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Three evolutionary pathways in bile acid conjugation
472A
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
1199
1200
DECREASED PLASMA TRIGLYCERIDE LEVELS AND NO CHANGE IN PLASMA CHOLESTEROL LEVELS IN CHOLESTEROL 7ol-HYDROXYLASE DEFICIENT MICE ON AN APOLIPOPROTEIN E3-LEIDEN BACKGROUND. Sabine M Post, Hans M Princen, TNO-prevention and health, Lei-
THREE EVOLUTIONARY PATHWAYS IN BILE ACID CONJUGATION.
den Netherlands Cholesterol 7a-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the conversion of cholesterol to bile acids. We cross-bred mice lacking CYP7A1 on an apolipoprotein E*3-Leiden (E3L) background, to increase the sensitivity of the effect of removal of CYP7A 1 on lipoprotein and lipid metabolism. E3L mice have a defective clearance of apolipoprotein B-containing lipoproteins and show therefore a more human-like lipoprotein profile. On chow diet, plasma triglyceride levels were -43% (P~0.05) decreased in the CYP7A1-/-/E3L mice as compared to their E3L littermates (1.57-+0.72 mmol/L versus 2.74---0.78 mmol/L). Plasma cholesterol levels in CYP7A1-/-/E3L mice and E3L littermates did not differ (2.55-+0.78 mmol/L versus 2.87-+0.64 mmol/L). To increase lipid levels the mice were fed a lipogenic (sucrose-rich) diet. Basal cholesterol levels in CYP7A1-/-/E3L mice increased to 4 . 4 1+- 1.12 mmolFL (P<0.05), whereas triglyceride levels did not change as compared to chow. Again, the absence of CYPTA1 decreased plasma triglyceride levels to the same extent (-40%) as on chow and showed no effect on cholesterol levels. Plasma free fatty acids (as marker of peripheral lipolysis) and ketonbodies (as marker of hepatic ]]-oxidation of fatty acids) did not differ between the groups, suggesting that the decreased plasma triglyceride levels result from a decreased VLDL-production, as a consequence of a strongly decreased bile acid synthesis in these mice. Fecal bile acid excretion (11.38-+ 1.79 (chow) and 7.13-+ 1.48 (lipogenic diet)/~mol/d/100 g BW) was decreased by -74% and -81% on both diets, respectively, as compared to their E3L littermates. Concommitantly, excretion of neutral sterols in the feces was 2-fold increased. To test our hypothesis VLDL-production experiments are currently underway.
Lee R Hagey, Edy A MacDonald, Zoological Society of San Diego, San Diego, CA Bile salts are generally secreted in conjugated form: bile alcohols with sulfate, and bile acids with glycine or taurine. Conjugation makes bile salts water soluble and membrane impermeable, permitting high, micellar concentrations in bile and intestinal content. To determine whether the mode of conjugation is purely random or follows a pattern based on evolutionary relationships, we determined the mode of conjugation in some 1100 vertebrate species using electrospray mass spectrometry and related the findings to currently accepted principles of vertebrate evolution. Results: Three pathways of biochemical evolution were identified: A) C27bile alcohol sulfates to taurine conjugated C27 bile acids, followed by evolution to C24 bile acids conjugated with taurine (frogs, turtles, lizards, snakes, birds, marsupials, monotremes, edentates); B) C27 bile alcohol sulfates directly to C24 taurine conjugated C24bile acids (fish); and C) C27bile alcohol sulfates to glycine conjugated C24bile acids followed by a gradual transition to taurine conjugated C24bile acids (primates, artiodactyla, Perissodactyla, cetacea, rodents, lagomorphs). Conclusions and implications: The patterns of bile acid conjugation follow evolutionary relationships and are not random. One of the most common bile acids, taurocholate, has arisen from convergence of multiple biochemical pathways of bile acid conjugation. The key enzyme for bile acid conjugation - amino acid transferase - appears to have evolved at a different rate than the enzymes involved in side chain modification. Whether the intermediate stage of glycine conjugation is caused by the inability of the transferase to use taurine or a scarcity of taurine in the hepatocyte remains to be clarified.
