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• W2135072015 abstract "We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile.These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes. We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile. These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes. Hepatic fatty acid (FA), triglyceride, and cholesterol metabolism are highly regulated processes determined by the concerted feedback regulation of genes governing their synthesis, lipoprotein uptake and production, and biliary lipid secretion (1Diestchy J.M. Turley S.D. Spady D.K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans.J. Lipid Res. 1993; 34: 1637-1659Google Scholar, 2Cohen D.E. Hepatocellular transport and secretion of biliary lipids.Curr. Opin. Lipidol. 1999; 10: 295-302Google Scholar, 3Rigotti A. Marzolo M.P. Nervi F. Lipid transport from the hepatocyte into the bile.in: Hoekstra D. Cell Lipids. Current Topics in Membranes. Academic Press, New York1994: 579-615Google Scholar, 4Vlahecic Z.R. Hylemon P.B. Chiang J.Y.L. Hepatic cholesterol metabolism.in: Arias J.M. Jakoby W.B. Popper H. Schachter D. Schafritz D.A. The Liver: Biology and Pathobiology. Raven, New York1988: 379-389Google Scholar, 5Shelness G.S. Sallers J.A. Very low-density lipoprotein assembly and secretion.Curr. Opin. Lipidol. 2001; 12: 151-157Google Scholar, 6Wang S-L. Du E. Martin T.D. Davis R.A. Coordinate regulation of lipogenesis and the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1.J. Biol. Chem. 1997; 272: 19351-19364Google Scholar, 7Kang S. Davis R.A. Cholesterol and hepatic lipoprotein assembly and secretion.Biochim. Biophys. Acta. 2000; 1529: 223-230Google Scholar). Regulation of FA and cholesterol synthesis are highly interrelated processes and share common transcription regulatory factors (8Brown M.S. Goldstein J.L. The SREBP pathway: Regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Google Scholar, 9Horton J.D. Shimomura I. Sterol regulatory element-binding proteins: Activators of cholesterol and fatty acid biosynthesis.Curr. Opin. Lipidol. 1999; 10: 143-150Google Scholar). Even though the physiological role(s) for sterol carrier protein-2 (SCP-2) gene products remain elusive, several studies suggest a number of important transport and catalytic functions of these proteins in the regulation of lipid metabolism (10Gallegos A. Atshaves B. Storey S. Starodud O. Petrescu A. Huang H. McIntosh A. Martin G. Chao H. Kier A. Schroeder F. Gene structure, intracellular localization, and functional roles of sterol carrier protein-2.Prog. Lipid Res. 2001; 40: 498-563Google Scholar, 11Seedorf U. Ellinghaus P. Nofer J.R. Sterol carrier protein-2.Biochim. Biophys. Acta. 2000; 1486: 45-54Google Scholar, 12Fielding P.E. Fielding C.J. 1999. Intracellular cholesterol transport.J. Lipid Res. 1997; 40: 781-796Google Scholar, 13Simons K. Ikonen E. How cells handle cholesterol.Science. 2000; 290: 1721-1726Google Scholar, 14Schroeder F. Frolov A. Schoer J. Gallegos A. Atshaves B.P. Stolwich N.J. Scott A.I. Kier A.B. Intracellular cholesterol binding proteins, cholesterol transport, and membrane domains.in: Freeman D. Chang T.Y. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 231-234Google Scholar). SCP-2 gene products not only bind cholesterol, FA, FA-acyl-CoA, and phospholipids, but also enhance trafficking of these lipids within cells (15Jackson S.M. Ericsoon J. Edwards P. Signaling molecules derived from the cholesterol biosynthetic pathway.in: Bittman R. Subcellular Biochemistry. Cholesterol functions and metabolism in biology and medicine. Volume 28. Plenum Press, New York1997: 1-21Google Scholar, 16Ohba T. Holt J.A. Billheimer J.T. Strauss 3rd J.F. Human sterol carrier protein 2 gene has two promoters.Biochemistry. 