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• W2024403197 abstract "Background & Aims: Microsomal triglyceride transfer protein (MTTP) is critical for the production of very-low-density lipoproteins (VLDL). The current studies were undertaken to examine the in vivo role of MTTP in hepatic cholesterol and fatty acid metabolism, as well as in biliary lipid secretion. We also tested whether MTTP plays a role in diet-induced cholelithiasis in mice. Methods: We used mice in which Mttp had been inactivated in the liver (MttpΔ/Δ mice). We measured several parameters of cholesterol metabolism, fatty acid synthesis, and biliary lipid levels in mice fed a normal or a lithogenic diet. We also assessed the incidence of diet-associated gallstones. Results: Hepatic Mttp inactivation markedly decreased plasma triglyceride and cholesterol levels and increased biliary cholesterol and bile acid output. Hepatic cholesterogenesis and fatty acid synthesis were significantly decreased in MttpΔ/Δ mice compared with control mice. The incidence of gallstones decreased from 90% in control mice to 33% in MttpΔ/Δ mice after 8 weeks of a lithogenic diet (P < .0001). The mechanism of the protective effect appears to be increased biliary phospholipid output in MttpΔ/Δ mice, leading to significant unsaturation of gallbladder bile. Conclusions: These results indicate that modulation of Mttp expression in the liver affects hepatic lipid synthesis and storage as well as biliary lipid secretion. Our findings further indicate that inhibition of hepatic MTTP activity decreases the risk of experimental cholelithiasis by favoring phospholipid output into the bile. Background & Aims: Microsomal triglyceride transfer protein (MTTP) is critical for the production of very-low-density lipoproteins (VLDL). The current studies were undertaken to examine the in vivo role of MTTP in hepatic cholesterol and fatty acid metabolism, as well as in biliary lipid secretion. We also tested whether MTTP plays a role in diet-induced cholelithiasis in mice. Methods: We used mice in which Mttp had been inactivated in the liver (MttpΔ/Δ mice). We measured several parameters of cholesterol metabolism, fatty acid synthesis, and biliary lipid levels in mice fed a normal or a lithogenic diet. We also assessed the incidence of diet-associated gallstones. Results: Hepatic Mttp inactivation markedly decreased plasma triglyceride and cholesterol levels and increased biliary cholesterol and bile acid output. Hepatic cholesterogenesis and fatty acid synthesis were significantly decreased in MttpΔ/Δ mice compared with control mice. The incidence of gallstones decreased from 90% in control mice to 33% in MttpΔ/Δ mice after 8 weeks of a lithogenic diet (P < .0001). The mechanism of the protective effect appears to be increased biliary phospholipid output in MttpΔ/Δ mice, leading to significant unsaturation of gallbladder bile. Conclusions: These results indicate that modulation of Mttp expression in the liver affects hepatic lipid synthesis and storage as well as biliary lipid secretion. Our findings further indicate that inhibition of hepatic MTTP activity decreases the risk of experimental cholelithiasis by favoring phospholipid output into the bile. Microsomal triglyceride transfer protein (MTTP) plays an obligatory role in the assembly and secretion of triglyceride-rich, apolipoprotein (apo-) B-containing lipoproteins, such as very-low-density lipoproteins (VLDL) and chylomicrons.1Shelness G.S. Sellers J.A. Very-low-density lipoprotein assembly and secretion.Curr Opin Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (233) Google Scholar, 2Gordon D.A. Jamil H. Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly.Biochim Biophys Acta. 2000; 1486: 72-83Crossref PubMed Scopus (191) Google Scholar, 3Davis R.A. 1999 Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver.Biochim Biophys Acta. 1999; 1440: 1-31Crossref PubMed Scopus (163) Google Scholar, 4Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apo B-lipoprotein assembly.J Lipid Res. 2003; 44: 22-32Crossref PubMed Scopus (443) Google Scholar The protein also catalyzes the transport of triglycerides into the lumen of the endoplasmic reticulum and facilitates the transfer of triglyceride, cholesterol, and phospholipids to apo-B, the key structural protein of VLDL particles.1Shelness G.S. Sellers J.A. Very-low-density lipoprotein assembly and secretion.Curr Opin Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (233) Google Scholar, 2Gordon D.A. Jamil H. Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly.Biochim Biophys Acta. 2000; 1486: 72-83Crossref PubMed Scopus (191) Google Scholar, 3Davis R.A. 1999 Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver.Biochim Biophys Acta. 1999; 1440: 1-31Crossref PubMed Scopus (163) Google Scholar, 4Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apo B-lipoprotein assembly.J Lipid Res. 2003; 44: 22-32Crossref PubMed Scopus (443) Google Scholar The concentration of MTTP within the endoplasmic reticulum of hepatocytes is an important determinant of the VLDL secretion rate.5Leung G.K. Veniant M.M. Kim S.K. Zlot C.H. Raabe M. Bjorkegren J. Neese R.A. Hellerstein M.K. Young S.G. A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion.J Biol Chem. 2000; 275: 7515-7520Crossref PubMed Scopus (66) Google Scholar The critical role of MTTP is evident from the rare disease abetalipoproteinemia, which is caused by mutations in the large subunit of MTTP. In abetalipoproteinemia, the absence of MTTP prevents the assembly and secretion of triglyceride-rich lipoproteins in both the liver and the intestine. Patients with abetalipoproteinemia have hypocholesterolemia, low levels of high-density lipoprotein (HDL) cholesterol, mild fatty liver, and intestinal fat malabsorption.6Gregg R.E. Wetterau J.R. The molecular basis of abetalipoproteinemia.Curr Opin Lipidol. 1994; 5: 81-86Crossref PubMed Scopus (118) Google Scholar, 7Sharp D. Blinderman L. Combs K.A. Kienzle B. Ricci B. Wager-Smith K. Gil C.M. Turck C.W. Bouma M.E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia.Nature. 1993; 365: 65-69Crossref PubMed Scopus (403) Google Scholar, 8Wetterau J.R. Aggerbeck L.P. Bouma M.E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.Science. 1992; 258: 999-1001Crossref PubMed Scopus (634) Google Scholar, 9Shoulders C.C. Brett D.J. Bayliss J.D. Narcisi T.M. Jarmuz A. Grantham T.T. Leoni P.R. Bhattacharya S. Pease R.J. Cullen P.M. Abetalipoproteinemia is caused by defects of the gene encoding the 97 kDa subunit of a microsomal triglyceride transfer protein.Hum Mol Genet. 1993; 2: 2109-2116Crossref PubMed Scopus (226) Google Scholar Hepatic VLDL production was almost completely abolished in liver-specific Mttp knockout mice,10Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J Clin Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar whereas hepatic over expression of Mttp has been reported to cause increased secretion of VLDL triglycerides and apo-B.11Tietge U.J. Bakillah A. Maugeais C. Tsukamoto K. Hussain M. Rader D.J. Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein.Br J Lipid Res. 1999; 40: 2134-2139PubMed Google Scholar The liver is essential for the maintenance of cholesterol, free fatty acid (FA), and triglyceride homeostasis. Hepatic FA, triglyceride, and cholesterol metabolism are highly interrelated processes and are coordinately regulated by genes governing lipid synthesis, lipoprotein uptake and production, and biliary lipid secretion.1Shelness G.S. Sellers J.A. Very-low-density lipoprotein assembly and secretion.Curr Opin Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (233) Google Scholar, 12Diestchy 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: 1637-1659PubMed Google Scholar, 13Rigotti 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: 579-615Crossref Scopus (29) Google Scholar, 14Jackson S.M. Ericsson J. Edwards P. Signaling molecules derived from the cholesterol biosynthetic pathway.in: Bittman R. Subcellular biochemistry. Vol 28. Plenum Press, New York1997: 1-21Google Scholar, 15Dixon J.L. Ginsberg H.N. Hepatic synthesis of lipoproteins and apolipoproteins.Semin Liver Dis. 1992; 12: 364-372Crossref PubMed Scopus (49) Google Scholar, 16Acton S. Rigotti A. Landschulz K.T. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high-density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (1997) Google Scholar We suspected that perturbations in VLDL secretion might affect the availability of endogenous and dietary cholesterol for bile acid synthesis and the availability for phosphatidylcholine and free cholesterol for secretion into bile.17Zanlungo S. Rigotti A. Nervi F. Hepatic cholesterol transport from plasma into bile: implications for gallstone disease.Curr Opin Lipidol. 