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• W2023379280 abstract "Leptin administration to obese C57BL/6J (ob/ob) mice results in weight loss by reducing body fat. Because adipose tissue is an important storage depot for cholesterol, we explored evidence that leptin-induced weight loss inob/ob mice was accompanied by transport of cholesterol to the liver and its elimination via bile. Consistent with mobilization of stored cholesterol, cholesterol concentrations in adipose tissue remained unchanged during weight loss. Plasma cholesterol levels fell sharply, and microscopic analyses of gallbladder bile revealed cholesterol crystals as well as cholesterol gallstones. Surprisingly, leptin reduced biliary cholesterol secretion rates without affecting secretion rates of bile salts or phospholipids. Instead, cholesterol supersaturation of gallbladder bile was due to marked decreases in bile salt hydrophobicity and not to hypersecretion of biliary cholesterolper se, such as occurs in humans during weight loss. In addition to regulating bile salt composition, leptin treatment decreased bile salt pool size. The smaller, more hydrophilic bile salt pool was associated with substantial decreases in intestinal cholesterol absorption. Within the liver, leptin treatment reduced the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, but it did not change activities of cholesterol 7α-hydroxylase or acyl-CoA:cholesterol acyltransferase. These data suggest that leptin regulates biliary lipid metabolism to promote efficient elimination of excess cholesterol stored in adipose tissue. Cholesterol gallstone formation during weight loss in ob/ob mice appears to represent a pathologic consequence of an adaptive response that prevents absorption of biliary and dietary cholesterol. Leptin administration to obese C57BL/6J (ob/ob) mice results in weight loss by reducing body fat. Because adipose tissue is an important storage depot for cholesterol, we explored evidence that leptin-induced weight loss inob/ob mice was accompanied by transport of cholesterol to the liver and its elimination via bile. Consistent with mobilization of stored cholesterol, cholesterol concentrations in adipose tissue remained unchanged during weight loss. Plasma cholesterol levels fell sharply, and microscopic analyses of gallbladder bile revealed cholesterol crystals as well as cholesterol gallstones. Surprisingly, leptin reduced biliary cholesterol secretion rates without affecting secretion rates of bile salts or phospholipids. Instead, cholesterol supersaturation of gallbladder bile was due to marked decreases in bile salt hydrophobicity and not to hypersecretion of biliary cholesterolper se, such as occurs in humans during weight loss. In addition to regulating bile salt composition, leptin treatment decreased bile salt pool size. The smaller, more hydrophilic bile salt pool was associated with substantial decreases in intestinal cholesterol absorption. Within the liver, leptin treatment reduced the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, but it did not change activities of cholesterol 7α-hydroxylase or acyl-CoA:cholesterol acyltransferase. These data suggest that leptin regulates biliary lipid metabolism to promote efficient elimination of excess cholesterol stored in adipose tissue. Cholesterol gallstone formation during weight loss in ob/ob mice appears to represent a pathologic consequence of an adaptive response that prevents absorption of biliary and dietary cholesterol. high density lipoprotein acyl-CoA:cholesterol acyltransferase cholesterol 7α-hydroxylase 3-hydroxy-3-methylglutaryl-CoA reductase high performance liquid chromatography low density lipoprotein very low density lipoprotein Bile is the route for cholesterol elimination from the body, and reverse cholesterol transport is the metabolic pathway for movement of cholesterol from peripheral tissues to the liver for biliary secretion (1Glomset J.A. J. Lipid Res. 1968; 9: 155-167Abstract Full Text PDF PubMed Google Scholar). Consistent with their central role in reverse cholesterol transport, high density lipoproteins (HDL)1 are the principal source of biliary cholesterol (2Carey M.C. Gut. 1997; 41: 721-722Crossref PubMed Scopus (17) Google Scholar, 3Ji Y. Wang N. Ramakrishnan R. Sehayek E. Huszar D. Breslow J.L. Tall A.R. J. Biol. Chem. 1999; 274: 33398-33402Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In response to biliary secretion of detergent-like bile salt molecules, HDL-derived cholesterol is secreted from hepatocytes into bile together with phospholipid molecules as vesicles (4Cohen D.E. Curr. Opin. Lipidol. 