The Pathogenic Role of Cystathionine γ-Lyase/Hydrogen Sulfide in Streptozotocin-Induced Diabetes in Mice
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.028
ISSN1525-2191
AutoresGuangdong Yang, Guanghua Tang, Ling Zhang, Lingyun Wu, Rui Wang,
Tópico(s)Cannabis and Cannabinoid Research
ResumoReduced β-cell mass and increased activities of ATP-sensitive K+ channels in pancreatic β cells are associated with the pathogenesis of diabetes. Cystathionine γ-lyase (CSE) is a major hydrogen sulfide (H2S)–producing enzyme in pancreatic β cells. Herein, we examine the effects of genetic and pharmacologic ablation of CSE on β-cell functions and their correlation with streptozotocin (STZ)-induced diabetes. Compared with wild-type mice, CSE knockout (CSE KO) mice that received STZ injections exhibited a delayed onset of diabetic status. The application of dl-propargylglycine (PPG) to inhibit CSE activity protected wild-type mice from STZ-induced hyperglycemia and hypoinsulinemia. STZ significantly increased pancreatic H2S production in wild-type mice but not in CSE KO mice. STZ induced more apoptotic β-cell death in wild-type mice than in CSE KO mice. STZ exposure decreased the viability of cultured INS-1E cells, which was partly reversed by PPG co-treatment. STZ also significantly stimulated H2S production in cultured INS-1E cells. In addition, STZ stimulated ATP-sensitive K+ currents in pancreatic β cells from wild-type mice but not in the presence of PPG or in β cells from CSE KO mice. Sodium hydrosulfide injection instantly increased blood glucose, decreased plasma insulin, and deteriorated glucose tolerance in mice. Take together, these results provide evidence that the CSE/H2S system plays a critical role in regulating β-cell functions. Reduced β-cell mass and increased activities of ATP-sensitive K+ channels in pancreatic β cells are associated with the pathogenesis of diabetes. Cystathionine γ-lyase (CSE) is a major hydrogen sulfide (H2S)–producing enzyme in pancreatic β cells. Herein, we examine the effects of genetic and pharmacologic ablation of CSE on β-cell functions and their correlation with streptozotocin (STZ)-induced diabetes. Compared with wild-type mice, CSE knockout (CSE KO) mice that received STZ injections exhibited a delayed onset of diabetic status. The application of dl-propargylglycine (PPG) to inhibit CSE activity protected wild-type mice from STZ-induced hyperglycemia and hypoinsulinemia. STZ significantly increased pancreatic H2S production in wild-type mice but not in CSE KO mice. STZ induced more apoptotic β-cell death in wild-type mice than in CSE KO mice. STZ exposure decreased the viability of cultured INS-1E cells, which was partly reversed by PPG co-treatment. STZ also significantly stimulated H2S production in cultured INS-1E cells. In addition, STZ stimulated ATP-sensitive K+ currents in pancreatic β cells from wild-type mice but not in the presence of PPG or in β cells from CSE KO mice. Sodium hydrosulfide injection instantly increased blood glucose, decreased plasma insulin, and deteriorated glucose tolerance in mice. Take together, these results provide evidence that the CSE/H2S system plays a critical role in regulating β-cell functions. Hydrogen sulfide (H2S) is a novel and important gasotransmitter.1Wang R. Hydrogen sulphide: the third gasotransmitter in biology and medicine.Antioxid Redox Signal. 2010; 12: 1061-1064Crossref PubMed Scopus (223) Google Scholar, 2Wang R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter?.FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1540) Google Scholar, 3Zhao W. Zhang J. Lu Y. Wang R. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1635) Google Scholar H2S has been reported to regulate cellular apoptosis and proliferation,4Yang G. Yang W. Wu L. Wang R. H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.J Biol Chem. 2007; 282: 16567-16576Crossref PubMed Scopus (175) Google Scholar protect the heart from ischemic damage,5Elrod J.W. Calvert J.W. Morrison J. Doeller J.E. Kraus D.W. Tao L. Jiao X. Scalia R. Kiss L. Szabo C. Kimura H. Chow C.W. Lefer D.J. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function.Proc Natl Acad Sci U S A. 2007; 104: 15560-15565Crossref PubMed Scopus (903) Google Scholar induce vasorelaxation and lower blood pressure,3Zhao W. Zhang J. Lu Y. Wang R. