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Lipoprotein-X formation in mouse models of erythropoietic protoporphyria
Transport, biliary disease, gallstones
I P/C07/09 I
[ P/C07/11 I
INDUCTION OF MDR2 P-GLYCOPROTEIN (PGP) BY FIBRATES IS MEDIATED BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR alpha (PPARa) IN THE MOUSE T. Kok, V. Bloks, H. Wolters, M. Mtlller, B. Staels t, E Kuipers University Hospital Groningen, Groningen, The Netherlands. qnstitut Pasteur, Lille, France. Fibrates are commonly used hypolipidemic drugs that exert their actions via PPARc~, a member of the nuclear hormone receptor superfamily. Fibrate treatment decreases secretion of bile salts and is associated with increased gallstone incidence in humans. Fibmtes induce expression of mdr2, encoding the canalicular phospholipid translocator mdr2 Pgp, in rodents. The mechamsm of fibrate-induced changes in bile formation are still unknown. We have studied bile formation in wildtypo (+/+) and PPARtxdeficient SV129 (-/-) mice, fed either normal chow or chow supplemented with 0.05% ciprofibrate (C). Bile was collected from gallbladder cannulated mice and expression of transporters involved in bile formation was evaluated by RT-PCR and Western analysis. Expression of mdr2 was reduced by ~50% in livers of -/- mice compared to +/+ mice. C did not induce expression of mdr2 in 4- mice, as it did in +/+ mice (+170%). No effect of PPAR~x-deficiency or C were found on expression of bsep (bile salt export pump). Yet, bsep protein levels were reduced in C-treated +/+ mice. Likewise, protein levels of uptake carriers ntcp and oatp were reduced in C-treated +/+ mice. Expression of mdrlb was undetectable in -/- mice, irrespective of treatment. Surprisingly, bile formation was hardly affected in -/- mice with or without C-treatment when compared to untreated +/+ mice. C-treatment of +/+ mice resulted in 3-fold increase in bile flow, presumably due to the presence of excessive amounts of lactate and (acetyl)carnitine in bile, but biliary secretion of bile salts and cholesterol was not affected. Conclusions: Induction of mdr2 expression by C is mediated by PPARct. Effects of C on bile formation in mice are, at least in part, related to altered hepatic glucose and fat metabolism.
LIPOPROTEIN-X FORMATION IN MOUSE MODELS OF ERYTHROPOIETIC PROTOPORPHYRIA V. Bloks, T. Pl6sch, J. Bailer, R. Havinga, P.L.M. Jansen, E Kuipers University Hospital Groningen, Groningen, The Netherlands. Erythropoietic Protoporphyria (EPP) is an inherited disorder of heme synthesis caused by deficiency of the mitochondrial enzyme ferroehelatase. EPP in humans is associated with liver disease, hypertriglyceridemia and low HDL cholesterol. To explore consequences of ferrochelatase-deficiency on lipid metabolism, we have analyzed hepatic lipid content, plasma lipoprotein levels and bile composition in mice homozygous (fch/fch) or heterozygous (fch/+) for a point mutation in the ferrochelatase gene and in wildtype controls (+/+). Livers offch/fch mice show bile duct proliferation and biliary fibrosis but bile formation is not impaired: in fact, biliary bile salt secretion is increased by 500%. The free cholesterol content and sphingomyelin contents of fch/fch livers are markedly increased. Plasma cholesterol in fch/feh rmce (9,9 ± 6.4 raM) is elevated when compared withfch/+ (2.9 ± 0.2 raM) and +/+ (2.5 ± 0.3 raM) mice, with excess free cholesterol present in fch/fch plasma. This is not due to liver disease-related reduction of LCAT activity, but to the presence of Lipoprotein-X (LPX), a particle composed of bile typelipids formed under cholestatic conditions. The presence of LPX is surprising since fch/fch mice are not cholestmic: however, biliary secretion rates of pliospholipids and cholesterol are reduced relative to that of bile salts in spite of the fact that expression of mdr2, encoding the phospholipid translocator mdr2 P-glycoprotein, is increased. LPX is also present in +/+ mice treated with griseofnlvin, an inhibitor of ferrochelatase providing a chemical model of EPP. Griseofnlvin-treated +/+ mice also display relatively reduced biliary cholesterol and phospholipid secretion rates and increased mdr2 expression. Griseofulvin-treated mdr2 Pgp-deficient mice do not form LPX, although protoporphyrins accumulate in livers of these animals to a similar extent as in +/+ mice. The data demonstrate that LPX can be formed under noncholestatic conditions associated with uncoupling of biliary cholesterol and phospholipid secretion from bile salt secretion in situations when mdr2 expression, crucial for LPX formation, is induced.
