- Email: [email protected]
Regulation of cholesterol and bile acid synthesis in Hep G2 cells: Effect of cholesterol availability and bile acids
240A
533
AASLD ABSTRACTS
REGULATION OF CHOLESTEROL AND BILE ACID SYNTHESIS IN HEF G2 CELLS: EFFECT OF CHOLESTEROL AVAILABILITY AND BILE ACIDS. F. Carubbi. F. Rasetti*. M.E Guicciardi, M. Concari, M. Bozzoli. G. Galli*. N: CamUi. Internal Medicine, Modena University * Pharmacology, Milan University, Italy To further investigate hepatic metabolism of cholesterol (C) and bile acids (BA) we have validated a new method for determination of C and BA neosynthesis rate by using deuterium oxide (D20) in the human hepatoblastoma celt llne HepG2. Aim of our study has been to determine: 1. the effect of cholesterol availability on C and BA synthesis, by addition of lipoprotein cholesterol or the HMG-CoA reduetase inhibitor Pravastatin; 2. BA negative feedback regulation of synthesis in our model. Methods: confluent cells were grown in MEM containing 25% D20 and 10% fetal calf serum (FCS) or delipidated FCS (DeI-M) with or without Pravastatin (@Squibb, 10'401xM) or different BA (10-1001£Vl) for 24-72 hours. C and BA both from ceils and expanded media were extracted and analyzed by GLC, isotope ratio mass spectrometry for neosynthesis rate, expressed as Ixg/107 cells/72 hours. Results: C and BA synthesis in the presence of D20 yields a spectrum of enriched molecules due to substitution of D for H. HepG2 cells synthesize and secrete conjugated BA, mainly Chenodeoxyeholic (CDC 22+8), Cholic (CA 15+g), 315)=5 Cholenoic acid (12+7). Neosynthesis of C is nigher when no C is present in the media (plus 32%) and lower (less 15-35%) when Pravastatin is added. BA synthesis is nigher in the presence of C (plus 40-55%) and is not lowered by Pravastatin, which reduces total BA concentration when exogenous C is not available. Deoxycholic (DCA 50-100 laM) and CDC inhibit BA neosynthesis (40-60%) to a greater extent than CA and Ursodeoxyeholic (UDCA). Conclusions: C availability modulates C and BA synthesis in HepG2 cells. Negative feedback regulation of BA synthesis oceurs in HepG2 cells, more hydrophobic BA (DCA) having greater inhibitory effect than hydrophilic BA (UDCA, CA). Pravastatin modulates BA synthesis by affecting cholesterol availability.
535 SERUM CONCENTRATIONS OF 7a-HYDROXY-4-CHOLESTEN-3ONE REFLECT BILE ACID SYNTHESIS IN MAN. G Sauter. F Bert. U Beuers, S Fischer. and G Paum~artner. Department of Medicine II, Klinikum Grosshadem,Universityof Munich, Munich, Germany Serum levels of 7a-hydroxy-4-cholasten-3-one(7a-OH-3-ON) have been shown to reflect the activity of cholesterol-7a-hydroxylasein man. The reliability of this parameter for the assessmentof bile acid synthesis,however, remains unclear, since bile acids may be formed through pathways which bypass cholesterol-7a-hydroxylase.We therefore studied the relationship of 7aOH-3-ON levels in serum with rates of synthesisof cholic acid (CA) and chenodeoxycholic acid (CDCA) as determined by an isotope dilution technique. Patients and Methods: Fasting serum levels of 7a-OH-3-ON and bile acid kinetics were determined in 20 patients (mean age 46± 15 yrs, females/males 15/5) with gallbladder stones but normal liver function tests (n=15), primary biliary cirrhosis (n=3), and primary sclerosing cholangitis (n=2). 7a-OH-3ON was extracted on a column of ODS-hondedsilica at 64 °C and determined by HPLC. Pool sizes and turnover rates (FTR) of CA and CDCA were deters mined from the decay curves of ]3C-CA and 13C-CDCA after oral administration of 50 mg of each label. Synthesis rates were calculated from the equation synthesis = pool size x FTR. Results: Serum levels of 7a-OH-3- [~800] ,~. ON closely correlated with rates ofJ ~ ] . "/ synthesis of CA (r=0.74, p<0.001)]~ 6°°] " (Spearman's r a n k correlation),1~ ,0o] • _ , - - ~ ' . CDCA (r=0.77, p<0.001), and. CA+ [<~ ]~.,-.-""~ • CDCA (r=0.87, p<0.001) (Flgure)./~ 2 o o ~ . . ~ . . ~ o ! ? . ~ d ! ~ There were no significant + i v=d~ronieehole~talieliwrdisease correlationsofserum7a-OH-3-ON ~ 0 - fo~ 2o s0 ~ o ~ 0 701-OH-3 -ON (ng/ml) with poo1sizes of CA or CDCA. L Conclusion: The results demonstrate a close relationship between 7a-OH-3ON levels and the rates of synthesis of CA and CDCA as determined by an isotope dilution technique. These data indicate that analysisof 7a-OH-3-ON in serum is a convenient method for assessing bile acid synthesis in patients with normal liver function and chronic cholestatic liver disease.
