Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina
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ABSTRACT |
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Tissue kallikrein specifically processes lowmolecular weight kininogen to produce potent vasoactive kinin peptides (1). Intact kinin binds to the bradykinin B2 receptor and transduces signals through nitric oxide (NO)-cGMP and prostacyclin-cAMP pathways, thereby modulating a broad spectrum of cellular functions (2). The B2 receptor can be blocked by the specific B2 receptor antagonist icatibant (also known as HOE140) (3). Previous reports have shown that the kallikrein-kinin system (KKS) components are locally expressed in the heart (4), and streptozotocin (STZ)-induced diabetes results in a decrease of active cardiac tissue kallikrein levels (5,6), resulting in increased thickness of the left ventricle wall and cardiac hypertrophy (7). The STZ animal model develops characteristic symptoms of diabetes such as hyperglycemia, hyperlipidemia, and increased water and food intake without body weight gain. In addition, STZ diabetes also induces key symptoms including increased glycogen storage in the myocardium, depressed ventricular performance, and cardiac hypertrophy (8). Our recent studies using gene transfer approaches have demonstrated that the KKS improves cardiac function in animal models of myocardial ischemia, chronic heart failure, and cardiac hypertrophy (911). In addition, transgenic rats overexpressing the human tissue kallikrein gene resulted in reduction of isoproterenol-induced cardiac hypertrophy and fibrosis, and these protective effects were abolished by icatibant (12). These findings indicate a potential protective role of the KKS in diabetic cardiomyopathy.
STZ-induced diabetes results in hyperglycemia and hyperlipidemia, and without insulin treatment, animals have poor control over glucose and circulating lipid levels. Previous studies have shown that the KKS is involved in glucose management by stimulating GLUT4 translocation (13), improving insulin stimulation of GLUT4 (14), and preventing dephosphorylation of insulin receptor substrate-1 (15). Whether the KKS plays a role in improving glucose utilization and lipid metabolism in STZ-induced diabetes has not been explored.
In this study, we used a gene transfer approach to determine the role and potential mechanisms of the KKS in diabetic cardiomyopathy, as well as glucose and lipid metabolism. We showed that kallikrein gene transfer improves myocardial contractility, reduced glycogen accumulation, and hyperlipidemia through increased phospholamban phosphorylation and sarco(endo)plasmic reticulum (Ca2+ + Mg2+)-ATPase (SERCA)-2a levels, GLUT4 translocation, and activation of the Aktglycogen synthase kinase (GSK) signaling pathway in STZ-induced diabetic rats.
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RESEARCH DESIGN AND METHODS |
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Preparation of replication-deficient adenoviral vectors Ad.CMV-TK and Ad.Null.
Adenoviral vectors containing the human tissue kallikrein cDNA (Ad.CMV-TK) under the control of the cytomegalovirus enhancer/promoter (CMV) and control adenovirus without a reporter gene (Ad.Null) were constructed and prepared as previously described (16).
Measurements of blood glucose, insulin levels, and physiological parameters.
Blood was collected via the tail vein after gene delivery and was then centrifuged to obtain serum. Serum samples were processed for glucose assay on a SYNCHRON LX System (Beckman Coulter) by the Department of Clinical Pathology, Medical University of South Carolina. Blood glucose levels were measured according to the clinical standards established for human insulin with a linear standard curve of 11,200 mg/dl. Blood insulin levels were determined by radioimmunoassay with a kit according to manufacturers instructions (Linco Research, St. Charles, MI). Animals were weighed and placed into separate cages and each supplied with 500 ml water and 30 g rat food at 5 days after gene delivery. Remaining water volume and food weight were measured 24 h later, and the differences were used to calculate water and food consumption. The epididymal fat pad and the gastrocnemius muscles from both the left and right hindlimbs were removed, briefly blotted, and weighed.
Expression of human tissue kallikrein in STZ-induced diabetic rats.
Twenty-fourhour urine collection was performed as previously described (17). Expression of recombinant human tissue kallikrein in rat serum and urine after gene delivery was monitored by a specific enzyme-linked immunosorbent assay (18).
Cardiac extract, plasma membrane, and cytosolic fraction preparation.