1999; 235: 111-120Google Scholar). In leptin-deficient obese C57BL/6J (ob/ob) mice, elevated plasma cholesterol levels are due to increased HDL concentrations (5Nishina P.M. Lowe S. Wang J. Paigen B. Metabolism. 1994; 43: 549-553Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Silver et al. (6Silver 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) have demonstrated that defective clearance of HDL particles from plasma by the liver in these animals is reversed by leptin administration. Moreover, hepatocytes cultured fromob/ob mice display alterations in HDL processing and cellular cholesterol distribution, which are also normalized by leptin (7Silver D.L. Wang N. Tall A.R. J. Clin. Invest. 2000; 105: 151-159Crossref PubMed Scopus (96) Google Scholar). We have reported abnormalities in biliary lipid secretion in Zucker (fa/fa) rats (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar), which become obese because of a missense mutation in the extracellular domain of the leptin receptor that sharply reduces responsiveness to leptin. Although bile salt secretion rates were preserved, biliary secretion rates of cholesterol and phospholipids were severely reduced. Acute (6 h) infusions of leptin at high doses partially restored biliary cholesterol secretion, and the same treatment in lean Zucker (Fa/−) rats promoted hypersecretion of biliary cholesterol (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar). Taken together, these observations suggest that leptin may promote biliary elimination of plasma cholesterol. In ob/ob mice, the expanded adipose tissue mass represents an important storage depot for cholesterol (9Angel A. Farkas J. J. Lipid Res. 1974; 15: 491-499Abstract Full Text PDF PubMed Google Scholar). Because chronic leptin administration to these animals reduces adiposity (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 (4256) 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 (3884) Google Scholar), excess cholesterol must be mobilized for delivery to the liver and secretion into bile. This study was designed to elucidate a regulatory role for leptin in hepatic cholesterol elimination during leptin-induced weight loss in ob/ob mice. Our results reveal that leptin administration leads to a marked increase in the proportion of hydrophilic bile salts in bile, as well as a sharp decline in the size of the circulating bile salt pool. These changes mechanistically account for reduced intestinal cholesterol absorption, which inhibits both assimilation of dietary cholesterol and reabsorption of biliary cholesterol. However, the reduced capacity of hydrophilic bile salts to solubilize cholesterol within the gallbladder results in cholesterol crystallization and gallstone formation. Recombinant murine leptin was a gift from Amgen (Thousand Oaks, CA). [4-14C]Cholesterol (50 mCi/mmol), [5,6-3H]β-sitostanol (50 Ci/mmol), and oleoyl-[1-14C]CoA (55 mCi/mmol) were obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO).dl-Hydroxy-[3-14C]methylglutaryl-CoA (57 mCi/mmol), dl-[5-3H]mevalonolactone (58 Ci/mmol), and [14C]cholesterol (49 mCi/mmol) were purchased from PerkinElmer Life Sciences. Cholesteryl-[1α, 2α-3H]oleate (24 Ci/mmol) was purchased from AmershamBiosciences. Aluminum and glass silica gel plates were purchased from EM Science (Gibbstown, NJ). The general chemical reagents were obtained from Sigma unless otherwise specified. Male 8-week-old C57BL/6J mice that were homozygous for theob mutation were obtained from The Jackson Laboratory (Bar