Cardiac Metallothionein Synthesis in Streptozotocin-Induced Diabetic Mice, and Its Protection against Diabetes-Induced Cardiac Injury
2005; Elsevier BV; Volume: 167; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)62949-5
ISSN1525-2191
AutoresYe Song, Jianxun Wang, Yan Li, Yibo Du, Gavin E. Arteel, Jack T. Saari, Yin Kang, Lu Cai,
Tópico(s)Muscle Physiology and Disorders
ResumoOxidative stress is involved in the pathogenesis of diabetes and its cardiovascular complications. Metallothionein (MT), a stress-response protein, is significantly increased in the liver and kidney of diabetic animals. We examined whether diabetes also induces cardiac MT synthesis through oxidative damage and whether MT overexpression protects the heart from injury. Diabetes was induced in mice by single injection of streptozotocin (STZ), and cardiac MT mRNA and protein levels were measured 2 weeks and 2 months after STZ treatment. Diabetes significantly increased cardiac MT synthesis 2 weeks and 2 months after STZ treatment, with no change in cardiac metals including zinc, copper, and iron. Serum and cardiac vasopeptide endothelin and inflammatory cytokine tumor necrosis factor-α were also significantly increased in diabetic hearts, as were the ratio of oxidized to reduced glutathione and the immunohistochemical staining of 3-nitrotyrosine and 4-hydroxynonenal. To explore the biological importance of increased MT synthesis in the heart, MT-overexpressing transgenic mice were treated with STZ and then examined 2 months later. A loss of inotropic reserve, uncovered during β-adrenergic stimulation, and the presence of cardiac fibrosis, shown by increased Sirius red staining of collagen, were evident in the wild-type diabetic mice but not in the MT-overexpressing transgenic diabetic mice. These results suggest that diabetes-induced cardiac MT expression likely associates with systemic increases in endothelin-1 and tumor necrosis factor-α and the resulting cardiac oxidative stress. Overexpressing cardiac MT significantly protects the heart from diabetes-induced injury. Oxidative stress is involved in the pathogenesis of diabetes and its cardiovascular complications. Metallothionein (MT), a stress-response protein, is significantly increased in the liver and kidney of diabetic animals. We examined whether diabetes also induces cardiac MT synthesis through oxidative damage and whether MT overexpression protects the heart from injury. Diabetes was induced in mice by single injection of streptozotocin (STZ), and cardiac MT mRNA and protein levels were measured 2 weeks and 2 months after STZ treatment. Diabetes significantly increased cardiac MT synthesis 2 weeks and 2 months after STZ treatment, with no change in cardiac metals including zinc, copper, and iron. Serum and cardiac vasopeptide endothelin and inflammatory cytokine tumor necrosis factor-α were also significantly increased in diabetic hearts, as were the ratio of oxidized to reduced glutathione and the immunohistochemical staining of 3-nitrotyrosine and 4-hydroxynonenal. To explore the biological importance of increased MT synthesis in the heart, MT-overexpressing transgenic mice were treated with STZ and then examined 2 months later. A loss of inotropic reserve, uncovered during β-adrenergic stimulation, and the presence of cardiac fibrosis, shown by increased Sirius red staining of collagen, were evident in the wild-type diabetic mice but not in the MT-overexpressing transgenic diabetic mice. These results suggest that diabetes-induced cardiac MT expression likely associates with systemic increases in endothelin-1 and tumor necrosis factor-α and the resulting cardiac oxidative stress. Overexpressing cardiac MT significantly protects the