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• W2032779971 abstract "Recently, we generated mice lacking microsomal triglyceride transfer protein (MTP) in the liver (Mttp Δ/Δ) and demonstrated that very low density lipoprotein secretion from hepatocytes was almost completely blocked. The blockade in lipoprotein production was accompanied by mild to moderate hepatic steatosis, but the mice appeared healthy. Although hepatic MTP deficiency appeared to be innocuous, we hypothesized that a blockade in very low density lipoprotein secretion and the accompanying steatosis might increase the sensitivity ofMttp Δ/Δ livers to additional hepatic insults. To address this issue, we compared the susceptibility ofMttp Δ/Δ mice andMttp flox/flox controls to hepatic injury fromEscherichia coli lipopolysaccharides, concanavalin A, andPseudomonas aeruginosa exotoxin A. At baseline, neither theMttp Δ/Δ nor theMttp flox/flox mice had elevated serum transaminases or histologic evidence of hepatic inflammation. After the administration of the toxins, however, theMttp Δ/Δ mice manifested higher levels of transaminases and, unlike the Mttp flox/floxmice, developed histologic evidence of hepatic inflammation. The toxic challenge induced tumor necrosis factor-α to a similar extent inMttp Δ/Δ andMttp flox/flox mice, but other parameters of injury (e.g. chemokine transcript levels and lipid peroxides) were disproportionately increased in theMttp Δ/Δ mice. Our results suggest that blocking lipoprotein secretion in the liver may increase the susceptibility of the liver to certain toxic challenges. Recently, we generated mice lacking microsomal triglyceride transfer protein (MTP) in the liver (Mttp Δ/Δ) and demonstrated that very low density lipoprotein secretion from hepatocytes was almost completely blocked. The blockade in lipoprotein production was accompanied by mild to moderate hepatic steatosis, but the mice appeared healthy. Although hepatic MTP deficiency appeared to be innocuous, we hypothesized that a blockade in very low density lipoprotein secretion and the accompanying steatosis might increase the sensitivity ofMttp Δ/Δ livers to additional hepatic insults. To address this issue, we compared the susceptibility ofMttp Δ/Δ mice andMttp flox/flox controls to hepatic injury fromEscherichia coli lipopolysaccharides, concanavalin A, andPseudomonas aeruginosa exotoxin A. At baseline, neither theMttp Δ/Δ nor theMttp flox/flox mice had elevated serum transaminases or histologic evidence of hepatic inflammation. After the administration of the toxins, however, theMttp Δ/Δ mice manifested higher levels of transaminases and, unlike the Mttp flox/floxmice, developed histologic evidence of hepatic inflammation. The toxic challenge induced tumor necrosis factor-α to a similar extent inMttp Δ/Δ andMttp flox/flox mice, but other parameters of injury (e.g. chemokine transcript levels and lipid peroxides) were disproportionately increased in theMttp Δ/Δ mice. Our results suggest that blocking lipoprotein secretion in the liver may increase the susceptibility of the liver to certain toxic challenges. Microsomal triglyceride transfer protein (MTP) 1The abbreviations used are:MTPmicrosomal triglyceride transfer proteinMttpthe mouse gene for the large subunit of microsomal triglyceride transfer proteinapoapolipoproteinVLDLvery low density lipoprotein(s)Scd1stearoyl-CoA desaturase 1pI-pCpolyinosinic-polycytidylic ribonucleic acidSREBPsterol regulatory element-binding proteinLPSlipopolysaccharidePEAP. aeruginosa exotoxin AMIPmacrophage inflammatory proteinILinterleukin: ALT, alanine aminotransferaseASTaspartate aminotransferaseLDHlactate dehydrogenaseTNFtumor necrosis factorTBARSthiobarbituric acid-reactive substancesConAconcanavalin A is critical for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins, both in the intestine and in the liver (1Gordon