Edwin P. Kwan

S100A4/Mts1 Produces Murine Pulmonary Artery Changes Resembling Plexogenic Arteriopathy and Is Increased in Human Plexogenic Arteriopathy

S100A4/Mts1 confers a metastatic phenotype in tumor cells and may also be related to resistance to apoptosis and angiogenesis. Approximately 5% of transgenic mice overexpressing S100A4/Mts1 develop pulmonary arterial changes resembling human plexogenic arteriopathy with intimal hyperplasia leading to occlusion of the arterial lumen. To assess the pathophysiological significance of this observation, immunohistochemistry was applied to quantitatively analyze S100A4/Mts1 expression in pulmonary arteries in surgical lung biopsies from children with pulmonary hypertension secondary to congenital heart disease. S100A4/Mts1 was not detected in pulmonary arteries with low-grade hypertensive lesions but was expressed in smooth muscle cells of lesions showing neointimal formation and with increased intensity in vessels with an occlusive neointima and plexiform lesions. Putative downstream targets of S100A4/Mts1 include Bax, which is pro-apoptotic, and the pro-angiogenic vascular endothelial growth factor (VEGF). The increase in S100A4/Mts1 expression precedes heightened expression of Bax in progressively severe neointimal lesions but in non-S100A4/Mts1-expressing cells. VEGF immunoreactivity did not correlate with severity of disease. The relationship of increased S100A4/Mts1 to pathologically similar lesions in the transgenic mice and patients occurs despite differences in localization (endothelial versus smooth muscle cells).

Pancreatic GLP-1 receptor activation is sufficient for incretin control of glucose metabolism in mice

Glucagon-like peptide-1 (GLP-1) circulates at low levels and acts as an incretin hormone, potentiating glucose-dependent insulin secretion from islet β cells. GLP-1 also modulates gastric emptying and engages neural circuits in the portal region and CNS that contribute to GLP-1 receptor-dependent (GLP-1R-dependent) regulation of glucose homeostasis. To elucidate the importance of pancreatic GLP-1R signaling for glucose homeostasis, we generated transgenic mice that expressed the human GLP-1R in islets and pancreatic ductal cells (Pdx1-hGLP1R:Glp1r-/- mice). Transgene expression restored GLP-1R-dependent stimulation of cAMP and Akt phosphorylation in isolated islets, conferred GLP-1R-dependent stimulation of β cell proliferation, and was sufficient for restoration of GLP-1-stimulated insulin secretion in perifused islets. Systemic GLP-1R activation with the GLP-1R agonist exendin-4 had no effect on food intake, hindbrain c-fos expression, or gastric emptying but improved glucose tolerance and stimulated insulin secretion in Pdx1-hGLP1R:Glp1r-/- mice. i.c.v. GLP-1R blockade with the antagonist exendin(9-39) impaired glucose tolerance in WT mice but had no effect in Pdx1-hGLP1R:Glp1r-/- mice. Nevertheless, transgenic expression of the pancreatic GLP-1R was sufficient to normalize both oral and i.p. glucose tolerance in Glp1r-/- mice. These findings illustrate that low levels of endogenous GLP-1 secreted from gut endocrine cells are capable of augmenting glucoregulatory activity via pancreatic GLP-1Rs independent of communication with neural pathways.

SNAREing voltage-gated K+ and ATP-sensitive K+ channels : Tuning β-cell excitability with syntaxin-1A and other exocytotic proteins

The three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa), and synaptobrevin, constitute the minimal machinery for exocytosis in secretory cells such as neurons and neuroendocrine cells by forming a series of complexes prior to and during vesicle fusion. It was subsequently found that these SNARE proteins not only participate in vesicle fusion, but also tether with voltage-dependent Ca2+ channels to form an excitosome that precisely regulates calcium entry at the site of exocytosis. In pancreatic islet β-cells, ATP-sensitive K+ (KATP) channel closure by high ATP concentration leads to membrane depolarization, voltage-dependent Ca2+ channel opening, and insulin secretion, whereas subsequent opening of voltage-gated K+ (Kv) channels repolarizes the cell to terminate exocytosis. We have obtained evidence that syntaxin-1A physically interacts with Kv2.1 (the predominant Kv in β-cells) and the sulfonylurea recep...