heart from diabetes-induced injury. Diabetes and its cardiovascular complications are related to multiple pathogenic factors, including hyperglycemia, hyperlipidemia, and inflammatory response.1Chatham JC Forder JR McNeill JH The Heart in Diabetes. ed 1. Kluwer Academic Publishers, Norwell1996Crossref Google Scholar, 2Ceriello A New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy.Diabetes Care. 2003; 26: 1589-1596Crossref PubMed Scopus (672) Google Scholar, 3Da Ros R Assaloni R Ceriello A Antioxidant therapy in diabetic complications: what is new?.Curr Vasc Pharmacol. 2004; 2: 335-341Crossref PubMed Scopus (55) Google Scholar However, the pivotal mediator for the pathogenesis of diabetes and its cardiovascular complications is oxidative stress, directly or indirectly derived from the multiple factors mentioned above.2Ceriello A New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy.Diabetes Care. 2003; 26: 1589-1596Crossref PubMed Scopus (672) Google Scholar, 3Da Ros R Assaloni R Ceriello A Antioxidant therapy in diabetic complications: what is new?.Curr Vasc Pharmacol. 2004; 2: 335-341Crossref PubMed Scopus (55) Google Scholar Oxidative stress is defined as the imbalance between the production of reactive oxygen and nitrogen species (ROS and RNS) and antioxidant capacity.3Da Ros R Assaloni R Ceriello A Antioxidant therapy in diabetic complications: what is new?.Curr Vasc Pharmacol. 2004; 2: 335-341Crossref PubMed Scopus (55) Google Scholar, 4Cai L Kang YJ Oxidative stress and diabetic cardiomyopathy: a brief review.Cardiovasc Toxicol. 2001; 1: 181-193Crossref PubMed Scopus (338) Google Scholar Diabetes impairs cardiac antioxidant capacity, showing decreases in enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and nonenzymatic (vitamin C, E, or A) antioxidant defenses, as well as total radical-trapping antioxidant capacity in the heart.4Cai L Kang YJ Oxidative stress and diabetic cardiomyopathy: a brief review.Cardiovasc Toxicol. 2001; 1: 181-193Crossref PubMed Scopus (338) Google Scholar, 5Joyeux M Faure P Godin-Ribuot D Halimi S Patel A Yellon DM Demenge P Ribuot C Heat stress fails to protect myocardium of streptozotocin-induced diabetic rats against infarction.Cardiovasc Res. 1999; 43: 939-946Crossref PubMed Scopus (26) Google Scholar, 6Ustinova EE Barrett CJ Sun SY Schultz HD Oxidative stress impairs cardiac chemoreflexes in diabetic rats.Am J Physiol. 2000; 279: H2176-H2187Google Scholar In addition to impaired defenses, diabetes also causes ROS and RNS overproduction in the hearts of diabetic animals and patients.2Ceriello A New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy.Diabetes Care. 2003; 26: 1589-1596Crossref PubMed Scopus (672) Google Scholar, 3Da Ros R Assaloni R Ceriello A Antioxidant therapy in diabetic complications: what is new?.Curr Vasc Pharmacol. 2004; 2: 335-341Crossref PubMed Scopus (55) Google Scholar, 4Cai L Kang YJ Oxidative stress and diabetic cardiomyopathy: a brief review.Cardiovasc Toxicol. 2001; 1: 181-193Crossref PubMed Scopus (338) Google Scholar In response to the diabetes-derived oxidative stress, however, cardiac compensatory mechanisms to protect the heart from these deleterious effects, such as induction of heat shock protein and metallothionein (MT), may take place7Atalay M Oksala NK Laaksonen DE Khanna S Nakao C Lappalainen J Roy S Hanninen O Sen CK Exercise training modulates heat shock protein response in diabetic rats.J Appl Physiol. 2004; 97: 605-611Crossref PubMed Scopus (162) Google Scholar, 8Gerhardinger C Costa MB Coulombe MC Toth I Hoehn T Grosu P Expression of acute-phase response proteins in retinal Muller cells in diabetes.Invest Ophthalmol Vis Sci. 2005; 46: 349-357Crossref PubMed Scopus (209) Google Scholar, 9Yin X Wu H Chen Y Kang YJ Induction of antioxidants by adriamycin in mouse heart.Biochem Pharmacol. 