Leptin Induces the Hepatic High Density Lipoprotein Receptor Scavenger Receptor B Type I (SR-BI) but Not Cholesterol 7α-Hydroxylase (Cyp7a1) in Leptin-deficient (ob/ob) Mice

Cholesterol elimination from the body involves reverse cholesterol transport from peripheral tissues in which the elimination of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by the liver and subsequent biliary excretion as free cholesterol and bile acids are important. In situations of peripheral fat and cholesterol accumulation, such as obesity, these pathways may be overloaded, contributing to increased cholesterol deposition. Leptin has an important role in obesity, suppressing food intake and increasing energy expenditure. This hormone, which is absent in genetically obese ob/ob mice, is also thought to be involved in the coordination of lipid excretion pathways, although available data are somewhat inconsistent. We therefore studied the expression of the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor as well as the rate-limiting enzyme in bile acid synthesis, cholesterol 7α-hydroxylase (Cyp7a1), in leptin-deficient ob/ob mice and their wild-type controls. In ob/ob mice, protein levels of both LDL receptor and SR-BI were reduced, whereas LDL receptor mRNA levels were increased and those of SR-BI were reduced, regardless of challenge with a 2% cholesterol diet. In ob/ob mice, the enzymatic activity and mRNA for Cyp7a1 were reduced, and the increase in response to dietary cholesterol was blunted. Upon short-term (2 days) treatment with leptin, a dose-dependent increase was seen in the SR-BI protein and mRNA, whereas the Cyp7a1 protein and mRNA were reduced. Our findings indicate that leptin is an important regulator of hepatic SR-BI expression and, thus, HDL cholesterol levels, whereas it does not stimulate Cyp7a1 and bile acid synthesis. Cholesterol elimination from the body involves reverse cholesterol transport from peripheral tissues in which the elimination of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by the liver and subsequent biliary excretion as free cholesterol and bile acids are important. In situations of peripheral fat and cholesterol accumulation, such as obesity, these pathways may be overloaded, contributing to increased cholesterol deposition. Leptin has an important role in obesity, suppressing food intake and increasing energy expenditure. This hormone, which is absent in genetically obese ob/ob mice, is also thought to be involved in the coordination of lipid excretion pathways, although available data are somewhat inconsistent. We therefore studied the expression of the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor as well as the rate-limiting enzyme in bile acid synthesis, cholesterol 7α-hydroxylase (Cyp7a1), in leptin-deficient ob/ob mice and their wild-type controls. In ob/ob mice, protein levels of both LDL receptor and SR-BI were reduced, whereas LDL receptor mRNA levels were increased and those of SR-BI were reduced, regardless of challenge with a 2% cholesterol diet. In ob/ob mice, the enzymatic activity and mRNA for Cyp7a1 were reduced, and the increase in response to dietary cholesterol was blunted. Upon short-term (2 days) treatment with leptin, a dose-dependent increase was seen in the SR-BI protein and mRNA, whereas the Cyp7a1 protein and mRNA were reduced. Our findings indicate that leptin is an important regulator of hepatic SR-BI expression and, thus, HDL cholesterol levels, whereas it does not stimulate Cyp7a1 and bile acid synthesis. Leptin is a circulating cytokine-like protein secreted mainly from adipose tissue. It acts to reduce food intake and increase energy expenditure by binding to its receptor in hypothalamic nuclei in the brain (1Friedman J.M. Nutr. Rev. 2002; 60: S1-S14Crossref PubMed Scopus (383) Google Scholar, 2Ahima R.S. Flier J.S. Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1485) Google Scholar). A number of additional functions in immune, reproductive, and hormonal systems have been ascribed to this protein (1Friedman J.M. Nutr. Rev. 2002; 60: S1-S14Crossref PubMed Scopus (383) Google Scholar, 2Ahima R.S. Flier J.S. Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1485) Google Scholar). ob/ob mice, with a mutation in the leptin gene (3Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11722) Google Scholar), display obesity, hypothermia, infertility, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. The increased level of plasma cholesterol in this mouse model is mainly attributed to higher plasma high density lipoprotein (HDL) 1The abbreviations used are: HDL, high density lipoprotein; Cyp7a1, cholesterol 7α-hydroxylase; SR-BI, scavenger receptor class B type I; LDL, low density lipoprotein; VLDL, very low density lipoprotein; C4, 7α-hydroxy-4-cholesten-3-one.1The abbreviations used are: HDL, high density lipoprotein; Cyp7a1, cholesterol 7α-hydroxylase; SR-BI, scavenger receptor class B type I; LDL, low density lipoprotein; VLDL, very low density lipoprotein; C4, 7α-hydroxy-4-cholesten-3-one. cholesterol (4Nishina P.M. Lowe S. Wang J. Paigen B. Metabolism. 