Pioglitazone but Not Glibenclamide Improves Cardiac Expression of Heat Shock Protein 72 and Tolerance Against Ischemia/Reperfusion Injury in
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《糖尿病学杂志》
1 Department of Cardiovascular Science, Faculty of Medicine, Oita University, Oita, Japan
2 Department of Internal Medicine 1, Faculty of Medicine, Oita University, Oita, Japan
CPP, coronary perfusion pressure; GGA, geranylgeranylacetone; HSP, heat shock protein; LVDP, left ventricular developed pressure; OGTT, oral glucose tolerance test; PI3, phosphatidylinositol 3; PPAR-, peroxisome proliferator–activated receptor-; STZ, streptozotocin
ABSTRACT
We tested the hypothesis that pioglitazone could restore expression of heat shock protein (HSP)72 in insulin-resistant rat heart. At 12 weeks of age, male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and control (LETO) rats were treated with pioglitazone (10 mg · kg–1 · day–1) or glibenclamide (5 mg · kg–1 · day–1) for 4 weeks. Thereafter, hyperthermia (43°C for 20 min) was applied. In response to hyperthermia, the activation of serine/threonine kinase Akt depending on phosphatidylinositol 3 (PI3) kinase was necessary for cardiac expression of HSP72. Hyperthermia-induced activation of Akt and HSP72 expression were depressed in OLETF rat hearts. Pioglitazone but not glibenclamide improved insulin sensitivity in OLETF rats, which was associated with the restoration of Akt activation and HSP72 expression. In experiments with isolated perfused heart, reperfusion-induced cardiac functional recovery was suppressed in OLETF rat hearts, which was improved by pioglitazone but not glibenclamide. Our results suggest that PI3 kinase–dependent Akt activation, an essential signal for HSP72 expression, is depressed in the heart in insulin-resistant OLETF rats, and the results suggest also that the restoration of HSP72 expression and tolerance against ischemia/reperfusion injury by treatment with pioglitazone might be due to an improvement of insulin resistance, leading to restoration of impaired PI3 kinase–dependent Akt activation in response to hyperthermia.
Heat shock protein (HSP)72, a member of the HSP family, has been reported to be involved predominantly in cardioprotection (1–4). We have shown that induction of HSP72 by either hyperthermia or oral administration of geranylgeranylacetone (GGA) leads to protection against ischemia/reperfusion injury (5–8). However, information is very limited with respect to the impact of insulin resistance and type 2 diabetes on the expression of HSP72. Kurucz et al. (9) reported that the expression of HSP72 mRNA in skeletal muscle of patients with type 2 diabetes is decreased, which correlates with the parameters of insulin resistance. Consistent with this, we have shown attenuated cardiac HSP72 expression in response to GGA in insulin-resistant rats produced by a high-fat diet (10). Skeletal muscle, liver, and heart are known to be targeted organs of insulin resistance (11–13). However, the mechanisms underlying the depressed expression of HSP72 in insulin-resistant organs are unclear. Recently, thiazolidinediones, including pioglitazone, have been demonstrated to improve insulin resistance experimentally and clinically (14,15). Whereas sulfonylurea including glibenclamide stimulates endogenous insulin secretion through blockade of ATP-sensitive potassium channels on pancreatic -cells, pioglitazone, a synthetic peroxisome proliferator–activated receptor- (PPAR-) agonist, does not stimulate insulin release from pancreatic -cells (14,15). It is therefore interesting to compare the effects of treatment with pioglitazone and treatment with glibenclamide on the expression of HSP72 in the insulin-resistant animal model.
Activation of the serine/threonine kinase Akt promotes survival of some cell types including cardiomyocytes (16). Hyperthermia is known to cause Akt activation, and experimental evidence indicates that hyperthermia-induced activation of Akt can involve either a phosphatidylinositol 3 (PI3) kinase–dependent or a PI3 kinase–independent pathway (17,18). However, little is known about the involvement of Akt in hyperthermia-induced HSP72 expression in the heart. We have demonstrated recently that PI3 kinase–dependent Akt activation is essential for expression of HSP72 (19). In the present study, therefore, we tested the hypothesis that 1) depressed cardiac HSP72 expression in insulin-resistant rats is related to depressed PI3 kinase–dependent Akt activation in response to hyperthermia and that 2) treatment with pioglitazone improves this depressed response, resulting in restoration of cardiac expression of HSP72, which is associated with the protection against ischemia/reperfusion injury.
RESEARCH DESIGN AND METHODS
All experimental procedures were carried out in accordance with the guidelines of the Physiological Society of Oita University, Japan, for the care and use of laboratory animals.
Antibody to HSP72 (mouse) was purchased from Stressgen Biotechnologies. Antibodies to Akt, phospho-Ser473-Akt, and phospho-Thr311-Akt were purchased from Cell Signaling Technology. Horseradish peroxidase–tagged secondary antibodies and enhanced chemiluminescence reagents were purchased from Amersham Pharmacia Biotech. Bradford protein assay kits were purchased from Bio-Rad. Wortmannin and other chemical agents were purchased from Sigma Chemical.
Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (70–80 g initial weight) were provided by the animal center of Tokushima Research Institute (Otsuka Pharmaceuticals). Age-matched Long-Evans Tokushima Otsuka (LETO) rats, which were developed from the same colony but do not develop insulin resistance, were used as normal controls. All rats were housed in a room illuminated daily from 700 to 1900 (12:12-h light/dark cycle) with temperature maintained at 21 ± 1°C. They were allowed free access to tap water and standard pellet rat diet. At 12 weeks of age, OLETF (n = 90) and LETO (n = 90) rats were introduced to be treated with pioglitazone (10 mg · kg–1 · day–1, OLETF-PIO or LETO-PIO group, n = 30 for each), glibenclamide (5 mg · kg–1 · day–1, OLETF-GLIB or LETO-GLIB group, n = 30 for each), or vehicle (OLETF-VEH or LETO-VEH group, n = 30 for each) orally for 4 weeks. At 16 weeks of age, each rat was anesthetized with pentobarbital (20 mg/kg i.p.) and immersed in a water bath at 43°C (hyperthermia) or 37°C (normothermia, NT) for 20 min. Rectal temperature was monitored throughout the thermo treatment to confirm changes in body temperature. In addition, some rats of the hyperthermia group (n = 4 for each group) were injected with either wortmannin (16 μg/kg i.v.) or saline (n = 4 for each group) into a tail vein 15 min before hyperthermia. Twenty-four hours after thermo treatment with normothermia or hyperthermia, following anesthesia with pentobarbital (20 mg/kg i.p.), rat hearts were isolated and prepared for Western blot analysis (n = 4 for each group) or isolated perfused heart experiments (n = 7 for each group).