1988; 56: 87-93Crossref Scopus (81) Google Scholar because MT is up-regulated in the heart challenged by other oxidative stresses.10Kang YJ The antioxidant function of metallothionein in the heart.Proc Soc Exp Biol Med. 1999; 222: 263-273Crossref PubMed Scopus (161) Google Scholar, 11Onody A Zvara A Hackler Jr, L Vigh L Ferdinandy P Puskas LG Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study.FEBS Lett. 2003; 536: 35-40Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar MT is a cysteine-rich, metal-binding protein. Four major isoforms of MT have been identified in mammalian tissues, but MT-I and MT-II are the major isoforms found in most tissues.10Kang YJ The antioxidant function of metallothionein in the heart.Proc Soc Exp Biol Med. 1999; 222: 263-273Crossref PubMed Scopus (161) Google Scholar, 12Cai L Satoh M Tohyama C Cherian MG Metallothionein in radiation exposure: its induction and protective role.Toxicology. 1999; 132: 85-98Crossref PubMed Scopus (185) Google Scholar Several physiological roles have been proposed for MT, including detoxification of potentially toxic heavy metals, such as cadmium (Cd), and homeostasis of essential metals, such as zinc (Zn) and copper (Cu). MT is also a potent antioxidant, interacting with various ROS and RNS more efficiently than do other antioxidants in cell-free systems.10Kang YJ The antioxidant function of metallothionein in the heart.Proc Soc Exp Biol Med. 1999; 222: 263-273Crossref PubMed Scopus (161) Google Scholar, 12Cai L Satoh M Tohyama C Cherian MG Metallothionein in radiation exposure: its induction and protective role.Toxicology. 1999; 132: 85-98Crossref PubMed Scopus (185) Google Scholar, 13Cai L Klein JB Kang YJ Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage.J Biol Chem. 2000; 275: 38957-38960Crossref PubMed Scopus (147) Google Scholar, 14Cai L Cherian MG Zinc-metallothionein protects from DNA damage induced by radiation better than glutathione and copper- or cadmium-metallothioneins.Toxicol Lett. 2003; 136: 193-198Crossref PubMed Scopus (79) Google Scholar MT exists in most organs, including in animal and human hearts, and is inducible to a high level by various oxidative or pathogenic stresses.9Yin X Wu H Chen Y Kang YJ Induction of antioxidants by adriamycin in mouse heart.Biochem Pharmacol. 1988; 56: 87-93Crossref Scopus (81) Google Scholar, 10Kang YJ The antioxidant function of metallothionein in the heart.Proc Soc Exp Biol Med. 1999; 222: 263-273Crossref PubMed Scopus (161) Google Scholar, 11Onody A Zvara A Hackler Jr, L Vigh L Ferdinandy P Puskas LG Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study.FEBS Lett. 2003; 536: 35-40Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Cai L Satoh M Tohyama C Cherian MG Metallothionein in radiation exposure: its induction and protective role.Toxicology. 1999; 132: 85-98Crossref PubMed Scopus (185) Google Scholar Zn and Cu, major metals that bind to MT under physiological conditions, significantly induce MT synthesis in various organs.10Kang YJ The antioxidant function of metallothionein in the heart.Proc Soc Exp Biol Med. 1999; 222: 263-273Crossref PubMed Scopus (161) Google Scholar, 12Cai L Satoh M Tohyama C Cherian MG Metallothionein in radiation exposure: its induction and protective role.Toxicology. 