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Poor glycemic control does not influence the oxidizability of circulating LDL in type 2 diabetic patients
297
XVIII S.I.S.A. National Congress
Different modifications of low-density lipoproteins in type 1 and 2 diabetes mellitus
Poor glycemic control does not influence the oxidizability of circulating LDL in type 2 diabetic patients.
Barbara Uberti, Roberto Gambino, Elisabetta Pisu, Gianfranco Pagano, Maurizio Cassader
Roberto Gambino, Gianluca Ruiu, Natalina Alemanno, Elisabetta Pisu, Gianfranco Pagano, Maurizio Cassader
Dipartimento di Medicina Interna, Universit~t di Torino, Corso A.M. Dogliotti 14, 10126 Torino, Italy.
Dipartimento di Medicina Interna, Universit~t di Torino, Corso A.M. Dogliotti 14, 10126 Torino, Italy
Background: Focus has turned to qualitative changes in lipoprotein metabolism rather than lipoprotein concentrations in diabetes. Modified or aberrant forms of lipoproteins favor the progression of atherosclerosis. The aim was to evaluate the degree of LDL modification related to the types of diabetes. Methods: 14 type 1 and 12 type 2 diabetic patients were included in this study. LDL was isolated by preparative sequential ultracentrifugation. The electrophoretic mobility and the UV absorption at 200 and 234 nm of LDL were measured by capillary electrophoresis. Student's t test was used to compare groups. Pearson's correlation coefficients were used to describe relationships between variables. Results: LDL from type 1 diabetes subjects showed the highest electrophoretic mobility (p=0.000). The diene conjugates contents were higher in the type 2 patients especially in those with HbAlc levels >8 % (p=0.003). In the group of type 1 diabetic subjects the electrophoretic mobility of LDL was correlated with the diene contents (r=0.55, p=0.040). In the group of type 2 we observed a significant correlation between diene conjugates and total cholesterol (r=0.68, p=0.015) or LDL-cholesterol (r=0.68, p=0.015). Conclusion: The increased diene content in type 2 subjects and the highest LDL mobility found in type 1 subjects show that the LDL undergoes different modifications. In type 2 patients electronegative LDL are in a state of higher susceptibility to oxidation, whereas in type 1 subjects the finding of electronegative lipoproteins could be a marker for atherosclerotic complications.
Background: Glycosylation is a process that increases the negative charge of LDL Modifications that increase the net negative charge may have important metabolic consequences and enhance LDL atherogenicity. Glyceted LDL is catabolized more slowly than normal LDL and is degraded by the scavenger pathway promoting foam cell formation. We aimed to determine if increased non-enzymatic glycosylation of the LDL was sufficient to increase the susceptibility to in vivo oxidation of the LDL particles. Methods: 22 type 2 diabetic patients were included in this study. They were enrolled on the basis of good and poor glycemic control. LDL were isolated by sequential ultracentrifugation and analyzed by capillary electrophoresis for the content of diene conjugated and for electronegativity. Results: The glyc-LDL levels were increased in all diabetic type 2 patients peaking in the diabetic subjects in poor diabetic control (17.3 +/- 8.07 %). The LDL content of diene conjugates was similar between the two groups (6.65 +/- 0.77 % for the patients with good glycemic control vs 6.88 +/- 0.74 % for those with poor glycemic control; p= 0.49) as well as the electrophoretic mobility was (-1.14544 +/- 0.089 x 10-4 x cm2 x V-lx s-1 vs -1.13666 +/- 0.073 x 10-4 x cm2 x V-lx s-l, p= 0.80). Conclusion: The susceptibility to in vivo oxidation of LDL from type 2 diabetic patients in poor glycemic control did not differ from that of well-controlled diabetic patients. LDL glycosylation was not able to increase the oxidizability of LDL in the diabetic patients with poor glycemic control.
Polymerized collagen partially reverts the synthetic phenotype of smooth muscle cells isolated from rabbit carotid arteries after collar insertion.
NUCLEAR RECEPTORS AND REGULATION SYNTHESIS IN HUMANS
Ferri N., Baetta R.,Comparato C., Gianolini M, Silva F., Corsini A., Boschini E.I, and Donetti E.?
M Bertolotti, C. Gabbi, C. Anzivino, M. Crestani*, N. Mitro*, C. Godio*, E. De Fabiani*, M. Del Puppo**, L. Carulli, A. Rossi, P. Loria, N. Carulli
Departments of Pharmacological Sciences and ?Human Morphology, University of Milan, Milan.