1995; 34: 10660-10668Google Scholar, 17Chanderbhan R. Noland B.J. Scallen T.J. Vahouny G.V. Sterol carrier protein 2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis.J. Biol. Chem. 1982; 257: 8928-8934Google Scholar, 18Frolov A. Woodford J.K. Murphy E.J. Billheimer J.T. Schroeder F.J. Spontaneous and protein-mediated sterol transfer between intracellular membranes.J. Biol. Chem. 1996; 271: 16075-16083Google Scholar). Experiments in cultured cell systems transfected with cDNAs encoding for the SCP-2/sterol carrier protein X (SCP-X) products demonstrated the role of these proteins in microsomal membrane utilization of FA for phospholipids, triglyceride, and cholesterol ester synthesis (19Gavey K.L. Noland B.J. Scallen T.J. The participation of sterol carrier protein 2 in the conversion of cholesterol ester by rat liver microsomes.J. Biol. Chem. 1981; 256: 2993-2999Google Scholar, 20Murphy E.J. Schroeder F.J. Sterol carrier protein-2 mediated cholesterol esterification in transfected L-cell fibroblasts.Biochim. Biophys. Acta. 1997; 1345: 283-292Google Scholar, 21Murphy E.J. Stiles T. Schroeder F. Sterol carrier protein-2 expression alters phospholipid content and fatty acyl composition in L-cell fibroblasts.J. Lipid Res. 2000; 41: 788-796Google Scholar, 22Tarodub O. Jolly C.A. Atshaves B.P. Roths J.B. Murphy E.J. Kier A.B. Schroeder F. Sterol carrier protein-2 localization in endoplasmic reticulum: role in phospholipid formation.Am. Cell Physiol. 2000; 279: C1259-C1269Google Scholar). Overexpression of SCP-2 in rat hepatoma cells enhanced intracellular cholesterol cycling, increased plasma membrane cholesterol content, and inhibited cholesterol esterification and HDL production (23Baum C.L. Reschly E.J. Gayen A.K. Groh M.E. Schadick K. Sterol carrier protein-2 overexpression enhances cholesterol cycling and inhibits cholesterol ester synthesis and high density lipoprotein cholesterol secretion.J. Biol. Chem. 1997; 272: 6490-6498Google Scholar). In addition, studies in mice with disruption of the SCP-2/SCP-X gene have shown a failure in the oxidation of 2-methyl-branched FA and the side chain of cholesterol. Furthermore, liver triglyceride and cholesterol ester concentrations significantly decreased in these SCP-2-knockout mice, suggesting that these animals have altered phospholipid, fatty acid, and triglyceride metabolism (24Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fokber M. Engel T. Denis S. Wouters F. Wirtz K.W.A. Wanders R.J.A. Maeda N. Assmann G. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function.Genes Dev. 1998; 12: 1189-1201Google Scholar, 25Kannenberg F. Ellinghaus P. Assman G. Seedorf U. Aberrant oxidation of the cholesterol side chain in bile acid synthesis of sterol carrier protein-2/sterol carrier protein-x knockout mice.J. Biol. Chem. 1999; 274: 35455-35460Google Scholar). We have recently shown that transient hepatic overexpression of SCP-2 in mice altered plasma LDL cholesterol (LDL-C) and HDL-C concentrations, and decreased hepatic apolipoprotein B (apoB), apoE, and apoAI, and LDL receptor (LDLR) expression (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar). These various changes were associated with a simultaneous enhancement of the enterohepatic circulation of cholesterol and bile acids and intestinal cholesterol absorption. These data, together with the studies of SCP-2 knockout mice, strongly support the concept that SCP-2 has a coordinate regulatory role in hepatic lipid metabolism by modulating expression of various genes involved in cholesterol, bile acid, FA, and triglyceride metabolism. Since SCP-2 proteins are located in peroxisomes as well as in endoplasmic reticulum and cytosol (22Tarodub O. Jolly C.A. Atshaves B.P. Roths J.B. Murphy E.J. Kier A.B. Schroeder F. Sterol carrier protein-2 localization in endoplasmic reticulum: role in phospholipid formation.Am. Cell Physiol. 2000; 279: C1259-C1269Google Scholar, 27Keller G.A. Scallen T.J. Clarke D. Maher P.A. Krisans S.K. Singer S.J. Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes.J. Cell Biol. 1989; 108: 1353-1361Google Scholar, 28Antokenov V.D. van Veldhoven P.P. Waelkensand E. Mannaerts G.P. Substrate specificities of 3-oxoacyl-CoA thiolase. A sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.J. Biol. Chem. 1997; 272: 26023-26031Google Scholar, 29van Amerongen A. van Noort M. van Beckhoven J.R. Rommerts F.F. Orly J. Wirtz K.W. The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland.Biochim. Biophys. Acta. 1989; 20: 243-248Google Scholar), we speculate here that the SCP-2 gene could have important regulatory functions on VLDL production. Specifically, we postulate that hepatic SCP-2 gene products inhibit VLDL production by preferentially channeling phospholipids and sterols to the canalicular pole rather than to the sinusoidal domain of the hepatocyte. The reciprocal relationship between plasma and biliary lipid secretion has been previously reported (3Rigotti A. Marzolo M.P. Nervi F. Lipid transport from the hepatocyte into the bile.in: Hoekstra D. Cell Lipids. Current Topics in Membranes. Academic Press, New York1994: 579-615Google Scholar, 30Nervi F. Marinovic I. Rigotti A. Ulloa N. Regulation of biliary cholesterol secretion. Functional relationship between the canalicular and sinusoidal cholesterol secretory pathways in the rat.J. Clin. Invest. 1988; 82: 1818-1825Google Scholar). To further examine whether SCP-2 indeed plays an interrelated physiological role in lipoprotein and biliary lipid metabolism in vivo, we determined the effect of adenovirus-mediated SCP-2 gene transfer into the mouse liver on FA synthesis and VLDL production, as well as biliary cholesterol and phospholipids secretion and composition. Adult male C57BL/6 mice over 8 weeks of age were used in all experiments as previously described (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar). Briefly, they had free access to commercial rodent diet Prolab RMH 3000 (PMI Nutritional International Inc., Brentwood, MO). Animals were subjected to experimental protocols approved by the Research Advisory Committee of our institution. All experiments were carried out during the dark phase of reversal diurnal cycle between 8 AM to 1 PM (dark phase, 8 AM to 8 PM). The recombinant adenovirus Ad.rSCP2 was generated by homologous recombination in 293 cells as described previously (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar, 31Kozarsky K.F. Mckinley D.R. Austin I.L. Raper S.E. Statford-Perricaudet L.D. Wilson J.M. In vivo correction of low density lipoprotein receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses.J. Biol. Chem. 1994; 269: 13695-13702Google Scholar, 32Engelhardt J.F. Yang Y. Statford-Perricaudet L.D. Allen E.D. Kozarsky K. Perricaudet M. Yankaskas J.R. Wilson J.M. Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses.Nat. Genet. 1993; 4: 27-34Google Scholar). Briefly, the adenoviral backbone used for the construction of the vector-containing rat scp2 cDNA under control of the CMV enhancer/promoter was derived from a replication-deficient first-generation type-5 adenovirus with deletions of E1 and E3 genes. The control adenovirus Ad.E1Δ contained the same E1 and E3 deletions without the transgene expression cassette. Large-scale production of recombinant adenoviruses was performed after purification from infected 293 cells (33Kozarsky K.F. Jooss K. Donahee M. Strauss J.F. Wilson J.M. Effective treatment of familial hypercholesterolemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene.Nat. Genet. 1996; 10: 54-62Google Scholar). Viruses were administered intravenously as previously described (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar). Animals were studied 7 days after adenoviral infection during the dark phase of a reversed diurnal cycle. Western blotting of liver homogenates for SCP2 was performed using a rabbit polyclonal anti-rat SCP2 serum from each sample as previously described (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar, 34Puglielli L.A. Rigotti A. Greco A. Santos M. Nervi F. Sterol carrier protein-2 is involved in cholesterol transfer from the reticulum to the plasma membrane in human fibroblasts.J. Biol. Chem. 1995; 270: 18723-18726Google Scholar, 35Puglielli L.A. Rigotti A. Amigo L. Núñez L. Greco A. Santos M. Nervi F. Modulation of intrahepatic cholesterol trafficking: evidence by in vivo antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transport into rat bile.Biochem. J. 1996; 317: 681-687Google Scholar). Bile was collected through a common bile duct fistula for 30 to 60 min in preweighed tubes and stored at −20°C. Blood was removed by puncture of the inferior vena cava with a heparinized syringe. Plasma was immediately separated by centrifugation at 10,000 rpm × 10 min at 4°C and stored at −20°C. Plasma lipoprotein separation was performed by Superose 6-fast protein liquid chromotography gel filtration of fresh plasma specimens (36Rigotti A. Trigatti B.L. Penman M. Rayburn H.M. Herz J. Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.Proc. Natl. Acad. Sci. USA. 1997; 94: 12610-12615Google Scholar). For other determinations, liver, bile, and plasma samples were frozen at −20°C until processing. Total plasma and lipoprotein cholesterol, and triglyceride concentrations were measured by enzymatic kits (Sigma Chemicals Co., St. Louis, MO). Hepatic triglycerides were extracted, solubilized, and measured as previously described (37Carr T.P. Andersen C.J. Rudel L.L. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts.Clin. Biochem. 1993; 26: 39-42Google Scholar). Hepatic and biliary cholesterol, biliary phospholipids, and bile acids were determined by routine methods (30Nervi F. Marinovic I. Rigotti A. Ulloa N. Regulation of biliary cholesterol secretion. Functional relationship between the canalicular and sinusoidal cholesterol secretory pathways in the rat.J. Clin. Invest. 1988; 82: 1818-1825Google Scholar, 38Nervi F. Del Pozo R. Covarrubias C. Ronco B. The effect of progesterone on the regulatory mechanisms of biliary cholesterol secretion in the rat.Hepatology. 1983; 3: 360-367Google Scholar, 39Amigo L. Mendoza H. Zanlungo S. Miquel J.F. Rigotti A. González S. Nervi F. Enrichment of canalicular membrane with cholesterol and sphingomyelin prevents bile salt-induced hepatic damage.J. Lipid Res. 1999; 40: 533-542Google Scholar). Molecular species of phosphatidylcholine (i.e., sn-1 and sn-2 fatty acyl compositions) were determined as previously described (40Patton G.M. Fasulo J.M. Robins S.J. Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography.J. Lipid Res. 1882; 23: 190-195Google Scholar, 41Cohen D.E. Carey M.C. Acyl chain unsaturation modulates distribution of lecithin molecular species between mixed micelles and vesicles in model bile. Implication for particle structure and metastable cholesterol solubilities.J. Lipid Res. 1991; 32: 1291-1302Google Scholar). Hydrophobic index is a concentration-weighted average of HPLC-determined hydrophobicities of individual phosphatidylcholines present in a mixture and was determined according to Hay et al. (41Cohen D.E. Carey M.C. Acyl chain unsaturation modulates distribution of lecithin molecular species between mixed micelles and vesicles in model bile. Implication for particle structure and metastable cholesterol solubilities.J. Lipid Res. 1991; 32: 1291-1302Google Scholar, 42Hay D.W. Cahalane M.J. Timofeyeva N. Carey M.C. Molecular species of lecithins in human gallbladder biles.J. Lipid Res. 1993; 34: 759-768Google Scholar). Conventional liver biopsies were performed in 4 Ad.E1Δ and 5 Ad.rSCP2 mice. Specimens were stained with hematoxilin-eosin and were blindly analyzed by a pathologist. After a 2-h fasting period, rates of hepatic fatty acid and cholesterol synthesis were measured at the mid-dark phase of the diurnal cycle (10 AM). Each mouse received 50 mCi [3H] water (Amersham Pharmacia Biotech, Piscataway, NJ) by intraperitoneal injection as previously described (43Lowestein J.M. Brunengraber H. Walke M. Measurement of rates of lipogenesis with deuterated and tritiated water.Methods Enzymol. 1975; 35B: 279-287Google Scholar, 44Dietschy J.M. Spady D.K. Measurement of rates of cholesterol synthesis using tritiated water.J. Lipid Res. 1984; 25: 1469-1476Google Scholar). One hour after radiolabel injection, animals were anesthetized and ∼0.5 ml of blood was obtained from the inferior vena cava for determination of water-specific activity in plasma. After liver removal, tissue specimens were saponified and digitonin-precipitable sterols were isolated (43Lowestein J.M. Brunengraber H. Walke M. Measurement of rates of lipogenesis with deuterated and tritiated water.Methods Enzymol. 