At the end of the experiment, all rats were anesthetized intraperitoneally with pentobarbital at a dose of 50 mg/kg body wt. Rats were then perfused with normal saline (0.9% NaCl) via the heart. The whole heart and left ventricle were removed, blotted, and weighed. Cardiac extracts and plasma membrane fraction from both heart and skeletal muscles were isolated as previously described (10,19). Briefly, heart or skeletal muscle were minced and homogenized at 4°C in lysis buffer and then centrifuged at 2,000g for 10 min. Total tissue extracts in the supernatant was collected and kept on ice. The pellet was resuspended in lysis buffer, rehomogenized for 10 s, and centrifuged for 10 min at 2,000g. Cytosolic fractions in the two supernatants were pooled. The plasma membrane fraction in the pellet was resuspended and loaded onto a 1030% (wt/wt) continuous sucrose gradient and centrifuged at 190,000g for 1 h. Protein concentrations were determined by Lowrys method.
Measurements of NO content and cAMP levels.
Nitrate and nitrite (NOx) levels in cardiac extracts were measured by a fluorimetric assay as previously described (20). Radioimmunoassay of cardiac cAMP levels was conducted according to previously described procedures (21).
Measurements for serum triglyceride and cholesterol levels.
Circulating triglycerides and cholesterol levels in rats were measured using a diagnostic kit following the manufacturers protocol (INFINITY Triglycerides Reagent TR22421, INFINITY Cholesterol Reagent TR13421, Triglyceride Standard TR22923, Cholesterol Standard TR13923; Thermo Electron, Woburn, MA). Briefly, serum samples were diluted 1:100 in either triglyceride or cholesterol reagent and incubated for 15 min, and 0.2 ml of the reaction mixture was used to measure at 500 nm on a Molecular Devices Emax plate reader.
Cardiac function.
Cardiac function was performed as previously described (22). Briefly, animals were anesthetized with pentobarbital sodium (50 mg/kg body wt). The femoral and carotid arteries were cannulated. Heart rate and arterial blood pressure were recorded. Fluorescent microspheres (FluoSpheres; Molecular Probes, Eugene, OR) were injected directly into the left ventricle, whereas arterial blood was collected for a total of 90 s from the femoral artery. The collected blood and one kidney were subjected to digestion to release the microspheres, which were then quantitated in a spectrofluorometer, with excitation at 570 nm and emission at 598 nm.
Morphological analysis.
Heart tissue sections embedded in paraffin were cut at 4 µm and stained with the periodic acid Schiff (PAS) reagent and then analyzed microscopically and morphometrically. Adobe Photoshop 5.5 (Adobe) was used for imaging and preparation of photomicrographs. Evaluation of sections was done under double-blind conditions.
Glycogen assay.
Quantitative analysis of cardiac glycogen content was determined as previously described (23). Briefly, 0.1 g cardiac tissue was dissolved in 30% KOH and then heated at 100°C for 10 min. The samples were diluted (1:10) with 30% KOH and precipitated by adding anhydrous ethanol and centrifuging at 5,700 rpm for 15 min. The pellet was resuspended in 0.5 ml H2O, and 1 ml 0.2% anthrone reagent (0.2 g in 100 ml 98% H2SO4) was added and then heated at 100°C for 10 min. The measurement was made using a Cary 3 UV-Visible Spectrophotometer (620 nm).
Western blot analysis.
Cardiac extracts (80100 µg) were subjected to Western blot analyses for SERCA2a, phospholamban, Akt, and GSK-3ß, and ß-actin and plasma membrane proteins from cardiac and skeletal muscle extracts were immunoblotted for GLUT4 as previously described (10). All blots immunoreacted with a primary antibody overnight at 4°C with dilutions as follows: SERCA2a 1:2,000 (Affinity BioReagents, Golden, CO), phospho-phospholamban, phospholamban 1:2,000 (Upstate Biotechnology, Lake Placid, NY), GLUT4 1:1,000 (Santa Cruz, Santa Cruz, CA), ß-actin 1:2,000 (Sigma), Akt 1:1,000 (Cell Signaling, Beverly MA), and GSK-3ß 1:1,000 (Cell Signaling, Beverly, MA). Chemiluminescence (Western Lightning; Perkin Elmer Life Sciences, Boston, MA) was used to a detect signal following the manufacturers instructions.
GSK-3ß activity assay.
GSK-3ß activity was measured using phospho-glycogen synthase peptide-2 (Upstate Biotechnology) according to a previously published method (24). Briefly, 10 µl cardiac extract (10 µg) was mixed with 10 µl GSK-3ß substrate peptide and 10 µl reaction buffer per assay, followed by 10 µl diluted [-32P]ATP (4,000 cpm) per sample. After incubating for 30 min at 37°C, 25 µl was spotted on the center of P81 paper. Assay papers were washed with 0.75% phosphoric acid and then with acetone. The assay papers were dried, and the radioactivity was then counted in a scintillation counter.