2004; 15: 279-286Crossref PubMed Scopus (38) Google Scholar, 18Marzolo M.P. Amigo L. Nervi F. Hepatic production of very-low-density lipoprotein, catabolism of low-density lipoprotein, biliary lipid secretion and bile salt synthesis in rats fed a bean (Phaseolus vulgaris) diet.J Lipid Res. 1993; 34: 804-814Google Scholar, 19Stone B.G. Evans C.D. Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.J Lipid Res. 1992; 33: 1665-1675PubMed Google Scholar, 20Nervi 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: 1818-1825Crossref PubMed Scopus (72) Google Scholar A significant fraction of VLDL particles is transformed into LDL, while a lesser fraction is left as cholesterol ester-rich particles that are rapidly removed by the liver. The remnant cholesterol esters contribute cholesterol to the metabolically active cholesterol pool in the liver. Therefore, VLDL metabolism could have a major regulatory role on biliary lipid secretion, and, conceivably, the activity of MTTP could be relevant to the pathogenesis of cholesterol gallstone disease. Unfortunately, studies examining biliary lipid physiology and pathophysiology in MTTP-deficient conditions in humans or animal models are lacking. To determine whether MTTP indeed plays a physiologic role in biliary lipid metabolism, we studied cholesterol, phospholipid, and bile acid metabolism in mice with an inactivating mutation in Mttp in the liver. We also determined whether the inactivation of Mttp, by altering hepatic lipid metabolism, favors the formation of diet-induced cholesterol gallstones in mice. Adult male C57BL/6 mice over 8 weeks of age and weighing 19–23 g were used in all experiments. Animals received humane care and were subjected to experimental protocols approved by the Research Advisory Committee of the Faculty of Medicine of the Pontificia Universidad Católica de Chile. Mice had free access to the commercial rodent food (Prolab RMH 3000, PMI Nutritional International, Brentwood, MO). Groups of animals were also fed an experimental lithogenic diet containing 1.25% cholesterol, 15% total fat, and 0.5% cholic acid (TD90221, Harlan Teklad, Madison, WI). The animals were housed at 25°C in a well-ventilated room with controlled reverse light cycling (mid-dark was set to be at 10 am and midlight at 10 pm). All experiments were carried out during the dark phase of the diurnal cycle. Mttpflox/floxMx1-Cre Mice: Liver-specific Mttp knockout mice were obtained as previously communicated.10Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J Clin Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar Mttpflox/flox mice were bred with Mx1-Cre transgenic mice to generate Mttpflox/floxMx1-Cre mice. To excise exon 1 of Mttp and thus eliminate MTTP expression in the liver, 21- to 28-day-old male Mttpflox/flox Mx1-Cre mice were injected with 500 μg polyinosinic-polycytidylic ribonucleic acid (pI-pC; Sigma Chemical, St. Louis, MO) every other day for 8 days.10Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J Clin Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar Littermate Mttpflox/flox mice lacking the Cre transgene were also injected with pI-pC. Excision of exon 1 was assessed by Southern blot analysis of SacI-digested genomic DNA with a 3′-flanking probe. The mice had a mixed genetic background (∼50% 129/SvJae and ∼50% C57BL/6). They were housed in a facility with a 12-hour light/12-hour dark cycle and were fed rodent chow containing 4.5% fat (Ralston Purina, St. Louis, MO). Genotypes were determined by Southern blots or by polymerase chain reaction (PCR) with genomic DNA from tail biopsy specimens. After 12 hours of fasting, mice were anesthetized as described previously.18Marzolo M.P. Amigo L. Nervi F. Hepatic production of very-low-density lipoprotein, catabolism of low-density lipoprotein, biliary lipid secretion and bile salt synthesis in rats fed a bean (Phaseolus vulgaris) diet.J Lipid Res. 1993; 34: 804-814Google Scholar The cystic duct was ligated, and a common bile duct fistula was performed with a PE 10 polyethylene catheter (Clay-Adams, New York, NY). Gallbladder bile was obtained by aspiration after ligation of the cystic duct. Hepatic bile specimens were collected for 30–60 minutes in preweighed tubes, during which time the animals’ bodies were maintained at 37°C with a heating lamp. At the end of the experiments, each animal’s blood was removed by puncture of the inferior vena cava with a heparinized syringe, and samples of livers were quickly frozen in liquid nitrogen for subsequent lipid determinations. Plasma