(2+)-triggered first phase exocytotic response as well as reduced granule mobilization-dependent second phase, compared to wild-type beta-cells. Using sub-cellular fractionation, confocal microscopy and transmission electron microscopy of isolated mouse islets and INS-1 (832/13) cells, we show that endogenous and overexpressed PDE3B is localized to insulin granules and plasma membrane. We conclude that PDE3B, through hydrolysis of cAMP in pools regulated by Ca(2+), plays a regulatory role in depolarization-induced insulin secretion and that the enzyme is associated with the exocytotic machinery in beta-cells.     

Beta-cell-targeted overexpression of phosphodiesterase 3B in mice causes impaired insulin secretion, glucose intolerance, and deranged islet morphology

The second messenger cAMP mediates potentiation of glucose-stimulated insulin release. Use of inhibitors of cAMP-hydrolyzing phosphodiesterase (PDE) 3 and overexpression of PDE3B in vitro have demonstrated a regulatory role for this enzyme in insulin secretion. In this work, the physiological significance of PDE3B-mediated degradation of cAMP for the regulation of insulin secretion in vivo and glucose homeostasis was investigated in transgenic mice overexpressing PDE3B in pancreatic beta-cells. A 2-fold overexpression of PDE3B protein and activity blunted the insulin response to intravenous glucose, resulting in reduced glucose disposal. The effects were "dose"-dependent because mice overexpressing PDE3B 7-fold failed to increase insulin in response to glucose and hence exhibited pronounced glucose intolerance. Also, the insulin secretory response to intravenous glucagon-like peptide 1 was reduced in vivo. Similarly, islets stimulated in vitro exhibited reduced insulin secretory capacity in response to glucose and glucagon-like peptide 1. Perifusion experiments revealed that the reduction specifically affected the first phase of glucose-stimulated insulin secretion. Furthermore, morphological examinations demonstrated deranged islet cytoarchitecture. In conclusion, these results are consistent with an essential role for PDE3B in cAMP-mediated regulation of insulin release and glucose homeostasis.     

Oleanolic acid enhances insulin secretion in pancreatic beta-cells

We investigated the effect of oleanolic acid, a plant-derived triterpenoid, on insulin secretion and content in pancreatic beta-cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS-1 832/13 cells and enhanced acute glucose-stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin-4 under glucose-stimulated conditions, yet neither Ca(2+) nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti-diabetic properties of this natural product.     

The calcium/calmodulin-dependent phosphodiesterase PDE1C down-regulates glucose-induced insulin secretion.

To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic beta-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in betaTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion.     

Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic beta-cell: a computational approach

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic beta-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic beta-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca(2+)) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca(2+) oscillations in the beta-cell. Slow Ca(2+) oscillations (<1 min(-1)) produce low-frequency cAMP oscillations, and faster Ca(2+) oscillations (>3-4 min(-1)) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca(2+) oscillations. In contrast, observed antiphasic Ca(2+) and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca(2+)-dependent AC and PDE activation in coupling of Ca(2+) and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 beta-cells.     

Total parenteral nutrition-stimulated activity of inducible nitric oxide synthase in rat pancreatic islets is suppressed by glucagon-like peptide-1.

Long-term total parenteral nutrition (TPN) is associated with elevated plasma lipids and a marked decrease of glucose-stimulated insulin release. Since nitric oxide (NO) has been shown to modulate negatively the insulin response to glucose, we investigated the influence of TPN-treatment on isoforms of islet NO-synthase (NOS) activities in relation to the effect of glucagon-like peptide-1 (GLP-1), a known activator of glucose-stimulated insulin release. Isolated islets from TPN rats incubated at basal glucose (1 mmol/l) showed a modestly increased insulin secretion accompanied by an enhanced accumulation of islet cAMP and cGMP. In contrast, TPN islets incubated at high glucose (16.7 mmol/l) displayed an impaired insulin secretion and a strong suppression of islet cAMP content. Moreover, islet inducible NOS (iNOS) as well as islet cGMP content were greatly increased in these TPN islets. A dose-response study of GLP-1 with glucose-stimulated islets showed that GLP-1 could overcome and completely restore the impaired insulin release in TPN islets, bringing about a marked increase in islet cAMP accumulation concomitant with heavy suppression of both glucose-stimulated increase in islet cGMP content and the activities of constitutive NOS (cNOS) and iNOS. These effects of GLP-1 were mimicked by dibutyryl-cAMP. The present results show that the impaired insulin response of glucose-stimulated insulin release seen after TPN treatment is normalized by GLP-1. This beneficial effect of GLP-1 is most probably exerted by a cAMP-induced suppression of both iNOS and cNOS activities in these TPN islets.     

