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In pancreatic ␤-cells, glucose metabolism signals insulin secretion by altering the cellular array of messenger molecules. ATP is particularly important, given its role in regulating cation channel activity, exocytosis, and events dependent upon its hydrolysis. Uncoupling protein (UCP)-2 is proposed to catalyze a mitochondrial inner-membrane H ؉ leak that bypasses ATP synthase, thereby reducing cellular ATP content. Previously, we showed that overexpression of UCP-2 suppressed glucose-stimulated insulin secretion (GSIS) in isolated islets (1). The aim of this study was to identify downstream consequences of UCP-2 overexpression and to determine whether insufficient insulin secretion in a diabetic model was correlated with increased endogenous UCP-2 expression. In isolated islets from normal rats, the degree to which GSIS was suppressed was inversely correlated with the amount of UCP-2 expression induced. Depolarizing the islets with KCl or inhibiting ATP-dependent K ؉ (K ATP ) channels with glybenclamide elicited similar insulin secretion in control and UCP-2-overexpressing islets. The glucose-stimulated mitochondrial membrane (⌿ m ) hyperpolarization was reduced in ␤-cells overexpressing UCP-2. ATP content of UCP-2-induced islets was reduced by 50%, and there was no change in the efflux of Rb ؉ at high versus low glucose concentrations, suggesting that low ATP led to reduced glucose-induced depolarization, thereby causing reduced insulin secretion. Sprague-Dawley rats fed a diet with 40% fat for 3 weeks were glucose intolerant, and in vitro insulin secretion at high glucose was only increased 8.5-fold over basal, compared with 28-fold in control rats. Islet UCP-2 mRNA expression was increased twofold. These studies provide further strong evidence that UCP-2 is an important negative regulator of ␤-cell insulin secretion and demonstrate that reduced ⌬⌿ m and increased activity of K ATP channels are mechanisms by which UCP-2-mediated effects are mediated. These studies also raise the possibility that a pathological upregulation of UCP-2 expression in the prediabetic state could contribute to the loss of glucose responsiveness observed in obesity-related type 2 diabetes in humans.

Knowledge of how the brain achieves its diverse central control of basic physiology is severely limited by the virtual absence of appropriate cell models. Isolation of clonal populations of unique peptidergic neurons from the hypothalamus will facilitate these studies. Herein we describe the mass immortalization of mouse primary hypothalamic cells in monolayer culture, resulting in the generation of a vast representation of hypothalamic cell types. Subcloning of the heterogeneous cell populations resulted in the establishment of 38 representative clonal neuronal cell lines, of which 16 have been further characterized by analysis of 28 neuroendocrine markers. These cell lines represent the first available models to study the regulation of neuropeptides associated with the control of feeding behavior, including neuropeptide Y, ghrelin, urocortin, proopiomelanocortin, melanin-concentrating hormone, neurotensin, proglucagon, and GHRH. Importantly, a representative cell line responds appropriately to leptin stimulation and results in the repression of neuropeptide Y gene expression. These cell models can be used for detailed molecular analysis of neuropeptide gene regulation and signal transduction events involved in the direct hormonal control of unique hypothalamic neurons, not yet possible in the whole brain. Such studies may contribute information necessary for the strategic design of therapeutic interventions for complex neuroendocrine disorders, such as obesity.

