Kv2.1 clusters on ß-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3.

The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic ß cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the ß-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.

Characterization of Zinc Influx Transporters (ZIPs) in Pancreatic ß Cells: ROLES IN REGULATING CYTOSOLIC ZINC HOMEOSTASIS AND INSULIN SECRETION.

Zinc plays an essential role in the regulation of pancreatic ß cell function, affecting important processes including insulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in the zinc efflux transport protein ZnT8 have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx transporters (ZIPs). In this study, we examined the ß cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human and mouse islets and MIN6 ß cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic ß cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this dysregulated cytosolic zinc homeostasis led to impaired insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast two-hybrid assay. Knock-down of ZIP6 but not ZIP7 in MIN6 ß cells impaired the protective effects of GLP-1 on fatty acid-induced cell apoptosis, possibly via reduced activation of the p-ERK pathway. Therefore, our data suggest that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in ß cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on ß cell survival.

LKB1 couples glucose metabolism to insulin secretion in mice.

AIMS/HYPOTHESIS: Precise regulation of insulin secretion by the pancreatic beta cell is essential for the maintenance of glucose homeostasis. Insulin secretory activity is initiated by the stepwise breakdown of ambient glucose to increase cellular ATP via glycolysis and mitochondrial respiration. Knockout of Lkb1, the gene encoding liver kinase B1 (LKB1) from the beta cell in mice enhances insulin secretory activity by an undefined mechanism. Here, we sought to determine the molecular basis for how deletion of Lkb1 promotes insulin secretion. METHODS: To explore the role of LKB1 on individual steps in the insulin secretion pathway, we used mitochondrial functional analyses, electrophysiology and metabolic tracing coupled with by gas chromatography and mass spectrometry. RESULTS: Beta cells lacking LKB1 surprisingly display impaired mitochondrial metabolism and lower ATP levels following glucose stimulation, yet compensate for this by upregulating both uptake and synthesis of glutamine, leading to increased production of citrate. Furthermore, under low glucose conditions, Lkb1(-/-) beta cells fail to inhibit acetyl-CoA carboxylase 1 (ACC1), the rate-limiting enzyme in lipid synthesis, and consequently accumulate NEFA and display increased membrane excitability. CONCLUSIONS/INTERPRETATION: Taken together, our data show that LKB1 plays a critical role in coupling glucose metabolism to insulin secretion, and factors in addition to ATP act as coupling intermediates between feeding cues and secretion. Our data suggest that beta cells lacking LKB1 could be used as a system to identify additional molecular events that connect metabolism to cellular excitation in the insulin secretion pathway.

Proinsulin intermolecular interactions during secretory trafficking in pancreatic ß cells.

Classically, exit from the endoplasmic reticulum (ER) is rate-limiting for secretory protein trafficking because protein folding/assembly occurs there. In this study, we have exploited "hPro-CpepSfGFP," a human proinsulin bearing "superfolder" green fluorescent C-peptide expressed in pancreatic ß cells where it is processed to human insulin and CpepSfGFP. Remarkably, steady-state accumulation of hPro-CpepSfGFP and endogenous proinsulin is in the Golgi region, as if final stages of protein folding/assembly were occurring there. The Golgi regional distribution of proinsulin is dynamic, influenced by fasting/refeeding, and increased with ß cell zinc deficiency. However, coexpression of ER-entrapped mutant proinsulin-C(A7)Y shifts the steady-state distribution of wild-type proinsulin to the ER. Endogenous proinsulin coprecipitates with hPro-CpepSfGFP and even more so with hProC(A7)Y-CpepSfGFP. Using Cerulean and Venus-tagged proinsulins, we find that both WT-WT and WT-mutant proinsulin pairs exhibit FRET. The data demonstrate that wild-type proinsulin dimerizes within the ER but accumulates at a poorly recognized slow step within the Golgi region, reflecting either slow kinetics of proinsulin hexamerization, steps in formation of nascent secretory granules, or other unknown molecular events. However, in the presence of ongoing misfolding of a subpopulation of proinsulin in ß cells, the rate-limiting step in transport of the remaining proinsulin shifts to the ER.

Characterization of Erg K+ channels in alpha- and beta-cells of mouse and human islets.

Voltage-gated eag-related gene (Erg) K(+) channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in alpha-TC6 and Min6 cells alpha- and beta-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in alpha- and beta-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca(2+)-imaging experiments were performed on isolated alpha- and beta-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca(2+) increase in both alpha- and beta-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of alpha- and beta-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.

Branch-specific Ca2+ influx from Na+-dependent dendritic spikes in olfactory granule cells.