1201
1202
THE EFFECTS OF TRANSHEPATIC BILE SALT FLUX ON HEPATIC PRODUCTION OF VERY LOW DENSITY LIPOPROTEIN TRIGLYCERIDE IN VIVO IN RATS AND MICE. Baukje M Elzinga, Rick Havinga, Julius F Bailer,
ASSOCIATION OF THE A-278C PROMOTER POLYMORPHISM IN THE CHOLESTEROL 7ALPIIA-HYDROXYLASE GENE W I T H HYPERLIPIDEMIC DISORDERS. Maaike K Hofman, TNO-PG Gaubius Lab, Leiden / Div
Vincent Bloks, Folkert Kuipers, Henkjan J Verkade, University Hospital Groningen, Groningen Netherlands
Hum Nutrition and Epidemiology, Wagening Netherlands; Martine Groenendijk, TNO-PG Gaubius Lab, Leiden Netherlands; Iris J Jonkers, Martina F Mohrschladt, Gnus A Smelt, Leiden Univ Medical Ctr, Leiden Netherlands; Hans M Princen, TNO-PG Gaubins Lab, Leiden Netherlands
Cholestyramine treatment for lowering of plasma cholesterol is associated with elevated plasma TG levels in humans, suggesting a relationship between bile salts and TG metabolism. In previous studies we showed that bile salts (BS) dose-dependently inhibit the secretion of Very Low Density Lipoprotein-Triglyceride (VLDL-TG)in rat and human hepatocytes in vitro (Hepatology 1996; 23: 218-28; Biochem J 1997; 316: 531-8). In the present study we aimed to determine whether BS affect VLDL-TG secretion in vivo: regulation of hepatic VLDL-TG secretion might represent a physiological role of BS. METHODS: Plasma TG concentration, hepatic TG secretion rate (Triton WR-1339 method) and plasma VLDL lipid composition (in fraction d< 1.019 g/ml) were determined in rats and mice under conditions of low or high transhepatic BS flux. In rats, the transhepatic BS flux was quantitatively manipulated by 1-wk chronic bile diversion (BD), followed by intraduodenal infusion of the bile salt taurocholate (TC, 93/.~mol.h-1.kg BW-1) or saline for 6 h. Results were compared with control rats with an intact enterohepatic circulation. In mice, the transhepatic BS flux was manipulated by dietary supplementation with either TC (0.5 wt%) or cholestyramine (2 wt%) for three weeks. RESULTS: Biliary BS secretion rate was decreased to 8% of control values in saline-infused BD rats and to 53% in TC-infused BD rats. Despite the profound differences in transhepatic BS fluxes, plasma TG concentration, hepatic TG secretion rate and VLDL lipid composition were similar in the three groups. In mice, 3-wk dietary supplementation with TC increased biliary BS secretion by 300%, compared with unsupplemented controls. Dietary cholestyramine supplementation decreased biliary BS secretion by 50%, compared with controlmice. Among the three groups of mice an inverse relationship was found between mean BS secretion rate into bile and either mean plasma TG concentration (R2=0.89) or mean hepatic TG production rate (R2=0.87). The decreased plasma TG concentrations in TC-fed mice could mainly be attributed to decreased TG content in the VLDL/IDL fraction (-36% compared to controls, p<0.01). The observed changes in the hepatic TG secretion rate were not accompanied by differences in mRNA levels of the microsomal triglyceride transfer protein, involved in VLDL assembly, or of apolipoprotein B in the three groups of mice. CONCLUSION: The present data support the concept that BS are able to inhibit VLDL-TG secretion in vivo. It is not likely, however, that the magnitude of the transhepatic BS flux exerts a major regulatory role on VLDL-TG secretion under physiological conditions. Only supraphysiological transhepatic BS fluxes are associated with significantly decreased plasma VLDL-TG concentrations and hepatic TG production rates in mice.
The first and rate-limiting step in the breakdown of cholesterol is catalysed by the enzyme cholesterol 7alpha-hydroxylase (CYP7A1) and serves as the major site of regulation of bile acid synthesis in the liver. An A to C substitution 278 bp upstream in the promoter of the CYP7A1 gene was found to be associated with variations in plasma lipoprotein levels. So far, the polymorphism has only been studied in healthy normolipidemic populations. Therefore, the aim of this study was to investigate the involvement of this polymorphism in lipid disorders, by using patients with four different types of hyperlipidaemia: hypertriglyceridaemia (HTG), familial combined hyperlipidaemia (ECH), familial dysbetalipoproteinaemia (FD) and familial hypercholesterolaemia (FH). In a control population no significant differences between plasma lipid levels and the CYP7A1 genotype were found. However, after adjustment for the apoE phenotype, a major modulator of plasma lipid levels, including only E3E3, homozygous A carriers had elevated levels of plasma triglycerides (+ 34%, p = 0,036). In HTG patients, the AA genotype was associated with significantly higher plasma concentrations of total cholesterol (+23%, p = 0.016) and VLDL-triglycerides (+48%, p = 0.05). Furthermore, there was a tendency towards increased levels of total triglycerides (+39%, p = 0.07) and VLDLcholesterol (+35%, p = 0,054). No significant associations were found between plasma lipid levels and the CYP7A1 genotype in patients with FCH, FD and FH. Our results show that the A-278C polymorphism in the CYP7A1 gene has a modulating effect in HTG patients, possibly by the involvement in overproduction of VLDL particles. No effect of this polymorphism was found in FCH, FD and FH patients.
