2626 Diabetes Volume 67, December 2018 Syntaxin 4 Expression in Pancreatic b-Cells Promotes Islet Function and Protects Functional b-Cell Mass Eunjin Oh,1 Miwon Ahn,1 Solomon Afelik,2 Thomas C. Becker,3 Bart O. Roep,4 and Debbie C. Thurmond1 Diabetes 2018;67:2626–2639 | https://doi.org/10.2337/db18-0259 Syntaxin 4 (Stx4) enrichment in human and mouse islet Complications Trial (DCCT) correlated with reduced inci- grafts improves the success of transplants in reversing dences of retinopathy, nephropathy, and hypoglycemic streptozotocin (STZ)-induced diabetes in mice, although complications (4). As such, there is great interest in de- the underlying molecular mechanisms remain elusive. veloping strategies to enhance the function of insulin Toward a further understanding of this, human islets secretion from residual b-cell mass in vivo and to protect and inducible transgenic mice that selectively overex- islet b-cells from targeted demise. b b press Stx4 in islet -cells ( TG-Stx4) were challenged Evidence suggests that b-cell dysfunction precedes and fl with proin ammatory stressors in vitro and in vivo. Re- acts as an early predictor of T1D (5). One of the rate- b b markably, TG-Stx4 mice resisted the loss of -cell mass limiting features of the insulin secretion process is the and the glucose intolerance that multiple low doses of abundance of soluble n-ethylmaleimide-sensitive fusion STZ induce. Under standard conditions, glucose toler- protein-attachment protein receptor (SNARE) proteins ance was enhanced and mice maintained normal fasting in each b-cell. Insulin granules mobilized and juxtaposed glycemia and insulinemia. Conversely, Stx4 heterozy- gous knockout mice succumbed rapidly to STZ-induced to the cell surface undergo docking and fusion before glucose intolerance compared with their wild-type litter- b-cells release insulin; docking entails the pairing of pro- mates. Human islet b-cells overexpressing Stx4 exhibited tein complexes on the granule (vesicle SNAREs [VAMPs]) ISLET STUDIES enhanced insulin secretory capability; resilience against with their cognate receptor complexes at the target mem- t t proinflammatory cytokine–induced apoptosis; and re- brane ( -SNAREs) (6,7). Two types of -SNAREs, syn- duced expression of the CXCL9, CXCL10,andCXCL11 taxins and SNAPs, and one v-SNARE combine to form genes coordinate with decreased activation/nuclear lo- a heterotrimeric SNARE core complex. Syntaxin 1A (Stx1A) calization of nuclear factor-kB. Finding ways to boost and syntaxin 4 (Stx4) oversee glucose-stimulated insulin Stx4 expression presents a novel potential therapeutic secretion (GSIS), pairing with VAMP2 or VAMP8 (8,9), avenue for promoting islet function and preserving b-cell along with SNAP25 (10). However, whereas Stx4 over- mass. expression prompts improved glucose tolerance and GSIS in vivo (11,12), Stx1A enrichment results in b-cell fail- ure, which is linked to Stx1A-specific regulation of ion Type 1 diabetes (T1D) is linked to inflammation related to channels (13). the damaging effects of proinflammatory cytokines on Stx4 is a T1D candidate protein, predicted by in silico pancreatic islet b-cells (1,2). Data suggest that b-cell phenome-interactome network analysis (14); the Stx4 gene preservation and recovery interventions are most effective is located within T1D susceptibility region Iddm10 in individuals with the highest levels of residual b-cell (www.T1Dbase.org). In addition to Stx4, several other function and insulin production (3). Even modest b-cell SNARE exocytosis proteins are notably deficient in T2D function upon entry into the Diabetes Control and islets, yet remarkably, restoration of just Stx4 to those 1Department of Molecular and Cellular Endocrinology, Diabetes and Metabolism Received 6 March 2018 and accepted 25 September 2018. Research Institute of City of Hope, Duarte, CA This article contains Supplementary Data online at http://diabetes 2 Department of Surgery/Division of Transplantation, University of Illinois at .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0259/-/DC1. Chicago, Chicago, IL E.O. and M.A. contributed equally to this work. 3Department of Internal Medicine, Duke Molecular Physiology Institute, Duke University, Durham, NC © 2018 by the American Diabetes Association. Readers may use this article as 4Department of Diabetes Immunology, Diabetes and Metabolism Research In- long as the work is properly cited, the use is educational and not for profit, and the stitute of City of Hope, Duarte, CA work is not altered. More information is available at http://www.diabetesjournals .org/content/license. Corresponding author: Debbie C. Thurmond, [email protected]. diabetes.diabetesjournals.org Oh and Associates 2627 islets resulted in the resumption of biphasic GSIS (9). peroxidase and antimouse–horseradish peroxidase second- Given the established role of Stx4 in both phases of GSIS, ary antibodies (Bio-Rad, Hercules, CA) were used for this restored islet function was presumed to be related immunoblotting. Enhanced