Statistical analysis.
Data are expressed as means ± SE. Comparisons among groups were made by ANOVA followed by Fishers protected least-significant difference or by an unpaired Students t test. Differences were considered significant at P < 0.05.
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RESULTS |
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Improvement in physiological parameters after kallikrein gene delivery.
STZ-induced diabetic rats injected with control virus showed stable signs of diabetes, including hyperglycemia, hypoinsulinemia, and increased food and water intake with no increase in body weight. Kallikrein gene delivery improves these physiological parameters. Most importantly, kallikrein reduced elevated blood glucose levels induced by STZ treatment (Table 1). Kallikrein has no effect on blood insulin levels but significantly improved body weight gain, food and water intake, epididymal fat pad, and gastrocnemius muscle weight in STZ-treated rats (data not shown). The improvement of kallikrein on epididymal fat pad weight and muscle weight was abrogated by icatibant, indicating a kinin B2 receptormediated event.
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Kallikrein gene delivery increases phospholamban phosphorylation and SERCA2a levels.
Contractile dysfunction in the diabetic state is related to an impaired sarcoplasmic reticulum function, leading to disturbed intracellular calcium handling. To further elucidate the potential mechanism of kallikrein in the improvement of cardiac contractility, we investigated the effect of kallikrein gene transfer on the sarcoplasmic reticulum calcium pump (SERCA2a) after STZ treatment. Western blot analysis showed that STZ treatment reduced SERCA2a levels compared with the control rats, whereas kallikrein gene transfer significantly increased SERCA2a levels (Fig. 3). ß-Actin levels remained the same among the three groups. Increased SERCA2a is due to phosphorylation of phospholamban, leading to increased Ca2+ transport. Similar to SERCA2a, kallikrein gene transfer significantly increased phosphorylated phospholamban in the left ventricle extracts compared with the Ad.Null group, whereas no change was observed in total phospholamban levels (Fig. 3). These results indicate that kallikrein improves cardiac contractility in diabetic cardiomyopathy by increased phospholamban phosphorylation leading to increased SERCA2a levels, thus improving the calcium sequestration of the sarcomeric reticulum. Increased phospholamban phosphorylation was accompanied by increased cAMP and NO levels after kallikrein gene transfer (Table 1).
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DISCUSSION |
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Increased accumulation of cardiac glycogen in STZ-treated animals is typical in both insulin-dependent and insulin-independent models of diabetes (25). Increased storage of glycogen in the myocardium results when there is a shift in energy substrate utilization, typically from a carbohydrate metabolism to a lipid metabolism (26). This switch in energy source produces an excessive accumulation of glycogen within the myocardium, which may have accelerated glycogen synthesis or an overall impairment in glycogenolysis, or a combination of the two. Our results showed that kallikrein gene transfer markedly reduced STZ-induced glycogen accumulation by nearly 50%, as identified by both PAS staining and quantitative glycogen as-say. To study the mechanism of kallikrein in glycogen regulation, we examined the intracellular signal proteins Akt and GSK-3ß. The phosphorylated form of Akt increases GSK-3ß phosphorylation, leading to decreased GSK-3ß activity and thus decreasing the rate of glycogen synthesis (27). Our results showed that kallikrein gene delivery significantly increased both phospho-Akt and phospho-GSK-3ß levels and decreased GSK-3ß activity in the STZ-induced diabetic rat. This study demonstrates a potential role for the kallikrein-kinin system through activation of Akt and GSK-3ß in overcoming the impairment in glycogenolysis and improving the use of myocardial glycogen.
Our results show that kallikrein gene delivery promotes a significant reduction in blood glucose levels independent of insulin levels. To examine the potential mechanisms of this significant drop in blood glucose, we examined the effect of kallikrein gene transfer on the glucose transporter GLUT4 translocation. Kinin has previously been shown to increase GLUT4 translocation in cardiac and skeletal muscles as well as in adipocytes (1315). Western blot analysis confirmed that kallikrein gene delivery significantly increases GLUT4 translocation into plasma membranes in both skeletal and cardiac muscle in STZ-induced diabetic rats. Increased GLUT4 translocation after kallikrein gene delivery resulted in improved glucose utilization in response to an increased glucose load resulting from the STZ treatment.