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
1199
1200
DECREASED PLASMA TRIGLYCERIDE LEVELS AND NO CHANGE IN PLASMA CHOLESTEROL LEVELS IN CHOLESTEROL 7ol-HYDROXYLASE DEFICIENT MICE ON AN APOLIPOPROTEIN E3-LEIDEN BACKGROUND. Sabine M Post, Hans M Princen, TNO-prevention and health, Lei-
THREE EVOLUTIONARY PATHWAYS IN BILE ACID CONJUGATION.
den Netherlands Cholesterol 7a-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the conversion of cholesterol to bile acids. We cross-bred mice lacking CYP7A1 on an apolipoprotein E*3-Leiden (E3L) background, to increase the sensitivity of the effect of removal of CYP7A 1 on lipoprotein and lipid metabolism. E3L mice have a defective clearance of apolipoprotein B-containing lipoproteins and show therefore a more human-like lipoprotein profile. On chow diet, plasma triglyceride levels were -43% (P~0.05) decreased in the CYP7A1-/-/E3L mice as compared to their E3L littermates (1.57-+0.72 mmol/L versus 2.74---0.78 mmol/L). Plasma cholesterol levels in CYP7A1-/-/E3L mice and E3L littermates did not differ (2.55-+0.78 mmol/L versus 2.87-+0.64 mmol/L). To increase lipid levels the mice were fed a lipogenic (sucrose-rich) diet. Basal cholesterol levels in CYP7A1-/-/E3L mice increased to 4 . 4 1+- 1.12 mmolFL (P<0.05), whereas triglyceride levels did not change as compared to chow. Again, the absence of CYPTA1 decreased plasma triglyceride levels to the same extent (-40%) as on chow and showed no effect on cholesterol levels. Plasma free fatty acids (as marker of peripheral lipolysis) and ketonbodies (as marker of hepatic ]]-oxidation of fatty acids) did not differ between the groups, suggesting that the decreased plasma triglyceride levels result from a decreased VLDL-production, as a consequence of a strongly decreased bile acid synthesis in these mice. Fecal bile acid excretion (11.38-+ 1.79 (chow) and 7.13-+ 1.48 (lipogenic diet)/~mol/d/100 g BW) was decreased by -74% and -81% on both diets, respectively, as compared to their E3L littermates. Concommitantly, excretion of neutral sterols in the feces was 2-fold increased. To test our hypothesis VLDL-production experiments are currently underway.
Lee R Hagey, Edy A MacDonald, Zoological Society of San Diego, San Diego, CA Bile salts are generally secreted in conjugated form: bile alcohols with sulfate, and bile acids with glycine or taurine. Conjugation makes bile salts water soluble and membrane impermeable, permitting high, micellar concentrations in bile and intestinal content. To determine whether the mode of conjugation is purely random or follows a pattern based on evolutionary relationships, we determined the mode of conjugation in some 1100 vertebrate species using electrospray mass spectrometry and related the findings to currently accepted principles of vertebrate evolution. Results: Three pathways of biochemical evolution were identified: A) C27bile alcohol sulfates to taurine conjugated C27 bile acids, followed by evolution to C24 bile acids conjugated with taurine (frogs, turtles, lizards, snakes, birds, marsupials, monotremes, edentates); B) C27 bile alcohol sulfates directly to C24 taurine conjugated C24bile acids (fish); and C) C27bile alcohol sulfates to glycine conjugated C24bile acids followed by a gradual transition to taurine conjugated C24bile acids (primates, artiodactyla, Perissodactyla, cetacea, rodents, lagomorphs). Conclusions and implications: The patterns of bile acid conjugation follow evolutionary relationships and are not random. One of the most common bile acids, taurocholate, has arisen from convergence of multiple biochemical pathways of bile acid conjugation. The key enzyme for bile acid conjugation - amino acid transferase - appears to have evolved at a different rate than the enzymes involved in side chain modification. Whether the intermediate stage of glycine conjugation is caused by the inability of the transferase to use taurine or a scarcity of taurine in the hepatocyte remains to be clarified.
