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208 THE ANTIATHEROGENIC PROPERTIES OF HDL AFTER BARIATRIC SURGERY
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Atherosclerosis Supplements 12, no. 1 (2011) 13–184
Results: Incubation of THP-1 cells with cLDL increased cellular free cholesterol and cholesteryl ester, ABCA1 and ABCG1 mRNA and protein (1.5 to 3 fold, p < 0.05) and [3 H] cholesterol efflux to apoA-I twofold (p < 0.05). The CEC of serum, HDL, and apoB depleted serum did not differ between patients and controls. The CEC of cHDL, however, was reduced to 50±10% of control (p < 0.05), whereas the specific cell association of [125 I]cHDL was increased. Conclusion: CRF does not appear to alter the CEC of HDL or serum. The increase of ABCA1/ABCG1 expression and cholesterol efflux to apoA-I in macrophages exposed to cLDL may represent a compensatory mechanism to reduce cholesterol accumulation. 206 SKIN AUTOFLUORESCENCE IS INVERSELY RELATED TO HDL ANTI-OXIDATIVE CAPACITY IN TYPE 2 DIABETES MELLITUS D.J. Mulder1 , J.F. De Boer2 , R. Graaff3 , R. De Vries4 , W. Annema2,5 , J.D. Lefrandt1 , A.J. Smit1 , U.J.F. Tietge2,5 , R.P.F. Dullaart4 . 1 Department of Internal Medicine, 2 Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, 3 Department of Biomedical Engineering, 4 Department of Endocrinology, University Medical Center Groningen, Groningen, 5 TI Food and Nutrition, Wageningen, The Netherlands Objectives: High density lipoprotein (HDL) particles protect lipoproteins from oxidative modification. An impaired anti-oxidative functionality of HDL in type 2 diabetes mellitus (T2DM) might contribute to enhanced formation of oxidative stress products, such as Advanced Glycation Endproducts (AGEs). We tested whether in T2DM the HDL anti-oxidative capacity is related to the accumulation of AGEs in the skin. Methods: Skin autofluorescence (AF), a non-invasive marker of AGEs, and HDL anti-oxidative capacity, i.e. the ability of HDL to protect against LDL oxidation in vitro, were assessed in 67 non-smoking T2DM patients without complications (median age: 60 (53−65), 60% males, 6.5 (5.2−8.5) years of diabetes duration). Results: HDL anti-oxidative capacity correlated inversely with plasma glucose, HbA1c , and triglycerides (P < 0.05 to <0.001) as well as with skin AF in univariate analysis (r = −0.306, P < 0.02). Multiple linear regression showed that skin AF remained inversely related to HDL anti-oxidative capacity (partial r = −0.324, P = 0.011) taking account of age, sex, plasma glucose, HbA1c, total cholesterol/HDL cholesterol ratio and triglycerides. Conclusions: Skin AF is inversely related to the anti-oxidative ability of HDL. This suggests that a decreased anti-oxidative functionality of HDL may contribute to the tissue accumulation of AGEs in tissues and thereby also to long-term diabetic complications. 207 A HIGH-FAT CHOLESTEROL-CONTAINING DIET PROMOTES REVERSE CHOLESTEROL TRANSPORT FROM MACROPHAGES TO FECES IN VIVO THAT DEPENDS ON ABCG5/G8 1,2 J.C. Escola-Gil ` , G. Llaverias1,2 , J. Julve1,2 , D. Santos2 , J. Mendez´ Gonzalez ´ 1 , F. Blanco-Vaca1,2 . 1 IIB Sant Pau, 2 CIBER de Diabetes y ´ Enfermedades Metabolicas Asociadas. CIBERDEM, Barcelona, Spain