Oral glucose tolerance test.
At 16 weeks of age, an oral glucose tolerance test (OGTT) was performed after an overnight fast (n = 8 for each group). Glucose solution (2 g/kg) was administered orally, and at 0, 30, 60, 90, and 120 min, blood was drawn from a tail vein. The plasma glucose concentration was measured with a commercial test kit (GR-102; Terumo). The plasma insulin concentration was quantified using an enzyme-linked immunosorbent assay insulin kit (Morinaga Seikagaku).
Western blot analysis.
Western blot analysis was performed as described (5,7,19). Briefly, rats were heparinized (500 IU/kg i.p.) and anesthetized with pentobarbital (50 mg/kg i.p.). Each heart was removed rapidly and frozen in liquid nitrogen. The frozen tissues were homogenized with lysis buffer (50 mmol/l Tris-HCl [pH 7.4], 10% glycerol, 2 mmol/l EDTA, 150 mmol/l NaCl, 1 mmol/l MgCl2, 50 mmol/l glycerophosphate, 2 mmol/l Na3VO4, 20 mmol/l NaF, 1 mmol/l phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1% Nonidet P-40). Samples were centrifuged and the concentration of protein was measured by the Bradford method (20). An equal amount of total protein in each fraction was subjected to SDS-PAGE and transferred electrophoretically onto a polyvinylidine fluoride membrane. After blocking with 0.5% nonfat milk, the membranes were incubated with antibodies. After repeated washing, the membranes were incubated with secondary antibodies. The proteins were detected by enhanced chemiluminescence following exposure to Hyperfilm. The amount of protein on the immunoblots was quantified using National Institutes of Health image analysis software.
Isolated perfused heart experiments.
Isolated perfused experiments using a Langendorff apparatus were performed as described (5,7,19). At 24 h after hyperthermia (n = 7 for each group) or normothermia (n = 7 for each group), the heart was isolated and perfused retrogradely with Krebs-Henseleit buffer (pH 7.4; 118 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 25.0 mmol/l Na2HCO3, 11.0 mmol/l glucose, 100 μU/ml insulin, and 3% BSA) equilibrated with 95% O2/5% CO2 gas mixture at 36.5°C at a constant pressure of 75 mmHg. A water-filled latex balloon was inserted through the mitral valve orifice into the left ventricle, and the left ventricular end-diastolic pressure was adjusted to 0 mmHg. During the initial 10 min of constant pressure perfusion, the perfusion flow rate was determined for each heart, which was then perfused at a determined perfusion rate using a microtube pump, while the heart was covered with water-jacketed glassware and the relative humidity was maintained at 90% or more. No-flow global ischemia was introduced for 20 min, followed by reperfusion for 30 min. The coronary effluent during the 30-min reperfusion period was collected for measurement of creatine kinase content. Creatine kinase activity was determined with a spectrophotometer by measuring NADPH production as the change in absorbance at 340 nm in the solution containing 2 mmol/l ADP, 20 mmol/l glucose, and 2 mmol/l NADP, along with 2 IU/ml of both hexokinase and glucose-6-phosphate dehydrogenase at a pH of 7.1. The reaction was initiated by addition of creatine phosphate (21). The ratio of released creatine kinase to left ventricular weight was calculated. Left ventricular pressure was monitored using a pressure transducer to obtain the peak positive and negative first derivatives of left ventricular pressure (dP/dtmax and dP/dtmin). Left ventricular developed pressure (LVDP) was defined as the difference between the left ventricular systolic and diastolic pressure. Left ventricular pressure, coronary perfusion pressure (CPP), and electrocardiogram were recorded continuously on a polygraph recorder (WS-681G; Nihon Kohden) and stored on a PCM data recorder (RD-111T; TEAC) for later analysis.
Statistical analysis.
Data are expressed as means ± SE. Serial changes in plasma concentrations of glucose and insulin during OGTT and LVDP, left ventricular end-diastolic pressure, dP/dt, CPP, and heart rate during the reperfusion period were analyzed by two-way ANOVA followed by the Bonferroni-Dunn test. Comparison of physiological and serum parameters, the relative intensity of each protein, and the ratio of released creatine kinase to left ventricular weight was analyzed using one-way ANOVA, followed by the Bonferroni-Dunn test. A P value <0.05 was considered statistically significant.
RESULTS
Physical and metabolic characteristics.
OGTT.
Cardiac HSP72 expression and PI3 kinase dependence.
Cardiac Akt phosphorylation and PI3 kinase dependence.
Reperfusion-induced left ventricular functional recovery.
Effects of treatment with pioglitazone or glibenclamide on cardiac HSP72 expression.
Effects of pioglitazone treatment on Akt phosphorylation.
Effects of treatment with pioglitazone or glibenclamide on reperfusion-induced left ventricular functional recovery.
DISCUSSION
The core findings of the present study are as follows. 1) In response to hyperthermia, the Akt activation depending on PI3 kinase was necessary for cardiac HSP72 expression. 2) Hyperthermia-induced Akt activation and HSP72 expression was depressed in OLETF rat hearts. 3) Pioglitazone but not glibenclamide improved insulin sensitivity in OLETF rats, which was associated with the restoration of Akt activation and HSP72 expression. 4) In isolated perfused heart experiments, reperfusion-induced left ventricular functional recovery was suppressed in OLETF rat hearts, which was restored by treatment with pioglitazone but not glibenclamide.