Syntaxin 4 facilitates biphasic glucose-stimulated insulin secretion from pancreatic beta-cells

Numerous overexpression studies have recently implicated Syntaxin 4 as an effector of insulin secretion, although its requirement in insulin granule exocytosis is unknown. To address this, islets from Syntaxin 4 heterozygous (-/+) knockout mice were isolated and compared with islets from wild-type mice. Under static incubation conditions, Syntaxin 4 (-/+) islets showed a 60% reduction in glucose-stimulated insulin secretion compared with wild-type islets. Perifusion analyses revealed that Syntaxin 4 (-/+) islets secreted 50% less insulin during the first phase of glucose-stimulated insulin secretion and that this defect could be fully restored by the specific replenishment of recombinant Syntaxin 4. This essential role for Syntaxin 4 in secretion from the islet was localized to the beta-cells because small interfering RNA-mediated depletion of Syntaxin 4 in MIN6 beta-cells abolished glucose-stimulated insulin secretion. Moreover, immunofluorescent confocal microscopy revealed that Syntaxin 4 was principally localized to the beta-cells and not the alpha-cells of the mouse islet. Remarkably, islets isolated from transgenic mice that express 2.4-fold higher levels of Syntaxin 4 relative to wild-type mice secreted approximately 35% more insulin during both phases of insulin secretion, suggesting that increased Syntaxin 4 may be beneficial for enhancing biphasic insulin secretion in a regulated manner. Taken together, these data support the notion that Syntaxin 4-based SNARE complexes are essential for biphasic insulin granule fusion in pancreatic beta-cells.     

Glucagon-like peptide-1 and islet lipolysis.

A role for glucagon-like peptide 1 (GLP-1) has been suggested in stimulating beta-cell lipolysis via elevation of cAMP and activation of protein kinase A, which in turn may activate hormone-sensitive lipase (HSL), thereby contributing to fatty acid generation (FFA) from intracellular triglyceride stores. FFAs may then be metabolized to a lipid signal, which is required for optimal glucose-stimulated insulin secretion. Since HSL is expressed in islet beta-cells, this effect could contribute to the stimulation of insulin secretion by GLP-1, provided that a lipid signal of importance for insulin secretion is generated. To examine this hypothesis, we have studied the acute effect of GLP-1 on isolated mouse islets from normal mice and from mice with high-fat diet induced insulin resistance. We found, however, that although GLP-1 (100 nM) markedly potentiated glucose-stimulated insulin secretion from islets of both feeding groups, the peptide was not able to stimulate islet palmitate oxidation or increase lipolysis measured as glycerol release. This indicates that a lipid signal does not contribute to the acute stimulation of insulin secretion by GLP-1. To test whether lipolysis might be involved in the islet effects of long-term GLP-1 action, mice from the two feeding groups were chronically treated with exendin-4, a peptide that lowers blood glucose by interacting with GLP-1 receptors, in order to stimulate insulin secretion, for 16 days before isolation of the islets. The insulinotropic effects of GLP-1 and forskolin were exaggerated in isolated islets from exendin-4 treated mice given a high-fat diet, with a augmented palmitate oxidation as well as islet lipolysis at high glucose levels in these islets. Exendin-4 treatment had less impact on mice fed a normal diet. From these results we conclude that while GLP-1 does not seem to induce beta-cell lipolysis acutely in mouse islets, the peptide affects beta-cell fat metabolism after long-term adaptation to GLP-1 receptor stimulation.     