Munc13-1 Deficiency Reduces Insulin Secretion and Causes Abnormal Glucose Tolerance

Munc13-1 is a diacylglycerol (DAG) receptor that is essential for synaptic vesicle priming. We recently showed that Munc13-1 is expressed in rodent and human islet β-cells and that its levels are reduced in islets of type 2 diabetic humans and rat models, suggesting that Munc13-1 deficiency contributes to the abnormal insulin secretion in diabetes. To unequivocally demonstrate the role of Munc13-1 in insulin secretion, we studied heterozygous Munc13-1 knockout mice (+/−), which exhibited elevated glucose levels during intraperitoneal glucose tolerance tests with corresponding lower serum insulin levels. Munc13-1+/− mice exhibited normal insulin tolerance, indicating that a primary islet β-cell secretory defect is the major cause of their hyperglycemia. Consistently, glucose-stimulated insulin secretion was reduced 50% in isolated Munc13-1+/− islets and was only partially rescued by phorbol ester potentiation. The corresponding alterations were minor in mice expressing one allele of a Munc13-1 mutant variant, which does not bind DAG (H567K/+). Capacitance measurements of Munc13-1+/− and Munc13-1H567k/+ islet β-cells revealed defects in granule priming, including the initial size and refilling of the releasable pools, which become accentuated by phorbol ester potentiation. We conclude that Munc13-1 plays an important role in glucose-stimulated insulin secretion and that Munc13-1 deficiency in the pancreatic islets as occurs in diabetes can reduce insulin secretion sufficient to cause abnormal glucose homeostasis.

Interaction Between Munc13-1 and RIM Is Critical for Glucagon-Like Peptide-1–Mediated Rescue of Exocytotic Defects in Munc13-1–Deficient Pancreatic β-Cells

OBJECTIVE—Glucagon-like peptide-1 (GLP-1) rescues insulin secretory deficiency in type 2 diabetes partly via cAMP actions on exchange protein directly activated by cAMP (Epac2) and protein kinase A (PKA)-activated Rab3A-interacting molecule 2 (Rim2). We had reported that haplodeficient Munc13-1+/− mouse islet β-cells exhibited reduced insulin secretion, causing glucose intolerance. Munc13-1 binds Epac2 and Rim2, but their functional interactions remain unclear. RESEARCH DESIGN AND METHODS—We used Munc13-1+/− islet β-cells to examine the functional interactions between Munc13-1 and Epac2 and PKA. GLP-1 stimulation of Munc13-1+/− islets normalized the reduced biphasic insulin secretion by its actions on intact islet cAMP production and normal Epac2 and Rim2 levels. RESULTS—To determine which exocytotic steps caused by Munc13-1 deficiency are rescued by Epac2 and PKA, we used patch-clamp capacitance measurements, showing that 1) cAMP restored the reduced readily releasable pool (RRP) and partially restored refilling of a releasable pool of vesicles in Munc13-1+/− β-cells, 2) Epac-selective agonist [8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate] partially restored the reduced RRP and refilling of a releasable pool of vesicles, and 3) PKA blockade by H89 (leaving Epac intact) impaired cAMP ability to restore the RRP and refilling of a releasable pool of vesicles. Conversely, PKA-selective agonist (N6-benzoyladenosine-cAMP) completely restored RRP and partially restored refilling of a releasable pool of vesicles. To determine specific contributions within Epac-Rim2–Munc13-1 interaction sites accounting for cAMP rescue of exocytosis caused by Munc13-1 deficiency, we found that blockade of Rim2–Munc13-1 interaction with Rim-Munc13-1–binding domain peptide abolished cAMP rescue, whereas blockade of Epac-Rim2 interaction with Rim2-PDZ peptide only moderately reduced refilling with little effect on RRP. CONCLUSIONS—cAMP rescue of priming defects caused by Munc13-1 deficiency via Epac and PKA signaling pathways requires downstream Munc13-1–Rim2 interaction.