chemiluminescence and ECL to the ability of Stx4 to overcome the need for the other Prime reagents were purchased from Thermo Fisher Sci- missing exocytosis factors in the islets. Although promis- entific. For immunohistochemistry, antibodies against ing, because of the limitations of previous studies it re- Stx4 were purchased from Chemicon International; mains unclear whether Stx4 function in b-cell GSIS is the against glucagon, from Abcam; and against insulin, from primary or sole mechanism underpinning the restored GSIS Dako. Proinflammatory cytokines were purchased from phenomenon; in those studies, the previously available Stx4 ProSpec-Tany Technogene Ltd (East Brunswick, NJ) as overexpression systems used in human islets and mice were described elsewhere (20). not b-cell specific. Hence, in this study we used b-cell-selective expression Intraperitoneal Glucose Tolerance Test and Insulin systems to demonstrate that Stx4 enrichment, in addition Tolerance Test to its known beneficial role in b-cell function, correlates Blood samples were collected after male and female knock- with ameliorating apoptosis, preserving b-cells from in- out or transgenic mice were deprived of food, then freshly flammation-induced destruction by tempering the nuclear made D-glucose was injected intraperitoneally into the factor (NF)-kB–induced CXCL9, CXCL10, and CXCL11 in- mice for intraperitoneal glucose tolerance tests (IPGTTs). flammatory program. For the insulin tolerance test, the same mice were de- prived of food for 6 h (0800–1400 h), and after blood was RESEARCH DESIGN AND METHODS collected, the mice were injected intraperitoneally with Humulin R (0.75 unit/kg body wt) (Eli Lilly & Co., Indian- Mice apolis, IN). All mouse studies were conducted per the guidelines and assurances of the City of Hope Institutional Animal Care Multiple Low-Dose Streptozotocin Protocol and Use Committee. Stx4 heterozygous (+/2) knockout Male mice were subjected to an IPGTT at 10 weeks of age, mice were generated as previously described (12,15). To before the multiple low-dose streptozotocin (MLD-STZ) generate bTG-Stx4 mice, we bred commercially available protocol was initiated (21). Mice were injected with STZ rat insulin promoter (RIP)–reverse tetracycline-controlled (35 mg/kg body wt) for 5 consecutive days. Five days later transactivator (rtTA) mice (no. 008250; The Jackson Lab- (10 days after the first injection), Stx4+/+ (wild-type) and 2 oratory, Bar Harbor, ME), with our custom-generated TRE- Stx4+/ mice were deprived of food for 6 h (0800 h–1400 h), Stx4 mice. The TRE-Stx4 mice were initially made through blood samples were collected to measure blood glucose, the use of B6 blastocyst injection (Indiana University and then the mice were injected intraperitoneally with School of Medicine Transgenic Core). Rat Stx4 cDNA glucose (1 g/kg body wt) and the blood sampled from the was subcloned into the 59 BamHI and 39 ClaI sites down- tail vein every 30 min through the use of a glucometer stream of the tetracycline response element promoter; (HemoCue, Mission Viejo, CA), to again measure blood 29 founder mice were obtained, and four lines among glucose. Male bTG-Stx4 mice were given Dox (2 mg/mL) in these mice were phenotyped to show similar glucose and drinking water for 3 weeks before the MLD-STZ injections insulin tolerances. These mice were crossed with RIP-rtTA were begun. A total of 24 days after initiating the MLD- mice (RIP confers b-cell selectivity; tetracycline/doxycy- STZ protocol, bTG-Stx4 or non-Dox-treated (control) mice cline [Dox] induces binding of the TRE to elicit expression were deprived of food for 6 h (0800 h–1400 h), then of the transgene). Mouse insulin promoter (MIP)–rtTA injected intraperitoneally with glucose (2 g/kg body wt), mice were a gift from Philip Scherer (University of Texas after which we performed the IPGTTs. Southwestern Medical Center), and we used them, where indicated, in place of the RIP-rtTA, as previously described Isolation, Immunoblotting, and Secretion Assays With (16,17). bTG-Stx4 transgenic mice were provided drinking the Use of Transgenic Mouse Islets water containing Dox (1–2 mg/mL acidified water kept in Pancreatic mouse islets were isolated, as previously de- dark red bottles to protect it from light) or regular drinking scribed (22), from 10- to 14-week-old male and female fi water for at least 3 weeks before experiments to yield mice, as noted in the gure legends. Islets cultured over- stabilized transgene expression. night were manually placed in microcentrifuge tubes con- taining Krebs-Ringer bicarbonate buffer (10 mmol/L Antibodies and Cytokines HEPES [pH 7.4], 134 mmol/L NaCl, 5 mmol/L NaHCO3, Stx4 antibody was generated for use in immunoblotting 4.8 mmol/L KCl, 1 mmol/L CaCl2, 1.2 mmol/L MgSO4, mouse islets, as previously described (18,19). Stx4 for 1.2 mmol/L KH2PO4) containing 2.8 mmol/L glucose and human islets (Chemicon International, Temecula, CA); 0.1% BSA and left for 1 h. Then the buffer was changed to tubulin (Sigma-Aldrich, St.