Two-photon laser scanning microscopy was used to correlate electrical events detected with whole-cell somatic recordings to Ca2+ transients in dendrites of olfactory bulb granule cells. A subset of spontaneous subthreshold depolarizing events recorded at the soma were shown to correspond to suprathreshold dendritic, Na-dependent action potentials [APs; dendritic spikes (D-spikes)]. These potentials were blocked by intracellular QX-314 (lidocaine N-ethyl bromide), hyperpolarizing current injection at the soma, and by partial inhibition of AMPA/kainate receptors with 0.75 microM DNQX. They were affected only slightly by 100 microM NiCl2. The majority of D-spikes recorded at the soma had a time to peak of <4 ms, comparable with somatic APs, a nonexponential decay, and amplitudes between 3 and 21 mV. Somatically recorded APs produced Ca2+ transients that were observed in spines and dendrites in all parts of the cell. Ca2+ transients from D-spikes were restricted to subsets of distal dendrites and their associated spines but were absent from the soma and dendrite within approximately 50-80 microm of the soma. Ca2+ transients in different branches could be correlated with different-sized D-spikes. D-spike and backpropagating AP-induced Ca2+ transients summed in dendrites, provided the interval between them was >5-6 ms. Generation of a D-spike in a particular dendrite <5-6 ms before a somatic AP blocked backpropagation of the somatic AP into that dendrite. The temporally specific interplay between D-spikes and backpropagating APs may play a role in regulating feedback and feedforward inhibition of groups of mitral cells synapsing on different granule cell dendrites.

The SNARE Protein Syntaxin-1a Plays an Essential Role in Biphasic Exocytosis of the Incretin Hormone Glucagon-Like Peptide 1.

Exocytosis of the hormone glucagon-like peptide 1 (GLP-1) by the intestinal L cell is essential for the incretin effect after nutrient ingestion and is critical for the actions of dipeptidyl peptidase 4 inhibitors that enhance GLP-1 levels in patients with type 2 diabetes. Two-photon microscopy revealed that exocytosis of GLP-1 is biphasic, with a first peak at 1-6 min and a second peak at 7-12 min after stimulation with forskolin. Approximately 75% of the exocytotic events were represented by compound granule fusion, and the remainder were accounted for by full fusion of single granules under basal and stimulated conditions. The core SNARE protein syntaxin-1a (syn1a) was expressed by murine ileal L cells. At the single L-cell level, first-phase forskolin-induced exocytosis was reduced to basal (P < 0.05) and second-phase exocytosis abolished (P < 0.05) by syn1a knockout. L cells from intestinal-epithelial syn1a-deficient mice demonstrated a 63% reduction in forskolin-induced GLP-1 release in vitro (P < 0.001) and a 23% reduction in oral glucose-stimulated GLP-1 secretion (P < 0.05) in association with impairments in glucose-stimulated insulin release (by 60%; P < 0.01) and glucose tolerance (by 20%; P < 0.01). The findings identify an exquisite mechanism of metered secretory output that precisely regulates release of the incretin hormone GLP-1 and hence insulin secretion after a meal.

Orexin A modulates mitral cell activity in the rat olfactory bulb: patch-clamp study on slices and immunocytochemical localization of orexin receptors.

Orexin A and B are involved in feeding behaviors, and recently fibers containing these peptides were found in the rat olfactory bulb. These fibers, which originate from the lateral and posterior hypothalamus and the perifornical area, are distributed in the glomerular, mitral cell, and granule cell layers. Orexin receptors are mainly expressed by mitral cells. In the present study, RT-PCR experiments were done to determine orexin receptor expression during the early postnatal life of rats, and immunocytochemical experiments were performed to further clarify the structural and ultrastructural localization of orexin receptors in the olfactory bulb. Furthermore, a functional electrophysiological approach examined the action of orexin A on mitral cell excitability and spontaneous activity using in vitro patch-clamp techniques. RT-PCR results show that mRNA of the two type receptors, type 1 orexin receptors and type 2 orexin receptors, are expressed in the olfactory bulb of rat from 10 d to the adult stage. At the same ages, immunocytochemical data show that orexin 1 receptors are localized in the cell bodies of periglomerular, mitral/tufted, and granule cells. Immunoreactivity was also demonstrated in mitral/tufted cell dendrites arborizing in the glomerulus and mitral/tufted and granule cell processes running in the external plexiform layer. Functionally, orexin A produced either a direct, tetrodotoxin-insensitive depolarization in one group of mitral cells (7%), or, in another group (30%), an indirect, tetrodotoxin-sensitive hyperpolarization. Both actions were mediated by type 1 orexin receptors because the response was antagonized by SB-334867-A, a selective antagonist. Mitral cell recordings performed under bicuculline [gamma-aminobutyric acid (GABA)A receptor antagonist], indicate that the orexin-induced indirect hyperpolarization was partly mediated through GABA(A) receptors. Because granule cells and periglomerular cells express orexin receptors and are GABAergic cells, they could be both involved in this hyperpolarization. Other mechanisms, which could support an indirect hyperpolarization of mitral cells through dopamine interneuron solicitation, are proposed. Our results provide data that should allow us to better understand neural communication and regulation mechanisms between the hypothalamic feeding centers and the olfactory bulb.