Distinct In Vivo Roles of Caspase-8 in β-Cells in Physiological and Diabetes Models

Inadequate pancreatic β-cell mass resulting from excessive β-cell apoptosis is a key defect in type 1 and type 2 diabetes. Caspases are the major molecules involved in apoptosis; however, in vivo roles of specific caspases in diabetes are unclear. The purpose of this study is to examine the role of Caspase (Casp)8 in β-cells in vivo. Using the Cre-loxP system, mice lacking Casp8 in β-cells (RIPcre+Casp8fl/fl mice) were generated to address the role of Casp8 in β-cells in physiological and diabetes models. We show that islets isolated from RIPcre+Casp8fl/fl mice were protected from Fas ligand (FasL)–and ceramide-induced cell death. Furthermore, RIPcre+Casp8fl/fl mice were protected from in vivo models of type 1 and type 2 diabetes. In addition to being the central mediator of apoptosis in diabetes models, we show that Casp8 is critical for maintenance of β-cell mass under physiological conditions. With aging, RIPcre+Casp8fl/fl mice gradually develop hyperglycemia and a concomitant decline in β-cell mass. Their islets display decreased expression of molecules involved in insulin/IGF-I signaling and show decreased pancreatic duodenal homeobox-1 and cAMP response element binding protein expression. At the level of individual islets, we observed increased insulin secretory capacity associated with increased expression of exocytotic proteins. Our results show distinct context-specific roles of Casp8 in physiological and disease states; Casp8 is essential for β-cell apoptosis in type 1 and type 2 diabetes models and in regulating β-cell mass and insulin secretion under physiological conditions.

The RalA GTPase Is a Central Regulator of Insulin Exocytosis from Pancreatic Islet Beta Cells

RalA is a small GTPase that is thought to facilitate exocytosis through its direct interaction with the mammalian exocyst complex. In this study, we report an essential role for RalA in regulated insulin secretion from pancreatic beta cells. We employed lentiviral-mediated delivery of RalA short hairpin RNAs to deplete endogenous RalA protein in mouse pancreatic islets and INS-1 beta cells. Perifusion of mouse islets depleted of RalA protein exhibited inhibition of both first and second phases of glucose-stimulated insulin secretion. Consistently, INS-1 cells depleted of RalA caused a severe inhibition of depolarization-induced insulin exocytosis determined by membrane capacitance, including a reduction in the size of the ready-releasable pool of insulin granules and a reduction in the subsequent mobilization and exocytosis of the reserve pool of granules. Collectively, these data suggest that RalA is a critical component in biphasic insulin release from pancreatic beta cells.

High β-cell mass prevents streptozotocin-induced diabetes in thioredoxin-interacting protein-deficient mice

Thioredoxin-interacting protein (TxNIP) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. Diabetic mice exhibit increased expression of TxNIP in pancreatic islets, and recent studies suggest that TxNIP is a proapoptotic factor in beta-cells that may contribute to the development of diabetes. Here, we examined the role of TxNIP deficiency in vivo in the development of insulin-deficient diabetes and whether it impacted on pancreatic beta-cell mass and/or insulin secretion. TxNIP-deficient (Hcb-19/TxNIP(-/-)) mice had lower baseline glycemia, higher circulating insulin concentrations, and higher total pancreatic insulin content and beta-cell mass than control mice (C3H). Hcb-19/TxNIP(-/-) did not develop hyperglycemia when injected with standard multiple low doses of streptozotocin (STZ), in contrast to C3H controls. Surprisingly, although beta-cell mass remained higher in Hcb-19/TxNIP(-/-) mice compared with C3H after STZ exposure, the relative decrease induced by STZ was as great or even greater in the TxNIP-deficient animals. Consistently, cultured pancreatic INS-1 cells transfected with small-interfering RNA against TxNIP were more sensitive to cell death induced by direct exposure to STZ or to the combination of inflammatory cytokines interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha. Furthermore, when corrected for insulin content, isolated pancreatic islets from TxNIP(-/-) mice exhibited reduced glucose-induced insulin secretion. These data indicate that TxNIP functions as a regulator of beta-cell mass and influences insulin secretion. In conclusion, the relative resistance of TxNIP-deficient mice to STZ-induced diabetes appears to be because of an increase in beta-cell mass. However, TxNIP deficiency is associated with sensitization to STZ- and cytokine-induced beta-cell death, indicating complex regulatory roles of TxNIP under different physiological and pathological conditions.