In the present study, we used OLETF rats, which have been established as an animal model of type 2 diabetes, characterized by late onset of hyperglycemia (after 20 weeks of age), mild course of diabetes, and conversion to insulin-dependent diabetes after 40 weeks of age (22,23). In our protocol, 12-week-old rats were introduced to receive oral pioglitazone or glibenclamide for 4 weeks. At 16 weeks, when compared with corresponding control LETO rats, while plasma glucose concentrations were not significantly different, plasma insulin concentrations were higher in OLETF rats. In addition, during OGTT, increases of plasma glucose and insulin concentrations were greater in OLETF rats. Thus, vehicle-treated OLETF rats of 16 weeks of age can be regarded as an insulin-resistant model but not a type 2 diabetic model. As shown in Table 1 and Fig. 1, in OLETF rats, both pioglitazone and glibenclamide suppressed the increase of plasma glucose during OGTT. However, their effects on plasma insulin were strikingly different. While pioglitazone improved insulin sensitivity, glibenclamide rather worsened it. These findings suggest that the differences of our observations between pioglitazone-treated and glibenclamide-treated OLETF rats may be, at least in part, due to differed alteration in insulin sensitivity despite the similar levels of plasma glucose.
Information is very limited with respect to the impact of insulin resistance and type 2 diabetes on HSP72 expression. In the study using human subjects, Kurucz et al. (9) provided the first evidence of decreased expression of the HSP72 mRNA in skeletal muscle from patients with type 2 diabetes, with this reduction being correlated with some markers of insulin resistance. Consistently, Bruce et al. (24) demonstrated that intramuscular HSP72 and heme oxygenase-1 mRNA were reduced in patients with type 2 diabetes. The authors reported the more convinced correlation between a reduction in HSP72 mRNA with insulin resistance (24). However, these clinical studies evaluated the HSP72 mRNA expression only in skeletal muscle and did not provide the underlying mechanism. In this regard, we have focused on PI3 kinase–dependent Akt phosphorylation as a triggering mechanism for hyperthermia-induced HSP72 expression. In fact, we have recently reported that transient activation of Akt in a PI3 kinase–dependent manner is required for hyperthermia-induced HSP72 expression and that streptozotocin (STZ)-induced diabetic rat heart showed an attenuation of this response, resulting in depressed HSP72 expression (19). Because insulin supplementation restored these abnormalities, we proposed insulin deficiency is a predominant etiology for depressed PI3 kinase–dependent Akt activation in response to hyperthermia in STZ-induced diabetic heart (19). It is noteworthy that OLETF rats, characterized by insulin resistance, also showed attenuated PI3 kinase–dependent Akt phosphorylation in response to hyperthermia in the present study. Although it appears difficult to explain the disparity, these two animal models may have common pathogenesis in a sense. The cardiac muscles in STZ-induced diabetic rat cannot utilize insulin due to insulin deficiency while those in OLETF rat cannot effectively utilize insulin because of insulin resistance. This hypothesis may be supported by the combination of our previous (19) and present observations; that is, both insulin supplementation in STZ-induced diabetic rat (19) and improvement in insulin resistance in OLETF rat by pioglitazone in the present study could restore the PI3 kinase–dependent Akt phosphorylation and resultant HSP72 expression.
Thiazolidinediones, including pioglitazone, are synthetic PPAR- agonists, acting as insulin sensitizers (14,15). In the last few years, it has been reported numerically that the therapeutic benefits of PPAR- agonists may reach far beyond their use as insulin sensitizers (25). It is noteworthy that pioglitazone reportedly leads to increased expression of HSP72 in the gastric mucosa in acetic-acid gastric ulcers in association with promoting ulcer healing (26). Regarding myocardial ischemia/reperfusion injury, chronic troglitazone administration improved left ventricular functional recovery and increased net myocardial lactate uptake, indicating that troglitazone enhances myocardial carbohydrate oxidation (27). It was also reported that rosiglitazone attenuated the increase in caspase-3 activity and apoptotic cell death (28). More recently, using Zucker diabetic fatty (ZDF) rats, Yue et al. (29) reported that oral rosiglitazone administration for 8 days reduced ischemia/reperfusion-induced infarct size and apoptosis, which was in association with an improvement of insulin sensitivity. It is noteworthy that Akt phosphorylation in ZDF rat heart subjected to ischemia/reperfusion was enhanced by rosiglitazone. In their experiments, the Akt phosphorylation might induce cell surviving cascade against apoptosis. Thus, the restoration of Akt phosphorylation in response to hyperthermia observed in pioglitazone-treated OLETF rats may be due to diverse effects derived from the activation of PPAR-.
Limitations.
There are several reservations in the present study. First, we discussed PI3 kinase–dependent Akt phosphorylation as a predominant signal for hyperthermia-induced HSP72 overexpression. However, in either OLETF or LETO rats, while the pretreatment with wortmannin completely inhibited Akt phosphorylation by hyperthermia (Fig. 3), it resulted in failure to completely suppress the HSP72 expression (Fig. 2). These observations suggest the working of operative mechanisms other than activation of PI3 kinase–dependent Akt activation for hyperthermia-induced HSP72 expression, which should be clarified in a future study. Second, our experiments could not establish whether pioglitazone-induced HSP72 expression improves reperfusion-induced cardiac functional recovery. To answer this issue appropriately, specific inhibition of expression of the HSP72 gene by, for instance, intracoronary injection of antisense HSP72 oligonucleotide, will be required. Third, in the clinical setting, it appears impossible to introduce the patients to temperatures as high as 43°C. It should be determined whether other HSP72 inducers, including GGA (5,6), lead to similar results. Fourth, our experiments were not performed in a single session under the same conditions. For instance, the absolute ratio of phospho-Akt/total Akt that appears in Fig. 3 is difficult to compare with what appears in Fig. 6. Finally, the amount of released creatine kinase did not correlate well with the LVDP recovery during reperfusion period (Figs. 4 and 7). Especially in hyperthermia-treated OLETF rat hearts, the reperfusion-induced left ventricular functional recovery appeared to be depressed compared with the reduction in released creatine kinase. The finding suggests the complicated functional damage of OLETF rat hearts in response to ischemia/reperfusion, which was not reflected as released creatine kinase.
Conclusions.