Zip4 mediated zinc influx stimulates insulin secretion in pancreatic beta cells.

Zinc has an important role in normal pancreatic beta cell physiology as it regulates gene transcription, insulin crystallization and secretion, and cell survival. Nevertheless, little is known about how zinc is transported through the plasma membrane of beta cells and which of the class of zinc influx transporters (Zip) is involved. Zip4 was previously shown to be expressed in human and mouse beta cells; however, its function there is still unknown. Therefore, the aim of this study was to define the zinc transport role of Zip4 in beta cells. To investigate this, Zip4 was over-expressed in MIN6 beta cells using a pCMV6-Zip4GFP plasmid. Organelle staining combined with confocal microscopy showed that Zip4 exhibits a widespread localization in MIN6 cells. Time-lapse zinc imaging experiments showed that Zip4 increases cytoplasmic zinc levels. This resulted in increased granular zinc content and glucose-stimulated insulin secretion. Interestingly, it is unlikely that the increased glucose stimulated insulin secretion was triggered by a modulation of mitochondrial function, as mitochondrial membrane potential remained unchanged. To define the role of Zip4 in-vivo, we generated a beta cell-specific knockout mouse model (Zip4BKO). Deletion of the Zip4 gene was confirmed in Zip4BKO islets by PCR, RT-PCR, and immuno-histochemistry. Zip4BKO mice showed slightly improved glucose homeostasis but no change in insulin secretion during an oral glucose tolerance test. While Zip4 was not found to be essential for proper glucose homeostasis and insulin secretion in vivo in mice, this study also found that Zip4 mediates increases in cytoplasmic and granular zinc pools and stimulates glucose dependant insulin secretion in-vitro.

The furan fatty acid metabolite CMPF is elevated in diabetes and induces ß cell dysfunction.

Gestational diabetes (GDM) results from failure of the ß cells to adapt to increased metabolic demands; however, the cause of GDM and the extremely high rate of progression to type 2 diabetes (T2D) remains unknown. Using metabolomics, we show that the furan fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is elevated in the plasma of humans with GDM, as well as impaired glucose-tolerant and T2D patients. In mice, diabetic levels of plasma CMPF induced glucose intolerance, impaired glucose-stimulated insulin secretion, and decreased glucose utilization. Mechanistically, we show that CMPF acts directly on the ß cell, causing impaired mitochondrial function, decreasing glucose-induced ATP accumulation, and inducing oxidative stress, resulting in dysregulation of key transcription factors and ultimately reduced insulin biosynthesis. Importantly, specifically blocking its transport through OAT3 or antioxidant treatment could prevent CMPF-induced ß cell dysfunction. Thus, CMPF provides a link between ß cell dysfunction and GDM/T2D that could be targeted therapeutically.

UCP2 regulates the glucagon response to fasting and starvation.

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2(-/-)) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell-specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia.

Serotonin (5-HT) activation of immortalized hypothalamic neuronal cells through the 5-HT1B serotonin receptor.

Serotonin [or 5-hydroxytryptamine or (5-HT)] has been implicated as a key modulator in energy homeostasis and a primary focus in the treatment of obesity. There is growing evidence that 5-HT, acting through the 5-HT 1B receptor (5-HT(1B)R) in the paraventricular nucleus of the hypothalamus (PVN), is important to this regulation. However, there is some contention as to whether 5-HT(1B)R action occurs directly on PVN neurons or indirectly via inhibitory inputs into the PVN. To address these questions, we used a novel clonal, hypothalamic neuronal cell model, adult mouse hypothalamic-2/30 (mHypoA-2/30), expressing a PVN-specific marker, single-minded homolog 1, as well as a complement of PVN neuropeptides, including TRH, vasopressin, ghrelin, nucleobindin-2, and galanin. Adult mouse hypothalamic-2/30 neurons were also found to express the 5-HT(1B)R and 5-HT 6 receptor, but not 2C, all previously linked to feeding regulation. Direct serotonergic stimulation (100 nm to 10 µm) of these neurons resulted in dose-dependent cFos activation. 5-HT (10 µm) suppressed forskolin-induced cAMP levels and induced a rise in intracellular Ca(2+) through ER Ca(2+) release, effects that were mimicked by the 5-HT(1B)R agonists, CGS12066B and CP93129, and that were attenuated in the presence of the 5-HT(1B)R-specific inhibitors, GR55562 and isamoltane hemifumarate. Modest transcriptional changes in ghrelin and nucleobindin-2 were also observed in response to 100 nm and 10 µm 5-HT, respectively. These findings support the model wherein 5-HT action through the 1B receptor subtype occurs directly on PVN neurons, leading to potential modification of neuronal transcriptional and secretory machinery.