HEPATOLOGYOctober 1995
534 HEPATIC TRANSPORT OF UNESTERIFIED CHOLESTEROL IN THE RAT IS TRACED BY THE PLANT STEROL, SITOSTANOL SJ Robins. JM Fasulo. CR Pritzker. and GM Patton. Dept. of Medicine, Boston VA and Boston University School of Medicine, Boston, MA The hepatic uptake, transport, and secretion into bile of unesterified cholesterol (UC) from a lipid particle cannot be directly quantitated because of extensive exchange and equilibration between different pools of UC. Plant sterols are structurally similar to cholesterol but because of poor intestinal absorption are ordinarily not present in the liver. We have found no exchange of the plant sterol, sitostanol (SIT) with UC when these sterols were admixed in HDL and emulsion particles or in liposomes and isolated hepatic plasma membranes. Thus, to quantitate hepatic sterol uptake and secretion in the absence of exchange with endogenous hepatic sterols, isolated rat livers were perfused with SIT, incorporated into phosphatidylcholine liposomes. Appreciable amounts of SIT were taken up by the liver and uptake was independent of the presence of bile salt. However, like UC, the secretion of SIT into bile required bile salt. SIT was detected in bile within 5 rain after a perfusion was begun and reached a plateau by about 20 rain. When both sterols were simultaneously peffused, the kinetics of secretion of SIT in bile were precisely the same as UC. With a constant portal vein perfusion of radiolabeled UC and SIT for 10 rain, the initial rate of biliary UC and SIT secretion was similarly linear from 4-12 rain and maximum at 16 min. Both sterols were secreted in bile at a similar rate (radioactivity increasing at 9.05:1.6%/rain for UC and 11.1+1.1%/re_in for SIT, P NS). At the peak of biliary SIT secretion, the amount of SIT relative to UC was much greater in bile (40-50% of sterols) than in the whole liver (11% of sterols). Selective biliary secretion of SIT was associated with much greater concentrations of SIT in canalicular membranes than in the interior membranes of the hepatocyte and ha newly-secreted HDL (derived from plasma membranes) compared to newly-secreted VLDL (derived from interior membranes). These results indicate that SIT parallels the transport into bile of UC from lipoproteins (J Biol Chem 260:6511, 1985) and suggests that SIT can be used as a physiologic analog of UC to quantitate the contribution of UC to bile from different kinds of lipid particles.
536 PREFERENTIAL STEROL TRANSPORT FROM HDL TO BILE. S._/J Robins. JM Fasulo. G Salen. and GM Patton. Dept, of Medicine, Boston and East Orange VA Medical Centers, Boston Univ. Sch. of Medicine, Boston, MA, and UMDNJ-NJ, Newark, NJ. Unesterified cholesterol (UC) that is taken up by the liver from the blood is rapidly mixed by exchange with liver UC. Thus, amounts of biliary UC that derive from different kinds of plasma lipoproteins cannot be quantitated. However, plant sterols (PS) do not exchange with UC and are secreted in bile with the same kinetics as UC. To compare the contribution to bile of sterols from different lipoproteins, we used PS as analogs of UC. VLDL, LDL, and HDL were isolated from the plasma of 3 patients with hereditary phytosterolemia. PS comprised 15-20% of total sterols in all lipoprotein fractions. Lipoproteins were separately added to isolated, perfused rat livers to provide approximately equal amounts of PS. In 30-rain recirculating perfusions, the uptake of PS was -10% of perfusate PS from all lipopoteins. However, amounts of PS secreted in bile were markedly different for different lipoproteins, With perfusion of either VLDL or LDL there was no increase in the biliary secretion of PS. In sharp contrast, with perfusion of HDL the secretion of biliary PS was markedly increased (29.9 to 69.1 nmol/h, P < 0.01). The increase in biliary PS was detected 5-10 rain after HDL was added to perfusates and was similarly large for each of 3 individual PS that were tracked. The Fig. compares perfusions with LDL and HDL. Perfusate • Campesterol -^ Peffusate ~.~
"
20 ~ 10 0 0
Sil~stanol
20 to 0 10
20
30 0 to 20 30 Durationof Perfasion (Min) Results show, when net sterol transport from lipoproteins into bile can be determined, only HDL provides a vehicle for UC elimination in bile that is consistent with its putative function in "reverse cholesterol transport".