1994; 43: 549-553Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Detailed metabolic studies have shown that ob/ob mice have defective HDL plasma clearance and disturbances in the intracellular distribution of cholesterol and its processing in the hepatocyte (5Silver D.L. Jiang X.C. Tall A.R. J. Biol. Chem. 1999; 274: 4140-4146Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 6Silver D.L. Wang N. Tall A.R. J. Clin. Invest. 2000; 105: 151-159Crossref PubMed Scopus (96) Google Scholar). These abnormalities in HDL metabolism are normalized upon treatment with leptin. An increase in plasma apoA-I was found concomitant with a reduced hepatic apoA-I mRNA, further supporting the concept of a reduced hepatic metabolism of HDL in leptin deficiency. A major pathway for the excretion of body cholesterol is through the hepatic conversion of this sterol into bile acids, which is controlled by the activity of the microsomal enzyme cholesterol 7α-hydroxylase (Cyp7a1). Abnormalities of hepatic cholesterol metabolism in ob/ob mice have been reported (7Hyogo H. Roy S. Paigen B. Cohen D.E. J. Biol. Chem. 2002; 277: 34117-34124Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), but the results are not consistent. Thus, increased Cyp7a1 mRNA levels were reported after 2 days of leptin treatment using transcriptional profiling (8Liang C.P. Tall A.R. J. Biol. Chem. 2001; 276: 49066-49076Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), whereas treatment for 2 weeks did not alter Cyp7a1 enzyme activity or bile acid synthesis (7Hyogo H. Roy S. Paigen B. Cohen D.E. J. Biol. Chem. 2002; 277: 34117-34124Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The leptin deficiency in ob/ob mice promotes overeating, leading to weight gain which can be counteracted by prolonged leptin substitution (9Campfield L.A. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3063) Google Scholar, 10Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4219) Google Scholar, 11Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3864) Google Scholar). Prolonged leptin treatment of ob/ob mice may therefore reflect the effects of weight reduction and the consequences thereof, such as e.g. increased insulin sensitivity and reduced hepatic fat accumulation. The present study was undertaken to investigate hepatic cholesterol metabolism in ob/ob mice. We evaluated the effects on cholesterol and bile acid metabolism following challenge with a diet rich in cholesterol, and we assessed the responsiveness to short term leptin treatment at increasing doses. We have found that the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor are suppressed in ob/ob mice. The activity and mRNA for Cyp7a1 were reduced and showed an attenuated response to cholesterol feeding. With leptin treatment, the SR-BI protein and mRNA increased dose-dependently, whereas the Cyp7a1 protein and mRNA were reduced. Animals—C57BL/6J-ob/ob male mice and littermate controls were purchased from Taconic, Ry, Denmark. The animals were allowed to adapt to the animal housing for approximately 2 weeks and fed a standard mouse chow (R36, Lactamin, Kimstad, Sweden). All experiments were started when animals were 10 weeks old. The lights were on between 7 a.m. and 7 p.m. The experiments were approved by the regional animal care and use committee. Treatments—For cholesterol feeding experiments, ob/ob and their wild-type controls were given either standard mouse chow or standard mouse chow supplemented with 2% cholesterol plus 10% oil (w/w) for 7 days. Each of the four groups consisted of four animals. At 9 a.m. the animals were anesthetized by ether inhalation, and blood was collected by cardiac puncture, after which they were killed by cervical dislocation. Livers were removed and frozen in liquid nitrogen. For the leptin treatment experiments, leptin (Peprotech, London, UK) was dissolved in sodium citrate buffer according to the manufacturer's instructions. Leptin was administrated intraperitoneally in a volume of 200 μl at 6 p.m. for 2 days at the indicated doses. Controls received sodium citrate buffer. The experiment was ended on the third day by sacrificing the animals at 10 a.m., 16 h after the last injection. Each