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Diazoxide increases cytosolic ATP: A new paradigm for preconditioning?
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
Material and methods: Two experimental groups (n = 12/ group)-GK, with Goto-Kakizaki rats (animal model of DM) and GK S, (GK rats treated with gliclazide 10 mg/kg/day between 12 and 16 weeks of age). At 12 and 16 weeks, insulin plasma levels were determined, using competitive enzyme-linked imunosorbent assay (ELISA) and used to calculate logarithm of the homeostasis model assessment [log(HOMA)], a measure of insulin-resistance, according to the formula log[(Glicemia (mmol/l) Insulinemia (AU/ml))/ 22.5]. At 16 weeks, 24 h urine was collected to determine levels of 8-hidroxy-2V-desoxyguanosine (8-OHdG), a measure of DNA oxidative stress, using competitive ELISA. Results: Log(HOMA) was significantly decreased in the GK S group between 12 and 16 weeks (0.07 T 0.01 versus 0.08 T 0.01; P < 0.05), so that at 16 weeks it was significantly lower than that of the GK group (0.08 T 0.01 versus 0.09 T 0.02; P < 0.05). Regarding 8-OHdG urine levels, there was a trend towards a lower value in the GK S group (155 T 42 versus 183 T 69 ng/24 h; P < 0.05)—Fig. 1. Conclusion: In diabetic animals, chronic therapy with gliclazide decreases of insulin-resistance and tends to lower nuclear oxidative stress level. doi:10.1016/j.yjmcc.2006.03.049
035. Oxidative capacity recovers in the unloaded heart with clenbuterol Kameljit K. Kalsi, Gopal K.R. Soppa, Cesare M.N. Terracciano, Ryszard T. Smolenski, Magdi H. Yacoub. Imperial College London, Heart Science Centre, Harefield, Middlesex, UK Long-term mechanical unloading of the heart by LV assist device (LVAD) causes left ventricular (LV) atrophy in patients with heart failure. It has been proposed that LV atrophy may be prevented by administration of the h2-agonist clenbuterol. Chronic administration of clenbuterol induces myocardial hypertrophy and improves calcium handling. However, its actions on the metabolism of the unloaded myocardium are unclear. We hypothesize that clenbuterol induces functional improvement by increasing oxidative metabolism. Hearts from Lewis rats were mechanically unloaded using heterotopic abdominal transplantation. Osmotic mini-pumps, inserted at the time of transplantation, administered either saline, control group (n = 4) or clenbuterol (CLEN) (2 mg/kg) (n = 5). After 1 week, the transplanted hearts were excised and cardiomyocytes isolated. Cells were incubated in Krebs buffer containing 11 mM 1 –13C glucose for 2 h. Glucose oxidation was determined by using liquid chromatography/mass spectrometry glutamate to alanine isotopomer ratios. Non-oxidative metabolism of glucose was measured by the release of 13C-enriched lactate in the media. Glutamate/alanine in control cardiomyocytes was 51 T 2% but decreased in unloaded cells 41 T 4% (P < 0.05). Glucose oxidation increased with CLEN in both recipient and unloaded cells to (62 T 3% and 64 T 9% respectively). Lactate enrichment increased in control unloaded cardiomyocytes to
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62 T 6% compared to recipient cells 48 T 1%. However, with CLEN lactate, levels were similar in recipient and unloaded cardiomyocytes (59 T 1% and 57 T 1%, respectively). In conclusion, clenbuterol improves glucose oxidation in the unloaded myocardium and this may be useful during LVAD treatment in patients with heart failure. doi:10.1016/j.yjmcc.2006.03.050
036. Diazoxide increases cytosolic ATP: A new paradigm for preconditioning? Sophie Pelloux a, Brigitte Chhin a, Carlos Ojeda b, Be Wieringa c, Michel Ovize a, Yves Tourneur a. a Inserm E0226, France. b Inserm Erit-M 107, France. c NCMLS, The Netherlands We developed a cardiac cell line model derived from the HL-1 to study the induction of pharmacological preconditioning by diazoxide (100 AM) against simulated ischemia – reperfusion. We reported earlier that HL-1 cells possess electrophysiological properties of cardiac myocytes, express cardiac specific proteins, and that diazoxide was able to attenuate HL-1 cell death following a prolonged hypoxia and reoxygenation. In the present study, cells were transfected to transiently express the firefly luciferase with a modified pCDNA3 plasmid. In the presence of luciferin (200 AM), cytoplasmic ATP could be monitored by photon counting. Diazoxide did not change the global ATP concentration measured by biochemical techniques (2.65 + 0.12 nmol/mg prot. vs. control 2.71 + 0.07, N = 8), which indicates that ATP is not homogeneous and that luciferase is sensitive to local concentration modifications. Luminescence rapidly responded to changes in oxidative phosphorylation or glycolysis. Application of diazoxide (100 AM) produced a rapid and prolonged increase in ATP. This increased ATP production was recorded even in the presence of azide (2 mM), a blocker of the electron transport chain, and was prevented by glybenclamide (10 AM). These data suggest that diazoxide may protect HL-1 cardiomyocytes rather by activating glycolysis than via a direct effect on mitochondria. doi:10.1016/j.yjmcc.2006.03.051
037. Cardiac performance is reduced following high-fat diet Ashwin Akki, Anne-Marie L. Seymour. Department Biological Sciences, University of Hull, UK Cardiac hypertrophy results in cellular remodelling of the heart and, in particular, in alterations in metabolism, which may underlie the development of cardiac dysfunction. One key characteristic of this remodelling is a marked reduction in fatty acid oxidation. We have investigated whether a mismatch between fatty acid supply and utilization occurs in cardiac hypertrophy that may predispose the heart to an