1999; 132: 85-98Crossref PubMed Scopus (185) Google Scholar, 14Cai L Cherian MG Zinc-metallothionein protects from DNA damage induced by radiation better than glutathione and copper- or cadmium-metallothioneins.Toxicol Lett. 2003; 136: 193-198Crossref PubMed Scopus (79) Google Scholar We have demonstrated that MT levels increase in response to chronic stress, such as allogenic and isogenic organ transplantation,15Cai L Deng DX Jiang J Chen S Zhong R Cherian MG Chakrabarti S Induction of metallothionein synthesis with preservation of testicular function in rats following long term renal transplantation.Urol Res. 2000; 28: 97-103Crossref PubMed Scopus (8) Google Scholar which may be induced by inflammation factors including interleukin (IL)-6 and tumor necrosis factor (TNF)-α.15Cai L Deng DX Jiang J Chen S Zhong R Cherian MG Chakrabarti S Induction of metallothionein synthesis with preservation of testicular function in rats following long term renal transplantation.Urol Res. 2000; 28: 97-103Crossref PubMed Scopus (8) Google Scholar, 16Courtade M Carrera G Paternain JL Martel S Carre PC Folch J Pipy B Metallothionein expression in human lung and its varying levels after lung transplantation. Toulouse Lung Transplantation Group.Chest. 1998; 113: 371-378Crossref PubMed Scopus (20) Google Scholar, 17Baba HA Schmid KW Takeda A Wichter T Gradaus R Erren M Plenz G Grabellus F Tjan TD Deng MC Metallothionein: localization in human transplant endomyocardium, relation to cytokines and allograft function.J Heart Lung Transplant. 1999; 18: 963-971Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Enhanced expression of hepatic and renal MT in diabetic rats was also indicated in our previous study.18Cai L Chen S Evans T Cherian MG Chakrabarti S Endothelin-1-mediated alteration of metallothionein and trace metals in the liver and kidneys of chronically diabetic rats.Int J Exp Diabetes Res. 2002; 3: 193-198Crossref PubMed Scopus (32) Google Scholar A literature search showed organ-specific patterns for diabetes-induced MT expression.19Cai L Metallothionein as an adaptive protein prevents diabetes and its toxicity.Nonlinearity Biol Toxicol Med. 2004; 2: 89-103Crossref PubMed Google Scholar, 20Chen ML Failla ML Metallothionein metabolism in the liver and kidney of the streptozotocin-diabetic rat.Comp Biochem Physiol B. 1998; 90: 439-445Google Scholar Diabetes-induced renal MT was metal-dependent, whereas hepatic MT was metal-independent.18Cai L Chen S Evans T Cherian MG Chakrabarti S Endothelin-1-mediated alteration of metallothionein and trace metals in the liver and kidneys of chronically diabetic rats.Int J Exp Diabetes Res. 2002; 3: 193-198Crossref PubMed Scopus (32) Google Scholar, 19Cai L Metallothionein as an adaptive protein prevents diabetes and its toxicity.Nonlinearity Biol Toxicol Med. 2004; 2: 89-103Crossref PubMed Google Scholar, 20Chen ML Failla ML Metallothionein metabolism in the liver and kidney of the streptozotocin-diabetic rat.Comp Biochem Physiol B. 1998; 90: 439-445Google Scholar MT plays a critical role in Zn and Cu homeostasis, which is required to maintain normal cellular functions in different tissues, and diabetes-derived abnormal homeostasis of Zn or Cu may be related to the development of diabetic cardiovascular complications.21Aguilar MV Laborda JM Martinez-Para MC Gonzalez MJ Meseguer I Bernao A Mateos CJ Effect of diabetes on the tissular Zn/Cu ratio.J Trace Elem Med Biol. 1998; 12: 155-158Crossref PubMed Scopus (17) Google Scholar, 22Karahan SC Deger O Orem A Ucar F Erem C Alver A Onder E The effects of impaired trace element status on polymorphonuclear leukocyte activation in the development of vascular complications in type 2 diabetes mellitus.Clin Chem Lab Med. 2001; 39: 109-115Crossref PubMed Scopus (29) Google Scholar To date, however, it remains unclear whether diabetes affects cardiac MT expression. The antioxidant function of MT in the heart has been extensively implicated.10Kang YJ The antioxidant function of metallothionein in the heart.Proc Soc Exp Biol Med. 1999; 222: 263-273Crossref PubMed Scopus (161) Google Scholar, 23Nath R Kumar D Li T Singal PK Metallothioneins, oxidative stress and the cardiovascular system.Toxicology. 