1975; 35B: 279-287Google Scholar, 44Dietschy J.M. Spady D.K. Measurement of rates of cholesterol synthesis using tritiated water.J. Lipid Res. 1984; 25: 1469-1476Google Scholar). For determination of fatty acid synthesis, liver homogenates were extracted twice with 10 ml of petroleum ether after acidification with 1 ml 1 N HCl (43Lowestein J.M. Brunengraber H. Walke M. Measurement of rates of lipogenesis with deuterated and tritiated water.Methods Enzymol. 1975; 35B: 279-287Google Scholar). Results were expressed as mmol of [3H]water incorporated into fatty acids, or digitonin-precipitable sterol nmol/h/g liver weight. To measure hepatic VLDL triglyceride production, we used the well-characterized method of Triton WR-1339 (tyloxapol) (Sigma Chemicals Co.) injection to block peripheral removal of newly secreted VLDL (45Li X. Catalina F. Grundy S.M. Oatel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48 relative to B-100-containing lipoproteins.J. Lipid Res. 1996; 37: 210-220Google Scholar, 46Ishikawa T. Fidge N. Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats.J. Lipid Res. 1979; 20: 254-264Google Scholar, 47Otway S. Robinson D.S. The use of a non-ionic detergent (Triton-WR 1339) to determine rates of triglyceride entry into the circulation of the rat under different physiological conditions.J. Physiol. 1967; 190: 321-332Google Scholar). Food was removed from the cages at 8:30 PM the day prior to the experiment at the beginning of the light phase of the diurnal cycle, when animals normally eat a scarce amount of food. (Rodents usually ingest the major proportion of their daily food during the dark phase of the diurnal cycle.) An aliquot of blood was drawn from a tail vein the next day for determination of basal triglyceride concentration in plasma. Triton WR-1339 was intravenously injected at a dose of 35 mg/kg body weight in 50 μl of saline solution. Three hours later, blood was drawn from the inferior vena cava. We chose this dose of Triton WR-1339 and the 3 h postinjection sampling period because preliminary experiments demonstrated that higher doses, or longer time periods after Triton WR-1339 injection, were deleterious for animals infected with the SCP-2 recombinant adenovirus. All animals participating in this series of experiments tolerated well the administration of Triton WR1339 and looked healthy during the period of anesthesia and bleeding. An aliquot of plasma post-Triton WR1339 injection was stored for total triglyceride determination, while plasma lipoproteins were immediately isolated for VLDL triglyceride measurements. For the calculation of VLDL triglyceride production, we assumed that intestinal contribution to the plasma VLDL pool was minimal. Plasma volume was calculated assuming a value of 0.071 ml/g body weight (45Li X. Catalina F. Grundy S.M. Oatel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48 relative to B-100-containing lipoproteins.J. Lipid Res. 1996; 37: 210-220Google Scholar). Data are presented as mean ± SE. The two-tailed unpaired Student’s t-test was used to compare the sets of data. Statistically significant differences were considered at P < 0.05. To evaluate the relevance of SCP-2 in hepatic lipid metabolism, we studied C57BL/6 mice that transiently overexpressed SCP-2 in the liver by adenovirus-mediated gene transfer. Controls included noninfected saline-injected animals or mice infected with a control adenovirus that lacks a cDNA transgene (Ad.E1Δ). Hepatic SCP-2 expression was increased by 8-fold in Ad.rSCP2-infected mice 7 days postinfection (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar). Mice looked healthy and body weight remained within the normal range in SCP-2-overexpressing mice. Similar mild elevations of alanine aminotransferase and aspartate aminotransferase were observed in the serum of both groups of infected mice. The acute phenotypic changes induced by Ad.rSCP2 on plasma lipid