I P/C07/10 I USE OF A MOLECULARLY BASED ASSAY FOR THE DETECTION OF ANTIMITOCHONDRIAL ANTIBODY M2 D.P. Bogdanos 1, E.T. Davies2, A. Grasso 3, M. Okamoto 1, P. Butler3, R. Williams l, A.K. Burroughs3, D. Vergani 1 qnstitute of Hepatology, Royal Free & University College London Medical School, UK. 2Department of Immunology, King's College, London, UK. 3Institute of Liver Transplantation and Hepatobiliary Medicine, Royal Free & University College, London Medical School, UK.
Amongst antimitochondrial antibodies (AMAs), that giving the immunofluorescence M2 pattern is the serological hallmark of Primary Biliary cirrhosis (PBC). Identification of the inner lipoyl domain of the E2 subunit of the Pyruvate Dehydrogenase Complex (PDC-E2) as the AMA-M2 main target has enabled the development of molecularly based immunoassays. In the present study, we have compared an ELISA detecting antibodies to solid phase PDC-E2, (Vareiisa, Pharmada & Upjohn, Germany) with AMA-M2 as detected by immunofluorescence(IFL) using serum samples from 50 PBC patients (median age 56, range 2684, 43 female), at various stages of their disease, all AMA positive at diagnosis, and stored at -70°C until tasted. Forty-seven of the 50 sera were positive by IFL (median 1/640, range 1/40-1/10240) and 45150 by ELISA (median 77 IU/ml, range 10.1-107.7 lU/ml, negative<5 IU/ml); two sera were negative by both assays, only 3 sera giving discrepant results (X2=10.3, p=0.004). The reciprocal of AMA-IFL titres (log transformed, base 2) and ELISA absorbance readings were strongly correlated (R=0.515, p<0,0001). These results show a high degree of concordance between the two assays. Use of additional PBC related antigens such as the PDC-X, OGDC-E2 and BCOADC-E2 may help in reducingthe outstanding6% discrepancy.
P/C07/12 ] BILIARY STRUCTURES UNDER THE LENS IN NEONATAL AND INFANTILE LIVERS C. Sergi, W.J. Hofmann Institute of Pathology, University Hospital, INF 220, D-69120 Heidelberg, Germany.
Aims: Neonatal cholestasis occurs in about 2.35 per 104 live births, but only 1/3 of these have disorders of the bile ducts, including biliary atresia, paucity of intrahepati¢ bile ducts (PIBD: BD/PT<0.5), and disorders associated with persistence of fetal biliary structures (PFBS). The difficulty to discriminate these disorders has induced to think that the infantile cholangiopathies may represent a disease specman. Methods: 95 liver biopsy specimens from infants < I year were studied by immunohistochemistry (CK-7) using several duetal plate remodeling patterns in a blind fashion. Neoductular proliferation (NDP) and fibrosis were graded. Results: 5 categories were outlined: A) marked NDP and prevalent BD/PT<0.5 (22), B) intrahepatic biliary atresia (BD/PT--0) and no NDP (16), C) Prod (0.1~BD/PT<0.5) (18), D) congenital delay of development of the BD and age > 5 months (0.5~BD/PT<0.9) (9), and E) normal biliary structures (BD/PS>_ 0.9) (30). Fibrosis was seen frequently in categories A and B, and occasionally in the remaining categories. P F B S was, however, seen in categories A, B, and C. Conclusions: A distinction b e t w e e n the various types o f infantile cholangiopathy m a y occur, but the P F B S should b e always taken into account.