2000; 155: 17-26Crossref PubMed Scopus (74) Google Scholar Previous induction of cardiac MT by various agents significantly prevented hearts from oxidative damage.23Nath R Kumar D Li T Singal PK Metallothioneins, oxidative stress and the cardiovascular system.Toxicology. 2000; 155: 17-26Crossref PubMed Scopus (74) Google Scholar, 24Naganuma A Satoh M Imura N Specific reduction of toxic side effects of adriamycin by induction of metallothionein in mice.Jpn J Cancer Res. 1988; 79: 406-411Crossref PubMed Scopus (78) Google Scholar, 25Satoh M Naganuma A Imura N Involvement of cardiac metallothionein in prevention of adriamycin induced lipid peroxidation in the heart.Toxicology. 1988; 53: 231-237Crossref PubMed Scopus (63) Google Scholar, 26Ali MM Frei E Straub J Breuer A Wiessler M Induction of metallothionein by zinc protects from daunorubicin toxicity in rats.Toxicology. 2002; 179: 85-93Crossref PubMed Scopus (60) Google Scholar Using a cardiac-specific MT-overexpressing transgenic mouse, significant protection of the heart from diabetes-induced injury was also observed in our previous studies and others.27Kang YJ Cai L Metallothionein suppression of diabetic cardiomyopathy by inhibition of hyperglycemia-induced oxidative stress.Free Radic Biol Med. 2001; 31: 33Google Scholar, 28Cai L Kang YJ Metallothionein prevents diabetic cardiomyopathy.Toxicol Sci. 2001; 60: 13Google Scholar, 29Cai L Li W Sun X Li Y Kang YJ Prevention of diabetic cardiomyopathy by metallothionein through suppression of hyperglycemia-induced oxidative stress and cell death.Toxicol Sci. 2002; 66: 288Google Scholar, 30Liang Q Carlson EC Donthi RV Kralik PM Shen X Epstein PN Overexpression of metallothionein reduces diabetic cardiomyopathy.Diabetes. 2002; 51: 174-181Crossref PubMed Scopus (164) Google Scholar, 31Ye G Metreveli NS Ren J Epstein PN Metallothionein prevents diabetes-induced deficits in cardiomyocytes by inhibiting reactive oxygen species production.Diabetes. 2003; 52: 777-783Crossref PubMed Scopus (168) Google Scholar However, in these transgenic mice, MT was overexpressed only in cardiac myocytes and was ∼20- or 100-fold higher than that of control mice. Such high level of cardiac-specific MT expression would be impossible to be manipulated by pharmaceutical approach such as administration of MT inducers. This study, therefore, was aimed: 1) to investigate the effect of diabetes on cardiac MT expression by measuring MT mRNA and protein contents; 2) to explore the mechanisms for cardiac MT induction by measuring cardiac metal levels (Zn, Cu, and iron, Fe), systemic and cardiac vasopeptide (endothelin-1, ET-1), and inflammation factor (TNF-α, IL-6) levels, and cardiac oxidative damage; and 3) to explore the biological importance of MT expression at an inducible level in preventing diabetes-induced cardiac injury by comparing MT-overexpressing transgenic (MT-TG) with their wild-type (WT) diabetic mice. FVB mice, obtained from Harlan Bioproducts for Science, Inc. (Indianapolis, IN), MT-TG mice, and WT control mice (C57BL/6), purchased from The Jackson Laboratory (Bar Harbor, ME), were housed in the University of Louisville Research Resources Center at 22°C with a 12-hour light/dark cycle and free access to rodent chow and tap water. All animal procedures were approved by the Institutional Animals Care and Use Committee, which is certified by the American Association for Accreditation of Laboratory Animals Care. Eight-week-old male mice were intraperitoneally given a single dose of streptozotocin (STZ; Sigma Chemical Co., St. Louis, MO) at 150 mg/kg body weight, dissolved in sodium citrate buffer (pH 4.5). On day 3 after STZ treatment, whole blood glucose obtained from mouse tail-vein was detected using a SureStep complete blood glucose monitor (LifeScan, Milpitas, CA). STZ-treated mice with whole blood glucose higher than 250 mg/dl were considered as diabetic. Mice serving as vehicle controls were given the same volume of sodium citrate.32Cai L Li W Wang G Guo L Jiang Y Kang YJ Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway.Diabetes. 