Background: A high saturated fatty acid- and cholesterol-containing (HFHC) diet is considered an important contributor to atherosclerosis. However, HFHC diet increases plasma cholesterol and HDL cholesterol levels. In the present study, we determined the effects of this atherogenic diet on reverse cholesterol transport (RCT) from macrophages to feces in vivo. Methods: [3 H]cholesterol-labeled mouse macrophages were injected intraperitoneally into mice fed a low-fat low-cholesterol (LFLC) diet, a HFHC diet or the HFHC diet without cholesterol added (HFLC) for 8 weeks, and radioactivity was determined in plasma, liver and feces. Results: As expected, HFHC diet caused a significantly increase in plasma cholesterol, HDLc and liver cholesterol. HFHC diet, but not LFLC or HFLC diets, enhanced macrophage-derived [3 H]cholesterol flux to feces of C57BL/6 mice. These changes were sex-independent and not associated with obesity or insulin resistance. Unlike LFLC or HFLC diets, HFHC diet increased liver ABCG1 expression and increased [3 H]cholesterol efflux to plasma by 40%. HFHC diet also upregulated liver ABCG5/G8, which was associated with a 3-fold increase in HDL-derived [3 H]tracer fecal excretion, while plasma HDL fractional catabolic rate remained unchanged. Interestingly, HFHC diet had no effect on fecal macrophage-derived [3 H]cholesterol excretion in ABCG5/G8deficient mice. Conclusion: Despite its deleterious effect on atherosclerosis, HFHC diet promotes macrophage-to-feces RCT. Our data provide direct in vivo evidence of the crucial role of dietary cholesterol and liver ABCG5/G8 in HFHC dietmediated induction of macrophage-specific RCT.
Poster presentations
208 THE ANTIATHEROGENIC PROPERTIES OF HDL AFTER BARIATRIC SURGERY O. Amin Hussein1,2 , I. Shams3,4 , K. Abu Jabal3 , I. Waksman5 , I. Dashkovsky5 , S. Szvalb6 , N. Volkova7 , B. Fuhrman7 , M. Aviram7 . 1 Internal Medicine a, Ziv Medical Center, Safed, 2 Faculty of Medicine, Technion, Haifa, 3 Internal Medicine A, 4 Laboratory for Atherosclerosis Research, 5 Surgery Department, 6 Pathology Department, Ziv Medical Center, Safed, 7 Lipid Research Laboratory, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Background: High-density lipoprotein cholesterol (HDL-C) may not always be atheroprotective and in some conditions, it paradoxically enhances the process of atherosclerosis. The cardiovascular protective effects of HDL have been mainly attributed to its role in reverse cholesterol transport and to many other protective properties towards atherosclerosis as anti-oxidative effect. Aim of the study: To investigate the functional properties of HDL in patients after bariatric surgery. Patients and Methods: Forty candidate patients for bariatric surgery were enrolled. Blood was drawn before, 3 weeks (n = 19) or 6 months (n = 21) after bariatric surgery. Serum PON arylesterase activity was measured by using phenylacetate as substrate. Serum lipid peroxides (PD) were measured spectrophotometrically at 340 l. PD was measured at baseline and after oxidation with 2,2 -Azobis(2-methylpropionamidine) dihydrochloride (AAPH). [3 H]-Cholesterol efflux was measured by incubating J744 macrophages with serum. Radioactivity was measured by b counter in the cell lysate and the medium. Statistical methods: Student’s t-test for paired samples was used. p < 0.05 was cosidered significant. Results: Serum mediated cholesterol efflux did not change after 3 weeks, but increased significantly by 14% after 6 months. Both basal and AAPH-oxidized peroxides were significantly decreased 3 weeks and 6 months after surgery. PON lactonase and arylesterase activities decreased and liver function tests normalized already within 3 weeks. Conclusion: Bariatric surgery reduced serum peroxides as early as 3 weeks while the increase in serum-mediated cholesterol efflux was observed after 6 months. 