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1635) Google Scholar and alter insulin secretion and inflammation.6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar, 7Li L. Bhatia M. Moore P.K. Hydrogen sulphide: a novel mediator of inflammation?.Curr Opin Pharmacol. 2006; 6: 125-129Crossref PubMed Scopus (196) Google Scholar, 8Kaneko Y. Kimura Y. Kimura H. Niki I. L-cysteine inhibits insulin release from the pancreatic β-cell: possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitter.Diabetes. 2006; 55: 1391-1397Crossref PubMed Scopus (254) Google Scholar Two pyridoxal-5′-phosphate–dependent enzymes, cystathionine β-synthase (CBS) (EC4.2.1.22) and cystathionine γ-lyase (CSE; also often named CTH) (EC 4.4.1.1), are responsible for most endogenous production of H2S in mammalian tissues, which use l-cysteine as the main substrate.2Wang R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter?.FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1540) Google Scholar, 9Stipanuk M.H. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine.Annu Rev Nutr. 2004; 24: 539-577Crossref PubMed Scopus (739) Google Scholar CSE seems to be the main H2S-forming enzyme in the pancreas.4Yang G. Yang W. Wu L. Wang R. H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.J Biol Chem. 2007; 282: 16567-16576Crossref PubMed Scopus (175) Google Scholar, 10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar Recently, it was found that H2S formation was significantly higher in the pancreas of Zuker diabetic fatty rats and streptozotocin (STZ)-induced diabetic rats compared with nondiabetic animals.10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar, 11Yusuf M. Kwong-Huat B.T. Hsu A. Whiteman M. Bhatia M. Moore P.K. Streptozotocin-induced diabetes in the rat is associated with enhanced tissue hydrogen sulfide biosynthesis.Biochem Biophys Res Commun. 2005; 333: 1146-1152Crossref PubMed Scopus (158) Google Scholar As a substrate for H2S production, cysteine level was also elevated in diabetic patients with diabetic nephropathy renal complications.12Herrmann W. Schorr H. Obeid R. Makowski J. Fowler B. Kuhlmann M.K. Disturbed homocysteine and methionine cycle intermediates S-adenosylhomocysteine and S-adenosylmethionine are related to degree of renal insufficiency in type 2 diabetes.Clin Chem. 2005; 51: 891-897Crossref PubMed Scopus (61) Google Scholar H2S and cysteine inhibited insulin secretion from insulin-secreting β-cell lines (INS-1E, MIN6, and HIT-T15) or from isolated rat islets.6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar, 8Kaneko Y. Kimura Y. Kimura H. Niki I. L-cysteine inhibits insulin release from the pancreatic β-cell: possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitter.Diabetes. 2006; 55: 1391-1397Crossref PubMed Scopus (254) Google Scholar, 13Ali M.Y. Whiteman M. Low C.M. Moore P.K. Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway.J Endocrinol. 2007; 195: 105-112Crossref PubMed Scopus (73) Google Scholar Overexpression of CSE inhibited insulin release from INS-1E cells, but lowering endogenous H2S production by dl-propargylglycine (PPG) or CSE-targeted small-interfering RNA had the opposite effect.6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar Among demonstrated cellular and molecular mechanisms for pathophysiologic effects of H2S on β cells are the induction of cell apoptosis and the activation of ATP-sensitive K+ (KATP) channels.4Yang G. Yang W. Wu L. Wang R. H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.J Biol Chem. 2007; 282: 16567-16576Crossref PubMed Scopus (175) Google Scholar, 6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar, 13Ali M.Y. Whiteman M. Low C.M. Moore P.K. Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway.J Endocrinol. 2007; 195: 105-112Crossref PubMed Scopus (73) Google Scholar We have shown that exogenously applied H2S or endogenously produced H2S derived from overexpressed CSE induced apoptosis of INS-1E cells, which suggests a novel role of the CSE/H2S system in regulating pancreatic functions under physiologic conditions and in diabetes by stimulating β-cell apoptosis.5Elrod J.W. Calvert J.W. Morrison J. Doeller J.E. Kraus D.W. Tao L. Jiao X. Scalia R. Kiss L. Szabo C. Kimura H. Chow C.W. Lefer D.J. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function.Proc Natl Acad Sci U S A. 2007; 104: 15560-15565Crossref PubMed Scopus (903) Google Scholar In addition, we and others have shown that H2S functions as an endogenous opener of KATP channels in β cells independent of activation of cytosolic second messengers.6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar, 13Ali M.Y. Whiteman M. Low C.M. Moore P.K. Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway.J Endocrinol. 2007; 195: 105-112Crossref PubMed Scopus (73) Google Scholar Basal KATP channel currents were significantly reduced by lowering the endogenous H2S level with CSE–small-interfering RNA transfection in INS-1E cells.6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar Interaction of H2S and KATP channels in insulin-secreting cells may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic β cells. However, this interaction during the development of diabetes is still unclear. Given that altered H2S production is involved in the development of diabetes, inhibition of the CSE/H2S pathway may protect pancreatic β cells from cytotoxic damage and suppress abnormal KATP channel activity. To test this hypothesis, we examined the relationship of pancreatic CSE/H2S activity and β-cell mass and functions using CSE knockout (CSE KO) mice14Yang G. Wu L. Jiang B. Yang W. Qi J. Cao K. Meng Q. Mustafa A.K. Mu W. Zhang S. Snyder S.H. Wang R. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine γ-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1917) Google Scholar or cultured β-cell lines. STZ was used to treat mice, and the development of diabetes was compared between CSE KO and wild-type mice. The effects of STZ on H2S production and β-cell apoptosis and KATP channel activities were also investigated to probe the role of H2S in STZ-induced diabetes. CSE KO mice were generated as previously described.14Yang G. Wu L. Jiang B. Yang W. Qi J. Cao K. Meng Q. Mustafa A.K. Mu W. Zhang S. Snyder S.H. Wang R. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine γ-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1917) Google Scholar The second and third generations of 10- to 16-week-old male CSE KO mice and age-matched male wild-type littermates on the C57BL/6J/129 background were used. PCR genotyping of CSE KO mice was performed using a three-primer assay in two reactions.14Yang G. Wu L. Jiang B. Yang W. Qi J. Cao K. Meng Q. Mustafa A.K. Mu W. Zhang S. Snyder S.H. Wang R. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine γ-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1917) Google Scholar All the animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the US NIH (publication No. 85-23, revised 1996) and approved by the Animal Health Care Committees of the University of Saskatchewan and Lakehead University. All the animals were maintained on standard rodent chow and had free access to food and water. Mice (10 to 12 weeks old) were injected i.p. with STZ (40 mg/kg) between 10:00 AM and 11:00 AM for 5 consecutive days (days 1 to 5) to induce hyperglycemia.15O'Brien B.A. Harmon B.V. Cameron D.P. Allan D.J. Beta-cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model.J Pathol. 1996; 178: 176-181Crossref PubMed Scopus (171) Google Scholar, 16Flodstrom M. Tyrberg B. Eizirik D.L. Sandler S. Reduced sensitivity of inducible nitric oxide synthase-deficient mice to multiple low-dose streptozotocin-induced diabetes.Diabetes. 1999; 48: 706-713Crossref PubMed Scopus (145) Google Scholar STZ was freshly dissolved in citrate buffer (pH 4.5); mice in the control group received an equal volume of citrate buffer alone. In some wild-type mice, PPG dissolved in PBS or PBS alone was injected i.p. at 40 mg/kg/day for 30 days (between 9:00 AM and 10:00 AM on days 1 to 30). Whole blood glucose concentration was measured in blood obtained from the tail vein of mice using OneTouch blood glucose strips (LifeScan, Milpitas, CA). Plasma insulin was measured using an enzyme-linked immunosorbent assay (ELISA) kit with mouse insulin as a standard (Mercodia AB, Uppsala, Sweden) according to the manufacturer's procedure. Blood samples were obtained from mice after overnight fasting unless otherwise specified. Blood plasma was prepared by spinning a tube of fresh blood containing EDTA (1500 × g for 15 minutes at 4°C). Body weight and blood glucose level were measured every 3 to 4 days between 9:00 AM and 10:00 AM, and food and water intake were measured daily between 10:00 AM and 11:00 AM on days 25 to 30. INS-1E cells derived from a rat insulinoma were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum, 2.2 mg/mL