Glucagon-like peptide 1 stimulates post-translational activation of glucokinase in pancreatic beta cells

Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin secretion from pancreatic β cells, yet does not directly stimulate secretion. The mechanisms underlying this phenomenon are incompletely understood. Here, we report that GLP-1 augments glucose-dependent rises in NAD(P)H autofluorescence in both βTC3 insulinoma cells and islets in a manner consistent with post-translational activation of glucokinase (GCK). GLP-1 treatment increased GCK activity and enhanced GCK S-nitrosylation in βTC3 cells. A 2-fold increase in S-nitrosylated GCK was also observed in mouse islets. Furthermore, GLP-1 activated a FRET-based GCK reporter in living cells. Activation of this reporter was sensitive to inhibition of nitric-oxide synthase (NOS), and incorporating the S-nitrosylation-blocking V367M mutation into this sensor prevented activation by GLP-1. GLP-1 potentiation of the glucose-dependent increase in islet NAD(P)H autofluorescence was also sensitive to a NOS inhibitor, whereas NOS inhibition did not affect the response to glucose alone. Expression of the GCK(V367M) mutant also blocked GLP-1 potentiation of the NAD(P)H response to glucose in βTC3 cells, but did not significantly affect metabolism of glucose in the absence of GLP-1. Co-expression of WT or mutant GCK proteins with a sensor for insulin secretory granule fusion also revealed that blockade of post-translational GCK S-nitrosylation diminished the effects of GLP-1 on granule exocytosis by ～40% in βTC3 cells. These results suggest that post-translational activation of GCK is an important mechanism for mediating the insulinotropic effects of GLP-1.     

Expression and regulation of cyclic nucleotide phosphodiesterases in human and rat pancreatic islets

As shown by transgenic mouse models and by using phosphodiesterase 3 (PDE3) inhibitors, PDE3B has an important role in the regulation of insulin secretion in pancreatic β-cells. However, very little is known about the regulation of the enzyme. Here, we show that PDE3B is activated in response to high glucose, insulin and cAMP elevation in rat pancreatic islets and INS-1 (832/13) cells. Activation by glucose was not affected by the presence of diazoxide. PDE3B activation was coupled to an increase as well as a decrease in total phosphorylation of the enzyme. In addition to PDE3B, several other PDEs were detected in human pancreatic islets: PDE1, PDE3, PDE4C, PDE7A, PDE8A and PDE10A. We conclude that PDE3B is activated in response to agents relevant for β-cell function and that activation is linked to increased as well as decreased phosphorylation of the enzyme. Moreover, we conclude that several PDEs are present in human pancreatic islets.     

Glucosamine-induced alterations of mitochondrial function in pancreatic beta-cells: possible role of protein glycosylation

Chronic exposure of rat pancreatic islets and INS-1 insulinoma cells to glucosamine (GlcN) produced a reduction of glucose-induced (22.2 mM) insulin release that was associated with a reduction of ATP levels and ATP/ADP ratio compared with control groups. To further evaluate mitochondrial function and ATP metabolism, we then studied uncoupling protein-2 (UCP2), F1-F0-ATP-synthase, and mitochondrial membrane potential, a marker of F1-F0-ATP-synthase activity. UCP2 protein levels were unchanged after chronic exposure to GlcN on both pancreatic islets and INS-1 beta-cells. Due to the high number of cells required to measure mitochondrial F1-F0-ATP-synthase protein levels and mitochondrial membrane potential, we used INS-1 cells, and we found that chronic culture with GlcN increased F1-F0-ATP-synthase protein levels but decreased glucose-stimulated changes of mitochondrial membrane potential. Moreover, F1-F0-ATP-synthase was highly glycosylated, as demonstrated by experiments with N-glycosidase F and glycoprotein staining. Tunicamycin (an inhibitor of protein N-glycosylation), when added with GlcN in the culture medium, was able to partially prevent all these negative effects on insulin secretion, adenine nucleotide content, mitochondrial membrane potential, and protein glycosylation. Thus we suggest that GlcN-induced pancreatic beta-cell toxicity might be mediated by reduced cell energy production. An excessive protein N-glycosylation of mitochondrial F1-F0-ATP-synthase might lead to cell damage and secretory alterations in pancreatic beta-cells.     