2002; 51: 1938-1948Crossref PubMed Scopus (550) Google Scholar Experimental measurements were performed in the control and diabetic mice 2 weeks and 2 months after STZ treatment because these will reflect both early and late response of the heart to diabetes. To avoid the direct effect of STZ on cardiac MT expression a group of insulin-treated diabetic mice was included for 2-week experiments, as described previously.32Cai L Li W Wang G Guo L Jiang Y Kang YJ Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway.Diabetes. 2002; 51: 1938-1948Crossref PubMed Scopus (550) Google Scholar For 2-month experiments, the insulin-treated diabetes group was not included because MT protein turnover is ∼3 or 4 days in most tissues and its expression in such late period after STZ treatment will be unlikely affected.10Kang YJ The antioxidant function of metallothionein in the heart.Proc Soc Exp Biol Med. 1999; 222: 263-273Crossref PubMed Scopus (161) Google Scholar, 23Nath R Kumar D Li T Singal PK Metallothioneins, oxidative stress and the cardiovascular system.Toxicology. 2000; 155: 17-26Crossref PubMed Scopus (74) Google Scholar, 24Naganuma A Satoh M Imura N Specific reduction of toxic side effects of adriamycin by induction of metallothionein in mice.Jpn J Cancer Res. 1988; 79: 406-411Crossref PubMed Scopus (78) Google Scholar, 25Satoh M Naganuma A Imura N Involvement of cardiac metallothionein in prevention of adriamycin induced lipid peroxidation in the heart.Toxicology. 1988; 53: 231-237Crossref PubMed Scopus (63) Google Scholar, 26Ali MM Frei E Straub J Breuer A Wiessler M Induction of metallothionein by zinc protects from daunorubicin toxicity in rats.Toxicology. 2002; 179: 85-93Crossref PubMed Scopus (60) Google Scholar Whole blood was collected from dorsal vena cava of the anesthetized animals. Noncoagulated whole blood was used to measure glycated Hb-A1. Serum was prepared using a serum separator apparatus (Becton Dickinson, Rutherford, NJ) to measure triglyceride level using a kit from Sigma Chemical Co. and ET-1, IL-6, and TNF-α levels using their corresponding enzyme-linked immunosorbent assay kits from Cayman Chemical Co. (Ann Arbor, MI).33Kang YJ Li Y Zhou Z Roberts AM Cai L Myers SR Wang L Schuchke DA Elevation of serum endothelins and cardiotoxicity induced by particulate matter (PM2.5) in rats with acute myocardial infarction.Cardiovasc Toxicol. 2002; 2: 253-261Crossref PubMed Scopus (43) Google Scholar, 34Song Y Song Z Zhang L McClain CJ Kang YJ Cai L Diabetes enhances lipopolysaccharide-induced cardiac toxicity in the mouse model.Cardiovasc Toxicol. 2003; 3: 363-372Crossref PubMed Scopus (16) Google Scholar Selection of IL-6 and TNF-α was because these inflammatory factors were found to be increased in diabetic mice in our previous studies34Song Y Song Z Zhang L McClain CJ Kang YJ Cai L Diabetes enhances lipopolysaccharide-induced cardiac toxicity in the mouse model.Cardiovasc Toxicol. 