Our results suggest that PI3 kinase–dependent Akt activation in response to hyperthermia, an essential signal for HSP72 expression, at least in part, is depressed in the heart of insulin-resistant OLETF rats, and the results also suggest that restorations of HSP72 expression and tolerance against ischemia/reperfusion injury with pioglitazone in OLETF heart might be due to a restoration of impaired PI3 kinase–dependent Akt activation in response to hyperthermia.
ACKNOWLEDGMENTS
This study was supported in part by a grant-in-aid 17590756 (T.S.) from the Japanese Ministry of Education, Science and Culture.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Benjamin IJ, McMillan DR: Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 83:117–132, 1998
Williams RS, Benjamin IJ: Protective responses in the ischemic myocardium. J Clin Invest 106:813–818, 2000
Latchman DS: Heat shock proteins and cardiac protection. Cardiovasc Res 51:637–646, 2001
Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann WH: Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95:1446–1456, 1995
Ooie T, Takahashi N, Saikawa T, Nawata T, Arikawa M, Yamanaka K, Hara M, Shimada T, Sakata T: Single oral dose of geranylgeranylacetone induces heat-shock protein 72 and renders protection against ischemia/reperfusion injury in rat heart. Circulation 104:1837–1843, 2001
Yamanaka K, Takahashi N, Ooie T, Kaneda K, Yoshimatsu H, Saikawa T: Role of protein kinase C in geranylgeranylacetone-induced expression of heat-shock protein 72 and cardioprotection in the rat heart. J Mol Cell Cardiol 35:785–794, 2003
Shinohara T, Takahashi N, Ooie T, Ichinose M, Hara M, Yonemochi H, Saikawa T, Yoshimatsu H: Estrogen inhibits hyperthermia-induced expression of heat-shock protein 72 and cardioprotection against ischemia/reperfusion injury in female rat heart. J Mol Cell Cardiol 37:1053–1061, 2004
Zhu Z, Takahashi N, Ooie T, Shinohara T, Yamanaka K, Saikawa T: Oral administration of geranylgeranylacetone blunts the endothelial dysfunction induced by ischemia and reperfusion in the rat heart. J Cardiovasc Pharmacol 45:555–562, 2005
Kurucz I, Morva A, Vaag A, Eriksson KF, Huang X, Groop L, Koranyi L: Decreased expression of heat shock protein 72 in skeletal muscle of patients with type 2 diabetes correlates with insulin resistance. Diabetes 51:1102–1109, 2002
Ooie T, Kajimoto M, Takahashi N, Shinohara T, Taniguchi Y, Kouno H, Wakisaka O, Yoshimatsu H, Saikawa T: Effects of insulin resistance on geranylgeranylacetone-induced expression of heat shock protein 72 and cardioprotection in high-fat diet rats. Life Sci 77:869–881, 2005
Folli F, Saad MJ, Backer JM, Kahn CR: Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus. J Clin Invest 92:1787–1794, 1993
Saad MJ, Folli F, Kahn JA, Kahn CR: Modulation of insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats. J Clin Invest 92:2065–2072, 1993
Abel ED: Myocardial insulin resistance and cardiac complications of diabetes. Curr Drug Targets Immune Endocr Metabol Disord 5:219–226, 2005
Kobayashi M, Iwanishi M, Egawa K, Shigeta Y: Pioglitazone increases insulin sensitivity by activating insulin receptor kinase. Diabetes 41:476–483, 1992
Hayakawa T, Shiraki T, Morimoto T, Shii K, Ikeda H: Pioglitazone improves insulin signaling defects in skeletal muscle from Wistar fatty (fa/fa) rats. Biochem Biophys Res Commun 223:439–444, 1996
Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K: Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation 101:660–667, 2000
Konishi H, Matsuzaki H, Tanaka M, Ono Y, Tokunaga C, Kuroda S, Kikkawa U: Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A 93:7639–7643, 1996
Shaw M, Cohen P, Alessi DR: The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2. Biochem J 336:241–246, 1998
Shinohara T, Takahashi N, Ooie T, Hara M, Shigematsu S, Nakagawa M, Yonemochi H, Saikawa T, Yoshimatsu H: Phosphatidylinositol 3-kinase-dependent activation of Akt, an essential signal for hyperthermia-induced heat shock protein 72, is attenuated in the streptozotocin-induced diabetic heart. Diabetes 55:1307–1315, 2006
Bradford MM: A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254, 1976
Mekhfi H, Veksler V, Mateo P, Maupoil V, Rochette L, Ventura-Clapier R: Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils. Circ Res 78:1016–1027,1996
Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T: Spontaneous long-term hyperglycemic rat with diabetic complications: Otsuka Long-Evans Tokushima Fatty (OLETF) strain. Diabetes 41:1422–1428, 1992
Sato T, Asahi Y, Toide K, Nakayama N: Insulin resistance in skeletal muscle of the male Otsuka Long-Evans Tokushima Fatty rat, a new model of NIDDM. Diabetologia 38:1033–1041, 1995
Bruce CR, Carey AL, Hawley JA, Febbraio MA: Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism Diabetes 52:2338–2345, 2003
Abdelrahman M, Sivarajah A, Thiemermann C: Beneficial effects of PPAR- ligands in ischemia-reperfusion injury, inflammation and shock. Cardiovasc Res 65:772–781, 2005
Konturek PC, Brzozowski T, Kania J, Konturek SJ, Kwiecien S, Pajdo R, Hahn EG: Pioglitazone, a specific ligand of peroxisome proliferator-activated receptor-gamma, accelerates gastric ulcer healing in rat. Eur J Pharmacol 472:213–220, 2003
Zhu P, Lu L, Xu Y, Schwartz GG: Troglitazone improves recovery of left ventricular function after regional ischemia in pigs. Circulation 101:1165–1171, 2000
Liu HR, Tao L, Gao E, Lopez BL, Christopher TA, Willette RN, Ohlstein EH, Yue TL, Ma XL: Anti-apoptotic effects of rosiglitazone in hypercholesterolemic rabbits subjected to myocardial ischemia and reperfusion. Cardiovasc Res 62:135–144, 2004
Yue TL, Bao W, Gu JL, Cui J, Tao L, Ma XL, Ohlstein EH, Jucker BM: Rosiglitazone treatment in Zucker diabetic Fatty rats is associated with ameliorated cardiac insulin resistance and protection from ischemia/reperfusion-induced myocardial injury. Diabetes 54:554–562, 2005(Yayoi Taniguchi, Tatsuhik)
2 Department of Internal Medicine 1, Faculty of Medicine, Oita University, Oita, Japan
CPP, coronary perfusion pressure; GGA, geranylgeranylacetone; HSP, heat shock protein; LVDP, left ventricular developed pressure; OGTT, oral glucose tolerance test; PI3, phosphatidylinositol 3; PPAR-, peroxisome proliferator–activated receptor-; STZ, streptozotocin
ABSTRACT
