- Email: [email protected]
53 Molecular regulation of cholesterol metabolism in cultured human hepatocytes
Abstracts X I X t h National Congress, Italian SocieO, f o r the Study o f A t h e m s c l e r o s i s subjects 52.81 mg/dl) and Blood pressure values (SBP mean in total subjects 131.9 mmHg; DBP mean in total subjects 80.3). Results suggested a statistical significant correlation (p < 0.05) between HDL-C and EEPA (p 0.040) in all aged no smoker males but not in famele; moreover this significance is more important in no-smoker male without CVD (p 0.035), wherease we have not found a significant difference of statistical dates related to age. Considering the other variable analysed (BP) our reserch suggested a statistical significant correlation between SBR DBP and EEPA (respectively p 0.001 ; p 0.019) in young famale but not in male and in elderly people. This correlation was more evident in no smoker young female but only between DBP and EEPA (p 0.12). The aim of our study is to underline and confirm the important role of EEPA on HDL-C levels and BP values to prevent CVD.
['~'] THE INCREASE OF DIETARY ANTIOXIDANT INTAKE IMPROVES ENDOTHELIAL FUNCTION IN HEALTHY SUBJECTS L. Franzini 1 , S. Valtuefia1 , D. Ardig61 , D. Del Rio 2, N. Pellegrini2, R Brighenti2, I. Zavaroni 1 . 1Dept. of Inte~Tml Medicine and Biomedical
Sciences. Unic,ersitv of Pa~vza. Pa~vza. Italy," 2Dept. of Public Health. [bfic,ersiO, of Pa~za. Pa~m. Italy E-mail: laura, franzini@ libero.it Background: Oxidative stress has been advocated as a major cause for endothelial dysfunction, and low levels of plasma antioxidants associated with impaired endothelial-dependent vasodilatation (EDV). Although intravenous administration of antioxidants improves EDV in acute, a chronic oral supplementation does not seem to be effective and the impact of antioxidaaat intake with the diet on endothelial function has not been clarified. Objective: Aim of the study was to investigate whether a diet rich in antioxidants can improve endothelial function, assessed by flow mediated dilation (FMD) test. Methods: In a crossover design, 24 apparently healthy subjects consumed in a randomized order 15-days high and 15-days low antioxidant diets, with a two weeks wash out between the two. Both diets L and H were designed to be comparable regarding fruit, vegetable, alcohol, fibre and macronutrient intake, and to be very different regarding the TAC (10 mmol Trolox equivalent/day difference approx.). Before and after each diet, an FMD test was performed and endothelial-independent vasodilatation (EIV) measured after sublingual nitroglycerin administration. Results: Whereas EIV did not change under any condition, FMD significantly improved after the high antioxidant diet compared to baseline (p <0.001) and trended to decrease in presence of low antioxidant intake (although not significant, p 0.07). Conclusion: These results indicate that a short-term diet rich in antioxidants significantly improves endothelial function in apparently healthy subjects, and support the view that the choice of food with higher antioxidant content can provide an effective strategy to prevent cardiovascular disease.
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MOLECULAR REGULATION OF CHOLESTEROL METABOLISM IN CULTURED HUMAN HEPATOCYTES
C. Gabbi, M. Bertolotti, C. Anzivino, M. Ricchi, E. Tagliafico, E. Tenedini, L. Caxulli, E Carubbi, R Loria, N. Caxulli. Unic,ersit~'l di Modena e Reggio
Emilia, Modena, Italy E-mail: [email protected] Cholesterol homeostasis in the liver cell is maintained by a balance of input and output pathways. The role of nuclear receptors has recently been underlined but the integration of the different metabolic pathways is largely unknown. AIM of the present study is to analyze the expression of a number of genes involved in cholesterol and bile acid metabolism in cultured human hepatocytes. Methods: HepG2 cells were incubated with 100 ~tmol concentrations of bile acids with different physical-chemical properties (DCA, CDCA, UDCA) for 24hr; mRNA levels of cholesterol 7o,-hydroxylase (CYP7A1), 27-hydroxylase (CYP27), LDL-receptor, HMG-CoA reductase and a number of nuclear receptors involved in sterol metabolism were assayed by quantitative real-time RT-PCR. Results: The expression of CYP7A1 was significantly reduced, and that of SHP was increased, after incubation with the hydrophobic bile acids DCA and CDCA, but not with UDCA (ANOVA); expression of SREBP-2, HMG-CoA reductase and LDL-receptor were also significantly increased by hydrophobic bile acids. No significant effects were detected on the expression of FXR, HNF-4, CPF/LHR-1, LXR,:*,, CYP27. Conclusions: Hydrophobic, but not hydrophilic bile acids suppress CYP7A1 expression, possibly via increased expression of SHE and on the other hand enhance the expression of SREBP-2 and of genes involved in both cholesterol synthesis and LDL uptake, mimicking a condition of cholesterol depletion. Knowledge of the relationships linking bile acid and cholesterol metabolism might provide useful information for the management of cholesterol accumulation conditions. Supported by COFINPRIN grant 2004 067491.