Enhanced cAMP protein kinase A signaling determines improved insulin secretion in a clonal insulin-producing beta-cell line (INS-1 832/13)

In type 2 diabetes, beta-cells become glucose unresponsive, contributing to hyperglycemia. To address this problem, we recently created clonal insulin-producing cell lines from the INS-1 insulinoma line, which exhibit glucose responsiveness ranging from poor to robust. Here, mechanisms that determine secretory performance were identified by functionally comparing glucose-responsive 832/13 beta-cells with glucose-unresponsive 832/2 beta-cells. Thus, insulin secretion from 832/13 cells maximally rose 8-fold in response to glucose, whereas 832/2 cells responded only 1.5-fold. Insulin content in both lines was similar, indicating that differences in stimulus-secretion coupling account for the differential secretory performance. Forskolin or isobutylmethylxanthine markedly enhanced insulin secretion from 832/13 but not from 832/2 cells, suggesting that cAMP is essential for the enhanced secretory performance of 832/13 cells. Indeed, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, rp-isomer (Rp-8-Br-cAMPS) an inhibitor of protein kinase A (PKA), inhibited insulin secretion in response to glucose with or without forskolin. Interestingly, whereas forskolin markedly increased cAMP in 832/2 cells, 832/13 cells exhibited only a marginal rise in cAMP. This suggests that 832/13 cells are more sensitive to cAMP. Indeed, the cAMP-induced exocytotic response in patch-clamped 832/13 cells was 2-fold greater than in 832/2 cells. Furthermore, immunoblotting revealed that expression of the catalytic subunit of PKA was 2-fold higher in 832/13 cells. Moreover, when the regulatory subunit of PKA was overexpressed in 832/13 cells, to reduce the level of unbound and catalytically active kinase, insulin secretion and PKA activity were blunted. Our findings show that cAMP-PKA signaling correlates with secretory performance in beta-cells.     

The malate-aspartate NADH shuttle member Aralar1 determines glucose metabolic fate, mitochondrial activity, and insulin secretion in beta cells

The NADH shuttle system, which transports reducing equivalents from the cytosol to the mitochondria, is essential for the coupling of glucose metabolism to insulin secretion in pancreatic beta cells. Aralar1 and citrin are two isoforms of the mitochondrial aspartate/glutamate carrier, one key constituent of the malate-aspartate NADH shuttle. Here, the effects of Aralar1 overexpression in INS-1E beta cells and isolated rat islets were investigated for the first time. We prepared a recombinant adenovirus encoding for human Aralar1 (AdCA-Aralar1), tagged with the small FLAG epitope. Transduction of INS-1E cells and isolated rat islets with AdCA-Aralar1 increased aralar1 protein levels and immunostaining revealed mitochondrial localization. Compared with control INS-1E cells, overexpression of Aralar1 potentiated metabolism secretion coupling stimulated by 15 mm glucose. In particular, there was an increase of NAD(P)H generation, of mitochondrial membrane hyperpolarization, ATP levels, glucose oxidation, and insulin secretion (+45%, p < 0.01). Remarkably, this was accompanied by reduced lactate production. Rat islets overexpressing Aralar1 secreted more insulin at 16.7 mm glucose (+65%, p < 0.05) compared with controls. These results show that aspartate-glutamate carrier capacity limits glucose-stimulated insulin secretion and that Aralar1 overexpression enhances mitochondrial metabolism.     

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E beta-cells

BACKGROUND: The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS: Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS: In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION: Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.     

The protein tyrosine phosphatase alpha modifies insulin secretion in INS-1E cells

Increasing evidence indicates a role of insulin signalling for insulin secretion from the pancreatic beta-cells. Therefore, regulators of insulin signalling, like protein tyrosine phosphatases, could also have an impact on insulin secretion. Here, we investigated a possible role of the negative regulator protein tyrosine phosphatase alpha (PTP alpha) for insulin secretion. RT-PCR analysis confirmed that both splice variants of the extracellular domain of PTP alpha that vary by an insert of 9 amino acids are expressed in human islets and insulinoma cells (INS-1E, RIN1046-38). Overexpression of the wild type PTP alpha splice variant containing the 9 amino acids reduced insulin secretion, as did a mutant form unable to bind Grb2 (Tyr798Phe). By contrast, overexpression of a phosphatase inactive mutant improved insulin secretion. These data reveal a functional relevance of PTP alpha for insulin secretion.     

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.     

Cell type-specific activation of metabolism reveals that beta-cell secretion suppresses glucagon release from alpha-cells in rat pancreatic islets

Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.