119
I P/C07/09 I
[ P/C07/11 I
INDUCTION OF MDR2 P-GLYCOPROTEIN (PGP) BY FIBRATES IS MEDIATED BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR alpha (PPARa) IN THE MOUSE T. Kok, V. Bloks, H. Wolters, M. Mtlller, B. Staels t, E Kuipers University Hospital Groningen, Groningen, The Netherlands. qnstitut Pasteur, Lille, France. Fibrates are commonly used hypolipidemic drugs that exert their actions via PPARc~, a member of the nuclear hormone receptor superfamily. Fibrate treatment decreases secretion of bile salts and is associated with increased gallstone incidence in humans. Fibmtes induce expression of mdr2, encoding the canalicular phospholipid translocator mdr2 Pgp, in rodents. The mechamsm of fibrate-induced changes in bile formation are still unknown. We have studied bile formation in wildtypo (+/+) and PPARtxdeficient SV129 (-/-) mice, fed either normal chow or chow supplemented with 0.05% ciprofibrate (C). Bile was collected from gallbladder cannulated mice and expression of transporters involved in bile formation was evaluated by RT-PCR and Western analysis. Expression of mdr2 was reduced by ~50% in livers of -/- mice compared to +/+ mice. C did not induce expression of mdr2 in 4- mice, as it did in +/+ mice (+170%). No effect of PPAR~x-deficiency or C were found on expression of bsep (bile salt export pump). Yet, bsep protein levels were reduced in C-treated +/+ mice. Likewise, protein levels of uptake carriers ntcp and oatp were reduced in C-treated +/+ mice. Expression of mdrlb was undetectable in -/- mice, irrespective of treatment. Surprisingly, bile formation was hardly affected in -/- mice with or without C-treatment when compared to untreated +/+ mice. C-treatment of +/+ mice resulted in 3-fold increase in bile flow, presumably due to the presence of excessive amounts of lactate and (acetyl)carnitine in bile, but biliary secretion of bile salts and cholesterol was not affected. Conclusions: Induction of mdr2 expression by C is mediated by PPARct. Effects of C on bile formation in mice are, at least in part, related to altered hepatic glucose and fat metabolism.
LIPOPROTEIN-X FORMATION IN MOUSE MODELS OF ERYTHROPOIETIC PROTOPORPHYRIA V. Bloks, T. Pl6sch, J. Bailer, R. Havinga, P.L.M. Jansen, E Kuipers University Hospital Groningen, Groningen, The Netherlands. Erythropoietic Protoporphyria (EPP) is an inherited disorder of heme synthesis caused by deficiency of the mitochondrial enzyme ferroehelatase. EPP in humans is associated with liver disease, hypertriglyceridemia and low HDL cholesterol. To explore consequences of ferrochelatase-deficiency on lipid metabolism, we have analyzed hepatic lipid content, plasma lipoprotein levels and bile composition in mice homozygous (fch/fch) or heterozygous (fch/+) for a point mutation in the ferrochelatase gene and in wildtype controls (+/+). Livers offch/fch mice show bile duct proliferation and biliary fibrosis but bile formation is not impaired: in fact, biliary bile salt secretion is increased by 500%. The free cholesterol content and sphingomyelin contents of fch/fch livers are markedly increased. Plasma cholesterol in fch/feh rmce (9,9 ± 6.4 raM) is elevated when compared withfch/+ (2.9 ± 0.2 raM) and +/+ (2.5 ± 0.3 raM) mice, with excess free cholesterol present in fch/fch plasma. This is not due to liver disease-related reduction of LCAT activity, but to the presence of Lipoprotein-X (LPX), a particle composed of bile typelipids formed under cholestatic conditions. The presence of LPX is surprising since fch/fch mice are not cholestmic: however, biliary secretion rates of pliospholipids and cholesterol are reduced relative to that of bile salts in spite of the fact that expression of mdr2, encoding the phospholipid translocator mdr2 P-glycoprotein, is increased. LPX is also present in +/+ mice treated with griseofnlvin, an inhibitor of ferrochelatase providing a chemical model of EPP. Griseofnlvin-treated +/+ mice also display relatively reduced biliary cholesterol and phospholipid secretion rates and increased mdr2 expression. Griseofulvin-treated mdr2 Pgp-deficient mice do not form LPX, although protoporphyrins accumulate in livers of these animals to a similar extent as in +/+ mice. The data demonstrate that LPX can be formed under noncholestatic conditions associated with uncoupling of biliary cholesterol and phospholipid secretion from bile salt secretion in situations when mdr2 expression, crucial for LPX formation, is induced.