Harbor, ME). The animals were maintained in a temperature-controlled room with 12-h day-night cycles (6 a.m. to 6 p.m. light) and were allowed to adapt to the environment for 2 weeks prior to the experiments. The mice were fed a chow diet (LabDiet 5001, PMI Nutrition International Inc, Brentwood, MO) that contained 4.5% fat and <0.02% cholesterol. Starting at 10 weeks of age, ob/ob mice (n = 80) were treated once daily with intraperitoneal injections of leptin dissolved in saline (10 μg/g of body weight) or an equal volume of saline. To achieve isocaloric intake, saline-injected mice were pair-fed to animals that were administered leptin. The mice were anesthetized with intraperitoneal injections of 87 mg/kg ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and 13 mg/kg xylazine (Lloyd Laboratories, Shenandoah, IA). Surgery commenced at 9 a.m. with a midline abdominal incision. After inspecting the gallbladder for the presence of gallstones, the common bile duct was ligated with silk sutures. Bile flow was diverted for collection by inserting a PE-10 polyethylene catheter (Becton Dickinson Primary Care Diagnostics, Becton Dickinson, Sparks, MD) into the gallbladder and securing it with silk sutures. The cannula was externalized, and the abdominal incision was closed. The first ∼10 μl containing concentrated gallbladder bile was collected onto a glass microscopy slide for microscopic analysis to determine the presence of cholesterol crystals (12Wang D.Q.-H. Carey M.C. J. Lipid Res. 1996; 37: 606-630Abstract Full Text PDF PubMed Google Scholar). Thereafter, hepatic bile was collected by gravity into preweighed Eppendorf tubes for 2-h periods. Bile volume was determined gravimetrically assuming a density of 1 g/ml. At the end of the experiment, the mice were euthanized by cardiac puncture. The livers were immediately excised, rinsed with 0.15 m NaCl to remove blood, weighed, and snap frozen in liquid nitrogen. Samples of visceral and peripheral fat were also excised and snap frozen in liquid nitrogen. The tissue samples were stored at −80 °C prior to analyses. The blood was anticoagulated with EDTA, and plasma was separated by centrifugation and maintained at 4 °C for analysis within 24 h. During surgery and hepatic bile collections, the body temperature was maintained at 37 ± 0.5 °C with a heat lamp. These procedures were approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine. Bile salt pool size was determined essentially as described by Mardones et al. (13Mardones P. Quinones V. Amigo L. Moreno M. Miquel J.F. Schwarz M. Miettinen H.E. Trigatti B. Krieger M. VanPatten S. Cohen D.E. Rigotti A. J. Lipid Res. 2001; 42: 170-180Abstract Full Text Full Text PDF PubMed Google Scholar) with minor modifications. Briefly, the mice were anesthetized as described above. The liver, gallbladder, common bile duct, and small intestine were then harvested and used to prepare ethanolic extracts. Prior to extraction, glycocholate was added as an internal recovery standard. Bile salt masses and compositions were determined by HPLC (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar). Cholesterol absorption was measured by a fecal dual isotope ratio method (14Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, a mixture of 1 μCi of [4-14C]cholesterol and 2 μCi of [5,6-3H]β-sitostanol in 150 μl of medium-chain triglyceride oil (Mead Johnson Nutritionals, Evansville, IN) was delivered by intragastric gavage. The mice were housed individually in cages, and the stools were collected daily for 7 days. The stools were dried, weighed, and ground into powdered form. Radiolabeled sterols were extracted from 1 g of stool samples. The percentage of cholesterol absorbed was calculated from the14C/3H ratio in the extracted sterol mixture (14Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). The fecal bile salts were quantified enzymatically (15Mashige F. Tanaka N. Maki A. Kamei S. Yamanaka M. Clin. Chem. 1981; 27: 1352-1356Crossref PubMed Scopus (260) Google Scholar) following extraction into t-butanol (16van der Meer R. de Vries