209 GLYCOXIDATION IMPAIRS THE HDL-MEDIATED OXYSTEROLS EFFLUX AND INDUCES STEROL ACCUMULATION IN MACROPHAGES R. Tallada Iborra1 , A. Machado-Lima1 , G. Castilho1 , V.S. Nunes1 , E.R. Nakandakare1 , D.S. Abdalla2 , M. Passarelli1 . 1 Lipids Laboratory (LIM-10), ˜ Paulo. Sao ˜ Paulo, Brazil., Faculty of Medical Sciences, University of Sao 2 Department of Clinical and Toxicology Analysis, Faculty of Pharmaceutical ˜ Paulo, Sao ˜ Paulo, Brazil Sciences, University of Sao Introduction: Advanced glycation reduces the expression of ABCA-1 and ABCG-1, inducing lipid accumulation in macrophages (macs). We analyzed the content of total sterols and oxysterol subfractions in macs submitted to glycoxidation. Methods: J774 macs were incubated with oxidized LDL (50 mg/mL of DMEM) for 43 h and then treated with 0.5 mM glycolaldehyde (GAD-macs) or DMEM alone (C-macs) for 5h, in the absence or presence of the LXR-agonist, T0901317 (1mM). Cells were carefully washed and incubated with 50 mg/mL of HDL (5 h) in the absence or presence of T0901317. Oxysterols and total cholesterol were determined by gas chromatography/mass spectrometry in cell and culture media lipid extracts by utilizing 5a cholestane as internal standard. Results: In incubations with HDL+T0901317 the total content of intracellular sterols (cholesterol + oxysterols) and of cholesterol-5a,6a-epoxide and cholesterol-5b,6b-epoxide was, respectively, 1.4, 2.0 and 2.2 times higher in GAD-macs as compared to C-macs. In culture media, total sterols content was similar between C and GAD-macs and increased similarly after addition of HDL or HDL+T0901317. A 20% and 23% reduction was observed in the amount of respectively 7-ketocholesterol and 7b-hydroxycholesterol in culture media from GAD-macs incubated with HDL alone. The addition of HDL+T0901317 increased these oxysterols similarly to C-macs. Conclusion: Glycoxidation reduces the exportation of 7-ketocholesterol and 7b-hydroxycholesterol to HDL and induces macrophage sterol accumulation. This may contribute to atherogenesis in DM. Funding: FAPESP (Brazil)
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
Results: Incubation of THP-1 cells with cLDL increased cellular free cholesterol and cholesteryl ester, ABCA1 and ABCG1 mRNA and protein (1.5 to 3 fold, p < 0.05) and [3 H] cholesterol efflux to apoA-I twofold (p < 0.05). The CEC of serum, HDL, and apoB depleted serum did not differ between patients and controls. The CEC of cHDL, however, was reduced to 50±10% of control (p < 0.05), whereas the specific cell association of [125 I]cHDL was increased. Conclusion: CRF does not appear to alter the CEC of HDL or serum. The increase of ABCA1/ABCG1 expression and cholesterol efflux to apoA-I in macrophages exposed to cLDL may represent a compensatory mechanism to reduce cholesterol accumulation. 206 SKIN AUTOFLUORESCENCE IS INVERSELY RELATED TO HDL ANTI-OXIDATIVE CAPACITY IN TYPE 2 DIABETES MELLITUS D.J. Mulder1 , J.F. De Boer2 , R. Graaff3 , R. De Vries4 , W. Annema2,5 , J.D. Lefrandt1 , A.J. Smit1 , U.J.F. Tietge2,5 , R.P.F. Dullaart4 . 