lipoprotein separation was performed by Superose 6-FPLC gel filtration of fresh plasma.21Amigo L. Zanlungo S. Miquel J.F. Glick J. Hyogo H. Cohen D. Nervi F. Overexpression of sterol carrier protein 2 gene inhibits VLDL production and reciprocally enhances biliary lipid secretion.J Lipid Res. 2003; 44: 399-407Crossref PubMed Scopus (38) Google Scholar For other determinations, liver, bile, and plasma samples were kept frozen at −20°C until processing. Total plasma and lipoprotein cholesterol and triglyceride concentrations were measured with appropriate enzymatic kits (Sigma Chemicals Co). Hepatic triglycerides were extracted, solubilized, and measured as previously described.22Carr 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: 39-42Crossref PubMed Scopus (483) Google Scholar Hepatic and biliary cholesterol, biliary phospholipids, and bile acid levels were determined by routine methods.20Nervi 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: 1818-1825Crossref PubMed Scopus (72) Google Scholar, 21Amigo L. Zanlungo S. Miquel J.F. Glick J. Hyogo H. Cohen D. Nervi F. Overexpression of sterol carrier protein 2 gene inhibits VLDL production and reciprocally enhances biliary lipid secretion.J Lipid Res. 2003; 44: 399-407Crossref PubMed Scopus (38) Google Scholar, 22Carr 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: 39-42Crossref PubMed Scopus (483) Google Scholar, 23Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C. Glick J.M. Rodriguez A. Kozarsky K. Quinones V. Rigotti A. Nervi F. Sterol carrier protein 2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.Gastroenterology. 2000; 119: 1708-1719Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 24Nervi 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: 360-367Crossref PubMed Scopus (41) Google Scholar Hepatic phospholipid species were separated by thin-layer chromatography in the solvent system chloroform//methanol/acetic acid/formic acid/water mixture (70:30:12:4:1, vol/vol/vol/vol/vol) as previously described.25Kulinski A. Vance D.E. Vance J.E. A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion.J Biol Chem. 2004; 279: 23916-23924Crossref PubMed Scopus (76) Google Scholar Gallbladder cholesterol saturation index values were calculated as previously described.26Carey M.C. Critical tables for calculating the cholesterol saturation of native bile.J Lipid Res. 1978; 19: 945-955Abstract Full Text PDF PubMed Google Scholar After a 2-hour fast, hepatic FA and cholesterol synthesis rates were measured during the mid-dark phase of the diurnal cycle (10 am). Each mouse received an intraperitoneal injection of 50 mCi [3H]water (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described.27Dietschy J.M. Spady D.K. Measurement of rates of cholesterol synthesis using tritiated water.J Lipid Res. 1984; 25: 1469-1476Abstract Full Text PDF PubMed Google Scholar One hour after the injection, the animals were anesthetized and approximately 0.5 mL blood was obtained from the inferior vena cava and used to determine the water-specific activity in the plasma. After the removal of the liver, tissue specimens were saponified, and digitonin-precipitable sterols were isolated as previously described.28Lowestein J.M. Brunengraber H. Walke M. Measurement of rates of lipogenesis with deuterated and tritiated water.Methods Enzymol. 1975; 35B: 279-287Crossref Scopus (79) Google Scholar For determination of FA synthesis, liver homogenates were extracted twice with 10 mL petroleum ether after acidification with 1 mL 1 N HCl.28Lowestein J.M. Brunengraber H. Walke M. Measurement of rates of lipogenesis with deuterated and tritiated water.Methods Enzymol. 1975; 35B: 279-287Crossref Scopus (79) Google Scholar Results were expressed as nanomoles of [3H] water incorporated into digitonin-precipitable sterols/h/g liver weight, or FA h/g liver weight. The total mass of bile acids extracted from the small intestine, liver, and gallbladder was quantified as a measure of bile acid pool size as previously described.29Turley S.D. Daggy B.P. Dietschy J.M. Effect of feeding psyllium and cholestyramine in combination on low-density lipoprotein metabolism and fecal bile acid excretion in hamsters with dietary-induced hypercholesterolemia.J Cardiovasc Pharmacol. 