H. Glatz F.C.J. Benyen A.C. Geeden M.J.H. Katan M.B. Schouten J.A. Cholesterol Metabolism in Health and Disease. Ponsen and Looyen, Wageningen, The Netherlands1985Google Scholar). Plasma cholesterol and triglyceride concentrations were determined by enzymatic assays using reagents from Sigma and Roche Molecular Biochemicals, respectively. The plasma lipoproteins were fractionated by fast performance liquid chromatography using a Superose 6 HR10/30 column (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar). The cholesterol concentrations in fractions (0.3 ml) were determined in individual wells of a 96-well microtiter plate by mixing 150 μl of each fraction plus 200 μl of cholesterol reagent (Sigma). The color was analyzed using a Titertek Multiskan Plus microplate reader (Eflab, Helsinki, Finland) set to 492 nm. Plasma concentrations of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and HDL cholesterol were calculated as products of total plasma cholesterol concentrations and relative fast performance liquid chromatography peak areas of respective lipoprotein fractions (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar). Hepatic and adipose tissue contents of triglycerides and total as well as free cholesterol were quantified as described previously (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar, 17Carr T.P. Andersen C.J. Rudel L.L. Clin. Biochem. 1993; 26: 39-42Crossref PubMed Scopus (485) Google Scholar). Biliary cholesterol concentrations were determined using the same enzymatic method as for plasma. Biliary bile salt concentrations and compositions were determined by HPLC (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar) utilizing glycocholate as an internal standard. The bile salt hydrophobic index was determined according to Heuman (18Heuman D.M. J. Lipid Res. 1989; 30: 719-730Abstract Full Text PDF PubMed Google Scholar). Phospholipid concentrations in bile were determined by an inorganic phosphorus procedure (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar). The molecular species of phosphatidylcholines in bile were quantified by HPLC (19Cohen D.E. Carey M.C. J. Lipid Res. 1991; 32: 1291-1302Abstract Full Text PDF PubMed Google Scholar,20Patton G.M. Robins S.J. Kuksis A. Chromatography Library: Lipids in Biomedical Research and Clinical Diagnosis. Elsevier, Amsterdam, The Netherlands1987: 311-347Google Scholar). The biliary secretion rates of cholesterol, phospholipid, and bile salts (nmol/h) were calculated as products of lipid concentrations and bile flow. Hepatic microsomes were prepared by differential ultracentrifugation (21Shefer S. Salen G. Batta A.K. Fears R. Sabine J.R. Methods of Assay. CRC Press, Boca Raton, FL1986: 43-49Google Scholar) and stored at −80 °C. The microsomal protein concentrations were determined according to the Bradford method (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using a Bio-Rad protein assay reagent and bovine serum albumin as a standard. Hepatic microsomes were used to measure enzyme activities as follows. Activity of HMG-CoA reductase was determined according to Shapiro and Rodwell (23Shapiro D.J. Rodwell V.W. Biochem. Biophys. Res. Commun. 1969; 37: 867-872Crossref PubMed Scopus (110) Google Scholar). Briefly, the microsomes (1 mg of protein) were incubated with 7.5 nmol (0.33 GBq/nmol) of [3-14C]HMG-CoA, 4.5 μmol of glucose-6-phosphate, 3.6 μmol of EDTA, 0.45 μmol of NADP, 0.3 IU of glucose-6-phosphate dehydrogenase for 15 min at 37 °C. [3H]Mevalonic acid (0.024 GBq) used as an internal recovery standard was added to stop the reaction. Unlabeled mevalonate (1.2 mg/ml) was added to assist with recovery. The samples were further incubated for 30 min at 37 °C to allow for conversion of mevalonic acid to mevalonolactone. After incubation, the microsomal protein was precipitated by centrifugation for 1 min, and an aliquot of the supernatant (100 μl) was applied to aluminum silica gel TLC plates. The plates were developed in acetone-benzene (1:1 v/v) and then subjected to autoradiography. The area containing mevalonate (Rf = 