We tested the hypothesis that pioglitazone could restore expression of heat shock protein (HSP)72 in insulin-resistant rat heart. At 12 weeks of age, male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and control (LETO) rats were treated with pioglitazone (10 mg · kg–1 · day–1) or glibenclamide (5 mg · kg–1 · day–1) for 4 weeks. Thereafter, hyperthermia (43°C for 20 min) was applied. In response to hyperthermia, the activation of serine/threonine kinase Akt depending on phosphatidylinositol 3 (PI3) kinase was necessary for cardiac expression of HSP72. Hyperthermia-induced activation of Akt and HSP72 expression were depressed in OLETF rat hearts. Pioglitazone but not glibenclamide improved insulin sensitivity in OLETF rats, which was associated with the restoration of Akt activation and HSP72 expression. In experiments with isolated perfused heart, reperfusion-induced cardiac functional recovery was suppressed in OLETF rat hearts, which was improved by pioglitazone but not glibenclamide. Our results suggest that PI3 kinase–dependent Akt activation, an essential signal for HSP72 expression, is depressed in the heart in insulin-resistant OLETF rats, and the results suggest also that the restoration of HSP72 expression and tolerance against ischemia/reperfusion injury by treatment with pioglitazone might be due to an improvement of insulin resistance, leading to restoration of impaired PI3 kinase–dependent Akt activation in response to hyperthermia.
Heat shock protein (HSP)72, a member of the HSP family, has been reported to be involved predominantly in cardioprotection (1–4). We have shown that induction of HSP72 by either hyperthermia or oral administration of geranylgeranylacetone (GGA) leads to protection against ischemia/reperfusion injury (5–8). However, information is very limited with respect to the impact of insulin resistance and type 2 diabetes on the expression of HSP72. Kurucz et al. (9) reported that the expression of HSP72 mRNA in skeletal muscle of patients with type 2 diabetes is decreased, which correlates with the parameters of insulin resistance. Consistent with this, we have shown attenuated cardiac HSP72 expression in response to GGA in insulin-resistant rats produced by a high-fat diet (10). Skeletal muscle, liver, and heart are known to be targeted organs of insulin resistance (11–13). However, the mechanisms underlying the depressed expression of HSP72 in insulin-resistant organs are unclear. Recently, thiazolidinediones, including pioglitazone, have been demonstrated to improve insulin resistance experimentally and clinically (14,15). Whereas sulfonylurea including glibenclamide stimulates endogenous insulin secretion through blockade of ATP-sensitive potassium channels on pancreatic -cells, pioglitazone, a synthetic peroxisome proliferator–activated receptor- (PPAR-) agonist, does not stimulate insulin release from pancreatic -cells (14,15). It is therefore interesting to compare the effects of treatment with pioglitazone and treatment with glibenclamide on the expression of HSP72 in the insulin-resistant animal model.
Activation of the serine/threonine kinase Akt promotes survival of some cell types including cardiomyocytes (16). Hyperthermia is known to cause Akt activation, and experimental evidence indicates that hyperthermia-induced activation of Akt can involve either a phosphatidylinositol 3 (PI3) kinase–dependent or a PI3 kinase–independent pathway (17,18). However, little is known about the involvement of Akt in hyperthermia-induced HSP72 expression in the heart. We have demonstrated recently that PI3 kinase–dependent Akt activation is essential for expression of HSP72 (19). In the present study, therefore, we tested the hypothesis that 1) depressed cardiac HSP72 expression in insulin-resistant rats is related to depressed PI3 kinase–dependent Akt activation in response to hyperthermia and that 2) treatment with pioglitazone improves this depressed response, resulting in restoration of cardiac expression of HSP72, which is associated with the protection against ischemia/reperfusion injury.
RESEARCH DESIGN AND METHODS
All experimental procedures were carried out in accordance with the guidelines of the Physiological Society of Oita University, Japan, for the care and use of laboratory animals.
Antibody to HSP72 (mouse) was purchased from Stressgen Biotechnologies. Antibodies to Akt, phospho-Ser473-Akt, and phospho-Thr311-Akt were purchased from Cell Signaling Technology. Horseradish peroxidase–tagged secondary antibodies and enhanced chemiluminescence reagents were purchased from Amersham Pharmacia Biotech. Bradford protein assay kits were purchased from Bio-Rad. Wortmannin and other chemical agents were purchased from Sigma Chemical.
Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (70–80 g initial weight) were provided by the animal center of Tokushima Research Institute (Otsuka Pharmaceuticals). Age-matched Long-Evans Tokushima Otsuka (LETO) rats, which were developed from the same colony but do not develop insulin resistance, were used as normal controls. All rats were housed in a room illuminated daily from 700 to 1900 (12:12-h light/dark cycle) with temperature maintained at 21 ± 1°C. They were allowed free access to tap water and standard pellet rat diet. At 12 weeks of age, OLETF (n = 90) and LETO (n = 90) rats were introduced to be treated with pioglitazone (10 mg · kg–1 · day–1, OLETF-PIO or LETO-PIO group, n = 30 for each), glibenclamide (5 mg · kg–1 · day–1, OLETF-GLIB or LETO-GLIB group, n = 30 for each), or vehicle (OLETF-VEH or LETO-VEH group, n = 30 for each) orally for 4 weeks. At 16 weeks of age, each rat was anesthetized with pentobarbital (20 mg/kg i.p.) and immersed in a water bath at 43°C (hyperthermia) or 37°C (normothermia, NT) for 20 min. Rectal temperature was monitored throughout the thermo treatment to confirm changes in body temperature. In addition, some rats of the hyperthermia group (n = 4 for each group) were injected with either wortmannin (16 μg/kg i.v.) or saline (n = 4 for each group) into a tail vein 15 min before hyperthermia. Twenty-four hours after thermo treatment with normothermia or hyperthermia, following anesthesia with pentobarbital (20 mg/kg i.p.), rat hearts were isolated and prepared for Western blot analysis (n = 4 for each group) or isolated perfused heart experiments (n = 7 for each group).