D.A. Wetterau J.R. Gregg R.E. Trends Cell Biol. 1995; 5: 317-321Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 2Kane J.P. Havel R.J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 2717-2752Google Scholar). A genetic absence of MTP causes abetalipoproteinemia, a disease characterized by intestinal fat malabsorption, a virtual absence of chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins in the plasma, and strikingly low plasma levels of triglycerides and cholesterol. The fact that a deficiency in MTP reduces the plasma levels of atherogenic lipoproteins has attracted the attention of the pharmaceutical industry. Many companies have established MTP programs, with the goal of identifying MTP inhibitors suitable for treating humans with hyperlipidemias (3Jamil H. Chu C.-H. Dickson J.K., Jr. Chen Y. Yan M. Biller S.A. Gregg R.E. Wetterau J.R. Gordon D.A. J. Lipid Res. 1998; 39: 1448-1454Abstract Full Text Full Text PDF PubMed Google Scholar, 4van Greevenbroek M.M.J. Robertus-Teunissen M.G. Erkelens D.W. de Bruin T.W.A. J. Lipid Res. 1998; 39: 173-185Abstract Full Text Full Text PDF PubMed Google Scholar). Thus far, however, the efficacy and safety of these compounds in humans has not been documented. microsomal triglyceride transfer protein the mouse gene for the large subunit of microsomal triglyceride transfer protein apolipoprotein very low density lipoprotein(s) stearoyl-CoA desaturase 1 polyinosinic-polycytidylic ribonucleic acid sterol regulatory element-binding protein lipopolysaccharide P. aeruginosa exotoxin A macrophage inflammatory protein interleukin: ALT, alanine aminotransferase aspartate aminotransferase lactate dehydrogenase tumor necrosis factor thiobarbituric acid-reactive substances concanavalin A To investigate the role of MTP in lipoprotein assembly and secretion, we inactivated the MTP gene (Mttp) in mice (5Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Véniant M.M. Hamilton R.L. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8686-8691Crossref PubMed Scopus (223) Google Scholar). Heterozygous knockout mice (Mttp +/−) manifested slightly reduced levels of lipoprotein secretion, reduced levels of apoB100-containing lipoproteins in the plasma, and slightly increased levels of neutral lipids (triglycerides and cholesterol esters) in the liver. Homozygous knockout mice (Mttp −/−) died during embryonic development. Subsequently, we usedCre/LoxP recombination techniques to produce mice lacking Mttp expression in the liver but not in the intestine (6Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). Those mice, designatedMttp Δ/Δ mice, exhibited strikingly reduced plasma levels of apoB100, sizable reductions in the plasma levels of cholesterol and triglycerides, and mild to moderate steatosis with increased levels of neutral lipids in the liver. TheMttp Δ/Δ mice were healthy and grew normally; their plasma transaminase levels were normal, and their livers were free of inflammatory infiltrates (6Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). The fact that it was possible to eliminate hepatic Mttpexpression in a mammalian model without noticeable side effects supported the concept that it might be possible to develop MTP inhibitors to treat hyperlipidemias. Also encouraging were studies by Wetterau et al. (7Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C.-H. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S.-J. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan J.V.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K., Jr. Biller S.A. Science. 1998; 282: 751-754Crossref PubMed Scopus (251) Google Scholar) that showed that MTP inhibitors could reduce plasma lipoprotein levels in low density lipoprotein receptor-deficient rabbits without causing elevated transaminases or histologic evidence of liver inflammation. In this study, we further investigated the notion that it might be possible, with impunity, to inhibit MTP and block hepatic lipoprotein production. We were suspicious, based on several observations, that MTP inhibition might not be as safe as our original studies and those of Wetterau et al. (7Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C.