Rat insulinoma-derived INS-1 cells constitute a widely used beta-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E beta-cell phenotype over 116 passages (no. 27-142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40-100 with average insulin contents of 2.30 +/- 0.11 microg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca(2+), and mitochondrial Ca(2+) levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A K(ATP) current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native beta-cells, tolbutamide induced electrical activity, indicating that the K(ATP)conductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable beta-cell model.
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...
Specific amino acids are now known to acutely and chronically regulate insulin secretion from pancreatic beta-cells in vivo and in vitro. Understanding the molecular mechanisms by which amino acids regulate insulin secretion may identify novel targets for future diabetes therapies. Mitochondrial metabolism is crucial for the coupling of amino acid and glucose recognition to the exocytosis of the insulin granules. This is illustrated by in vitro and in vivo observations discussed in the present review. Mitochondria generate ATP, which is the main coupling factor in insulin secretion; however, the subsequent Ca2+ signal in the cytosol is necessary, but not sufficient, for full development of sustained insulin secretion. Hence mitochondria generate ATP and other coupling factors serving as fuel sensors for the control of the exocytotic process. Numerous studies have sought to identify the factors that mediate the amplifying pathway over the Ca2+ signal in nutrient-stimulated insulin secretion. Predominantly, these factors are nucleotides (GTP, ATP, cAMP and NADPH), although metabolites have also been proposed, such as long-chain acyl-CoA derivatives and the key amino acid glutamate. This scenario highlights further the importance of the key enzymes or transporters, glutamate dehydrogenase, the aspartate and alanine aminotransferases and the malate/aspartate shuttle, in the control of insulin secretion. Therefore amino acids may play a direct or indirect (via generation of putative messengers of mitochondrial origin) role in insulin secretion.
In peripheral tissues, dopamine is released from neuronal cells and is synthesized within specific parenchyma. Dopamine released from sympathetic nerves predominantly contributes to plasma dopamine levels. Despite growing evidence for peripheral source and action of dopamine and the widespread expression of dopamine receptors in peripheral tissues, most studies have focused on functions of dopamine in the central nervous system. Symptoms of several brain disorders, including schizophrenia, Parkinson's disease, attention-deficit hyperactivity disorder, and depression, are alleviated by pharmacological modulation of dopamine transmission. Regarding systemic disorders, the role of dopamine is still poorly understood. Here we describe the pioneering and recent evidence for functional roles of peripheral dopamine. Peripheral and central dopamine systems are sensitive to environmental stress, such as a high-fat diet, suggesting a basis of covariance of peripheral and central actions of dopaminergic agents. Given the extended use of such medications, it is crucial to better understand the integrated effects of dopamine in the whole organism. Delineation of peripheral and central dopaminergic mechanisms would facilitate targeted and safer use of drugs modulating dopamine action. We discuss the increasing evidence for a link between peripheral dopamine and obesity. This review also describes the recently uncovered protective actions of dopamine on energy metabolism and proliferation in tumoral 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 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...
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