group in the leptin treatment experiment consisted of five ob/ob mice, except the control group, which consisted of six ob/ob mice. Plasma Lipids and Lipoproteins—Blood was centrifuged, and the plasma was analyzed for total cholesterol and triglycerides using the IL Test™ cholesterol 181618-10 and triglyceride 181610-60 kits (IL Scandinavia, Gothenburg, Sweden) on the Monark 2000 system (IL Scandinavia, Gothenburg, Sweden). Lipoprotein cholesterol profiles were obtained by separation of 10 μl of plasma using a micro fast protein liquid chromatography system for the generation of lipoprotein profiles as described elsewhere (12Gullberg H. Rudling M. Forrest D. Angelin B. Vennstrom B. Mol. Endocrinol. 2000; 14: 1739-1749Crossref PubMed Google Scholar). Plasma Leptin and Insulin—Plasma leptin levels were determined by a mouse leptin enzyme-linked immunosorbent assay kit (R&D Systems, Oxon, UK) according to the manufacturer's instructions. Plasma insulin levels were analyzed using a rodent insulin RIA kit according to manufacturer's instructions (Linco, St. Charles, MI). Plasma 7α-Hydroxy-4-cholesten-3-one Assay—As an indirect measurement of cholesterol-7α-hydroxylase activity, the plasma levels of 7α-hydroxy-4-cholesten-3-one (C4) were analyzed from pooled plasma samples by high pressure liquid chromatography as described elsewhere (13Galman C. Arvidsson I. Angelin B. Rudling M. J. Lipid Res. 2003; 44: 859-866Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Liver Preparations—Pooled livers (0.5 g) were homogenized in 50 mm Tris, pH 6.8, 2 mm CaCl2, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 0.5 mm leupeptin. Samples were sonicated for 15 s and then centrifuged at 14,000 rpm for 10 min. The supernatant was centrifuged in an Airfuge® (Beckman Coulter AB, Bromma, Sweden) for 7 min, aliquoted, and stored at –80 °C until further analysis. Protein concentrations were determined with Lowry DC kit (Bio-Rad). All work was performed on ice. Protein Blot Analysis—LDL receptor expression was assayed using liver membrane proteins that were electrophoresed under non-reducing conditions on 6% SDS-PAGE (14Rudling M. Norstedt G. Olivecrona H. Reihner E. Gustafsson J.A. Angelin B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6983-6987Crossref PubMed Scopus (235) Google Scholar). Proteins were transferred to nitrocellulose membranes and incubated with 125I-labeled rabbit β-VLDL. The LDL receptor bands (≈120 kDa) were analyzed and quantified using a Fuji BAS 1800 analyzer (Fuji Photo Film Co.) and Image Gauge software (Science Lab, 98, version 3.12, Fuji Photo Film Co.). SR-BI was assayed by immunoblot using liver membrane proteins and a rodent polyclonal antibody (Novus Biologicals Inc., Littleton, CO), as described previously (15Galman C. Angelin B. Rudling M. Endocrinology. 2002; 143: 1809-1816Crossref PubMed Scopus (15) Google Scholar). The Cyp7a1 antibody was generated by immunizing rabbits with the peptide NH2-KLH-Cys-Tyr-Lys-Leu-Lys-His-COOH, as used previously (16Brassil P.J. Edwards R.J. Davies D.S. Biochem. Pharmacol. 1995; 50: 311-316Crossref PubMed Scopus (17) Google Scholar), corresponding to the last five amino acids in the mouse and rat Cyp7a1 proteins. The peptide was coupled to an affinity column to purify Cyp7a1 antibodies from rabbit serum. A Cyp7a1 Western blot was performed on hepatic microsomal proteins prepared as described previously (17Einarsson K. Angelin B. Ewerth S. Nilsell K. Bjorkhem I. J. Lipid Res. 1986; 27: 82-88Abstract Full Text PDF PubMed Google Scholar). In brief, 25 μg of microsomal protein was electrophoresed on a 10% Criterion Tris-HCl gel (Bio-Rad) and then transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked for 1 h at room temperature in blocking solution (phosphate-buffered saline, 0.2% Tween, and 5% fat-free dry milk powder) and thereafter probed with the Cyp7a1 antibody (2 μg/ml) in blocking solution for 2 h at room temperature. After extensive washing, the membrane was incubated with a horseradish peroxidase-conjugated sheep anti-rabbit antibody (The Binding Site Ltd., Birmingham, UK) in phosphate-buffered saline plus 0.2% Tween for 1 h at room temperature followed by washing in phosphate-buffered saline plus 0.2% Tween. Supersignal (Pierce) was used to develop the activity was assayed in hepatic microsomal membranes as described previously (17Einarsson K. Angelin B. Ewerth S. Nilsell K. Bjorkhem I. J. Lipid Res. 1986; 27: 82-88Abstract Full Text PDF PubMed Google Scholar). was from frozen livers with according to the manufacturer's instructions. The was with μg of total was incubated with for at the enzyme was then at °C for 15 min, and, after the of solution at 25 °C for 10 min. For synthesis, μg of total was synthesis was with according to the manufacturer's was performed with the assay on an system following the in the 2 on the An assay was used as The and used were as and are as The of were evaluated using Plasma Lipids and Lipoprotein of ob/ob I plasma total cholesterol and triglyceride concentrations with body at the of the experiment when ob/ob mice and controls were fed either a diet or 2% cholesterol for 7 days. (4Nishina P.M. Lowe S. Wang J. Paigen B. Metabolism. 