Material and methods: Two experimental groups (n = 12/ group)-GK, with Goto-Kakizaki rats (animal model of DM) and GK S, (GK rats treated with gliclazide 10 mg/kg/day between 12 and 16 weeks of age). At 12 and 16 weeks, insulin plasma levels were determined, using competitive enzyme-linked imunosorbent assay (ELISA) and used to calculate logarithm of the homeostasis model assessment [log(HOMA)], a measure of insulin-resistance, according to the formula log[(Glicemia (mmol/l) Insulinemia (AU/ml))/ 22.5]. At 16 weeks, 24 h urine was collected to determine levels of 8-hidroxy-2V-desoxyguanosine (8-OHdG), a measure of DNA oxidative stress, using competitive ELISA. Results: Log(HOMA) was significantly decreased in the GK S group between 12 and 16 weeks (0.07 T 0.01 versus 0.08 T 0.01; P < 0.05), so that at 16 weeks it was significantly lower than that of the GK group (0.08 T 0.01 versus 0.09 T 0.02; P < 0.05). Regarding 8-OHdG urine levels, there was a trend towards a lower value in the GK S group (155 T 42 versus 183 T 69 ng/24 h; P < 0.05)—Fig. 1. Conclusion: In diabetic animals, chronic therapy with gliclazide decreases of insulin-resistance and tends to lower nuclear oxidative stress level. doi:10.1016/j.yjmcc.2006.03.049
035. Oxidative capacity recovers in the unloaded heart with clenbuterol Kameljit K. Kalsi, Gopal K.R. Soppa, Cesare M.N. Terracciano, Ryszard T. Smolenski, Magdi H. Yacoub. Imperial College London, Heart Science Centre, Harefield, Middlesex, UK Long-term mechanical unloading of the heart by LV assist device (LVAD) causes left ventricular (LV) atrophy in patients with heart failure. It has been proposed that LV atrophy may be prevented by administration of the h2-agonist clenbuterol. Chronic administration of clenbuterol induces myocardial hypertrophy and improves calcium handling. However, its actions on the metabolism of the unloaded myocardium are unclear. We hypothesize that clenbuterol induces functional improvement by increasing oxidative metabolism. Hearts from Lewis rats were mechanically unloaded using heterotopic abdominal transplantation. Osmotic mini-pumps, inserted at the time of transplantation, administered either saline, control group (n = 4) or clenbuterol (CLEN) (2 mg/kg) (n = 5). After 1 week, the transplanted hearts were excised and cardiomyocytes isolated. Cells were incubated in Krebs buffer containing 11 mM 1 –13C glucose for 2 h. Glucose oxidation was determined by using liquid chromatography/mass spectrometry glutamate to alanine isotopomer ratios. Non-oxidative metabolism of glucose was measured by the release of 13C-enriched lactate in the media. Glutamate/alanine in control cardiomyocytes was 51 T 2% but decreased in unloaded cells 41 T 4% (P < 0.05). Glucose oxidation increased with CLEN in both recipient and unloaded cells to (62 T 3% and 64 T 9% respectively). Lactate enrichment increased in control unloaded cardiomyocytes to
933
62 T 6% compared to recipient cells 48 T 1%. However, with CLEN lactate, levels were similar in recipient and unloaded cardiomyocytes (59 T 1% and 57 T 1%, respectively). In conclusion, clenbuterol improves glucose oxidation in the unloaded myocardium and this may be useful during LVAD treatment in patients with heart failure. doi:10.1016/j.yjmcc.2006.03.050
036. Diazoxide increases cytosolic ATP: A new paradigm for preconditioning? Sophie Pelloux a, Brigitte Chhin a, Carlos Ojeda b, Be Wieringa c, Michel Ovize a, Yves Tourneur a. a Inserm E0226, France. b Inserm Erit-M 107, France. c NCMLS, The Netherlands We developed a cardiac cell line model derived from the HL-1 to study the induction of pharmacological preconditioning by diazoxide (100 AM) against simulated ischemia – reperfusion. We reported earlier that HL-1 cells possess electrophysiological properties of cardiac myocytes, express cardiac specific proteins, and that diazoxide was able to attenuate HL-1 cell death following a prolonged hypoxia and reoxygenation. In the present study, cells were transfected to transiently express the firefly luciferase with a modified pCDNA3 plasmid. In the presence of luciferin (200 AM), cytoplasmic ATP could be monitored by photon counting. Diazoxide did not change the global ATP concentration measured by biochemical techniques (2.65 + 0.12 nmol/mg prot. vs. control 2.71 + 0.07, N = 8), which indicates that ATP is not homogeneous and that luciferase is sensitive to local concentration modifications. Luminescence rapidly responded to changes in oxidative phosphorylation or glycolysis. Application of diazoxide (100 AM) produced a rapid and prolonged increase in ATP. This increased ATP production was recorded even in the presence of azide (2 mM), a blocker of the electron transport chain, and was prevented by glybenclamide (10 AM). These data suggest that diazoxide may protect HL-1 cardiomyocytes rather by activating glycolysis than via a direct effect on mitochondria. doi:10.1016/j.yjmcc.2006.03.051
037. Cardiac performance is reduced following high-fat diet Ashwin Akki, Anne-Marie L. Seymour. Department Biological Sciences, University of Hull, UK Cardiac hypertrophy results in cellular remodelling of the heart and, in particular, in alterations in metabolism, which may underlie the development of cardiac dysfunction. One key characteristic of this remodelling is a marked reduction in fatty acid oxidation. We have investigated whether a mismatch between fatty acid supply and utilization occurs in cardiac hypertrophy that may predispose the heart to an