-H. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S.-J. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan J.V.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K., Jr. Biller S.A. Science. 1998; 282: 751-754Crossref PubMed Scopus (251) Google Scholar) had implied. First, other human conditions associated with hepatic steatosis (e.g. diabetes mellitus, excessive consumption of ethanol, and obesity) increase the risk of developing hepatic inflammation and advanced liver disease (8Diehl A.M. Semin. Liver Dis. 1999; 19: 221-229Crossref PubMed Scopus (220) Google Scholar, 9Lieber C.S. J. Hepatol. 2000; 32: 113-128Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 10Tsukamoto H. Lu S.C. FASEB J. 2001; 15: 1335-1349Crossref PubMed Scopus (328) Google Scholar). Second, severe liver disease has been reported in humans with abetalipoproteinemia (11Partin J.S. Partin J.C. Schubert W.K. McAdams A.J. Gastroenterology. 1974; 67: 107-118Abstract Full Text PDF PubMed Google Scholar, 12Illingworth D.R. Connor W.E. Miller R.G. Arch. Neurol. 1980; 37: 659-662Crossref PubMed Scopus (65) Google Scholar). Although treatment with short-chain triglycerides might have contributed to the liver disease in those cases, it is also possible that the inability of those livers to secrete lipoproteins caused them to be susceptible to steatohepatitis and advanced liver disease. Normal human livers are required to face toxic insults. For example, intermittent lapses in the intestinal mucosal barrier can allow bacterial products to reach the liver (13Berg R.D. Adv. Exp. Med. Biol. 1999; 473: 11-30Crossref PubMed Google Scholar). Normal livers from healthy individuals can cope with these challenges effectively, without inflammation or tissue injury. The livers of susceptible individuals, however, cannot effectively deal with these challenges, either because of genetic differences or metabolic derangements (14Chitturi S. Farrell G.C. Semin. Liver Dis. 2001; 21: 27-41Crossref PubMed Scopus (602) Google Scholar). This failure of normal protective mechanisms can lead to hepatic inflammation and, in some cases, to advanced liver disease. We hypothesized that the blockade of hepatic lipoprotein production and resultant hepatic steatosis might render the liver more susceptible to toxic liver injury. To test this hypothesis, we compared the susceptibility of liver-specific MTP knockout mice and littermate controls to hepatic injury following challenges with exogenous toxins. A conditional Mttp allele, Mttp flox, in which exon 1 of Mttp is flanked by loxP sites, has been described previously (6Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). Mttp flox/floxmice were bred with Mx1-Cre transgenic mice (15Rajewsky K., Gu, H. Kühn R. Betz U.A.K. Müller W. Roes J. Schwenk F. J. Clin. Invest. 1996; 98: 600-603Crossref PubMed Scopus (244) Google Scholar) to generateMttp flox/floxMx1-Cre mice. To excise exon 1 of Mttp and thus eliminate MTP expression in the liver, 21–28-day-old maleMttp flox/floxMx1-Cre mice (16Gu H. Marth J.D. Orban P.C. Mossmann H. Rajewsky K. Science. 