Voltage-dependent (Kv) outward K ؉ currents repolarize ␤-cell action potentials during a glucose stimulus to limit Ca 2؉ entry and insulin secretion. Dominant-negative "knockout" of Kv2 family channels enhances glucose-stimulated insulin secretion. Here we show that a putative Kv2.1 antagonist (C-1) stimulates insulin secretion from MIN6 insulinoma cells in a glucose-and dosedependent manner while blocking voltage-dependent outward K ؉ currents. C-1-blocked recombinant Kv2.1-mediated currents more specifically than currents mediated by Kv1, -3, and -4 family channels (Kv1.4, 3.1, 4.2). Additionally, C-1 had little effect on currents recorded from MIN6 cells expressing a dominant-negative Kv2.1 ␣-subunit. The insulinotropic effect of acute Kv2.1 inhibition resulted from enhanced membrane depolarization and augmented intracellular Ca 2؉ responses to glucose. Immunohistochemical staining of mouse pancreas sections showed that expression of Kv2.1 correlated highly with insulin-containing ␤-cells, consistent with the ability of C-1 to block voltage-dependent outward K ؉ currents in isolated mouse ␤-cells. Antagonism of Kv2.1 in an ex vivo perfused mouse pancreas model enhanced first-and second-phase insulin secretion, whereas glucagon secretion was unaffected. The present study demonstrates that Kv2.1 is an important component of ␤-cell stimulus-secretion coupling, and a compound that enhances, but does not initiate, ␤-cell electrical activity by acting on Kv2

A short course of Cat-PAD improves the ocular and nasal components of rhinoconjunctivitis symptoms in subjects with cat allergy, with the treatment effect persisting 1 year after the start of treatment.

The role nitric oxide (NO) plays in physiological insulin secretion has been controversial. Here we present evidence that exogenous NO stimulates insulin secretion, and that endogenous NO production occurs and is involved in the regulation of insulin release. Radioimmunoassay measurement of insulin release and a dynamic assay of exocytosis using the dye FM1-43 demonstrated that three different NO donors-hydroxylamine (HA), sodium nitroprusside, and 3-morpholinosydnonimine (SIN-1)-each stimulated a marked increase in insulin secretion from INS-1 cells. Pharmacological manipulation of the guanylate cyclase/guanosine 3,5-cyclic monophosphate pathway indicated that this pathway was involved in mediating the effect of the intracellular NO donor, HA, which was used to simulate endogenous NO production. This effect was further characterized as involving membrane depolarization and intracellular T he endogenous synthesis of nitric oxide (NO) from L-arginine (L-arg) has been shown to play a critical role in a wide variety of physiological functions including neurotransmission, vascular tone, platelet aggregation, immunological reactions, penile erection, and endocrine and exocrine function (1). Cellular NO is synthesized by a family of NO synthase (NOS) enzymes, comprised of constitutively expressed, Ca 2ϩ / calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS), and a Ca 2ϩ /calmodulin-independent inducibly expressed NOS (iNOS) (2).Pancreatic ␤-cells are able to express the iNOS enzyme in response to inflammatory stimuli, leading to high cytotoxic levels of NO production, which appear to be involved in ␤-cell damage, dysfunction, and death and the pathogenesis of type 1 diabetes (3,4). The presence of a constitutive NOS (cNOS) enzyme in ␤-cells is substantiated by considerable evidence, including biochemical, histochemical, immunohistochemical, immunofluorescence, RT-PCR, and protein immunoblot analyses (5-11). Given that the amino acid NO precursor L-arg has long been known to stimulate insulin release (12), it has been postulated that a low level of NO produced from the ␤-cell cNOS isoform functions in the regulation of insulin release. However, several reports in which cNOS activity was manipulated or exogenous NO was applied have yielded seemingly conflicting results. NOS inhibition has been reported to produce an inhibitory effect (5,13-15), a stimulatory effect (6,9,16 -18), and no effect at all (19,20) on insulin release. Similarly, exogenously applied NO has been reported to exert a stimulatory (5,13,14,21,22) and an inhibitory effect (6,18,23-27) on insulin release. These discrepant data may be the result of variations in the specifics of the experimental conditions, including differences in the agents used (e.g., NOS substrate, NOS inhibitors, NO donors), the concentration of these agents, whether other stimulatory pathways were activated concurrently (e.g., with glucose), the experimental model used (e.g., ␤-cell line, islets, pancreas), and the species. These critical differences may result in d...

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؊8mol/l) in rat ␤-cells antagonized K V currents by 43.3 ؎ 6.3%, whereas the GLP-1 receptor antagonist exendin 9-39 had no effect. The effect of GLP-1 receptor activation on K V currents could be replicated (current reduction of 55.7 ؎