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LEVELS OF ADIPONECTIN CORRELATE WITH THE SEVERITY OF NONALCOHOLIC STEATOHEPATITIS AFTER AN ORAL FAT LOAD
R. Gambino, G. Musso, M. Durazzo, G. Biroli, M. Carello, E. Fag/l, G. Pacini, E De Michieli, L. Rabbione, A. Premoli, G. Pagano, M. Cassader.
Dipartimento di Medic.ina Inte~Tm, Unic,ersit~'l di Torino, Italy E-maih [email protected] Background: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease, encountered in individuals without significant alcohol consumption; it is part of a spectrum of liver damage, ranging from simple steatosis to advanced fibrosis and cirrhosis. Adiponectin has been related to hepatic steatosis and inflammation, but not to fibrosis in NASH. Methods: Circulating levels of adiponectin, TNF-% leptin, resistin and the postprandial lipid and adiponectin responses to an oral fat load were assessed in 25 non-obese non-diabetic patients with biopsy-proven NASH and correlated with metabolic indices and liver histology. Results: Circulating adiponectin was lower in NASH compared to controls and correlated negatively with liver steatosis, necroinftammation and fibrosis. The magnitude of postprandial lipemia was significantly higher in NASH than in controls and was related to fasting adiponectin. Controls showed a significant increase in serum adiponectin in response to the fat load, while patients with NASH showed a slight decrease. Postprandial FFA response correlated inversely with adiponectin response in both groups and independently predicted the severity of liver steatosis in NASH. Conclusion: hypoadiponectinemia is present before overt diabetes and obesity appears and correlates with the severity of the metabolic syndrome and of liver histology in NASH. The mechanisms underlying these differences are unknown, but the type of dietary fat seems to play a role.
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INCREASED ACTIVATION OF NF-KB AND ERKI/2 AFTER PERMANENT FOCAL ISCHEMIA IS ABOLISHED BY SIMVASTATI N PRE-TREATMENT
P. Gelosa, M. Cimino, C. Banff, M. Brioschi, E. Nobili, A. GiaJnella, E. Tremoli, L. Sironi. Dept. Pha~zacol. Sciences. Unic,ersiO, of Mihmo; Inst.
of Pha~mcol. Pha~zacognosv. [bfic,ersiO, of [b'bino. Italy E-maih [email protected] Since statins exert an anti-inflammatory activity we sought to investigate the effect of simvastatin treatment on the expression of IL-lbeta, MCP-1, NF-kB activity and on signalling pathways related with NF-kB activation in a rat model of cerebral ischemia (MCAO). Expression of IL-lb and MCP-1 was enhanced by pMCAO and this effect was inhibited by administration of simvastatin before ischemia. We then investigated the ability of NF-kB p65 subunit to bind DNA at different time after pMCAO. The cerebral cortex ipsilateral to the occlusion displayed an increase in binding activity which reached its peak 16 hours after the ischemic insult. In order to determine whether simvastatin could interfere with pMCAO-induced activation of NF-kB, animals were treated for 3 days with 20 mg/kg of statin before permanent occlusion of the artery or 2 hours after pMCAO. Pre-treatment with simvastatin abolished the activation of NF-kB observed in vehicle-treated animals. The effect of simvastatin was specific for NF-kB since other transcription factors such as Jun, Fos and NF-YA were not affected by the treatments. We have also evaluated the modulation of different signal transduction pathways but only the expression of ERK1/2 was enhanced by ischemia and this activation was prevented by simvastatin administration. U0126, a specific MEK inhibitor, reduced the ischemic damage and reduced the activation of inflammatory markers. These results provide evidence for the role of simvastatin in the protection of ischemic brain damage and suggests that this effect is mediated by inhibition of ERK1/2 pathway.
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AEROBIC EXERCISE TRAINING REDUCES AORTIC STIFFNESS IN UNTREATED PATIENTS WITH MILD ESSENTIAL HYPERTENSION
F. Gemelli, L. Pasqualini, G. Savaxese, M.R. Mannaxino, K. Mboumi, G. Pucci, E Coscia, G. Schillaci. Medicina Inte~Tm. Angiologia e Malattie da
Arteriosclerosi. Unic,ersitgt di Perugia. Italy E-maih [email protected] Arterial stiffness exerts a number of adverse effects on cardiovascular function and disease risk, and is associated with a greater rate of mortality in patients with essential hypertension. Available evidence indicates that aerobic exercise may be effective in preventing and treating arterial stiffening in healthy adults. The present study characterized large-artery properties in hypertensive patients and determined the efficacy of aerobic exercise training in modifying these properties. In 16 patients (11 men, mean age 41±7 years) with grade 1 hypertension, aortic pulse wave velocity (PWV) was determined noninvasively (SphygmoC or) before and after 8 weeks of moderate-intensity aerobic exercise.