533
AASLD ABSTRACTS
REGULATION OF CHOLESTEROL AND BILE ACID SYNTHESIS IN HEF G2 CELLS: EFFECT OF CHOLESTEROL AVAILABILITY AND BILE ACIDS. F. Carubbi. F. Rasetti*. M.E Guicciardi, M. Concari, M. Bozzoli. G. Galli*. N: CamUi. Internal Medicine, Modena University * Pharmacology, Milan University, Italy To further investigate hepatic metabolism of cholesterol (C) and bile acids (BA) we have validated a new method for determination of C and BA neosynthesis rate by using deuterium oxide (D20) in the human hepatoblastoma celt llne HepG2. Aim of our study has been to determine: 1. the effect of cholesterol availability on C and BA synthesis, by addition of lipoprotein cholesterol or the HMG-CoA reduetase inhibitor Pravastatin; 2. BA negative feedback regulation of synthesis in our model. Methods: confluent cells were grown in MEM containing 25% D20 and 10% fetal calf serum (FCS) or delipidated FCS (DeI-M) with or without Pravastatin (@Squibb, 10'401xM) or different BA (10-1001£Vl) for 24-72 hours. C and BA both from ceils and expanded media were extracted and analyzed by GLC, isotope ratio mass spectrometry for neosynthesis rate, expressed as Ixg/107 cells/72 hours. Results: C and BA synthesis in the presence of D20 yields a spectrum of enriched molecules due to substitution of D for H. HepG2 cells synthesize and secrete conjugated BA, mainly Chenodeoxyeholic (CDC 22+8), Cholic (CA 15+g), 315)=5 Cholenoic acid (12+7). Neosynthesis of C is nigher when no C is present in the media (plus 32%) and lower (less 15-35%) when Pravastatin is added. BA synthesis is nigher in the presence of C (plus 40-55%) and is not lowered by Pravastatin, which reduces total BA concentration when exogenous C is not available. Deoxycholic (DCA 50-100 laM) and CDC inhibit BA neosynthesis (40-60%) to a greater extent than CA and Ursodeoxyeholic (UDCA). Conclusions: C availability modulates C and BA synthesis in HepG2 cells. Negative feedback regulation of BA synthesis oceurs in HepG2 cells, more hydrophobic BA (DCA) having greater inhibitory effect than hydrophilic BA (UDCA, CA). Pravastatin modulates BA synthesis by affecting cholesterol availability.
535 SERUM CONCENTRATIONS OF 7a-HYDROXY-4-CHOLESTEN-3ONE REFLECT BILE ACID SYNTHESIS IN MAN. G Sauter. F Bert. U Beuers, S Fischer. and G Paum~artner. Department of Medicine II, Klinikum Grosshadem,Universityof Munich, Munich, Germany Serum levels of 7a-hydroxy-4-cholasten-3-one(7a-OH-3-ON) have been shown to reflect the activity of cholesterol-7a-hydroxylasein man. The reliability of this parameter for the assessmentof bile acid synthesis,however, remains unclear, since bile acids may be formed through pathways which bypass cholesterol-7a-hydroxylase.We therefore studied the relationship of 7aOH-3-ON levels in serum with rates of synthesisof cholic acid (CA) and chenodeoxycholic acid (CDCA) as determined by an isotope dilution technique. Patients and Methods: Fasting serum levels of 7a-OH-3-ON and bile acid kinetics were determined in 20 patients (mean age 46± 15 yrs, females/males 15/5) with gallbladder stones but normal liver function tests (n=15), primary biliary cirrhosis (n=3), and primary sclerosing cholangitis (n=2). 7a-OH-3ON was extracted on a column of ODS-hondedsilica at 64 °C and determined by HPLC. Pool sizes and turnover rates (FTR) of CA and CDCA were deters mined from the decay curves of ]3C-CA and 13C-CDCA after oral administration of 50 mg of each label. Synthesis rates were calculated from the equation synthesis = pool size x FTR. Results: Serum levels of 7a-OH-3- [~800] ,~. ON closely correlated with rates ofJ ~ ] . "/ synthesis of CA (r=0.74, p<0.001)]~ 6°°] " (Spearman's r a n k correlation),1~ ,0o] • _ , - - ~ ' . CDCA (r=0.77, p<0.001), and. CA+ [<~ ]~.,-.-""~ • CDCA (r=0.87, p<0.001) (Flgure)./~ 2 o o ~ . . ~ . . ~ o ! ? . ~ d ! ~ There were no significant + i v=d~ronieehole~talieliwrdisease correlationsofserum7a-OH-3-ON ~ 0 - fo~ 2o s0 ~ o ~ 0 701-OH-3 -ON (ng/ml) with poo1sizes of CA or CDCA. L Conclusion: The results demonstrate a close relationship between 7a-OH-3ON levels and the rates of synthesis of CA and CDCA as determined by an isotope dilution technique. These data indicate that analysisof 7a-OH-3-ON in serum is a convenient method for assessing bile acid synthesis in patients with normal liver function and chronic cholestatic liver disease.