I P/C07/10 I USE OF A MOLECULARLY BASED ASSAY FOR THE DETECTION OF ANTIMITOCHONDRIAL ANTIBODY M2 D.P. Bogdanos 1, E.T. Davies2, A. Grasso 3, M. Okamoto 1, P. Butler3, R. Williams l, A.K. Burroughs3, D. Vergani 1 qnstitute of Hepatology, Royal Free & University College London Medical School, UK. 2Department of Immunology, King's College, London, UK. 3Institute of Liver Transplantation and Hepatobiliary Medicine, Royal Free & University College, London Medical School, UK.
Amongst antimitochondrial antibodies (AMAs), that giving the immunofluorescence M2 pattern is the serological hallmark of Primary Biliary cirrhosis (PBC). Identification of the inner lipoyl domain of the E2 subunit of the Pyruvate Dehydrogenase Complex (PDC-E2) as the AMA-M2 main target has enabled the development of molecularly based immunoassays. In the present study, we have compared an ELISA detecting antibodies to solid phase PDC-E2, (Vareiisa, Pharmada & Upjohn, Germany) with AMA-M2 as detected by immunofluorescence(IFL) using serum samples from 50 PBC patients (median age 56, range 2684, 43 female), at various stages of their disease, all AMA positive at diagnosis, and stored at -70°C until tasted. Forty-seven of the 50 sera were positive by IFL (median 1/640, range 1/40-1/10240) and 45150 by ELISA (median 77 IU/ml, range 10.1-107.7 lU/ml, negative<5 IU/ml); two sera were negative by both assays, only 3 sera giving discrepant results (X2=10.3, p=0.004). The reciprocal of AMA-IFL titres (log transformed, base 2) and ELISA absorbance readings were strongly correlated (R=0.515, p<0,0001). These results show a high degree of concordance between the two assays. Use of additional PBC related antigens such as the PDC-X, OGDC-E2 and BCOADC-E2 may help in reducingthe outstanding6% discrepancy.
P/C07/12 ] BILIARY STRUCTURES UNDER THE LENS IN NEONATAL AND INFANTILE LIVERS C. Sergi, W.J. Hofmann Institute of Pathology, University Hospital, INF 220, D-69120 Heidelberg, Germany.
Aims: Neonatal cholestasis occurs in about 2.35 per 104 live births, but only 1/3 of these have disorders of the bile ducts, including biliary atresia, paucity of intrahepati¢ bile ducts (PIBD: BD/PT<0.5), and disorders associated with persistence of fetal biliary structures (PFBS). The difficulty to discriminate these disorders has induced to think that the infantile cholangiopathies may represent a disease specman. Methods: 95 liver biopsy specimens from infants < I year were studied by immunohistochemistry (CK-7) using several duetal plate remodeling patterns in a blind fashion. Neoductular proliferation (NDP) and fibrosis were graded. Results: 5 categories were outlined: A) marked NDP and prevalent BD/PT<0.5 (22), B) intrahepatic biliary atresia (BD/PT--0) and no NDP (16), C) Prod (0.1~BD/PT<0.5) (18), D) congenital delay of development of the BD and age > 5 months (0.5~BD/PT<0.9) (9), and E) normal biliary structures (BD/PS>_ 0.9) (30). Fibrosis was seen frequently in categories A and B, and occasionally in the remaining categories. P F B S was, however, seen in categories A, B, and C. Conclusions: A distinction b e t w e e n the various types o f infantile cholangiopathy m a y occur, but the P F B S should b e always taken into account.
119