1994; 265: 103-106Crossref PubMed Scopus (1169) Google Scholar) were injected with polyinosinic-polycytidylic ribonucleic acid (pI-pC; Sigma; 500 μg every other day for 8 days) (6Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). LittermateMttp flox/flox mice lacking the Cretransgene were also injected with pI-pC. Excision of exon 1 was assessed by Southern blot analysis of SacI-digested genomic DNA using a 3′-flanking probe. The mice had a mixed genetic background (∼50% 129/SvJae and ∼50% C57BL/6). They were housed in a pathogen-free barrier facility with a 12-h light/12-h dark cycle and were fed rodent chow containing 4.5% fat (Ralston Purina, St. Louis, MO). Genotypes were determined by Southern blots or by PCR with genomic DNA from tail biopsies. Plasma glucose levels were measured with a glucose (Trinder) 100 kit from Sigma. Plasma insulin levels were measured with a 1-2-3 ultra-sensitive rat insulin enzyme-linked immunosorbent assay from Alpco (Windham, NH). Murine 11K GeneChips (Affymetrix, Santa Clara, CA) were used to assess hepatic gene expression patterns. Total RNA was isolated from liver biopsies with TRizol Reagent (Invitrogen) and purified further with a RNeasy Midi kit (Qiagen, Los Angeles, CA). cDNA was synthesized from the RNA with the Superscript Choice System (Invitrogen) and T7-(dT)24primers (Genset, La Jolla, CA). Biotin-labeled cRNA was transcribed from the cDNA in the presence of biotin-labeled nucleotides (RNA Transcript Labeling kit for nucleic acid arrays, Enzo Diagnostics, Farmingdale, NY). The integrity of the total RNA and the cRNA was assessed by electrophoresis on a 1% agarose/formaldehyde gel. Fragmented cRNA was mixed with control oligonucleotides Bio B, C, D, and Cre (American Type Culture Collection, Manassas, VA) and hybridized to the GeneChip at 45 °C for 16 h. The GeneChip Fluidics Station 400 (Affymetrix) was used to stain the GeneChips with R-phycoerythrin streptavidin (Molecular Probes, Eugene, OR), and the signal was amplified with a biotin-labeled anti-streptavidin antibody (Vector Laboratories, Burlingame, CA). The expression data were obtained by scanning the arrays in a GeneArray Scanner (Hewlett-Packard, Palo Alto, CA); data were analyzed with GeneChip 3.1 software (Affymetrix). Expression of the stearoyl-CoA desaturase 1 gene (Scd1) was assessed by Northern blotting with a probe described previously (17Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Briefly, 25 μg of total liver RNA was denatured and separated on 1% agarose/formaldehyde gel electrophoresis. The integrity of the total RNA was confirmed on ethidium bromide-stained gels before transfer to a Nytran SuPerCharge membrane (Schleicher & Schuell). Prehybridization, hybridization, and washing procedures were performed as described previously (18Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. J. Biol. Chem. 2000; 275: 16390-16399Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Membranes were probed with [α-32P]dCTP-labeled cDNA fragments, and signals were visualized by autoradiography (Hyperfilm ECL, Amersham Biosciences). Band intensity was quantified by densitometry (Molecular Imager FX, Bio-Rad). An 18 S probe (Ambion, Austin, TX) was used to normalize Scd1 expression levels. Levels of Scd1 protein were determined by Western blotting of whole-liver homogenates. Levels of sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 were determined by Western blotting of nuclear extracts (19Sheng Z. Otani H. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 935-938Crossref PubMed Scopus (279) Google Scholar). To prepare the nuclear extracts, livers from four mice were pooled (∼1.5 g) and homogenized in 30 ml of buffer A (10 mm Hepes, pH 7.6, 25 mm KCl, 1 mm sodium EDTA, 2 msucrose, 10% (v/v) glycerol, 150 μm spermine, 2 μm spermidine) and protease inhibitors (Complete-Mini,Roche Molecular Biochemicals). The homogenate was subjected to several strokes with a Teflon pestle and filtered through three layers of cheesecloth. To isolate the nuclear pellet, a 25-ml portion of the homogenate was then layered over 10 ml of buffer A and spun in an SW28 Ti rotor (Beckman Instruments, Palo Alto, CA) at 24,000 rpm for 1 h at 4 °C. The pellet was resuspended in 1 ml of buffer (10 mm Hepes, pH 7.6, 100 mm KCl, 2 mmMgCl2, 1 mm sodium EDTA, 1 mmdithiothreitol, 10% glycerol), and protease inhibitors (Complete-Mini), 0.1 