Voltage-dependent K ϩ (Kv) 1 channels are important regulators of membrane potential in excitable tissues where they generally mediate action potential repolarization (1). In pancreatic islet ␤-cells, Kv channels repolarize glucose-stimulated action potentials, limit entry of Ca 2ϩ through voltage-dependent Ca 2ϩ channels, and therefore act as negative regulators of insulin secretion (2). Recent work by us and others (3-5) demonstrates the importance of Kv channels, particularly Kv2.1, in the regulation of ␤-cell excitability and insulin secretion. Importantly, because ␤-cell Kv channels are closed under resting conditions, the excitatory and insulinotropic effects of Kv channel antagonists are glucose-dependent (6, 7). Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L-cells in response to nutrient ingestion (8). Although known to exert effects on cell growth and proliferation, satiety, and intestinal motility, the most well recognized action of GLP-1 is to enhance insulin secretion from pancreatic islet ␤-cells (9). GLP-1 and its analogues are under intense investigation as potential treatments for type-2 diabetes, because their insulinotropic effect is dependent upon elevated glucose, avoiding the potentially dangerous complication of hypoglycemia (10). The actions of GLP-1 are mediated by the G protein-coupled GLP-1 receptor and result from effects on many targets within the ␤-cell, the most well characterized being the cAMP and PKAdependent inhibition of K ATP channels (9). Recent evidence also demonstrates a cAMP-dependent and PKA-independent component to the insulinotropic effect of GLP-1, mediated by enhanced Ca 2ϩ -induced Ca 2ϩ release from the endoplasmic reticulum via activation of cAMP guanine nucleotide exchange factor II (Epac2) (11,12).Recently (13), we reported that GLP-1 and the GLP-1 receptor agonist exendin 4 antagonizes Kv currents in rat ␤-cells (13). Because this likely contributes to the insulinotropic effect of GLP-1, particularly the glucose dependence, we investigated the mechanism of Kv current reduction and the signal transduction pathway(s) involved. We show that exendin 4 antagonizes Kv currents in rat ␤-cells by causing a hyperpolarizing shift in the voltage dependence of steady-state inactivation. We further demonstrate that antagonism of Kv channels by exendin 4 depends on activation of both the cAMP/PKA and phosphatidylinositol 3 (PI3)-kinase/PKC signaling pathways. Activation of PI3 kinase can result from an Src kinase-mediated trans-activation of the EGF receptor. This study provides

Aims/hypothesis: The antioxidant compound α-lipoic acid (α-LA) possesses antidiabetic and anti-obesity properties. In the hypothalamus, α-LA suppresses appetite and prevents obesity by inhibiting AMP-activated protein kinase (AMPK). Given the therapeutic potential of α-LA for the treatment of type 2 diabetes and obesity, and the importance of AMPK in beta cells, we examined the effect of α-LA on pancreatic beta cell function. Materials and methods: Isolated rat islets and MIN6 beta cells were treated acutely (15-90 min) or chronically (18-24 h) with α-LA or the known AMPK-activating compounds 5′-amino-imidazole-4-carboxamide ribonucleoside (AICAR) and metformin. Insulin secretion, the AMPK-signalling pathway, mitochondrial function and cell growth were assessed. Results: Acute or chronic treatment of islets and MIN6 cells with α-LA led to dose-dependent rises in phosphorylation of the AMPK α-subunit and acetyl CoA carboxylase. Chronic exposure to α-LA, AICAR or metformin caused a reduction in insulin secretion. α-LA inhibited the p70 s6 kinase translational control pathway, and inhibited MIN6 growth in a manner similar to rapamycin. Unlike AICAR and metformin, α-LA also acutely inhibited insulin secretion. Examination of the effect of α-LA on mitochondrial function showed that acute treatment with this compound elevated reactive oxygen species (ROS) production and enhanced mitochondrial depolarisation induced by Ca 2+ . Conclusions/ interpretation: This study is the first to demonstrate that α-LA directly affects beta cell function. The chronic effects of α-LA include AMPK activation and reductions in insulin secretion and content, and cell growth. Acutely, α-LA also inhibits insulin secretion, an effect probably involving the ROS-induced impairment of mitochondrial function.

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ABSTRACT:We report the encapsulation of MIN6 cells, a pancreatic b-cell line, using thermally induced gelable materials. This strategy uses aqueous solvent and mild temperatures during encapsulation, thereby minimizing adverse effects on cell function and viability. Using a 2:1 mixture of PNIPAAm-PEG-PNIPAAm tri-block copolymer and PNIPAAm homopolymer that exhibit reversible sol-to-gel transition at $308C, gels were formed that exhibit mechanical integrity, and are stable in H 2 O, PBS and complete DMEM with negligible mass loss at 378C for 60 days. MTT assays showed undetectable cytotoxicity of the polymers towards MIN6 cells. A simple microencapsulation process was developed using vertical co-extrusion and a 378C capsule collection bath containing a paraffin layer above DMEM. Spherical capsules with diameters ranging from 500 to 900 mm were formed. SEM images of freeze-dried capsules with PBS as the core solution showed homogenous gel capsule membranes. Confocal microscopy revealed that the encapsulated cells tended to form small aggregates over 5 days, and staining for live and dead cells showed high viability post-encapsulation. A static glucose challenge with day-5 cultured microencapsulated cells exhibited glucosedependent insulin secretion comparable to controls of free MIN6 cells grown in monolayers. These results demonstrate the potential use of these thermo-responsive polymers as cell encapsulation membranes.