Oral glucose tolerance test.
At 16 weeks of age, an oral glucose tolerance test (OGTT) was performed after an overnight fast (n = 8 for each group). Glucose solution (2 g/kg) was administered orally, and at 0, 30, 60, 90, and 120 min, blood was drawn from a tail vein. The plasma glucose concentration was measured with a commercial test kit (GR-102; Terumo). The plasma insulin concentration was quantified using an enzyme-linked immunosorbent assay insulin kit (Morinaga Seikagaku).
Western blot analysis.
Western blot analysis was performed as described (5,7,19). Briefly, rats were heparinized (500 IU/kg i.p.) and anesthetized with pentobarbital (50 mg/kg i.p.). Each heart was removed rapidly and frozen in liquid nitrogen. The frozen tissues were homogenized with lysis buffer (50 mmol/l Tris-HCl [pH 7.4], 10% glycerol, 2 mmol/l EDTA, 150 mmol/l NaCl, 1 mmol/l MgCl2, 50 mmol/l glycerophosphate, 2 mmol/l Na3VO4, 20 mmol/l NaF, 1 mmol/l phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1% Nonidet P-40). Samples were centrifuged and the concentration of protein was measured by the Bradford method (20). An equal amount of total protein in each fraction was subjected to SDS-PAGE and transferred electrophoretically onto a polyvinylidine fluoride membrane. After blocking with 0.5% nonfat milk, the membranes were incubated with antibodies. After repeated washing, the membranes were incubated with secondary antibodies. The proteins were detected by enhanced chemiluminescence following exposure to Hyperfilm. The amount of protein on the immunoblots was quantified using National Institutes of Health image analysis software.
Isolated perfused heart experiments.
Isolated perfused experiments using a Langendorff apparatus were performed as described (5,7,19). At 24 h after hyperthermia (n = 7 for each group) or normothermia (n = 7 for each group), the heart was isolated and perfused retrogradely with Krebs-Henseleit buffer (pH 7.4; 118 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 25.0 mmol/l Na2HCO3, 11.0 mmol/l glucose, 100 μU/ml insulin, and 3% BSA) equilibrated with 95% O2/5% CO2 gas mixture at 36.5°C at a constant pressure of 75 mmHg. A water-filled latex balloon was inserted through the mitral valve orifice into the left ventricle, and the left ventricular end-diastolic pressure was adjusted to 0 mmHg. During the initial 10 min of constant pressure perfusion, the perfusion flow rate was determined for each heart, which was then perfused at a determined perfusion rate using a microtube pump, while the heart was covered with water-jacketed glassware and the relative humidity was maintained at 90% or more. No-flow global ischemia was introduced for 20 min, followed by reperfusion for 30 min. The coronary effluent during the 30-min reperfusion period was collected for measurement of creatine kinase content. Creatine kinase activity was determined with a spectrophotometer by measuring NADPH production as the change in absorbance at 340 nm in the solution containing 2 mmol/l ADP, 20 mmol/l glucose, and 2 mmol/l NADP, along with 2 IU/ml of both hexokinase and glucose-6-phosphate dehydrogenase at a pH of 7.1. The reaction was initiated by addition of creatine phosphate (21). The ratio of released creatine kinase to left ventricular weight was calculated. Left ventricular pressure was monitored using a pressure transducer to obtain the peak positive and negative first derivatives of left ventricular pressure (dP/dtmax and dP/dtmin). Left ventricular developed pressure (LVDP) was defined as the difference between the left ventricular systolic and diastolic pressure. Left ventricular pressure, coronary perfusion pressure (CPP), and electrocardiogram were recorded continuously on a polygraph recorder (WS-681G; Nihon Kohden) and stored on a PCM data recorder (RD-111T; TEAC) for later analysis.
Statistical analysis.
Data are expressed as means ± SE. Serial changes in plasma concentrations of glucose and insulin during OGTT and LVDP, left ventricular end-diastolic pressure, dP/dt, CPP, and heart rate during the reperfusion period were analyzed by two-way ANOVA followed by the Bonferroni-Dunn test. Comparison of physiological and serum parameters, the relative intensity of each protein, and the ratio of released creatine kinase to left ventricular weight was analyzed using one-way ANOVA, followed by the Bonferroni-Dunn test. A P value <0.05 was considered statistically significant.
RESULTS
Physical and metabolic characteristics.
OGTT.
Cardiac HSP72 expression and PI3 kinase dependence.
Cardiac Akt phosphorylation and PI3 kinase dependence.
Reperfusion-induced left ventricular functional recovery.
Effects of treatment with pioglitazone or glibenclamide on cardiac HSP72 expression.
Effects of pioglitazone treatment on Akt phosphorylation.
Effects of treatment with pioglitazone or glibenclamide on reperfusion-induced left ventricular functional recovery.
DISCUSSION
The core findings of the present study are as follows. 1) In response to hyperthermia, the Akt activation depending on PI3 kinase was necessary for cardiac HSP72 expression. 2) Hyperthermia-induced Akt activation and HSP72 expression was depressed in OLETF rat hearts. 3) Pioglitazone but not glibenclamide improved insulin sensitivity in OLETF rats, which was associated with the restoration of Akt activation and HSP72 expression. 4) In isolated perfused heart experiments, reperfusion-induced left ventricular functional recovery was suppressed in OLETF rat hearts, which was restored by treatment with pioglitazone but not glibenclamide.