volume of 4 m(NH4)2SO4, pH 7.9, were added. The resuspended pellet was gently mixed and then centrifuged at 85,000 rpm in a TLA-100.2 rotor (Beckman Instruments) for 45 min at 4 °C. Aliquots of the supernatant containing the nuclear extracts (150 μg) and the whole-liver homogenates (100 μg) were then size-fractionated on 8% polyacrylamide gels. Western blots were performed with rabbit antisera against mouse SREBP-1 (20Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar) and mouse SREBP-2 (21Shimano H. Shimomura I. Hammer R.E. Herz J. Goldstein J.L. Brown M.S. Horton J.D. J. Clin. Invest. 1997; 100: 2115-2124Crossref PubMed Scopus (356) Google Scholar) and a rabbit antiserum against rat Scd1 (22Mziaut H. Korza G. Ozols J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8883-8888Crossref PubMed Scopus (54) Google Scholar). The binding of the primary antibodies was assessed by a horseradish peroxidase-labeled donkey anti-rabbit antibody and ECL Western blotting detection reagents (Amersham Biosciences). Liver pieces (∼100 mg) were homogenized with a Polytron, Ultra-Turbax T8 (VWR, San Francisco, CA), and lipids were extracted with chloroform/methanol, 2:1 (v/v). Plasma lipids were extracted by hexane/isopropyl alcohol, 3:2 (v/v). Before the lipid extraction, known amounts of tri- and pentadecanoic acid (Sigma) were added as internal standards (23Leung G.K. Véniant M.M. Kim S.K. Zlot C.H. Raabe M. Björkegren J. Neese R.A. Hellerstein M.K. Young S.G. J. Biol. Chem. 2000; 275: 7515-7520Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Triglycerides, phospholipids, and fatty acids were identified by thin-layer chromatography, transesterified with methanolic HCl (Aldrich), and quantified by gas chromatography (23Leung G.K. Véniant M.M. Kim S.K. Zlot C.H. Raabe M. Björkegren J. Neese R.A. Hellerstein M.K. Young S.G. J. Biol. Chem. 2000; 275: 7515-7520Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). One week after the last pI-pC injection, Mttp Δ/Δ mice andMttp flox/flox littermate controls were given an intraperitoneal injection of Escherichia colilipopolysaccharide (LPS, Sigma; 1.0 μg/g) or intravenous injections of concanavalin A (ConA; 400 μg) (Sigma) or Pseudomonas aeruginosa exotoxin A (PEA, Sigma, 600 μg/kg). Plasma triglycerides and liver injury-associated enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH)) were determined in the clinical chemistry laboratory of San Francisco General Hospital 12 h before and 4 (LPS, ConA, and PEA) and 24 h (LPS) after the injections of the toxins. Plasma tumor necrosis factor-α (TNF-α) was determined by a commercial immunoassay with antibodies against mouse TNF-α (R & D Systems, Minneapolis, MN). All procedures were approved by the Committee on Animal Research at the University of California, San Francisco. Hepatic levels of mRNAs for a variety of cytokines were quantified by RNase protection assays (24Gilman M. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. John Wiley & Sons, Inc., New York1993: 4.7.1-4.7.8Google Scholar) with a multiprobe cDNA template kit (mCK1b, mCK3, mCK5; PharMingen, San Diego, CA). Briefly, cRNA probes were transcribed with [α-32P]UTP (>800 Ci/mmol, Amersham Biosciences). Radiolabeled cRNA (5 × 105 Cerenkov cpm) was combined with 20 μg of liver RNA in 10 μl of hybridization buffer. The mixture was incubated at 55 °C for 16 h, and unhybridized RNA was digested by adding ribonuclease A and T1. RNase digestion was terminated with proteinase K and SDS, and the RNA-RNA hybrids were purified by phenol/chloroform extraction and ethanol precipitation. The double-stranded RNA was resuspended in electrophoresis buffer, denatured at 100 °C, and separated through 5% polyacrylamide/urea gels. RNA bands were visualized by autoradiography, and band intensity was quantified by densitometry (Hoefer Scientific Instruments, San Francisco, CA). Signals for specific cytokines were normalized to control RNAs (L32 or glyceraldehyde-3-phosphate dehydrogenase). Thiobarbituric acid-reactive substances (TBARS), frequently used to estimate levels of lipid peroxides (25Ohkawa H. Ohishi N. Yagi K. Anal. Biochem. 