a microfluidic device at 37uC and 5% CO2. The imaging buffer consisted of 125 NaCl, 5.7 KCl, 2.5 CaCl2N2 H2O, 1.5 MgCl2, 
10 HEPES, and 0.1% bovine serum albumin at pH 7.4. NADH autofluorescence was imaged with a LSM710 microscope using a Plan-Apochromat 20x/ 0.8 NA objective and a Coherent Chameleon laser tuned to 710 nm, as previously described. The laser power at the sample was below 3.5 mW to prevent damage to the islet. NADH autofluorescence was measured in intact islets as a function of glucose concentration with and without Kisspeptin-10 and GLP-1 736 amide. Addition of increasing glucose concentrations with and without the GPCR ligands occurred at 8 min intervals to allow NADH levels to plateau, after which Zstacks were collected. Results Kisspeptin and GLP-1 potentiate insulin secretion at elevated glucose levels We first investigated the effect of kisspeptin-10 and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19691102/ GLP-1 on insulin secretion from intact islets. Although KP is thought to play a role in modulation of insulin secretion, conflicting data have been published as to its effect on b-cell exocytosis. Here, intact islets were statically incubated with KP or GLP-1 at 2.8 mM, 10 mM, and 16.7 mM glucose concentrations to validate previous results and confirm that KP and GLP-1 stimulate insulin secretion in our system. At sub-stimulatory glucose concentrations, KP and GLP-1 do not increase insulin secretion compared to the untreated control, as shown GLP-1 and KP Signaling in b-Cells in Kisspeptin increases cellular redox Rutoside potential, but GLP-1 does not We next evaluated the effects on cellular metabolism during KP or GLP-1 exposure. While GLP-19s metabolic effects have been examined, little has been reported about the effect of KP on cellular metabolism, except that kisspeptin gene products may play an important role in sensing body energy status. To determine whether KP or GLP-1 effects on insulin secretion are correlated with changes in the metabolic processing of glucose, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19689163 we used NADH autofluorescence as a measure of cellular redox potential. NADH levels from intact islets were measured as a function of glucose in the presence and absence of KP or GLP-1. In untreated control islets, increasing concentrations of glucose produce a rise in the NADH autofluorescence and cellular redox potential; this is consistent with previously published data from our lab. Treatment with KP, which stimulates insulin secretion through activation of the PLC pathway, resulted in a significant, additional increase in NADH compared to untreated control islets over a range of glucose concentrations. However, treatment with GLP-1 did not significantly increase NADH autofluorescence compared to untreated control islets. These GLP-1 data are consistent with a previously published study. . We used a calcium indicator dye, Fluo4, to measure the b-cell Ca2+ response in isolated islets upon treatment with KP. At 10 mM glucose, an increase in the calcium signal is initially detected, followed by oscillations in i, in intact islets. No oscillations were detected at low glucose concentrations. We measured intact islet i oscillations at 10 mM glucose and found no significant effect of KP on either the frequency or amplitude oscillating component. In order to distinguish the effects of these GPCR ligands on i activity, these experiments also were performed in dispersed b-cells for ease of comparability between our study and others. In dispersed bcells, i oscillations were measured at 10 mM glucose in the pre