Universit~l di Modena e Reggio Emilia; *Universit& di Milano; **Universit~l di Milano Bicocce
The transition of vascular smooth muscle cells (SMC) from contractile to synthetic phenotype is a key event during atherogenesis. Development of 3D in vitro system, utilizing polymerized collagen gels, has been shown to partially prevent and revert this phenotypic switch. In this study, we investigate the influence of polymerized type I collagen on the phenotype of cultured SMC obtained from collared (SMCc) and sham-operated (SMCs) rabbit carotid arteries. Ultrastructural analysis shows that SMCc express an epitheliod shape and an enrichment of synthetic organelles. The expression of a-smooth muscle actin, a marker of the contractile phenotype, is reduced by 27% in SMCc vs SMCs. SMCs adhere to a higher extent than SMCc either on monomer (77%+3 vs 56%+1 after 15'), or on polymerized (81%+5 vs 54%+1 after 15') type I collagen. On the other hand, migration through monomer type I collagen, stimulated by 20 ng/ml of PDGF-BB, is more pronounced in SMCc than in SMCs (243% after 8 h). Ultrastructural results show a partially reverted phenotypic activation of SMCc when these cells are plated on polymerized type I collagen. However, SMCc are always more activated than SMCs. In conclusion, we show that synthetic phenotype of SMC, isolated from an in vivo model of atherosclerosis, may be maintained in vitro and partially reverted to a more contractile phenotype after culture on polymerized collagen. This data further support the notion that SMC phenotype is reversible and demonstrate the importance of cell-collagen interaction in this phenomenon.
The role of nuclear receptors in the transcriptional regulation of cholesterol 7alpha-hydroxylase (CYP7A1), the limiting enzyme of bile acid synthesis, has been highlighted in cellular and animal models. AIM of the present study was to analyze the expression of CYP7A1 and related nuclear receptors in human livers. METHODS. Surgical liver biopsies were obtained in 32 patients; 12 with gallbladder gallstones; 10 with abdominal cancer; 10 treated, either with cholestyramine (2), CDCA (3), or UDCA (5). mRNA levels of CYP7A1 and related nuclear receptors were assayed by real-time quantitative RT-PCR. Serum levels of 7alphahydroxycholest-3-one (7A-3-ONE), a marker of bile acid synthesis, were assayed by GC-MS. RESULTS. CYP7A1 mRNA varied widely. In gallstone patients PGC-1 was significantly (p < 0.05 on a log scale) less expressed; other genes were unaffected. In untreated patients serum levels of 7A-3-ONE showed an inverse correlation trend with age (r = 0.48, p = 0.05); no such correlation was present with CYP7A1 or other gene expression. Stepwise regression analysis with CYP7A1 mRNA as the dependent variable showed the strongest correlation with HNF-4 (r = 0.471 on a log scale, p < 0.05), all other genes (including SliP) bringing non-significant further contribution. CONCLUSIONS. HNF-4 might play a relevant role in the regulation of CYP7A1 transcription in humans; no evidence for an inhibitory role of SliP is present. Post-transcriptional control of CYP7A1 regulation should however be considered. GRANT SUPPORT. EU grant QLGI-CT-2001-01513 and COFIN-PRIN grant 2002062991.
OF BILE ACID
XVIII S.I.S.A. National Congress
Different modifications of low-density lipoproteins in type 1 and 2 diabetes mellitus
Poor glycemic control does not influence the oxidizability of circulating LDL in type 2 diabetic patients.
Barbara Uberti, Roberto Gambino, Elisabetta Pisu, Gianfranco Pagano, Maurizio Cassader
Roberto Gambino, Gianluca Ruiu, Natalina Alemanno, Elisabetta Pisu, Gianfranco Pagano, Maurizio Cassader
Dipartimento di Medicina Interna, Universit~t di Torino, Corso A.M. Dogliotti 14, 10126 Torino, Italy.
Dipartimento di Medicina Interna, Universit~t di Torino, Corso A.M. Dogliotti 14, 10126 Torino, Italy
Background: Focus has turned to qualitative changes in lipoprotein metabolism rather than lipoprotein concentrations in diabetes. Modified or aberrant forms of lipoproteins favor the progression of atherosclerosis. The aim was to evaluate the degree of LDL modification related to the types of diabetes. Methods: 14 type 1 and 12 type 2 diabetic patients were included in this study. LDL was isolated by preparative sequential ultracentrifugation. The electrophoretic mobility and the UV absorption at 200 and 234 nm of LDL were measured by capillary electrophoresis. Student's t test was used to compare groups. Pearson's correlation coefficients were used to describe relationships between variables. Results: LDL from type 1 diabetes subjects showed the highest electrophoretic mobility (p=0.000). The diene conjugates contents were higher in the type 2 patients especially in those with HbAlc levels >8 % (p=0.003). In the group of type 1 diabetic subjects the electrophoretic mobility of LDL was correlated with the diene contents (r=0.55, p=0.040). In the group of type 2 we observed a significant correlation between diene conjugates and total cholesterol (r=0.68, p=0.015) or LDL-cholesterol (r=0.68, p=0.015). Conclusion: The increased diene content in type 2 subjects and the highest LDL mobility found in type 1 subjects show that the LDL undergoes different modifications. In type 2 patients electronegative LDL are in a state of higher susceptibility to oxidation, whereas in type 1 subjects the finding of electronegative lipoproteins could be a marker for atherosclerotic complications.