1 Department of Internal Medicine, 2 Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, 3 Department of Biomedical Engineering, 4 Department of Endocrinology, University Medical Center Groningen, Groningen, 5 TI Food and Nutrition, Wageningen, The Netherlands Objectives: High density lipoprotein (HDL) particles protect lipoproteins from oxidative modification. An impaired anti-oxidative functionality of HDL in type 2 diabetes mellitus (T2DM) might contribute to enhanced formation of oxidative stress products, such as Advanced Glycation Endproducts (AGEs). We tested whether in T2DM the HDL anti-oxidative capacity is related to the accumulation of AGEs in the skin. Methods: Skin autofluorescence (AF), a non-invasive marker of AGEs, and HDL anti-oxidative capacity, i.e. the ability of HDL to protect against LDL oxidation in vitro, were assessed in 67 non-smoking T2DM patients without complications (median age: 60 (53−65), 60% males, 6.5 (5.2−8.5) years of diabetes duration). Results: HDL anti-oxidative capacity correlated inversely with plasma glucose, HbA1c , and triglycerides (P < 0.05 to <0.001) as well as with skin AF in univariate analysis (r = −0.306, P < 0.02). Multiple linear regression showed that skin AF remained inversely related to HDL anti-oxidative capacity (partial r = −0.324, P = 0.011) taking account of age, sex, plasma glucose, HbA1c, total cholesterol/HDL cholesterol ratio and triglycerides. Conclusions: Skin AF is inversely related to the anti-oxidative ability of HDL. This suggests that a decreased anti-oxidative functionality of HDL may contribute to the tissue accumulation of AGEs in tissues and thereby also to long-term diabetic complications. 207 A HIGH-FAT CHOLESTEROL-CONTAINING DIET PROMOTES REVERSE CHOLESTEROL TRANSPORT FROM MACROPHAGES TO FECES IN VIVO THAT DEPENDS ON ABCG5/G8 1,2 J.C. Escola-Gil ` , G. Llaverias1,2 , J. Julve1,2 , D. Santos2 , J. Mendez´ Gonzalez ´ 1 , F. Blanco-Vaca1,2 . 1 IIB Sant Pau, 2 CIBER de Diabetes y ´ Enfermedades Metabolicas Asociadas. CIBERDEM, Barcelona, Spain
Background: A high saturated fatty acid- and cholesterol-containing (HFHC) diet is considered an important contributor to atherosclerosis. However, HFHC diet increases plasma cholesterol and HDL cholesterol levels. In the present study, we determined the effects of this atherogenic diet on reverse cholesterol transport (RCT) from macrophages to feces in vivo. Methods: [3 H]cholesterol-labeled mouse macrophages were injected intraperitoneally into mice fed a low-fat low-cholesterol (LFLC) diet, a HFHC diet or the HFHC diet without cholesterol added (HFLC) for 8 weeks, and radioactivity was determined in plasma, liver and feces. Results: As expected, HFHC diet caused a significantly increase in plasma cholesterol, HDLc and liver cholesterol. HFHC diet, but not LFLC or HFLC diets, enhanced macrophage-derived [3 H]cholesterol flux to feces of C57BL/6 mice. These changes were sex-independent and not associated with obesity or insulin resistance. Unlike LFLC or HFLC diets, HFHC diet increased liver ABCG1 expression and increased [3 H]cholesterol efflux to plasma by 40%. HFHC diet also upregulated liver ABCG5/G8, which was associated with a 3-fold increase in HDL-derived [3 H]tracer fecal excretion, while plasma HDL fractional catabolic rate remained unchanged. Interestingly, HFHC diet had no effect on fecal macrophage-derived [3 H]cholesterol excretion in ABCG5/G8deficient mice. Conclusion: Despite its deleterious effect on atherosclerosis, HFHC diet promotes macrophage-to-feces RCT. Our data provide direct in vivo evidence of the crucial role of dietary cholesterol and liver ABCG5/G8 in HFHC dietmediated induction of macrophage-specific RCT.