HEPATOLOGYOctober 1995
534 HEPATIC TRANSPORT OF UNESTERIFIED CHOLESTEROL IN THE RAT IS TRACED BY THE PLANT STEROL, SITOSTANOL SJ Robins. JM Fasulo. CR Pritzker. and GM Patton. Dept. of Medicine, Boston VA and Boston University School of Medicine, Boston, MA The hepatic uptake, transport, and secretion into bile of unesterified cholesterol (UC) from a lipid particle cannot be directly quantitated because of extensive exchange and equilibration between different pools of UC. Plant sterols are structurally similar to cholesterol but because of poor intestinal absorption are ordinarily not present in the liver. We have found no exchange of the plant sterol, sitostanol (SIT) with UC when these sterols were admixed in HDL and emulsion particles or in liposomes and isolated hepatic plasma membranes. Thus, to quantitate hepatic sterol uptake and secretion in the absence of exchange with endogenous hepatic sterols, isolated rat livers were perfused with SIT, incorporated into phosphatidylcholine liposomes. Appreciable amounts of SIT were taken up by the liver and uptake was independent of the presence of bile salt. However, like UC, the secretion of SIT into bile required bile salt. SIT was detected in bile within 5 rain after a perfusion was begun and reached a plateau by about 20 rain. When both sterols were simultaneously peffused, the kinetics of secretion of SIT in bile were precisely the same as UC. With a constant portal vein perfusion of radiolabeled UC and SIT for 10 rain, the initial rate of biliary UC and SIT secretion was similarly linear from 4-12 rain and maximum at 16 min. Both sterols were secreted in bile at a similar rate (radioactivity increasing at 9.05:1.6%/rain for UC and 11.1+1.1%/re_in for SIT, P NS). At the peak of biliary SIT secretion, the amount of SIT relative to UC was much greater in bile (40-50% of sterols) than in the whole liver (11% of sterols). Selective biliary secretion of SIT was associated with much greater concentrations of SIT in canalicular membranes than in the interior membranes of the hepatocyte and ha newly-secreted HDL (derived from plasma membranes) compared to newly-secreted VLDL (derived from interior membranes). These results indicate that SIT parallels the transport into bile of UC from lipoproteins (J Biol Chem 260:6511, 1985) and suggests that SIT can be used as a physiologic analog of UC to quantitate the contribution of UC to bile from different kinds of lipid particles.
536 PREFERENTIAL STEROL TRANSPORT FROM HDL TO BILE. S._/J Robins. JM Fasulo. G Salen. and GM Patton. Dept, of Medicine, Boston and East Orange VA Medical Centers, Boston Univ. Sch. of Medicine, Boston, MA, and UMDNJ-NJ, Newark, NJ. Unesterified cholesterol (UC) that is taken up by the liver from the blood is rapidly mixed by exchange with liver UC. Thus, amounts of biliary UC that derive from different kinds of plasma lipoproteins cannot be quantitated. However, plant sterols (PS) do not exchange with UC and are secreted in bile with the same kinetics as UC. To compare the contribution to bile of sterols from different lipoproteins, we used PS as analogs of UC. VLDL, LDL, and HDL were isolated from the plasma of 3 patients with hereditary phytosterolemia. PS comprised 15-20% of total sterols in all lipoprotein fractions. Lipoproteins were separately added to isolated, perfused rat livers to provide approximately equal amounts of PS. In 30-rain recirculating perfusions, the uptake of PS was -10% of perfusate PS from all lipopoteins. However, amounts of PS secreted in bile were markedly different for different lipoproteins, With perfusion of either VLDL or LDL there was no increase in the biliary secretion of PS. In sharp contrast, with perfusion of HDL the secretion of biliary PS was markedly increased (29.9 to 69.1 nmol/h, P < 0.01). The increase in biliary PS was detected 5-10 rain after HDL was added to perfusates and was similarly large for each of 3 individual PS that were tracked. The Fig. compares perfusions with LDL and HDL. Perfusate • Campesterol -^ Peffusate ~.~
"
20 ~ 10 0 0
Sil~stanol
20 to 0 10
20
30 0 to 20 30 Durationof Perfasion (Min) Results show, when net sterol transport from lipoproteins into bile can be determined, only HDL provides a vehicle for UC elimination in bile that is consistent with its putative function in "reverse cholesterol transport".