['~'] THE INCREASE OF DIETARY ANTIOXIDANT INTAKE IMPROVES ENDOTHELIAL FUNCTION IN HEALTHY SUBJECTS L. Franzini 1 , S. Valtuefia1 , D. Ardig61 , D. Del Rio 2, N. Pellegrini2, R Brighenti2, I. Zavaroni 1 . 1Dept. of Inte~Tml Medicine and Biomedical
Sciences. Unic,ersitv of Pa~vza. Pa~vza. Italy," 2Dept. of Public Health. [bfic,ersiO, of Pa~za. Pa~m. Italy E-mail: laura, franzini@ libero.it Background: Oxidative stress has been advocated as a major cause for endothelial dysfunction, and low levels of plasma antioxidants associated with impaired endothelial-dependent vasodilatation (EDV). Although intravenous administration of antioxidants improves EDV in acute, a chronic oral supplementation does not seem to be effective and the impact of antioxidaaat intake with the diet on endothelial function has not been clarified. Objective: Aim of the study was to investigate whether a diet rich in antioxidants can improve endothelial function, assessed by flow mediated dilation (FMD) test. Methods: In a crossover design, 24 apparently healthy subjects consumed in a randomized order 15-days high and 15-days low antioxidant diets, with a two weeks wash out between the two. Both diets L and H were designed to be comparable regarding fruit, vegetable, alcohol, fibre and macronutrient intake, and to be very different regarding the TAC (10 mmol Trolox equivalent/day difference approx.). Before and after each diet, an FMD test was performed and endothelial-independent vasodilatation (EIV) measured after sublingual nitroglycerin administration. Results: Whereas EIV did not change under any condition, FMD significantly improved after the high antioxidant diet compared to baseline (p <0.001) and trended to decrease in presence of low antioxidant intake (although not significant, p 0.07). Conclusion: These results indicate that a short-term diet rich in antioxidants significantly improves endothelial function in apparently healthy subjects, and support the view that the choice of food with higher antioxidant content can provide an effective strategy to prevent cardiovascular disease.
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MOLECULAR REGULATION OF CHOLESTEROL METABOLISM IN CULTURED HUMAN HEPATOCYTES
C. Gabbi, M. Bertolotti, C. Anzivino, M. Ricchi, E. Tagliafico, E. Tenedini, L. Caxulli, E Carubbi, R Loria, N. Caxulli. Unic,ersit~'l di Modena e Reggio
Emilia, Modena, Italy E-mail: [email protected] Cholesterol homeostasis in the liver cell is maintained by a balance of input and output pathways. The role of nuclear receptors has recently been underlined but the integration of the different metabolic pathways is largely unknown. AIM of the present study is to analyze the expression of a number of genes involved in cholesterol and bile acid metabolism in cultured human hepatocytes. Methods: HepG2 cells were incubated with 100 ~tmol concentrations of bile acids with different physical-chemical properties (DCA, CDCA, UDCA) for 24hr; mRNA levels of cholesterol 7o,-hydroxylase (CYP7A1), 27-hydroxylase (CYP27), LDL-receptor, HMG-CoA reductase and a number of nuclear receptors involved in sterol metabolism were assayed by quantitative real-time RT-PCR. Results: The expression of CYP7A1 was significantly reduced, and that of SHP was increased, after incubation with the hydrophobic bile acids DCA and CDCA, but not with UDCA (ANOVA); expression of SREBP-2, HMG-CoA reductase and LDL-receptor were also significantly increased by hydrophobic bile acids. No significant effects were detected on the expression of FXR, HNF-4, CPF/LHR-1, LXR,:*,, CYP27. Conclusions: Hydrophobic, but not hydrophilic bile acids suppress CYP7A1 expression, possibly via increased expression of SHE and on the other hand enhance the expression of SREBP-2 and of genes involved in both cholesterol synthesis and LDL uptake, mimicking a condition of cholesterol depletion. Knowledge of the relationships linking bile acid and cholesterol metabolism might provide useful information for the management of cholesterol accumulation conditions. Supported by COFINPRIN grant 2004 067491.