0.6–0.9) was scraped and quantified by liquid scintillation counting using Ecolume (ICN Radiochemicals, Irvine, CA). HMG-CoA reductase activity was expressed as pmol of [14C]mevalonate produced per min per mg of microsomal protein. Recoveries of [3H]mevalonate ranged from 60 to 90%. Activity of Cyp7A1 was measured according to Jelineket al. (24Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Abstract Full Text PDF PubMed Google Scholar). [14C]Cholesterol was used as a substrate and delivered as cholesterol-phosphatidylcholine liposomes (1:8 by weight) that were prepared by sonication. An NADPH-regenerating system (glucose-6-phosphate dehydrogenase, NADP, and glucose-6-phosphate) was included in the assay as a source of NADPH. After addition of glucose-6-phosphate dehydrogenase (0.3 IU), samples containing 1 mg of microsomal protein were incubated for 30 min at 37 °C. The reaction was stopped by addition of 5 ml of chloroform-methanol (2:1 v/v) and 1 ml of 0.05% sulfuric acid. The lower phase was dried under nitrogen, redissolved in chloroform, and then applied to glass silica gel TLC plates together with 7α- and 7β-hydroxycholesterol standards. TLC plates were developed with ethyl acetate-toluene (3:2 v/v), exposed to iodine vapor to identify standards, and then subjected to autoradiography overnight using XAR-5 film (Kodak). Using the developed film as a guide, the locations of [14C]7α-hydroxycholesterol spots were determined, scraped, and quantified by liquid scintillation counting as described above. Hepatic Acat activity was measured by incorporation of [14C]oleoyl-CoA into cholesteryl esters in hepatic microsomes according to Smith et al. (25Smith J.L. de Jersey J. Pillay S.P. Hardie I.R. Clin. Chim. Acta. 1986; 158: 271-282Crossref PubMed Scopus (46) Google Scholar). Microsomes (1 mg of protein) were preincubated at a final volume of 180 μl with albumin (84 mg/ml) in buffer (50 mmKH2PO4, 100 mm sucrose, 50 mm KCl, 50 mm NaCl, 30 mm EDTA, 2 mm dithiothreitol, pH 7.2) for 5 min at 37 °C. This was followed by the addition of 20 μl of oleoyl [1-14C]coenzyme A (0.15 GBq/pmol). The reaction was continued for 15 min at 37 °C and then stopped by the addition of 2.5 ml of chloroform-methanol (2:1 v/v). After adding [3H]cholesteryl oleate (0.045 GBq) as an internal standard, the reaction mixture was extracted overnight using 2.5 ml of chloroform-methanol (2:1 v/v) and 1 ml of acidified water. The lower phase was then dried under nitrogen, resuspended in 150 μl of chloroform containing 30 μg of unlabeled cholesteryl oleate, and applied to a glass silica gel TLC plate. The plates were developed in hexane-diethyl ether (9:1 v/v). Cholesteryl oleate was visualized using iodine vapor, scraped from the plate, and quantified by liquid scintillation counting as described above. Recoveries of [3H]cholesteryl oleate ranged from 70 to 90%. The data are expressed as means ± S.E. The statistical significance of the differences between means of the experimental groups was tested by Student’s t test. A difference was considered statistically significant for a two-tailed p< 0.05. Fig. 1 shows trends in body weight during treatment with saline or leptin (10 μg/g). Consistent with well established effects of leptin on body weight in ob/obmice (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 (4256) 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 (3884) Google Scholar), we observed progressive weight loss over the 28-day treatment period (Fig. 1). Mice that were treated with saline lost weight because of pair feeding: Their dietary intake was restricted by 70–80% during the first 7 days and by 40–60% thereafter. For both groups of mice, weight loss was most rapid during the first 14 days. The final body weights (means ± S.E.) were 43.6 ± 0.3 g following saline treatment and 33.6 ± 0.3 g following leptin treatment. Table I presents lipid concentrations in hepatic bile expressed in absolute (mm) and relative (mol %) terms. Because lean body mass and water content of ob/obmice do not change under the experimental conditions of this experiment (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 (4256) 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 (3884) Google Scholar), secretion rates of biliary lipids (nmol/h) in Table I, as well as bile salt pool size (μmol) and fecal bile salt excretion rates (μmol/d) (see below) are presented without normalization to body weight (26Bennion L.J. Grundy S.M. J. Clin. Invest. 