1201
1202
THE EFFECTS OF TRANSHEPATIC BILE SALT FLUX ON HEPATIC PRODUCTION OF VERY LOW DENSITY LIPOPROTEIN TRIGLYCERIDE IN VIVO IN RATS AND MICE. Baukje M Elzinga, Rick Havinga, Julius F Bailer,
ASSOCIATION OF THE A-278C PROMOTER POLYMORPHISM IN THE CHOLESTEROL 7ALPIIA-HYDROXYLASE GENE W I T H HYPERLIPIDEMIC DISORDERS. Maaike K Hofman, TNO-PG Gaubius Lab, Leiden / Div
Vincent Bloks, Folkert Kuipers, Henkjan J Verkade, University Hospital Groningen, Groningen Netherlands
Hum Nutrition and Epidemiology, Wagening Netherlands; Martine Groenendijk, TNO-PG Gaubius Lab, Leiden Netherlands; Iris J Jonkers, Martina F Mohrschladt, Gnus A Smelt, Leiden Univ Medical Ctr, Leiden Netherlands; Hans M Princen, TNO-PG Gaubins Lab, Leiden Netherlands
Cholestyramine treatment for lowering of plasma cholesterol is associated with elevated plasma TG levels in humans, suggesting a relationship between bile salts and TG metabolism. In previous studies we showed that bile salts (BS) dose-dependently inhibit the secretion of Very Low Density Lipoprotein-Triglyceride (VLDL-TG)in rat and human hepatocytes in vitro (Hepatology 1996; 23: 218-28; Biochem J 1997; 316: 531-8). In the present study we aimed to determine whether BS affect VLDL-TG secretion in vivo: regulation of hepatic VLDL-TG secretion might represent a physiological role of BS. METHODS: Plasma TG concentration, hepatic TG secretion rate (Triton WR-1339 method) and plasma VLDL lipid composition (in fraction d< 1.019 g/ml) were determined in rats and mice under conditions of low or high transhepatic BS flux. In rats, the transhepatic BS flux was quantitatively manipulated by 1-wk chronic bile diversion (BD), followed by intraduodenal infusion of the bile salt taurocholate (TC, 93/.~mol.h-1.kg BW-1) or saline for 6 h. Results were compared with control rats with an intact enterohepatic circulation. In mice, the transhepatic BS flux was manipulated by dietary supplementation with either TC (0.5 wt%) or cholestyramine (2 wt%) for three weeks. RESULTS: Biliary BS secretion rate was decreased to 8% of control values in saline-infused BD rats and to 53% in TC-infused BD rats. Despite the profound differences in transhepatic BS fluxes, plasma TG concentration, hepatic TG secretion rate and VLDL lipid composition were similar in the three groups. In mice, 3-wk dietary supplementation with TC increased biliary BS secretion by 300%, compared with unsupplemented controls. Dietary cholestyramine supplementation decreased biliary BS secretion by 50%, compared with controlmice. Among the three groups of mice an inverse relationship was found between mean BS secretion rate into bile and either mean plasma TG concentration (R2=0.89) or mean hepatic TG production rate (R2=0.87). The decreased plasma TG concentrations in TC-fed mice could mainly be attributed to decreased TG content in the VLDL/IDL fraction (-36% compared to controls, p<0.01). The observed changes in the hepatic TG secretion rate were not accompanied by differences in mRNA levels of the microsomal triglyceride transfer protein, involved in VLDL assembly, or of apolipoprotein B in the three groups of mice. CONCLUSION: The present data support the concept that BS are able to inhibit VLDL-TG secretion in vivo. It is not likely, however, that the magnitude of the transhepatic BS flux exerts a major regulatory role on VLDL-TG secretion under physiological conditions. Only supraphysiological transhepatic BS fluxes are associated with significantly decreased plasma VLDL-TG concentrations and hepatic TG production rates in mice.
The first and rate-limiting step in the breakdown of cholesterol is catalysed by the enzyme cholesterol 7alpha-hydroxylase (CYP7A1) and serves as the major site of regulation of bile acid synthesis in the liver. An A to C substitution 278 bp upstream in the promoter of the CYP7A1 gene was found to be associated with variations in plasma lipoprotein levels. So far, the polymorphism has only been studied in healthy normolipidemic populations. Therefore, the aim of this study was to investigate the involvement of this polymorphism in lipid disorders, by using patients with four different types of hyperlipidaemia: hypertriglyceridaemia (HTG), familial combined hyperlipidaemia (ECH), familial dysbetalipoproteinaemia (FD) and familial hypercholesterolaemia (FH). In a control population no significant differences between plasma lipid levels and the CYP7A1 genotype were found. However, after adjustment for the apoE phenotype, a major modulator of plasma lipid levels, including only E3E3, homozygous A carriers had elevated levels of plasma triglycerides (+ 34%, p = 0,036). In HTG patients, the AA genotype was associated with significantly higher plasma concentrations of total cholesterol (+23%, p = 0.016) and VLDL-triglycerides (+48%, p = 0.05). Furthermore, there was a tendency towards increased levels of total triglycerides (+39%, p = 0.07) and VLDLcholesterol (+35%, p = 0,054). No significant associations were found between plasma lipid levels and the CYP7A1 genotype in patients with FCH, FD and FH. Our results show that the A-278C polymorphism in the CYP7A1 gene has a modulating effect in HTG patients, possibly by the involvement in overproduction of VLDL particles. No effect of this polymorphism was found in FCH, FD and FH patients.