Ya-Chi Huang

POU Homeodomain Protein Oct-1 Functions as a Sensor for Cyclic AMP

Cyclic AMP is a fundamentally important second messenger for numerous peptide hormones and neurotransmitters that control gene expression, cell proliferation, and metabolic homeostasis. Here we show that cAMP works with the POU homeodomain protein Oct-1 to regulate gene expression in pancreatic and intestinal endocrine cells. This ubiquitously expressed transcription factor is known as a stress sensor. We found that it also functions as a repressor of Cdx-2, a proglucagon gene activator. Through a mechanism that involves the activation of exchange protein activated by cyclic AMP, elevation of cAMP leads to enhanced phosphorylation and nuclear exclusion of Oct-1 and reduced interactions between Oct-1 or nuclear co-repressors and the Cdx-2 gene promoter, detected by chromatin immunoprecipitation. In rat primary pancreatic islet cells, cAMP elevation also reduces nuclear Oct-1 content, which causes increased proglucagon and proinsulin mRNA expression. Our study therefore identifies a novel mechanism by which cAMP regulates hormone-gene expression and suggests that ubiquitously expressed Oct-1 may play a role in metabolic homeostasis by functioning as a sensor for cAMP.

Unperturbed islet α-cell function examined in mouse pancreas tissue slices

Critical investigation into pancreatic islet α‐cell biology in health and diabetes has been sparse and inconsistent because of technical difficulties in islet isolation and dispersion into single cells. We have circumvented these difficulties by employing the pancreas slice preparation. We functionally characterized (electrophysiologically) the α‐cells in their in situ native state, then loaded the tested cells with biocytin dye to subsequently confirm the cell identities by immunocytochemistry. We characterized a very large number of α‐cells, which showed a wide‐range distribution in the electrophysiological parameters of several ion channels (ATP‐sensitive K+, Na+ and Ca2+ currents) and capacitance changes as measure of exocytosis of glucagon granules. This could explain the apparent inconsistency of previous reports on α‐cells that inadvertently showed skewed data due to insufficient sampling of α‐cells. Our innovative approaches will enable future studies into elucidating α‐cell dysregulation in diabetes.

Truncation of SNAP-25 reduces the stimulatory action of cAMP on rapid exocytosis in insulin-secreting cells

Synaptosomal protein of 25 kDa (SNAP-25) is important for Ca(2+)-dependent fusion of large dense core vesicles (LDCVs) in insulin-secreting cells. Exocytosis is further enhanced by cAMP-increasing agents such as glucagon-like peptide-1 (GLP-1), and this augmentation includes interaction with both PKA and cAMP-GEFII. To investigate the coupling between SNAP-25- and cAMP-dependent stimulation of insulin exocytosis, we have used capacitance measurements, protein-binding assays, and Western blot analysis. In insulin-secreting INS-1 cells overexpressing wild-type SNAP-25 (SNAP-25(WT)), rapid exocytosis was stimulated more than threefold by cAMP, similar to the situation in nontransfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-25(1-197)) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in the presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the readily releasable pool, especially from the immediately releasable pool, whereas cAMP enhanced mobilization of granules from the reserve pool in both SNAP-25(1-197) (P < 0.01) and SNAP-25(WT) (P < 0.05) cells. This was supported by hormone release measurements. Augmentation of the immediately releasable pool by cAMP has been suggested to act through the cAMP-GEFII-dependent, PKA-independent pathway. Indeed, we were able to verify an interaction between SNAP-25 with both cAMP-GEFII and RIM2, two proteins involved in the PKA-independent pathway. Thus we hypothesize that SNAP-25 is a necessary partner in the complex mediating cAMP-enhanced rapid exocytosis in insulin-secreting cells.

In Situ Electrophysiological Examination of Pancreatic α Cells in the Streptozotocin-Induced Diabetes Model, Revealing the Cellular Basis of Glucagon Hypersecretion

Early-stage type 1 diabetes (T1D) exhibits hyperglucagonemia by undefined cellular mechanisms. Here we characterized α-cell voltage-gated ion channels in a streptozotocin (STZ)-induced diabetes model that lead to increased glucagon secretion mimicking T1D. GYY mice expressing enhanced yellow fluorescence protein in α cells were used to identify α cells within pancreas slices. Mice treated with low-dose STZ exhibited hyperglucagonemia, hyperglycemia, and glucose intolerance, with 71% reduction of β-cell mass. Although α-cell mass of STZ-treated mice remained unchanged, total pancreatic glucagon content was elevated, coinciding with increase in size of glucagon granules. Pancreas tissue slices enabled in situ examination of α-cell electrophysiology. α cells of STZ-treated mice exhibited the following: 1) increased exocytosis (serial depolarization-induced capacitance), 2) enhanced voltage-gated Na+ current density, 3) reduced voltage-gated K+ current density, and 4) increased action potential (AP) amplitude and firing frequency. Hyperglucagonemia in STZ-induced diabetes is thus likely due to increased glucagon content arising from enlarged glucagon granules and increased AP firing frequency and amplitude coinciding with enhanced Na+ and reduced K+ currents. These alterations may prime α cells in STZ-treated mice for more glucagon release per cell in response to low glucose stimulation. Thus, our study provides the first insight that STZ treatment sensitizes release mechanisms of α cells.

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

Aims/hypothesisOf the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4’s role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells.MethodsEndogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)–RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes.ResultsSyn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs.Conclusions/interpretationSyn-4 is involved in distinct molecular machineries that influence exocytosis of both pre-docked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4’s role in mediating portions of first-phase and second-phase GSIS.