Background: Glycosylation is a process that increases the negative charge of LDL Modifications that increase the net negative charge may have important metabolic consequences and enhance LDL atherogenicity. Glyceted LDL is catabolized more slowly than normal LDL and is degraded by the scavenger pathway promoting foam cell formation. We aimed to determine if increased non-enzymatic glycosylation of the LDL was sufficient to increase the susceptibility to in vivo oxidation of the LDL particles. Methods: 22 type 2 diabetic patients were included in this study. They were enrolled on the basis of good and poor glycemic control. LDL were isolated by sequential ultracentrifugation and analyzed by capillary electrophoresis for the content of diene conjugated and for electronegativity. Results: The glyc-LDL levels were increased in all diabetic type 2 patients peaking in the diabetic subjects in poor diabetic control (17.3 +/- 8.07 %). The LDL content of diene conjugates was similar between the two groups (6.65 +/- 0.77 % for the patients with good glycemic control vs 6.88 +/- 0.74 % for those with poor glycemic control; p= 0.49) as well as the electrophoretic mobility was (-1.14544 +/- 0.089 x 10-4 x cm2 x V-lx s-1 vs -1.13666 +/- 0.073 x 10-4 x cm2 x V-lx s-l, p= 0.80). Conclusion: The susceptibility to in vivo oxidation of LDL from type 2 diabetic patients in poor glycemic control did not differ from that of well-controlled diabetic patients. LDL glycosylation was not able to increase the oxidizability of LDL in the diabetic patients with poor glycemic control.
Polymerized collagen partially reverts the synthetic phenotype of smooth muscle cells isolated from rabbit carotid arteries after collar insertion.
NUCLEAR RECEPTORS AND REGULATION SYNTHESIS IN HUMANS
Ferri N., Baetta R.,Comparato C., Gianolini M, Silva F., Corsini A., Boschini E.I, and Donetti E.?
M Bertolotti, C. Gabbi, C. Anzivino, M. Crestani*, N. Mitro*, C. Godio*, E. De Fabiani*, M. Del Puppo**, L. Carulli, A. Rossi, P. Loria, N. Carulli
Departments of Pharmacological Sciences and ?Human Morphology, University of Milan, Milan.
Universit~l di Modena e Reggio Emilia; *Universit& di Milano; **Universit~l di Milano Bicocce
The transition of vascular smooth muscle cells (SMC) from contractile to synthetic phenotype is a key event during atherogenesis. Development of 3D in vitro system, utilizing polymerized collagen gels, has been shown to partially prevent and revert this phenotypic switch. In this study, we investigate the influence of polymerized type I collagen on the phenotype of cultured SMC obtained from collared (SMCc) and sham-operated (SMCs) rabbit carotid arteries. Ultrastructural analysis shows that SMCc express an epitheliod shape and an enrichment of synthetic organelles. The expression of a-smooth muscle actin, a marker of the contractile phenotype, is reduced by 27% in SMCc vs SMCs. SMCs adhere to a higher extent than SMCc either on monomer (77%+3 vs 56%+1 after 15'), or on polymerized (81%+5 vs 54%+1 after 15') type I collagen. On the other hand, migration through monomer type I collagen, stimulated by 20 ng/ml of PDGF-BB, is more pronounced in SMCc than in SMCs (243% after 8 h). Ultrastructural results show a partially reverted phenotypic activation of SMCc when these cells are plated on polymerized type I collagen. However, SMCc are always more activated than SMCs. In conclusion, we show that synthetic phenotype of SMC, isolated from an in vivo model of atherosclerosis, may be maintained in vitro and partially reverted to a more contractile phenotype after culture on polymerized collagen. This data further support the notion that SMC phenotype is reversible and demonstrate the importance of cell-collagen interaction in this phenomenon.
The role of nuclear receptors in the transcriptional regulation of cholesterol 7alpha-hydroxylase (CYP7A1), the limiting enzyme of bile acid synthesis, has been highlighted in cellular and animal models. AIM of the present study was to analyze the expression of CYP7A1 and related nuclear receptors in human livers. METHODS. Surgical liver biopsies were obtained in 32 patients; 12 with gallbladder gallstones; 10 with abdominal cancer; 10 treated, either with cholestyramine (2), CDCA (3), or UDCA (5). mRNA levels of CYP7A1 and related nuclear receptors were assayed by real-time quantitative RT-PCR. Serum levels of 7alphahydroxycholest-3-one (7A-3-ONE), a marker of bile acid synthesis, were assayed by GC-MS. RESULTS. CYP7A1 mRNA varied widely. In gallstone patients PGC-1 was significantly (p < 0.05 on a log scale) less expressed; other genes were unaffected. In untreated patients serum levels of 7A-3-ONE showed an inverse correlation trend with age (r = 0.48, p = 0.05); no such correlation was present with CYP7A1 or other gene expression. Stepwise regression analysis with CYP7A1 mRNA as the dependent variable showed the strongest correlation with HNF-4 (r = 0.471 on a log scale, p < 0.05), all other genes (including SliP) bringing non-significant further contribution. CONCLUSIONS. HNF-4 might play a relevant role in the regulation of CYP7A1 transcription in humans; no evidence for an inhibitory role of SliP is present. Post-transcriptional control of CYP7A1 regulation should however be considered. GRANT SUPPORT. EU grant QLGI-CT-2001-01513 and COFIN-PRIN grant 2002062991.
OF BILE ACID