1996; 27: 71-79Crossref PubMed Scopus (47) Google Scholar Dr Stephen Turley (Southwestern Medical Center, Dallas, TX) kindly performed the bile acid pool composition analysis. Intestinal cholesterol absorption was measured by the “dual-isotope ratio” method, as previously described.22Carr 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: 39-42Crossref PubMed Scopus (483) Google Scholar, 30Sehayek E. Ono J.G. Shefer S.M. Nguyen L.B. Wang N. Batta A.K. Salen G. Smith J.D. Tall A.R. Breslow J.L. Biliary cholesterol excretion: a novel mechanism that regulates dietary cholesterol absorption.Proc Natl Acad Sci U S A. 1998; 95: 10194-10199Crossref PubMed Scopus (152) Google Scholar Intestinal cholesterol absorption was calculated as the percentage of cholesterol absorbed per day according to the following formula:% absorption= {1 − [fecal (C14/H3)]/[administered (C/14H3)]} × 100 To measure hepatic VLDL triglyceride production, we used the Triton WR-1339 (Sigma Chemicals Co) method to block peripheral removal of newly secreted VLDL.31Li 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: 210-220Abstract Full Text PDF PubMed Google Scholar, 32Ishikawa 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: 254-264PubMed Google Scholar, 33Otway S. Robinson D.S. 1967 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: 321-332Crossref PubMed Scopus (217) Google Scholar Plasma volume was calculated assuming a value of 0.071 mL/g body weight.32Ishikawa 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: 254-264PubMed Google Scholar Hepatic phosphatidylcholine synthesis was measured as previously described34Buccoliero R. Bodennec J. van Echten-Deckert G. Sandhoff K. Futerman A.H. Phospholipid synthesis is decreased in neuronal tissue in a mouse model of Sandhoff disease.J Neurochem. 2004; 90: 80-88Crossref PubMed Scopus (29) Google Scholar with some modifications. Briefly, mice were injected intraperitoneally with 0.4 μCi [methyl-14C]choline per gram of body weight. Twenty-four hours after the injection, the mice were killed, and their livers were removed, weighed, and homogenized in 40 mL chloroform/methanol (2:1, vol/vol) with a polytron homogenizer. The protein concentration of the homogenized samples was then determined. Phospholipids were extracted and washed,25Kulinski A. Vance D.E. Vance J.E. A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion.J Biol Chem. 2004; 279: 23916-23924Crossref PubMed Scopus (76) Google Scholar the chloroform lower phase was evaporated under N2, and the phospholipids were separated by thin-layer chromatography (TLC).25Kulinski A. Vance D.E. Vance J.E. A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion.J Biol Chem. 2004; 279: 23916-23924Crossref PubMed Scopus (76) Google Scholar The TLC plate was subsequently air-dried, phospholipids visualized by I2, and scraped into scintillation vials to which 2 mL methanol and 10 mL Optima Gold (Packard Instrument Co, Downers Grove, IL) scintillation fluid were added. Phospholipids were eluted from the silica gel and acetolyzed at 140°C in 2 mL acetic anhydride/acetic acid (2:3, vol/vol) for 16 hours; the mixture was then evaporated and separated into aqueous and organic phases by addition of chloroform/methanol/water (8:4:3, vol/vol/vol). Results are expressed as the amount of [methyl-14C]choline incorporated into hepatic phosphatidylcholine/total liver. We used quantitative reverse transcriptase-mediated PCR to measure messenger RNA levels of some genes involved in cholesterol and bile acid secretion into bile in MttpΔ/Δ and the Mttpflox/flox mice were fed a control chow or a lithogenic diet. Single-strand complementary DNA (cDNA) synthesis was performed with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) using random hexamers and starting from 5 μg total RNA. The PCR primer sequences were, for Cyp7a1 (7α-hydroxylase), 5’-GTGAGCTGTTGCATATGGTTT-3’ and 5’-GATCAGTTCAGAGACCTGGTC-3’; for Abcb 11 (bile salt export pump), 5’-CAGACACCATGTCTGACTCAGTGA-3’ and 5’-GGCCACACTCAGACCTATGACGGG-3’; for the canalicular cholesterol transporters Abcg5, 5’-AGAGTCAGGATGGCCTGTAT-3’ and 5’-ATGCTGAGCAGGGCCACTAT-3’; and, for Abcg8, 5’-GCACTGGTCATGGCTGAGAA-3’ and 5’-CACAGGAGTCTTGGCTGCTA-3’. An aliquot of the cDNA was subjected to PCR amplification using gene-specific primers, together with a “competimer”/primer mix specific for 18S ribosomal RNA (18S Internal Standards; Ambion, Austin, TX) employed to compensate for tube-to-tube variations. The ratio of 18S “competimers” to primers was adjusted such that the amount of 18S RNA product was within the same range as that of the messenger RNA (mRNA) species