1994; 43: 549-553Abstract Full Text PDF PubMed Scopus (101) Google Scholar, D.L. Jiang X.C. Tall A.R. J. Biol. Chem. 1999; 274: 4140-4146Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the ob/ob mice were which was to increased LDL and HDL levels of cholesterol feeding on total cholesterol and triglycerides in plasma of ob/ob mice to their 2% from 2% from from in a (12Gullberg H. Rudling M. Forrest D. Angelin B. Vennstrom B. Mol. Endocrinol. 2000; 14: 1739-1749Crossref PubMed Google Scholar), cholesterol feeding to control mice increased and density lipoprotein cholesterol but reduced HDL when ob/ob mice were fed the diet they with a of cholesterol in of LDL and SR-BI in ob/ob the increased LDL and HDL cholesterol seen in ob/ob mice be at in to a reduced expression of the hepatic for LDL and HDL M. Annu. Rev. Biochem. 1999; PubMed Scopus Google Scholar, B. A. M. 2000; PubMed Scopus Google Scholar, J.L. Science. 1986; PubMed Scopus Google Scholar), we the expression of the LDL receptor in the liver by blot assay with 125I-labeled rabbit on liver membranes from ob/ob and control mice The LDL receptor expression was reduced in both groups of ob/ob mice as with controls In both controls and ob/ob mice, the expression of LDL to increase in response to cholesterol feeding. of the mRNA expression of the LDL receptor with that the were in the ob/ob mice although both groups with a reduction when fed cholesterol The expression of the hepatic HDL receptor, SR-BI, was thereafter analyzed by Western blot of liver membranes was a reduction of the SR-BI protein in the livers of ob/ob mice with the control mice, and cholesterol feeding did not alter this and the hepatic mRNA levels of SR-BI were reduced in ob/ob mice both on the and the diet Cyp7a1 to Cholesterol in ob/ob the of bile acids from hepatic cholesterol cholesterol we the rate-limiting enzyme in the bile acid synthesis 2 the enzyme for control and ob/ob mice on and 2% cholesterol A Cyp7a1 response be found in the control the activity increased by following dietary in the ob/ob livers this response of Cyp7a1 to cholesterol was ob/ob groups also their control group This was when the mRNA levels of Cyp7a1 were that this in the ob/ob mice is at the transcriptional with Leptin of ob/ob then to investigate the metabolic effects of leptin treatment in the of weight of the animals. For this we a experiment in which ob/ob animals received day for 2 with from to μg of body weight injection. the of the animal body did not between the and the Plasma total cholesterol or triglycerides were not by leptin treatment, which was with fast protein liquid chromatography in which was found in of the lipoprotein The plasma levels of leptin in the animals increased with increasing of leptin the activity of the leptin, we then analyzed plasma insulin levels, studies have shown that leptin the peripheral insulin sensitivity in ob/ob mice, leading to plasma insulin levels (1Friedman J.M. Nutr. Rev. 2002; 60: S1-S14Crossref PubMed Scopus (383) Google Scholar, L.A. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3063) Google Scholar, 10Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4219) Google Scholar, 11Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3864) Google Scholar). It was found that plasma insulin levels decreased with increasing leptin at higher plasma levels of leptin, were effects on plasma insulin with Leptin of SR-BI but a Western blot of SR-BI on liver membranes from the ob/ob mice with increasing of leptin. The hepatic SR-BI protein expression well to the plasma insulin levels A and that the mRNA levels of liver SR-BI were also to a as the SR-BI protein levels B and short-term treatment with leptin the hepatic SR-BI expression in ob/ob mice under the cholesterol feeding experiments, it was that ob/ob mice have a Cyp7a1 activity, to a reduced of the leptin treatment has been reported to increase the Cyp7a1 mRNA levels in ob/ob mice (8Liang C.P. Tall A.R. J. Biol. Chem. 2001; 276: 49066-49076Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), we the effects of leptin on bile acid synthesis