In the present study, we used OLETF rats, which have been established as an animal model of type 2 diabetes, characterized by late onset of hyperglycemia (after 20 weeks of age), mild course of diabetes, and conversion to insulin-dependent diabetes after 40 weeks of age (22,23). In our protocol, 12-week-old rats were introduced to receive oral pioglitazone or glibenclamide for 4 weeks. At 16 weeks, when compared with corresponding control LETO rats, while plasma glucose concentrations were not significantly different, plasma insulin concentrations were higher in OLETF rats. In addition, during OGTT, increases of plasma glucose and insulin concentrations were greater in OLETF rats. Thus, vehicle-treated OLETF rats of 16 weeks of age can be regarded as an insulin-resistant model but not a type 2 diabetic model. As shown in Table 1 and Fig. 1, in OLETF rats, both pioglitazone and glibenclamide suppressed the increase of plasma glucose during OGTT. However, their effects on plasma insulin were strikingly different. While pioglitazone improved insulin sensitivity, glibenclamide rather worsened it. These findings suggest that the differences of our observations between pioglitazone-treated and glibenclamide-treated OLETF rats may be, at least in part, due to differed alteration in insulin sensitivity despite the similar levels of plasma glucose.
Information is very limited with respect to the impact of insulin resistance and type 2 diabetes on HSP72 expression. In the study using human subjects, Kurucz et al. (9) provided the first evidence of decreased expression of the HSP72 mRNA in skeletal muscle from patients with type 2 diabetes, with this reduction being correlated with some markers of insulin resistance. Consistently, Bruce et al. (24) demonstrated that intramuscular HSP72 and heme oxygenase-1 mRNA were reduced in patients with type 2 diabetes. The authors reported the more convinced correlation between a reduction in HSP72 mRNA with insulin resistance (24). However, these clinical studies evaluated the HSP72 mRNA expression only in skeletal muscle and did not provide the underlying mechanism. In this regard, we have focused on PI3 kinase–dependent Akt phosphorylation as a triggering mechanism for hyperthermia-induced HSP72 expression. In fact, we have recently reported that transient activation of Akt in a PI3 kinase–dependent manner is required for hyperthermia-induced HSP72 expression and that streptozotocin (STZ)-induced diabetic rat heart showed an attenuation of this response, resulting in depressed HSP72 expression (19). Because insulin supplementation restored these abnormalities, we proposed insulin deficiency is a predominant etiology for depressed PI3 kinase–dependent Akt activation in response to hyperthermia in STZ-induced diabetic heart (19). It is noteworthy that OLETF rats, characterized by insulin resistance, also showed attenuated PI3 kinase–dependent Akt phosphorylation in response to hyperthermia in the present study. Although it appears difficult to explain the disparity, these two animal models may have common pathogenesis in a sense. The cardiac muscles in STZ-induced diabetic rat cannot utilize insulin due to insulin deficiency while those in OLETF rat cannot effectively utilize insulin because of insulin resistance. This hypothesis may be supported by the combination of our previous (19) and present observations; that is, both insulin supplementation in STZ-induced diabetic rat (19) and improvement in insulin resistance in OLETF rat by pioglitazone in the present study could restore the PI3 kinase–dependent Akt phosphorylation and resultant HSP72 expression.
Thiazolidinediones, including pioglitazone, are synthetic PPAR- agonists, acting as insulin sensitizers (14,15). In the last few years, it has been reported numerically that the therapeutic benefits of PPAR- agonists may reach far beyond their use as insulin sensitizers (25). It is noteworthy that pioglitazone reportedly leads to increased expression of HSP72 in the gastric mucosa in acetic-acid gastric ulcers in association with promoting ulcer healing (26). Regarding myocardial ischemia/reperfusion injury, chronic troglitazone administration improved left ventricular functional recovery and increased net myocardial lactate uptake, indicating that troglitazone enhances myocardial carbohydrate oxidation (27). It was also reported that rosiglitazone attenuated the increase in caspase-3 activity and apoptotic cell death (28). More recently, using Zucker diabetic fatty (ZDF) rats, Yue et al. (29) reported that oral rosiglitazone administration for 8 days reduced ischemia/reperfusion-induced infarct size and apoptosis, which was in association with an improvement of insulin sensitivity. It is noteworthy that Akt phosphorylation in ZDF rat heart subjected to ischemia/reperfusion was enhanced by rosiglitazone. In their experiments, the Akt phosphorylation might induce cell surviving cascade against apoptosis. Thus, the restoration of Akt phosphorylation in response to hyperthermia observed in pioglitazone-treated OLETF rats may be due to diverse effects derived from the activation of PPAR-.
Limitations.
There are several reservations in the present study. First, we discussed PI3 kinase–dependent Akt phosphorylation as a predominant signal for hyperthermia-induced HSP72 overexpression. However, in either OLETF or LETO rats, while the pretreatment with wortmannin completely inhibited Akt phosphorylation by hyperthermia (Fig. 3), it resulted in failure to completely suppress the HSP72 expression (Fig. 2). These observations suggest the working of operative mechanisms other than activation of PI3 kinase–dependent Akt activation for hyperthermia-induced HSP72 expression, which should be clarified in a future study. Second, our experiments could not establish whether pioglitazone-induced HSP72 expression improves reperfusion-induced cardiac functional recovery. To answer this issue appropriately, specific inhibition of expression of the HSP72 gene by, for instance, intracoronary injection of antisense HSP72 oligonucleotide, will be required. Third, in the clinical setting, it appears impossible to introduce the patients to temperatures as high as 43°C. It should be determined whether other HSP72 inducers, including GGA (5,6), lead to similar results. Fourth, our experiments were not performed in a single session under the same conditions. For instance, the absolute ratio of phospho-Akt/total Akt that appears in Fig. 3 is difficult to compare with what appears in Fig. 6. Finally, the amount of released creatine kinase did not correlate well with the LVDP recovery during reperfusion period (Figs. 4 and 7). Especially in hyperthermia-treated OLETF rat hearts, the reperfusion-induced left ventricular functional recovery appeared to be depressed compared with the reduction in released creatine kinase. The finding suggests the complicated functional damage of OLETF rat hearts in response to ischemia/reperfusion, which was not reflected as released creatine kinase.
Conclusions.
Our results suggest that PI3 kinase–dependent Akt activation in response to hyperthermia, an essential signal for HSP72 expression, at least in part, is depressed in the heart of insulin-resistant OLETF rats, and the results also suggest that restorations of HSP72 expression and tolerance against ischemia/reperfusion injury with pioglitazone in OLETF heart might be due to a restoration of impaired PI3 kinase–dependent Akt activation in response to hyperthermia.