2003; 3: 363-372Crossref PubMed Scopus (16) Google Scholar and are also a potent MT inducer.15Cai L Deng DX Jiang J Chen S Zhong R Cherian MG Chakrabarti S Induction of metallothionein synthesis with preservation of testicular function in rats following long term renal transplantation.Urol Res. 2000; 28: 97-103Crossref PubMed Scopus (8) Google Scholar, 16Courtade M Carrera G Paternain JL Martel S Carre PC Folch J Pipy B Metallothionein expression in human lung and its varying levels after lung transplantation. Toulouse Lung Transplantation Group.Chest. 1998; 113: 371-378Crossref PubMed Scopus (20) Google Scholar, 17Baba HA Schmid KW Takeda A Wichter T Gradaus R Erren M Plenz G Grabellus F Tjan TD Deng MC Metallothionein: localization in human transplant endomyocardium, relation to cytokines and allograft function.J Heart Lung Transplant. 1999; 18: 963-971Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Selection of ET-1 is because we have demonstrated that ET-1 plays important roles in mediating the enhanced MT expression in the liver and kidney of diabetic rats,18Cai L Chen S Evans T Cherian MG Chakrabarti S Endothelin-1-mediated alteration of metallothionein and trace metals in the liver and kidneys of chronically diabetic rats.Int J Exp Diabetes Res. 2002; 3: 193-198Crossref PubMed Scopus (32) Google Scholar and also the development of diabetic cardiomyopathy.35Chen S Evans T Mukherjee K Karmazyn M Chakrabarti S Diabetes-induced myocardial structural changes: role of endothelin-1 and its receptors.J Mol Cell Cardiol. 2000; 32: 1621-1629Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 36Hileeto D Cukiernik M Mukherjee S Evans T Barbin Y Downey D Karmazyn M Chakrabarti S Contributions of endothelin-1 and sodium hydrogen exchanger-1 in the diabetic myocardium.Diabetes Metab Res Rev. 2002; 18: 386-394Crossref PubMed Scopus (42) Google Scholar Total cellular RNA was extracted from frozen tissues using the Trizol reagent. Random-primed first-strand cDNA was prepared from total cellular RNA using Superscript II and amplified by polymerase chain reaction (PCR) using mouse MT II primers (sense primer, 5′tctcgtcgatcttcaacc-3′; anti-sense, 5′-ggcttctacatggtctatttac-3′). The purified PCR fragment was subcloned into a PCR II vector as a template for synthesis of the cRNA probe. The plasmid was linearized with NCO I, and the anti-sense cRNA probe was produced using the T7 RNA polymerase Maxiscript kit (Ambion, Austin, TX) in the presence of α32-UTP (Amersham, Piscataway, NJ). Total cellular RNA extracted from heart samples (15 μg/lane) was fractionated by electrophoresis through denaturing (0.66 mol/L formaldehyde in 1% agarose) gel, downward transferred onto nylon membranes, and vacuum-dried at 80°C for 2 hours. Membranes were prehybridized for 1 hour and then hybridized with the anti-sense cRNA probes, as described above, in the hybridization solution (Ambion) at 68°C for 16 hours. Membranes were then washed under high-stringency conditions (final wash 0.1× standard saline citrate at 68°C) while monitored with a Geiger counter. Radioactivity was recorded on X-ray film. Expression of cardiac MT, 3-nitrotyrosine (3-NT), and 4-hydroxynonenal (4-HNE) was examined by immunohistochemical staining with monoclonal mouse anti-MT antibody (Zymed Laboratories, Inc., San Francisco, CA), rabbit anti-NT antibody (Upstate Biotechnology, Lake Plaid, NY), and rabbit anti-HNE antibody (Alpha Diagnostic Int., San Antonio, TX). Cardiac tissues were collected from diabetic FVB mice 2 weeks and 2 months after STZ treatment. After deparaffinization and redehydration, the slides were subjected to quenching endogenous peroxidase activity using 3% H2O2 for 10 minutes at room temperature. Nonspecific binding sites were blocked by 5% normal rabbit serum for 30 minutes. Sections were incubated with primary antibodies overnight at 4°C. The sections were then incubated with biotinylated rabbit anti-mouse IgG for 20 minutes, followed by incubation with streptavidin-horseradish