of interest. Amplification reactions were performed in the presence of [α-32P] dCTP, and general conditions were as follows: 23 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds. Amplification products were fractionated on 5% acrylamide gels, and bands were subsequently visualized using the Cyclone system (Packard, PerkinElmer Life Sciences, Inc.) and quantified with the MultiAnalyst software (Bio-Rad, Hercules, CA). Intensities of bands were corrected by division of the gene-specific signal by the signal of the 18S amplicon. All samples were run twice. Total RNA was prepared from mouse liver with the acid guanidinium thiocyanate-phenol-chloroform method.35Folch J. Lees M. Stanley G.H.S. A simple method for the isolation and purification of total lipids from animal tissues.J Biol Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar cDNA probes for the murine Abcb4 transporter, phosphatidylcholine transfer protein (PC-TP) and 18S rRNA were prepared from total liver RNA by a standard reverse transcription-PCR (RT-PCR) procedure with primers based on cDNA sequences obtained from GeneBank databases. Equal amounts of total RNA (30 μg per mouse) were size fractionated by agarose-formaldehyde gel electrophoresis and transferred to nylon membranes. Probes were labelled by the random primer method (Promega, Madison, WI) and used for hybridization as previously described.21Amigo L. Zanlungo S. Miquel J.F. Glick J. Hyogo H. Cohen D. Nervi F. Overexpression of sterol carrier protein 2 gene inhibits VLDL production and reciprocally enhances biliary lipid secretion.J Lipid Res. 2003; 44: 399-407Crossref PubMed Scopus (38) Google Scholar Radioactive bands were quantified by phosphorimaging with the GS-525 Molecular Image System (Bio-Rad). Results were normalized to the signal generated from hybridization of a 32P-labeled mouse 18S rRNA probe on the same filter. Data are presented as means ± SE. The 2-tailed unpaired Student t test or ANOVAs were used to compare the functional sets of data. The χ2 test was used for bivariate tabular analysis. Significant differences were considered at a P value < .05. To generate mice lacking MTTP in the liver (MttpΔ/Δ mice), Cre expression in Mttpflox/floxMx1-Cre mice was induced with pI-pC. Hepatic MTTP expression was abolished in MttpΔ/Δ mice, whereas MTTP expression in the small intestine remained normal relative to Mttpflox/flox mice as previously communicated10Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J Clin Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar (results not shown). The MttpΔ/Δ mice appeared healthy, and their body weights remained within the range of Mttpflox/flox mice. Consistent with previous studies,10Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J Clin Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar the plasma triglyceride and cholesterol levels in MttpΔ/Δ mice were far lower than in Mttpflox/flox mice fed either the control chow or the lithogenic diets as shown in Table 1. Serum alanine aminotransferase (ALT) and alkaline phosphatase concentrations were within normal ranges in MttpΔ/Δ mice fed the control chow and the lithogenic diets, indicating that hepatic deletion of the mttp gene did not increase the susceptibility for liver damage after prolonged feeding of the diets (Table 1, group B). Hepatic histology was similar in both groups of mice (moderate steatosis).Table 1Effect of Liver-Specific Mttp Gene Deletion on Body and Liver Weight and Serum Lipid and Enzyme Concentrations in Mice Fed a Control Chow or a Lithogenic DietGroupBody weight (g)Liver weight (g)Serum cholesterol (mg/dL)Serum triglycerides (mg/dL)Serum enzymes (IU/L)aThere were 6 mice for plasma enzyme determination in each group.ALTAPA. Control chow diet Mttpflox/flox (16Acton S. Rigotti A. Landschulz K.T. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high-density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (1997) Google Scholar)29 ± 0.41.56 ± 0.04124 ± 3.860 ± 47.4 ± 1.065 ± 4 MttpΔ/Δ (23Zanlungo S. Amigo L. Mendoza H. Miquel J.F. Vio C." @default.
• W2024403197 created "2016-06-24" @default.
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• W2024403197 date "2006-12-01" @default.
• W2024403197 modified "2023-10-13" @default.
• W2024403197 title "Inactivation of Hepatic Microsomal Triglyceride Transfer Protein Protects Mice From Diet-Induced Gallstones" @default.
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