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LEVELS OF ADIPONECTIN CORRELATE WITH THE SEVERITY OF NONALCOHOLIC STEATOHEPATITIS AFTER AN ORAL FAT LOAD
R. Gambino, G. Musso, M. Durazzo, G. Biroli, M. Carello, E. Fag/l, G. Pacini, E De Michieli, L. Rabbione, A. Premoli, G. Pagano, M. Cassader.
Dipartimento di Medic.ina Inte~Tm, Unic,ersit~'l di Torino, Italy E-maih [email protected] Background: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease, encountered in individuals without significant alcohol consumption; it is part of a spectrum of liver damage, ranging from simple steatosis to advanced fibrosis and cirrhosis. Adiponectin has been related to hepatic steatosis and inflammation, but not to fibrosis in NASH. Methods: Circulating levels of adiponectin, TNF-% leptin, resistin and the postprandial lipid and adiponectin responses to an oral fat load were assessed in 25 non-obese non-diabetic patients with biopsy-proven NASH and correlated with metabolic indices and liver histology. Results: Circulating adiponectin was lower in NASH compared to controls and correlated negatively with liver steatosis, necroinftammation and fibrosis. The magnitude of postprandial lipemia was significantly higher in NASH than in controls and was related to fasting adiponectin. Controls showed a significant increase in serum adiponectin in response to the fat load, while patients with NASH showed a slight decrease. Postprandial FFA response correlated inversely with adiponectin response in both groups and independently predicted the severity of liver steatosis in NASH. Conclusion: hypoadiponectinemia is present before overt diabetes and obesity appears and correlates with the severity of the metabolic syndrome and of liver histology in NASH. The mechanisms underlying these differences are unknown, but the type of dietary fat seems to play a role.
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INCREASED ACTIVATION OF NF-KB AND ERKI/2 AFTER PERMANENT FOCAL ISCHEMIA IS ABOLISHED BY SIMVASTATI N PRE-TREATMENT
P. Gelosa, M. Cimino, C. Banff, M. Brioschi, E. Nobili, A. GiaJnella, E. Tremoli, L. Sironi. Dept. Pha~zacol. Sciences. Unic,ersiO, of Mihmo; Inst.
of Pha~mcol. Pha~zacognosv. [bfic,ersiO, of [b'bino. Italy E-maih [email protected] Since statins exert an anti-inflammatory activity we sought to investigate the effect of simvastatin treatment on the expression of IL-lbeta, MCP-1, NF-kB activity and on signalling pathways related with NF-kB activation in a rat model of cerebral ischemia (MCAO). Expression of IL-lb and MCP-1 was enhanced by pMCAO and this effect was inhibited by administration of simvastatin before ischemia. We then investigated the ability of NF-kB p65 subunit to bind DNA at different time after pMCAO. The cerebral cortex ipsilateral to the occlusion displayed an increase in binding activity which reached its peak 16 hours after the ischemic insult. In order to determine whether simvastatin could interfere with pMCAO-induced activation of NF-kB, animals were treated for 3 days with 20 mg/kg of statin before permanent occlusion of the artery or 2 hours after pMCAO. Pre-treatment with simvastatin abolished the activation of NF-kB observed in vehicle-treated animals. The effect of simvastatin was specific for NF-kB since other transcription factors such as Jun, Fos and NF-YA were not affected by the treatments. We have also evaluated the modulation of different signal transduction pathways but only the expression of ERK1/2 was enhanced by ischemia and this activation was prevented by simvastatin administration. U0126, a specific MEK inhibitor, reduced the ischemic damage and reduced the activation of inflammatory markers. These results provide evidence for the role of simvastatin in the protection of ischemic brain damage and suggests that this effect is mediated by inhibition of ERK1/2 pathway.
[•
AEROBIC EXERCISE TRAINING REDUCES AORTIC STIFFNESS IN UNTREATED PATIENTS WITH MILD ESSENTIAL HYPERTENSION
F. Gemelli, L. Pasqualini, G. Savaxese, M.R. Mannaxino, K. Mboumi, G. Pucci, E Coscia, G. Schillaci. Medicina Inte~Tm. Angiologia e Malattie da
Arteriosclerosi. Unic,ersitgt di Perugia. Italy E-maih [email protected] Arterial stiffness exerts a number of adverse effects on cardiovascular function and disease risk, and is associated with a greater rate of mortality in patients with essential hypertension. Available evidence indicates that aerobic exercise may be effective in preventing and treating arterial stiffening in healthy adults. The present study characterized large-artery properties in hypertensive patients and determined the efficacy of aerobic exercise training in modifying these properties. In 16 patients (11 men, mean age 41±7 years) with grade 1 hypertension, aortic pulse wave velocity (PWV) was determined noninvasively (SphygmoC or) before and after 8 weeks of moderate-intensity aerobic exercise.