Activation of exchange protein directly activated by cyclic adenosine monophosphate and protein kinase A regulate common and distinct steps in promoting plasma membrane exocytic and granule-to-granule fusions in rat islet beta cells.

Objectives: Using FM1-43 epifluorescence imaging and electron microscopy, we recently reported that glucagon-like peptide (GLP-1)-mediated cyclic adenosine monophosphate (cAMP) potentiation of insulin secretion markedly promotes the number of plasma membrane (PM) exocytic sites and insulin secretory granule (SG)-to-granule fusions underlying compound and sequential exocytosis. Methods: Here, we used FM1-43 imaging to dissect the distinct contributions of putative GLP-1/cAMP activated substrates-exchange protein directly activated by cAMP (EPAC) and protein kinase A (PKA)-in mediating these exocytic events. Results: Like GLP-1, cAMP activation by forskolin increased the number of PM exocytic sites (2.3-fold), which were mainly of the robust-sustained (55.8%) and stepwise-multiphasic (37.7%) patterns corresponding to compound and sequential SG-SG exocytosis, respectively, with few monophasic hotspots (6.5%) corresponding to single-granule exocytosis. Direct activation of EPAC by 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cAMP also increased the number of exocytic sites, but which were mainly multiphasic (60%) and monophasic (40%) hotspots. Protein kinase A inhibition by H89 blocked forskolin-evoked robust-sustained hotspots, while retaining multiphasic (47%) and monophasic (53%) hotspots. Consistently, PKA activation (N6-benzoyladenosine-3′,5′-cAMP) evoked only multiphasic (60%) and monophasic (40%) hotspots. These results suggested that PKA activation is required but alone is insufficient to promote compound SG-SG fusions. 8-(4-Chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cAMP plus N6- benzoyladenosine-3′,5′-cAMP stimulation completely reconstituted the effects of forskolin, including increasing the number of exocytic sites, with a similar pattern of robust-sustained (42.6%) and stepwise (39.6%) hotspots and few monophasic (17.8%) hotspots. Conclusions: The EPAC and PKA modulate both distinct and common exocytic steps to potentiate insulin exocytosis where (a) EPAC activation mobilizes SGs to fuse at the PM, thereby increasing number of PM exocytic sites; and (b) PKA and EPAC activation synergistically modulate SG-SG fusions underlying compound and sequential exocytoses.Abbreviations: CICR - calcium-induced calcium release, 8-CPT-2′-O-Me-cAMP - 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, EPAC - exchange protein directly activated by cAMP, IBMX - 3-isobutyl-1-methylxanthine, GLP-1 - glucagon-like peptide 1, N6-Bnz-cAMP - N6-benzoyladenosine-3′,5′-cyclic monophosphate, PKA - protein kinase A, PM - plasma membrane, RRP - readily releasable pool, SG - secretory granule, SUR-1 - sulfonylurea receptor 1

Rescuing the Subprime Meltdown in Insulin Exocytosis in Diabetes

Neuroendocrine pancreatic islet β‐cells secrete the hormone insulin in response to glucose stimulation and adapt efficiently to increased demand by peripheral tissues to maintain glucose homeostasis. Insulin is packed within dense‐core granules, which traffic and dock onto the plasma membrane whereby a Ca2+ stimulus evokes exocytosis by soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE), complex‐mediated, membrane fusion. Recent studies have unveiled postdocking steps mediated by “priming” factors that influence SNARE complex assembly to confer fusion readiness to the docked granules. This review will summarize recent insights into the priming role for Munc13 in the exocytosis of insulin granules. We present evidence for the interaction of Munc13‐1 with exocytotic substrates involved in cAMP‐mediated potentiation of insulin release, the latter we show to mediate enhanced granule‐to‐granule fusion events underlying compound exocytosis. We thus also further review the current understanding of granule‐to‐granule fusion. As agents acting on cAMP signaling are clinically used to augment insulin release in diabetes, this better understanding of priming steps may reveal additional novel therapeutic strategies to increase the capacity for insulin release to improve the treatment of diabetes.