1975; 56: 996-1011Crossref PubMed Scopus (269) Google Scholar). In the hepatic bile of saline-treated mice, we observed increases in both absolute and relative cholesterol concentrations at 14 and 28 days. These concentrations were increased to a lesser extent in leptin-treated mice at 14 days, and at 28 days, absolute and relative cholesterol concentrations fell to and below base line, respectively. Absolute concentrations of biliary phospholipids rose to similar extents in leptin- and saline-treated mice at 14 days and decreased to base-line values at 28 days, whereas the relative concentrations did not change. No differences were observed in either absolute or relative biliary bile salt concentrations. Whereas the biliary secretion rates of bile salts and phospholipids decreased to the same degree in leptin- and saline-treated mice, the cholesterol secretion rates decreased only with leptin treatment.Table IBiliary lipid compositions and secretion ratesDayConcentration[TL]Moles percentSecretion rateChPLBSChPLBSChPLBSmmg/dl%nmol/h 00.63 ± 0.031-aThe values are the means ± S.E. and were determined from at least five mice/group at each time point.7.21 ± 0.7620.63 ± 1.381.60 ± 0.112.28 ± 0.1325.03 ± 1.5972.69 ± 1.51105 ± 131155 ± 1473346 ± 361Saline 141.47 ± 0.121-bp <0.05, compared with base line.11.16 ± 1.331-bp <0.05, compared with base line.27.26 ± 3.312.24 ± 0.213.57 ± 0.211-bp <0.05, compared with base line.23.03 ± 2.0173.40 ± 2.13117 ± 10769 ± 1011-bp <0.05, compared with base line.2477 ± 2911-bp <0.05, compared with base line. 280.90 ± 0.121-bp <0.05, compared with base line.7.64 ± 0.8421.19 ± 2.901.68 ± 0.214.10 ± 0.661-bp <0.05, compared with base line.25.74 ± 1.1170.16 ± 1.49112 ± 21720 ± 861-bp <0.05, compared with base line.1987 ± 2791-bp <0.05, compared with base line.Leptin (10 μg/g) 140.98 ± 0.121-bp <0.05, compared with base line.1-cp <0.05, compared with saline treatment.10.13 ± 0.381-bp <0.05, compared with base line.23.34 ± 2.081.98 ± 0.112.53 ± 0.211-cp <0.05, compared with saline treatment.26.94 ± 1.7270.53 ± 1.8769 ± 121-bp <0.05, compared with base line.1-cp <0.05, compared with saline treatment.679 ± 1001-bp <0.05, compared with base line.1705 ± 2841-bp <0.05, compared with base line. 280.57 ± 0.031-cp <0.05, compared with saline treatment.7.98 ± 0.1921.18 ± 0.541.68 ± 0.041.93 ± 0.061-bp <0.05, compared with base line.1-cp <0.05, compared with saline treatment.26.85 ± 0.3971.22 ± 0.3653 ± 31-bp <0.05, compared with base line.1-cp <0.05, compared with saline treatment.737 ± 341-bp <0.05, compared with base line.1961 ± 1141-bp <0.05, compared with base line.Ch, cholesterol; PL, phospholipid; BS, bile salt; [TL], total lipid concentration.1-a The values are the means ± S.E. and were determined from at least five mice/group at each time point.1-b p <0.05, compared with base line.1-c p <0.05, compared with saline treatment. Open table in a new tab Ch, cholesterol; PL, phospholipid; BS, bile salt; [TL], total lipid concentration. Microscopic analyses of gallbladder bile from ob/ob mice (n = 14) prior to saline or leptin treatment revealed that none contained cholesterol crystals or gallstones. However, following the period of rapid weight loss at 14 days (Fig. 1), abundant cholesterol monohydrate crystals and cholesterol gallstones were detected in 8 of 18 (44%) and 2 of 18 (11%) of leptin-treated mice, respectively. In mice treated with saline (n = 18), no cholesterol crystals or gallstones were observed. These findings were unchanged at 28 days. To gain mechanistic insights into the physicochemical events observed by microscopic analysis of gallbladder bile, we analyzed the molecular species of bile salts