of sodium bicarbonate, 1 mmol/L sodium pyruvate, 50 μmol/L 2-mercaptoethanol, 100 U/mL of penicillin, and 100 μg/mL of streptomycin, as previously described.6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar The glucose concentration in the culture media was consistent at 3 mmol/L. The experiments were performed when the cells reached 70% to 80% confluence between passages 56 and 70. The cells were maintained overnight in serum-free media before treatment. The cultured INS-1E cells were serum starved overnight and then were subjected to 30 minutes of STZ exposure (2 mmol/L) the next day. After STZ exposure, the medium was replaced with fresh normal medium (10% fetal bovine serum) without or with PPG (200 μmol/L) for another 24 hours. Quantification of cellular viability was made by using the MTT assay.4Yang G. Yang W. Wu L. Wang R. H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.J Biol Chem. 2007; 282: 16567-16576Crossref PubMed Scopus (175) Google Scholar Mouse islets were isolated by collagenase digestion as described elsewhere.10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar, 17Stiles B.L. Kuralwalla-Martinez C. Guo W. Gregorian C. Wang Y. Tian J. Magnuson M.A. Wu H. Selective deletion of Pten in pancreatic β cells leads to increased islet mass and resistance to STZ-induced diabetes.Mol Cell Biol. 2006; 26: 2772-2781Crossref PubMed Scopus (110) Google Scholar Briefly, after the mice were anesthetized by i.p. injection of ketamine HCl and xylazine (80 mg/kg and 5 mg/kg body weight, respectively), pancreatic islets were digested with collagenase and were handpicked under a microscope. Ca2+-free medium consisting of 120 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L MgSO4, 4 mmol/L glucose, 1 mmol/L EGTA, 25 mmol/L HEPES-NaOH (pH 7.4), and 1% (w/v) bovine serum albumin was used for islet isolation. Isolated islets were washed and preincubated with glucose-free RPMI 1640 medium in 24-well plates (10 islets per well).10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar After 30 minutes of preincubation with 3 mmol/L glucose, the islets were incubated for 30 minutes at 37°C in the presence of 20 mmol/L glucose in a humidified atmosphere of 5% CO2 and 95% air. At the end of each incubation period, the medium was collected and centrifuged for 10 minutes at 1500 × g to remove islet debris. Insulin levels in the supernatants were determined using the mouse insulin ELISA kit (Mercodia AB). The patch clamp technique was used to record whole cell KATP currents of isolated single β cells.6Yang W. Yang G. Jia X. Wu L. Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.J Physiol. 2005; 569: 519-531Crossref PubMed Scopus (419) Google Scholar, 10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar, 18Gier B. Krippeit-Drews P. Sheiko T. Aguilar-Bryan L. Bryan J. Düfer M. Drews G. Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress.J Clin Invest. 2009; 119: 3246-3256PubMed Google Scholar Only cells in which KATP channel activity was significantly decreased by a high glucose level or by the KATP channel blocker glibenclamide were used in this study. Mouse pancreases from each treatment group were dissected on day 30 after the first STZ injection and were immediately fixed in 4% buffered formalin.19Wu L. Noyan Ashraf M.H. Facci M. Wang R. Paterson P.G. Ferrie A. Juurlink B.H. Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system.Proc Natl Acad Sci U S A. 2004; 101: 7094-7099Crossref PubMed Scopus (242) Google Scholar After the tissues were embedded in 22-oxacalcitriol compounds, 8-μm-thick sections were cut using a cryostat and were picked up on poly-l-lysine–coated slides. H&E staining was performed as previously described.19Wu L. Noyan Ashraf M.H. Facci M. Wang R. Paterson P.G. Ferrie A. Juurlink B.H. Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system.Proc Natl Acad Sci U S A. 2004; 101: 7094-7099Crossref PubMed Scopus (242) Google Scholar Primary antibody (guinea pig against swine insulin) was incubated in a humid chamber for 1 hour at 1:5000 dilution (DakoCytomation, Mississauga, ON, Canada). Second antibody fluorescein isothiocyanate–donkey anti-guinea pig (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was diluted at 1:500. Images were observed and analyzed using an inverted Olympus IX70 microscope (Olympus, Tokyo, Japan). An in situ cell death detection kit (Hoffmann-La Roche Ltd., Mississauga, ON, Canada) was used to visualize the apoptotic pancreatic β cells.20Yang G. Sun X. Wang R. Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3.FASEB J. 2004; 18: 1782-1784Crossref PubMed Scopus (265) Google Scholar, 21Mathis D. Vence L. Benoist C. β-Cell death during progression to diabetes.Nature. 2001; 414: 792-798Crossref PubMed Scopus (758) Google Scholar Dual staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and insulin was performed. The nuclei were counterstained using DAPI (0.3 μmol/L; Molecular Probes Inc., Eugene, OR). Percentage of apoptosis was calculated by dividing the number of TUNEL-positive β cells by the total number of β-cell stained nuclei. At least 500 nuclei per pancreas were counted, and four mice per group were used. Pancreatic tissues or isolated islets were obtained and lysed.10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar The extracts were separated by centrifugation at 14,000 × g for 15 minutes at 4°C. SDS-PAGE and Western blot analysis were performed as described previously.4Yang G. Yang W. Wu L. Wang R. H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.J Biol Chem. 2007; 282: 16567-16576Crossref PubMed Scopus (175) Google Scholar, 10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar The primary antibodies were diluted at 1:1000 for CSE (Novus Biologicals, Littleton, CO) and at 1:10,000 for β-actin (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). Horseradish peroxidase–conjugated secondary antibody was used at 1:5000 dilution. Immunoreactions were visualized by electrochemiluminescence and were exposed to X-ray film (Kodak scientific imaging film; Fisher Scientific Company, Ottawa, ON, Canada). Endogenous H2S productions from mouse pancreas tissues and INS-1E cells were measured as previously reported.3Zhao W. Zhang J. Lu Y. Wang R. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1635) Google Scholar, 10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar The mRNA expressions of CBS and glucose transporter 2 (Glut-2) in islets were measured by real-time PCR using an iCycler iQ3 apparatus (Bio-Rad Laboratories, Hercules, CA) associated with the iCycler optical system software (version 3.1) using SYBR green PCR master mix (Molecular Probes Inc.), as described previously.4Yang G. Yang W. Wu L. Wang R. H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.J Biol Chem. 2007; 282: 16567-16576Crossref PubMed Scopus (175) Google Scholar Briefly, all PCRs were performed in a volume of 20 μL using 96-well optical-grade PCR plates and optical sealing tape. The cycling was conducted at 95°C for 90 seconds followed by 38 cycles at 95°C for 10 seconds and at 60°C for 20 seconds. The primers of CBS (GenBank accession number NM_144855) were 5′-GGTGGTGGCGTCTGCGTGTTCA-3′ (sense, position 1211–1232) and 5′-GCCCAGCGTGTCGGTCAGGT-3′ (antisense, position 1460–1480). These primers produced a product of 270 bp. The primers of Glut-2 (GenBank accession number NM_031197) were 5′-GGATGCCAATTACCGACAGC-3′ (sense, position 885–904) and 5′-AGGCGAATTTATCCAGCAGCACAA-3′ (antisense, position 1157–1180). These primers produced a product of 296 bp. The primers of β-actin were purchased from Ambion (Austin, TX) and produced a product of 295 bp. A standard curve was constructed using a series of dilutions of total RNA (Ambion) transcribed to cDNA using the same protocol outlined previously herein to confirm the same amplifying efficiency in the PCR. A standard melting curve analysis was performed using the following thermal cycling profile: 95°C for 10 seconds, 55°C for 15 seconds, and ramping to 95°C in 1° increments to confirm the absence of primer dimers. Relative mRNA quantification was calculated by using the following arithmetic formula: 2−ΔΔCT, where ΔCT is the difference between the threshold cycle of a given target cDNA and an endogenous reference β-actin cDNA. Glucose tolerance tests were performed in overnight-fasting mice on day 25 after STZ treatment; mice were injected i.p. with glucose (in saline) at 2 g/kg body weight. Areas under the curve of the glucose tolerance test were calculated using trapezoidal integration.22Allison D.B. Paultre F. Maggio C. Mezzitis N. Pi-Sunyer F.X. The use of areas under curves in diabetes research.Diabetes Care. 1995; 18: 245-250Crossref PubMed Scopus (277) Google Scholar Insulin sensitivity tests were performed by i.p. injection of 2-hour-fasting mice (10 to12 weeks old) with insulin (1 U/kg body weight). Blood glucose concentrations and plasma insulin levels were assessed before and after injection.10Wu L. Yang W. Jia X. Yang G. Duridanova D. Cao K. Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.Lab Invest. 2009; 89: 59-67Crossref PubMed Scopus (169) Google Scholar Insulin was from Eli Lilly & Co. (Indianapolis, IN). Chemicals were all obtained from Sigma-Aldrich unless otherwise mentioned. Data are presented as mean ± SEM, representing at least three independent experiments. Statistical comparisons were made using the Excel 2003 spreadsheet program (Microsoft Corp., Redmond, WA), with the Student's t-test to evaluate the difference between two groups; the differences between multiple groups were analyzed using SPSS 10.0 software (SPSS Inc., Chicago, IL), with analysis of variance and the post hoc Tukey's test. The significance level was set at P < 0.05. CSE protein was not detected in pancreases and islets from CSE KO mice (Figure 1, A and B), and CSE protein expression in pancreases from heterozygotes was estimated to be half that in wild-type mice (Figure 1A). The H2S production rate was significantly lowered in pancreases from CSE KO mice versus age-matched wild-type mice (8.1 ± 2.8 nmol/g/minute versus 27.2 ± 2.7 nmol/g/minute; P < 0.05) (Figure 1C). As shown in Figure 2A, the body weight of STZ-treated wild-type mice was significantly lower than that of vehicle-treated wild-type mice or STZ-treated CSE KO mice (P < 0.05), and the weight loss of STZ-treated wild-type mice was partly reversed by PPG treatment. STZ treatment had little effect on the body weight of CSE KO mice. Food and water intake were significantly higher in STZ-treated mice than in vehicle-treated mice (Figure 2, B and C). However, food and water intake were significantly lower in STZ-treated CSE KO mice than in STZ-treated wild-type mice (P < 0.05). Blood glucose (Figure 3A) and plasma insulin (Figure 3B) levels were similar between wild-type and CSE KO mice. Injection of wild-type mice with STZ for 5 days resulted in progressive hyperglycemia (Figure 3A). At day 30, STZ-treated wild-type mice had a significantly higher blood glucose level (28.2 ± 2.6 mmol/L) compared with vehicle-treated wild-type mice (5.7 ± 0.4 mmol/L). Diabetes development in CSE KO mice was significantly delayed after STZ treatment, with the blood glucose level on day 30 being only 14.4 ± 3.9 mmol/L. Although the plasma insulin level in STZ-treated CSE KO mice was lower than that in vehicle-treated CSE KO mice (1.98 ± 0.69 ng/mL versus 5.95 ± 0.91 ng/mL; P < 0.05), it was significantly higher than that of STZ-treated wild-type mice (0.33 ± 0.22 ng/mL) (Figure 3B). By day 11 after STZ injection, none of the CSE KO mice were diabetic (fasting blood glucose level >7.8 mmol/L) compared with all 16 wild-type animals. All 14 CSE KO mice developed diabetes by day 24 after STZ injection (Figure 3C). By day 30 after STZ injection, all 14 CSE KO mice survived, but only 16 of 18 wild-type mice were alive. Treatment of wild-type mice with the CSE inhibitor PPG for 30 days partially protected the mice against STZ-induced hyperglycemia (fasting blood glucose level, 16.3 ± 2.8 mmol/L) and hypoinsulinemia (plasma insulin level, 2.34 ± 0.41 ng/mL) compared with STZ-treated wild-type mice (28.2 ± 2.6 mmol/L and 0.33 ± 0.22 ng/mL). PPG treatment alone did not affect glucose and insulin levels in wild-type mice (Figure 3, A and B). To explore the role of CSE in STZ-induced diabetes, pancreatic tissues from STZ-treated mice at day 30 were subjected to H&E staining and immunohistochemical (IHC) analysis. The morphologic features of islets in vehicle-treated wild-type mice and CSE KO mice were similar, both showing a round shape and clearly identifiable islets with strong insulin-positive staining (Figure 3D, left). In wild-type mice treated with STZ, the number of islets was greatly reduced, and the remaining islets were smaller and distorted in appearance, with fewer cells stained for insulin (Figure 3D). In striking contrast, CSE KO islets were partly protected from this damage, displaying more insulin staining, with the overall morphologic appearance essentially comparable with that of wild-type mice treated with STZ (Figure 3D, right). Furthermore, 20 mmol/L glucose stimulated greater insulin release (24.8 ± 1.5 μg/μ
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