in this we analyzed the plasma levels of C4, which is a of hepatic Cyp7a1 activity (13Galman C. Arvidsson I. Angelin B. Rudling M. J. Lipid Res. 2003; 44: 859-866Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). we not increased levels, but a reduced in groups with leptin A was when the hepatic microsomal Cyp7a1 protein was assayed by Western blot The level of Cyp7a1 protein expression was in the ob/ob mice. of hepatic mRNA from these ob/ob animals showed a reduction or of the mRNA levels of Cyp7a1 Thus, it is that leptin treatment does not but Cyp7a1 when to ob/ob mice in a The of cholesterol and lipoprotein metabolism in animal of is The of abnormalities in response to insulin fat in the and increased food intake through with the for and the of studies in The leptin-deficient ob/ob mouse is a model for the of leptin. The present work abnormalities in hepatic cholesterol metabolism in ob/ob mice, of which are by leptin. The ob/ob mice increased plasma cholesterol as the mainly of an increase in but also in with the ob/ob mice a reduced expression of hepatic LDL findings have been seen in Angelin B. Rudling M. Endocrinology. PubMed Scopus Google and mice, T. B. and M. which display leptin receptor Y. L. R.L. Science. PubMed Scopus Google Scholar, M. T. K. A. M. K. Biochem. Res. PubMed Scopus Google Scholar, P.J. PubMed Scopus Google Scholar, I. Y. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the of LDL receptor in following dietary cholesterol challenge S. F. M. A. L.A. P.J. PubMed Scopus (69) Google Scholar, M. Angelin B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google was in ob/ob mice, whereas mRNA levels for the LDL receptor were higher in ob/ob mice and reduced by cholesterol challenge to a as that found in wild-type controls. The of the protein has been shown to be increased in ob/ob mice with type whereas that of is not I. Y. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Our of a increase in LDL receptor mRNA levels is with the of I. Y. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google and may indicate a between mRNA levels and protein expression for the LDL receptor, of of the LDL receptor M. Angelin B. J. Clin. Invest. PubMed Scopus Google Scholar, A. R.L. T. J.L. J. Lipid Res. Full Text PDF PubMed Google Scholar). by Western blot and of hepatic SR-BI expression we found a in ob/ob mice with wild-type animals. This is in with the increase in plasma HDL cholesterol and is also in with the results reported by D.L. Wang N. Tall A.R. J. Clin. Invest. 2000; 105: 151-159Crossref PubMed Scopus (96) Google decreased and lipid from HDL in ob/ob In a (5Silver D.L. Jiang X.C. Tall A.R. J. Biol. Chem. 1999; 274: 4140-4146Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), these were not to in hepatic SR-BI expression between ob/ob and wild-type livers using Western and blot analysis. We have for this but have results in in mice we have found that the hepatic expression of SR-BI is reduced. B. and M. After 2 days of leptin treatment was a very dose-dependent increase in hepatic SR-BI both as protein and as The in plasma of insulin sensitivity, the increase in This experiment further the concept that SR-BI is to leptin deficiency. we that is by we the data as further for the concept that SR-BI is an important for the increased HDL cholesterol in ob/ob mice. It is that the in SR-BI expression were at an when the in HDL cholesterol be Thus, in an additional experiment a of five ob/ob mice were μg of body weight for a reduction of total plasma cholesterol was to which is in with the results of (5Silver D.L. Jiang X.C. Tall A.R. J. Biol. Chem. 1999; 274: 4140-4146Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Cyp7a1 enzyme activity and mRNA were reduced in ob/ob mice, and was a Cyp7a1 response to dietary we found that Cyp7a1 was reduced in response to leptin treatment in ob/ob mice by using of Cyp7a1 mRNA, and plasma These experiments were performed at a when was in body weight between the and the of was not using in with followed by and Tall have reported an increase in Cyp7a1 mRNA after 2 days of leptin treatment of ob/ob mice (8Liang C.P. Tall A.R. J. Biol. Chem. 2001; 276: 49066-49076Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). the in with Cyp7a1 activity and bile acid excretion were not by leptin treatment for when weight reduction was Thus, it that leptin is a regulator of insulin sensitivity by leptin be an to insulin has been shown to Cyp7a1 D. M. M. G. J. Lipid Res. Full Text PDF PubMed Google Scholar, J. P. 1995; Google Scholar). The hormonal of Cyp7a1 in to be and further studies on the of leptin on bile acid metabolism are