In addition to hyperglycemia, diabetic patients also commonly suffer from dyslipidemia, which can lead to increased atherogenesis and incidence of heart disease (28). To determine if kallikrein gene delivery affects lipid metabolism, we examined serum triglyceride and cholesterol levels. STZ treatment resulted in markedly elevated serum triglyceride and cholesterol levels compared with nondiabetic control animals, but both were reduced to those of control animals after kallikrein gene delivery. It is of interest to note that use of the kinin B2 receptor antagonist icatibant abrogated the reductions in triglyceride and cholesterol levels in animals receiving kallikrein gene transfer, indicating that kinin receptors have an essential role in mediating the lipid-lowering effect in STZ-induced diabetic rats. Insulin action has a significant role in lipid biosynthesis and regulation by inhibiting VLDL production in the liver and the clearance and breakdown of LDLs in circulation (29,30). Because no change occurred in insulin levels in the diabetic rats after kallikrein gene delivery, it is possible that kallikrein/kinin is promoting an insulin-like effect, similar to GLUT4 translocation, on the management of serum lipid profiles. Currently, the mechanism of action for kallikrein in lipid production and management is unknown and must be further explored.
Along with decreased glucose and lipid levels, water and food intake was also significantly decreased in animals receiving kallikrein gene delivery. It has been previously observed that animals treated with STZ without insulin have increased food and water intake (31). The potential effect of kallikrein gene delivery on reduction of water and food consumption could be attributed to better glucose management through increased GLUT4 translocation into plasma membrane, thus resulting in reduction of blood glucose levels. These results indicate that kallikrein gene expression in the heart can reduce the impact of diabetes on the development of cardiomyopathy by reducing glycogen accumulation and hyperlipidemia through increased glucose utilization.
Diabetic cardiomyopathy is a well-characterized pathophysiological condition that develops throughout the life of the diabetic patient. The underlying dysfunction of the diabetic heart can be linked to two important proteins: phospholamban and SERCA2a. A previous report has noted that a decrease in protein content of phospho-phospholamban and SERCA2a results in a significant reduction in heart function (32). Increased phospho-phospholamban and SERCA2a levels were observed after kallikrein gene delivery, indicating a beneficial role of kallikrein in cardiac function. Kallikrein, through kinin formation, triggers activation of second messengers, such as cAMP and NO/cGMP. Increased cAMP contributes to the phosphorylation of phospholamban by binding to and activating protein kinase A. Phosphorylation of phospholamban leads to the dissociation of the phospholamban pentameric structure and results in the release of free SERCA2a, thus increasing affinity for Ca2+. Therefore, a significant influx of Ca2+ transients into cardiomyocytes, via SERCA2a, leads to an increased relaxation process, which is normally depressed in this animal model (33). The expression of human tissue kallikrein mRNA in the heart after intravenous injection of Ad.CMV-TK was detected by RT-PCR followed by Southern blot analysis, whereas human kallikrein mRNA was not detected in rats injected with Ad.Null, as published in several of our previous studies (17,26). In this study, our results showed that expression of recombinant kallikrein in the diabetic heart increases cardiac cAMP levels and can improve cardiac output and ±P/
t through the regulation of phospholamban and SERCA2a.
The present study demonstrates that adenovirus-mediated delivery of human tissue kallikrein leads to significant improvements in cardiac function, glucose utilization, and lipid metabolism in the STZ model of diabetes. Kallikrein/kinin, through second messengers cAMP and NO/cGMP, protects diabetic hearts from severe contractile dysfunction though increased phospho-phospholamban and SERCA2a levels, inhibits glycogen accumulation, and improves glucose utilization and lipid metabolism through activation of the Akt-GSK-3ß signaling pathway and increased GLUT4 translocation.
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ACKNOWLEDGMENTS |
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We thank Dr. Jo Anne Simpson for critical evaluation of histological changes in the heart.
Address correspondence and reprint requests to Lee Chao, PhD, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425-2211. E-mail: chaol{at}musc.edu
Received for publication August 27, 2004 and accepted in revised form February 2, 2005
+P/
t, maximum speed of contraction;
P/
t, maximum speed of relaxation; GSK, glycogen synthase kinase; KKS, kallikrein-kinin system; PAS, periodic acid Schiff; SERCA, sarco(endo)plasmic reticulum (Ca2+ + Mg2+)-ATPase; STZ, streptozotocin
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REFERENCES |
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