Somatostatin Receptor Type 2 Antagonism Improves Glucagon Counterregulation in Biobreeding Diabetic Rats

Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly attributable to inadequate glucagon secretion. Intra-islet somatostatin (SST) suppression of hypoglycemia-stimulated α-cell glucagon release plays an important role. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by SST receptor type 2 (SSTR2) antagonism of α-cells, which relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes-prone (BBDP) rats mimic insulin-dependent human autoimmune T1D, whereas nondiabetic BBDP rats mimic prediabetes. Diabetic and nondiabetic rats underwent a 3-h infusion of vehicle compared with SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycemia clamped at 3 ± 0.5 mmol/L. Diabetic rats treated with SSTR2a needed little or no glucose infusion compared with untreated rats. We attribute this effect to SSTR2a restoration of the attenuated glucagon response. Direct effects of SSTR2a on α-cells was assessed by resecting the pancreas, which was cut into fine slices and subjected to perifusion to monitor glucagon release. SSTR2a treatment enhanced low-glucose–stimulated glucagon and corticosterone secretion to normal levels in diabetic rats. SSTR2a had similar effects in vivo in nondiabetic rats and promoted glucagon secretion from nondiabetic rat and human pancreas slices. We conclude that SST contributes to impaired glucagon responsiveness to hypoglycemia in autoimmune T1D. SSTR2a treatment can fully restore hypoglycemia-stimulated glucagon release sufficient to attain normoglycemia in both diabetic and prediabetic stages.

Elevation in Intracellular Long-Chain Acyl-Coenzyme A Esters Lead to Reduced β-Cell Excitability via Activation of Adenosine 5′-Triphosphate-Sensitive Potassium Channels

Closure of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate K(ATP) channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced beta-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced beta-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic beta-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse beta-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on beta-cell K(ATP) channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K(ATP) channel activity in AdACSL-1-infected beta-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected beta-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mm glucose. These results suggest a direct link between intracellular fatty ester accumulation and K(ATP) channel activation, which may contribute to beta-cell dysfunction in type 2 diabetes.

Munc18c mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose stimulated insulin secretion in human pancreatic beta-cells

Objective Pancreatic beta-cells express three Munc18 isoforms. Much is known about the roles of Munc18a (pre-docked secretory granules-SGs) and Munc18b (newcomer SGs and SG–SG fusion) in insulin exocytosis. Although shown to influence glucose-stimulated insulin secretion (GSIS) in rodents the precise role of Munc18c in insulin SG exocytosis has not been elucidated. We here examined the role of Munc18c in human pancreatic beta-cells. Methods Munc18c-shRNA/RFP lenti-virus (versus control virus) was used to knock down the expression level of Munc18c in human islets or single beta-cells. Insulin secretion and granule exocytosis were measured by performing islets perifusion, single-cell patch clamp capacitance measurements and total internal reflection fluorescence microscopy (TIRFM). Results Munc18c is most abundant in the cytosol of human beta-cells. Endogenous function of Munc18c was assessed by knocking down (KD) its islet expression by 70% employing lenti-shRNA virus. Munc18c-KD caused reduction in cognate syntaxin-4 islet expression but not in other exocytotic proteins, resulting in the reduction in GSIS in first- (by 42%) and second phases (by 35%). Single cell analyses of RFP-tagged Munc18c-KD beta-cells by patch clamp capacitance measurements showed inhibition in both readily-releasable pool (by 52%) and mobilization from the reserve pool (by 57%). TIRFM to assess single SG behavior showed that Munc18c-KD inhibition of first phase GSIS was attributed to reduction in exocytosis of pre-docked and newcomer SGs, and second phase inhibition attributed entirely to reduction in newcomer SG fusion (SGs that undergo minimal residence or docking time at the plasma membrane before fusion). Conclusion Munc18c is involved in the distinct molecular machineries that affect exocytosis of both predocked and newcomer SG pools that underlie Munc18cs role in first and second phases of GSIS, respectively.

Ex Vivo Human Pancreatic Slice Preparations Offer a Valuable Model for Studying Pancreatic Exocrine Biology

A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathologic responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis.

Botulinum neurotoxin A and neurotoxin E cleavage products of synaptosome-associated protein of 25 kd exhibit distinct actions on pancreatic islet beta-cell Kv2.1 channel gating.

Objectives: Synaptosome-associated protein of 25 kd (SNAP-25) regulates pancreatic islet &bgr;-cell-delayed rectifier K+ channels (Kv2.1) in addition to insulin exocytosis. Botulinum neurotoxin A (BoNT/A) and E (BoNT/E) cleavage and presumed deletion of SNAP-25 have been used to examine SNAP-25 function. We hypothesized that proteolytic products of SNAP-25 (206 amino acids) resulting from BoNT/A and BoNT/E cleavage, SNAP-251-197 and SNAP-251-180, have independent actions on &bgr;-cell Kv gating. Methods: We examined by confocal microscopy and immunoblotting BoNT/A and BoNT/E cleavage of SNAP-25 to these N-terminal fragments, and the consequent effects of these BoNTs and SNAP-25 fragments on Kv currents in rat &bgr; cells and MIN6 cells by patch clamp electrophysiology. Results: Confocal microscopy and immunoblotting showed that MIN6 cells transfected with BoNT/A or BoNT/E generated SNAP-251-197 and SNAP-251-180 fragments that were retained in the cytosol. Both BoNTs caused increased rate of channel activation and slowed channel inactivation, mimicked by these SNAP-25 fragments, but not full-length SNAP-25. These SNAP-25 fragments potentiated tetraethylammonium block of &bgr;-cell Kv currents. Conclusions: BoNT/A or BoNT/E treatment of &bgr; cells generates N-terminal SNAP-25 fragments that are retained in &bgr; cells to directly influence Kv channel gating in a manner distinct from full-length SNAP-25, contributing to overall actions of these BoNTs on insulin secretion.