ACKNOWLEDGMENTS
This study was supported in part by a grant-in-aid 17590756 (T.S.) from the Japanese Ministry of Education, Science and Culture.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Benjamin IJ, McMillan DR: Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 83:117–132, 1998
Williams RS, Benjamin IJ: Protective responses in the ischemic myocardium. J Clin Invest 106:813–818, 2000
Latchman DS: Heat shock proteins and cardiac protection. Cardiovasc Res 51:637–646, 2001
Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann WH: Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95:1446–1456, 1995
Ooie T, Takahashi N, Saikawa T, Nawata T, Arikawa M, Yamanaka K, Hara M, Shimada T, Sakata T: Single oral dose of geranylgeranylacetone induces heat-shock protein 72 and renders protection against ischemia/reperfusion injury in rat heart. Circulation 104:1837–1843, 2001
Yamanaka K, Takahashi N, Ooie T, Kaneda K, Yoshimatsu H, Saikawa T: Role of protein kinase C in geranylgeranylacetone-induced expression of heat-shock protein 72 and cardioprotection in the rat heart. J Mol Cell Cardiol 35:785–794, 2003
Shinohara T, Takahashi N, Ooie T, Ichinose M, Hara M, Yonemochi H, Saikawa T, Yoshimatsu H: Estrogen inhibits hyperthermia-induced expression of heat-shock protein 72 and cardioprotection against ischemia/reperfusion injury in female rat heart. J Mol Cell Cardiol 37:1053–1061, 2004
Zhu Z, Takahashi N, Ooie T, Shinohara T, Yamanaka K, Saikawa T: Oral administration of geranylgeranylacetone blunts the endothelial dysfunction induced by ischemia and reperfusion in the rat heart. J Cardiovasc Pharmacol 45:555–562, 2005
Kurucz I, Morva A, Vaag A, Eriksson KF, Huang X, Groop L, Koranyi L: Decreased expression of heat shock protein 72 in skeletal muscle of patients with type 2 diabetes correlates with insulin resistance. Diabetes 51:1102–1109, 2002
Ooie T, Kajimoto M, Takahashi N, Shinohara T, Taniguchi Y, Kouno H, Wakisaka O, Yoshimatsu H, Saikawa T: Effects of insulin resistance on geranylgeranylacetone-induced expression of heat shock protein 72 and cardioprotection in high-fat diet rats. Life Sci 77:869–881, 2005
Folli F, Saad MJ, Backer JM, Kahn CR: Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus. J Clin Invest 92:1787–1794, 1993
Saad MJ, Folli F, Kahn JA, Kahn CR: Modulation of insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats. J Clin Invest 92:2065–2072, 1993
Abel ED: Myocardial insulin resistance and cardiac complications of diabetes. Curr Drug Targets Immune Endocr Metabol Disord 5:219–226, 2005
Kobayashi M, Iwanishi M, Egawa K, Shigeta Y: Pioglitazone increases insulin sensitivity by activating insulin receptor kinase. Diabetes 41:476–483, 1992
Hayakawa T, Shiraki T, Morimoto T, Shii K, Ikeda H: Pioglitazone improves insulin signaling defects in skeletal muscle from Wistar fatty (fa/fa) rats. Biochem Biophys Res Commun 223:439–444, 1996
Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K: Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation 101:660–667, 2000
Konishi H, Matsuzaki H, Tanaka M, Ono Y, Tokunaga C, Kuroda S, Kikkawa U: Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A 93:7639–7643, 1996
Shaw M, Cohen P, Alessi DR: The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2. Biochem J 336:241–246, 1998
Shinohara T, Takahashi N, Ooie T, Hara M, Shigematsu S, Nakagawa M, Yonemochi H, Saikawa T, Yoshimatsu H: Phosphatidylinositol 3-kinase-dependent activation of Akt, an essential signal for hyperthermia-induced heat shock protein 72, is attenuated in the streptozotocin-induced diabetic heart. Diabetes 55:1307–1315, 2006
Bradford MM: A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254, 1976
Mekhfi H, Veksler V, Mateo P, Maupoil V, Rochette L, Ventura-Clapier R: Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils. Circ Res 78:1016–1027,1996
Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T: Spontaneous long-term hyperglycemic rat with diabetic complications: Otsuka Long-Evans Tokushima Fatty (OLETF) strain. Diabetes 41:1422–1428, 1992
Sato T, Asahi Y, Toide K, Nakayama N: Insulin resistance in skeletal muscle of the male Otsuka Long-Evans Tokushima Fatty rat, a new model of NIDDM. Diabetologia 38:1033–1041, 1995
Bruce CR, Carey AL, Hawley JA, Febbraio MA: Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism Diabetes 52:2338–2345, 2003
Abdelrahman M, Sivarajah A, Thiemermann C: Beneficial effects of PPAR- ligands in ischemia-reperfusion injury, inflammation and shock. Cardiovasc Res 65:772–781, 2005
Konturek PC, Brzozowski T, Kania J, Konturek SJ, Kwiecien S, Pajdo R, Hahn EG: Pioglitazone, a specific ligand of peroxisome proliferator-activated receptor-gamma, accelerates gastric ulcer healing in rat. Eur J Pharmacol 472:213–220, 2003
Zhu P, Lu L, Xu Y, Schwartz GG: Troglitazone improves recovery of left ventricular function after regional ischemia in pigs. Circulation 101:1165–1171, 2000
Liu HR, Tao L, Gao E, Lopez BL, Christopher TA, Willette RN, Ohlstein EH, Yue TL, Ma XL: Anti-apoptotic effects of rosiglitazone in hypercholesterolemic rabbits subjected to myocardial ischemia and reperfusion. Cardiovasc Res 62:135–144, 2004
Yue TL, Bao W, Gu JL, Cui J, Tao L, Ma XL, Ohlstein EH, Jucker BM: Rosiglitazone treatment in Zucker diabetic Fatty rats is associated with ameliorated cardiac insulin resistance and protection from ischemia/reperfusion-induced myocardial injury. Diabetes 54:554–562, 2005(Yayoi Taniguchi, Tatsuhik)