in mouse hepatic biles (Fig.2A). As described previously for rat bile, HPLC resolved five major bile salt species comprising >90% of biliary bile salts, with tauro-α-, tauro-β-, and tauro-ω-muricholate eluting in one peak (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar). Leptin treatment was associated with substantial increases in the proportions of tauromuricholates and decreases in the proportions of taurocholate. In saline-treated mice, similar trends were observed, but they were much less pronounced. At 14 days, leptin also reduced the contents of the hydrophobic bile salts taurochenodeoxycholate and taurodeoxycholate. Fig. 2B displays the hydrophobic index of bile salts. The hydrophobic index is a concentration-weighted average of HPLC-determined hydrophobicities of individual bile salts present in a mixture (18Heuman D.M. J. Lipid Res. 1989; 30: 719-730Abstract Full Text PDF PubMed Google Scholar). This parameter allows the overall hydrophobicity of a mixture of bile salts to be represented by a single value. The bile salt hydrophobic index decreased markedly in leptin-treated mice compared with saline-treated mice. HPLC resolved nine major peaks corresponding to ten phosphatidylcholine molecular species that accounted for >95% of phosphatidylcholines (8VanPatten S. Ranginani N. Shefer S. Nguyen L.B. Rossetti L. Cohen D.E. Am. J. Physiol. 2001; 281: G393-G404Crossref PubMed Google Scholar). Compared with saline treatment at 28 days, leptin increased the proportion (mol %, means ± S.E.) of 16:0–18:2 (saline, 55.5 ± 0.5; leptin, 60.5 ± 0.4) phosphatidylcholine molecular species in bile. Decreases were observed in the proportions of 16:1–16:1 (saline, 0.76 ± 0.06; leptin, 0.41 ± 0.04), 16:1–20:4 (saline, 5.73 ± 0.31; leptin, 2.86 ± 0.26), 16:0–20:4 (saline, 9.87 ± 0.18; leptin, 8.61 ± 0.39), 18:0–18:2 (saline, 5.49 ± 0.12; leptin, 4.56 ± 0.15), and 18:0–18:1 (saline, 0.50 ± 0.01; leptin, 0.27 ± 0.07) phosphatidylcholines. There were no changes in the proportions of 16:1–18:2 (saline, 3.32 ± 0.04; leptin, 3.06 ± 0.30), 16:0–22:6 (saline, 4.63 ± 0.36; leptin, 5.40 ± 0.25), and 16:0–18:1 plus 18:0–20:4 (saline, 12.49 ± 0.37; leptin, 12.42 ± 0.41) phosphatidylcholine molecular species. Fig. 3A shows the effect of leptin on bile salt pool size. Compared with more modest decreases that were observed at 14 days in saline-treated mice, leptin markedly decreased bile salt pool sizes at 14 days. At 28 days, there were further decreases in the pool sizes of saline-treated mice. However, the values did not fall to those observed with leptin administration. The bile salt species and hydrophobic index of bile salts comprising the bile salt pool (data not shown) were similar to those of hepatic bile in Fig. 2. The rates of fecal bile salt excretion (Fig.3B) were decreased in both leptin- and saline-treated mice compared with base line. The fecal bile salt excretion rate did not differ in leptin-treated mice compared with saline-treated mice. As evidenced by reduced fecal bile salt excretion during the period spanning 14–21 days, the reduction in bile salt pool size because of leptin treatment was largely completed within the first 14 days. Because cholesterol absorption is regulated by both bile salt hydrophobicity (14Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar, 27Sehayek E. Ono J.G. Duncan E.M. Batta A.K. Salen G. Shefer S. Nguyen L.B. Yang K. Lipkin M. Breslow J.L. J. Lipid Res. 2001; 42: 1250-1256Abstract Full Text Full Text PDF PubMed Google Scholar, 28Akiyoshi T. Uchida K. Takase H. Nomura" @default.
• W2023379280 created "2016-06-24" @default.
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• W2023379280 creator A5016110387 @default.
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• W2023379280 date "2002-09-01" @default.
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• W2023379280 title "Leptin Promotes Biliary Cholesterol Elimination during Weight Loss in ob/ob Mice by Regulating the Enterohepatic Circulation of Bile Salts" @default.
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