Poster presentations
208 THE ANTIATHEROGENIC PROPERTIES OF HDL AFTER BARIATRIC SURGERY O. Amin Hussein1,2 , I. Shams3,4 , K. Abu Jabal3 , I. Waksman5 , I. Dashkovsky5 , S. Szvalb6 , N. Volkova7 , B. Fuhrman7 , M. Aviram7 . 1 Internal Medicine a, Ziv Medical Center, Safed, 2 Faculty of Medicine, Technion, Haifa, 3 Internal Medicine A, 4 Laboratory for Atherosclerosis Research, 5 Surgery Department, 6 Pathology Department, Ziv Medical Center, Safed, 7 Lipid Research Laboratory, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Background: High-density lipoprotein cholesterol (HDL-C) may not always be atheroprotective and in some conditions, it paradoxically enhances the process of atherosclerosis. The cardiovascular protective effects of HDL have been mainly attributed to its role in reverse cholesterol transport and to many other protective properties towards atherosclerosis as anti-oxidative effect. Aim of the study: To investigate the functional properties of HDL in patients after bariatric surgery. Patients and Methods: Forty candidate patients for bariatric surgery were enrolled. Blood was drawn before, 3 weeks (n = 19) or 6 months (n = 21) after bariatric surgery. Serum PON arylesterase activity was measured by using phenylacetate as substrate. Serum lipid peroxides (PD) were measured spectrophotometrically at 340 l. PD was measured at baseline and after oxidation with 2,2 -Azobis(2-methylpropionamidine) dihydrochloride (AAPH). [3 H]-Cholesterol efflux was measured by incubating J744 macrophages with serum. Radioactivity was measured by b counter in the cell lysate and the medium. Statistical methods: Student’s t-test for paired samples was used. p < 0.05 was cosidered significant. Results: Serum mediated cholesterol efflux did not change after 3 weeks, but increased significantly by 14% after 6 months. Both basal and AAPH-oxidized peroxides were significantly decreased 3 weeks and 6 months after surgery. PON lactonase and arylesterase activities decreased and liver function tests normalized already within 3 weeks. Conclusion: Bariatric surgery reduced serum peroxides as early as 3 weeks while the increase in serum-mediated cholesterol efflux was observed after 6 months. 209 GLYCOXIDATION IMPAIRS THE HDL-MEDIATED OXYSTEROLS EFFLUX AND INDUCES STEROL ACCUMULATION IN MACROPHAGES R. Tallada Iborra1 , A. Machado-Lima1 , G. Castilho1 , V.S. Nunes1 , E.R. Nakandakare1 , D.S. Abdalla2 , M. Passarelli1 . 1 Lipids Laboratory (LIM-10), ˜ Paulo. Sao ˜ Paulo, Brazil., Faculty of Medical Sciences, University of Sao 2 Department of Clinical and Toxicology Analysis, Faculty of Pharmaceutical ˜ Paulo, Sao ˜ Paulo, Brazil Sciences, University of Sao Introduction: Advanced glycation reduces the expression of ABCA-1 and ABCG-1, inducing lipid accumulation in macrophages (macs). We analyzed the content of total sterols and oxysterol subfractions in macs submitted to glycoxidation. Methods: J774 macs were incubated with oxidized LDL (50 mg/mL of DMEM) for 43 h and then treated with 0.5 mM glycolaldehyde (GAD-macs) or DMEM alone (C-macs) for 5h, in the absence or presence of the LXR-agonist, T0901317 (1mM). Cells were carefully washed and incubated with 50 mg/mL of HDL (5 h) in the absence or presence of T0901317. Oxysterols and total cholesterol were determined by gas chromatography/mass spectrometry in cell and culture media lipid extracts by utilizing 5a cholestane as internal standard. Results: In incubations with HDL+T0901317 the total content of intracellular sterols (cholesterol + oxysterols) and of cholesterol-5a,6a-epoxide and cholesterol-5b,6b-epoxide was, respectively, 1.4, 2.0 and 2.2 times higher in GAD-macs as compared to C-macs. In culture media, total sterols content was similar between C and GAD-macs and increased similarly after addition of HDL or HDL+T0901317. A 20% and 23% reduction was observed in the amount of respectively 7-ketocholesterol and 7b-hydroxycholesterol in culture media from GAD-macs incubated with HDL alone. The addition of HDL+T0901317 increased these oxysterols similarly to C-macs. Conclusion: Glycoxidation reduces the exportation of 7-ketocholesterol and 7b-hydroxycholesterol to HDL and induces macrophage sterol accumulation. This may contribute to atherogenesis in DM. Funding: FAPESP (Brazil)