Dopamine is a neurotransmitter that plays a critical role in neurological and psychiatric disorders, such as schizophrenia, Parkinson disease, and drug addiction (1). Increasing evidence also shows implication of dopamine in various physiological functions such as cell proliferation (2), gastrointestinal protection (3), and inhibition of prolactin secretion (4). Effects of dopamine on insulin secretion in general and on pancreatic beta cell function in particular have been poorly studied. Insulin exocytosis from the beta cell is primarily controlled by metabolismsecretion coupling. First, glucose equilibrates across the plasma membrane and is phosphorylated by glucokinase, initiating glycolysis (5). Subsequently, mitochondrial metabolism generates ATP, which promotes the closure of ATP-sensitive potassium channels and, as a consequence, depolarization of the plasma membrane (6). This leads to calcium influx through voltage-gated calcium channels and a rise in cytosolic calcium, triggering insulin exocytosis (6, 7). Additional signals participating in the amplifying pathway (8) are necessary to reproduce the sustained secretion elicited by glucose. Insulin secretion evoked by glucose metabolism can be further modulated by parasympathetic and sympathetic neurotransmitters (9).Treatment with dopamine precursor L-dopa in humans suffering from Parkinson disease reduces insulin secretion upon oral glucose tolerance test (10). In rodents, a single injection with L-dopa results in the accumulation of dopamine in beta cells and inhibition of the insulin secretory responses (11,12). In isolated islets, analogues of dopamine inhibit glucose-stimulated insulin release (13), whereas one study reports potentiation of insulin secretion upon acute dopamine accumulation (14). Taken as a whole, these previous studies suggest that beta cells might be directly responsive to dopamine. Here, we investigated the molecular mechanisms implicated in beta cell responses to dopamine action. In particular, the present data demonstrate the presence of dopamine receptors in beta cells. Moreover, the inhibitory effects of dopamine are predominantly ascribed to activation of the D2-like receptor family members. MATERIALS AND METHODS INS-1E Cells and Pancreatic Islets-INS-1Ecells, used as a well differentiated beta cell clone (15), were cultured in a humidified atmosphere containing 5% CO 2 in a medium composed of RPMI 1640 supplemented with 10 mM Hepes, 5% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 1 mM sodium pyruvate, and 50 M 2-mercaptoethanol. For rodent islets, Wistar rats or BALB/c mice weighing 200 -250 g and 25-30 g, respectively, were obtained from in-house breeding (CMU-Zootechnie, Geneva, Switzerland). We followed the principles of laboratory animal care, and the study was approved by the responsible ethics committee. Pancreatic islets were isolated by collagenase digestion and handpicking from male Wistar rats or BALB/c mice as described previously (16). Isolated islets w...

It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage‐sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta‐cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin‐containing secretory granules. Both glucose and 2‐ketoisocaproate (KIC) raised [Ca2+]i in a dose‐dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell‐to‐cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage‐sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.

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 malateaspartate 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 metabolism, through glycolysis and mitochondria, drives stimulation of insulin secretion in pancreatic beta cells (1, 2). According to low lactate dehydrogenase activity in beta cells, glycolysis-derived electrons carried by NADHϩH ϩ are mostly transferred to mitochondria through the NADH shuttle system. Therefore, NADH shuttles couple glycolysis to activation of mitochondrial energy metabolism, leading to insulin secretion. Moreover, low activity of NADH shuttles in beta cells has been found in type 2 diabetes models (3) and is also the cause of impaired glucose-stimulated insulin secretion (GSIS) 1 in fetal islets (4).In beta cells, the NADH shuttle system is composed essentially of the glycerophosphate and the malate-aspartate shuttles (5). The respective importance of these shuttles is illustrated in pancreatic islets of mice with abrogation of NADH shuttle activities. Mice lacking mitochondrial glycerol-phosphate dehydrogenase exhibit normal GSIS (6). However, additional inhibition of the malate-aspartate shuttle with aminooxyacetate strongly impairs the secretory response to glucose (6). This suggested that the malate-aspartate shuttle might play a key role in both mitochondrial metabolism and cytosolic redox state. Besides glycerophosphate and malate-aspartate shuttles, pyruvate-citrate shuttle also regenerates NAD ϩ necessary to maintain glycolysis. Pyruvate-citrate shuttle (7) contributes to the formation of malonyl-CoA and cytosolic NADPH, two molecules proposed as candidate coupling factors in GSIS (8, 9).In the mitochondria, NADH electrons are transferred to the electron transport chain, which in turn supplies th...

Aims/hypothesis Saturated fatty acids augment endoplasmic reticulum (ER) stress in pancreatic beta cells and this is implicated in the loss of beta cell mass that accompanies type 2 diabetes. However, the mechanisms underlying the induction of ER stress are unclear. Our aim was to establish whether saturated fatty acids cause defects in ER-to-Golgi protein trafficking, which may thereby contribute to ER stress via protein overload. Methods Cells of the mouse insulinoma cell line MIN6 were transfected with temperature-sensitive vesicular stomatitis virus G protein (VSVG) tagged with green fluorescent protein to quantify the rate of ER-to-Golgi protein trafficking. I14 antibody, which detects only correctly folded VSVG, was employed to probe the folding environment of the ER. ER stress markers were monitored by western blotting.Results Pretreatment with palmitate, but not oleate, significantly reduced the rate of ER-to-Golgi protein trafficking assessed using VSVG. This was not secondary to ER stress, since thapsigargin, which compromises chaperone function by depletion of ER calcium, markedly inhibited VSVG folding and promoted strong ER stress but only slightly reduced protein trafficking. Blockade of ER-to-Golgi protein trafficking with brefeldin A (BFA) was sufficient to trigger ER stress, but neither BFA nor palmitate compromised VSVG folding. Conclusions/interpretation Reductions in ER-to-Golgi protein trafficking potentially contribute to ER stress during lipoapoptosis. In this case ER stress would be triggered by protein overload, rather than a disruption of the proteinfolding capacity of the ER.

Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic β-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 β-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic β-cells.