concentration, biliary lipid secretion and hepatic cholesterol, phospholipids, and triglyceride concentrations are shown in Table 1. Liver weight significantly increased in Ad.rSCP2-infected mice compared with control saline-injected mice and Ad.E1Δ-infected mice. In both adenovirus-infected groups of mice, liver histology showed minor changes with scarce parenchymal cell ballooning, scattered necrotic hepatocytes around the periportal areas, and absence of significant inflammation. No differences were found between Ad.E1Δ and Ad.rSCP2 groups of animals. When compared with control Ad.E1Δ-infected mice, total plasma cholesterol and triglyceride levels were unchanged in Ad.rSCP2 animals, whereas biliary lipid outputs were significantly increased. An unexpected finding was the remarkable 4-fold decrease of hepatic triglyceride concentration and the significant increase in total hepatic cholesterol and phospholipids concentrations found in Ad.rSCP2 mice. Taken together, these results confirmed that hepatic SCP-2 expression has a significant role in the regulation of hepatic cholesterol, phospholipids, and triglyceride metabolism, as well as biliary lipid secretion.TABLE 1Acute phenotypic differences among sterol carrier protein-2 recombinant adenoviral infection, control adenoviral infection and noninfected saline-injected miceParameterSalineAd.E1ΔAd.rSCP2Body weight (g)22 ± 0.522 ± 0.621 ± 0.5Liver weight (g)1.1 ± 0.031.0 ± 0.031.3 ± 0.04Total plasma lipid concentration (mg/dl)Cholesterol87 ± 2.080 ± 2.190 ± 4.2Triglycerides18 ± 3.229 ± 3.425 ± 4.0Biliary lipid secretion (nmol/min ×100 g bw)Bile acids248 ± 31232 ± 29387 ± 44aSignificantly different at P < 0.01 compared with control groups.Phospholipids50 ± 546 ± 378 ± 3aSignificantly different at P < 0.01 compared with control groups.Cholesterol5.5 ± 0.55.1 ± 0.711 ± 0.4aSignificantly different at P < 0.01 compared with control groups.Hepatic lipid content (mg/g liver wt)Total cholesterol2.0 ± 0.32.4 ± 0.23.9 ± 0.6aSignificantly different at P < 0.01 compared with control groups.Cholesterol esters0.32 ± 0.30.39 ± 0.10.51 ± 0.2Triglycerides22.4 ± 5.713.1 ± 5.53.1 ± 1.0aSignificantly different at P < 0.01 compared with control groups.Phospholipids (organic P)1.0 ± 0.061.0 ± 0.041.4 ± 0.06aSignificantly different at P < 0.01 compared with control groups.Determinations were performed 7 days after IV administration of adenoviruses or saline. Values represent the mean ± SE. There were 6–8 fasted mice in each group. All experiments were performed during the mid-dark phase of the reversed diurnal cycle.a Significantly different at P < 0.01 compared with control groups. Open table in a new tab Determinations were performed 7 days after IV administration of adenoviruses or saline. Values represent the mean ± SE. There were 6–8 fasted mice in each group. All experiments were performed during the mid-dark phase of the reversed diurnal cycle. As previously reported (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar), plasma lipoprotein cholesterol distribution was altered in Ad.rSCP2 mice compared with the Ad.E1Δ mice: plasma LDL-C concentration increased by 100%, whereas HDL-C levels decreased by 25%. VLDL-C was not changed (results not shown). Since we previously found a significant decrease in hepatic apoB and apoE expression in Ad.rSCP2-infected mice (26Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodríguez A. Kozarsky K. Quiñones V. Rigotti A. Nervi F. Sterol carrier protein-2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Google Scholar), we hypothesized that lipid availability for VLDL assembly and VLDL secretion by the liver was impaired. We therefore studied hepatic FA and cholesterol synthesis, as well as VLDL production in vivo. As shown in Table 2, SCP-2 overexpression decreased in vivo hepatic synthesis of cholesterol and free fatty acid (FFA) by 40% and 56%" @default.
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• W2135072015 title "Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion" @default.
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