peroxidase for 20 minutes. The antibody binding sites were visualized by incubation with a diaminobenzidine-H2O2 solution. Methyl green counterstaining for MT staining and no counterstaining was used for 3-NT and 4-HNE staining. Semiquantitative analysis for 3-NT and 4-HNE was performed by computerization of the percentage of the positive staining from five samples in each group with two sections for each sample and five images for each section. Cardiac tissues were homogenized with a lysis buffer containing 25 mmol/L Tris-HCl (pH 7.4), 0.5 mol/L EDTA, 0.5 mol/L EGTA, 1 mmol/L phenylmethyl sulfonyl fluoride, 25 μg/ml leupeptin, and 0.1% Chaps and centrifuged at 12,000 rpm at 4°C in a Beckman GS-6R centrifuge for 10 minutes. The suspension was collected and protein concentration was determined. The sample was diluted in loading buffer (40 mmol/L Tris-HCl, pH 6.8, 1% sodium dodecyl sulfate, 50 mmol/L dithiothreitol, 7.5% glycerol, 0.003% bromophenol blue), heated at 95°C for 5 minutes, and then subjected to electrophoresis on 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel at 120 V. Proteins were transferred from gels to polyvinylidene difluoride membrane. Membranes were incubated in 2.5% glutaraldehyde solution for 1 hour, and then were rinsed briefly with Tris-buffered saline buffer and blocked in blocking buffer (5% milk and 0.5% bovine serum albumin) for 1 hour, followed by three washes with Tris-buffered saline containing 0.1% Tween 20. The rabbit anti-MT antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:300 was used and antigen-antibody complexes were then visualized by ECL kit (Amersham). MT levels in the heart were determined by a cadmium-hemoglobin (Cd-hem) assay.15Cai L Deng DX Jiang J Chen S Zhong R Cherian MG Chakrabarti S Induction of metallothionein synthesis with preservation of testicular function in rats following long term renal transplantation.Urol Res. 2000; 28: 97-103Crossref PubMed Scopus (8) Google Scholar, 18Cai L Chen S Evans T Cherian MG Chakrabarti S Endothelin-1-mediated alteration of metallothionein and trace metals in the liver and kidneys of chronically diabetic rats.Int J Exp Diabetes Res. 2002; 3: 193-198Crossref PubMed Scopus (32) Google Scholar Briefly, an aliquot of the cardiac protein suspension was diluted with 10 mmol/L Tris-HCl buffer (pH 7.4) and incubated with 10 ppm 109Cd solution with known specific activity to saturate the metal-binding sites of MT. Excess Cd was removed by adding hemoglobin and centrifugation. The Cd concentrations in the final supernatant were calculated from the radioactivity of the 109Cd, which were measured by a γ counter and converted to MT concentration on the basis of 7 g-atoms of Cd/MT. Total hepatic MT concentration was expressed as μg/g wet tissue. Cardiac metal concentrations were determined by using inductively coupled argon plasma emission spectroscopy (model 35608; Thermo ARL-VG Elemental, Franklin, MA) after lyophilization and digestion of the tissues with nitric acid and hydrogen peroxide.37Elsherif L Ortines RV Saari JT Kang YJ Congestive heart failure in copper-deficient mice.Exp Biol Med (Maywood). 2003; 228: 811-817PubMed Google Scholar Metal levels were expressed as μg/g dry tissue. Heart samples for measurements of ET-1 and TNF-α were prepared according to our previous publication.34Song Y Song Z Zhang L McClain CJ Kang YJ Cai L Diabetes enhances lipopolysaccharide-induced cardiac toxicity in the mouse model.Cardiovasc Toxicol. 2003; 3: 363-372Crossref PubMed Scopus (16) Google Scholar Briefly, heart samples were homogenized in 3 vol of ice-cold Ripa buffer. After incubation on ice for 30 minutes, samples were centrifuge
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