1979; 95: 351-358Crossref PubMed Scopus (23531) Google Scholar, 26Leclercq I.A. Farrell G.C. Field J. Bell D.R. Gonzalez F.J. Robertson G.R. J. Clin. Invest. 2000; 105: 1067-1075Crossref PubMed Scopus (670) Google Scholar), were determined with 50-mg liver fragments. To prevent the peroxidation of lipids during the procedure, liver fragments were homogenized in a 1.15% KCl solution containing 50 mm desferroxamine (Sigma). To generate mice lacking MTP in the liver (i.e.MttpΔ/Δ mice), Cre expression inMttp flox/floxMx1-Cre mice was induced with pI-pC. Consistent with previous studies (6Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar), the plasma triglyceride levels were lower in Mttp Δ/Δmice than in Mttp flox/flox mice (TableI). The reduction in plasma triglyceride levels in Mttp Δ/Δ mice was accompanied by an increase in hepatic lipid stores, which was evident both from the gross appearance of the liver (Fig. 1,A and B) and from histology (Fig. 1,C–F). Biochemical studies revealed that the liver triglyceride stores were 3-fold higher inMttp Δ/Δ mice than in littermateMttp flox/flox mice (Table I). The amount of lipid accumulation in this model was modest in comparison to some other genetic models of lipid accumulation. For example, the livers of mice expressing a truncated SREBP-1a synthesize high levels of fatty acids and have a 21-fold increase in liver triglyceride stores (20Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar).Table ICharacteristics of MttpΔ/Δ and littermate control Mttpflox/flox miceMouse phenotypeMttpflox/flox (n = 24)MttpΔ/Δ (n = 23)pvalueAge (days)49 ± 849 ± 90.93Body weight (g)19.9 ± 4.322.7 ± 4.50.09Liver weight (g)1.24 ± 0.131-an = 8.1.56 ± 0.231-bn = 12.<0.0001Plasma triglycerides (mg/dl)65.8 ± 18.237.2 ± 13.2<0.0001Plasma fatty acids (mmol/liter)0.21 ± 0.091-an = 8.0.25 ± 0.041-bn = 12.0.38Liver triglycerides (μmol/g)107 ± 871-cn = 7.333 ± 1651-an = 8.0.005Data represent means and S.D. p values calculated by two-tailed, unpaired t test. Liver weights and body weights determined in two separate groups of mice.1-a n = 8.1-b n = 12.1-c n = 7. Open table in a new tab Data represent means and S.D. p values calculated by two-tailed, unpaired t test. Liver weights and body weights determined in two separate groups of mice. We predicted that the microarray experiments might uncover many perturbations in the expression of genes affecting lipid metabolism. To address this issue, we compared hepatic gene expression inMttp Δ/Δ andMttp flox/flox mice with Affymetrix GeneChips. Remarkably, most genes involved in lipid metabolism were unchanged (e.g. acetyl-CoA carboxylase, acyl-coenzyme A:cholesterol acyltransferase, apoE, ATP-citrate lyase, cholesterol 7-α-hydroxylase, fatty-acid synthase, fatty acid transport protein, 3-hydroxy-3-methylglutaryl-coenzyme A lyase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, low density lipoprotein receptor, lipoprotein lipase, and peroxisome proliferator-activated receptor-α) (definition of unchanged: fold change <30%,p > 0.15). However, there were two noteworthy exceptions. First, Mttp expression was undetectable in the livers of Mttp Δ/Δ mice (n = 5), whereas Mttp expression inMttp flox/flox mice was 6-fold higher than the threshold detection level (n = 7) (p = 0.00000002). Second, Scd1 expression in the livers of Mttp Δ/Δ mice was reduced by 69% compared with the livers of Mttp flox/floxmice (p < 0.0005). Northern blots and Western blots confirmed the reduction in Scd1 expressionMttp Δ/Δ livers (Fig.2, A and B).Scd1 expression is up-regulated by SREBP-1 (27Tabor D.E. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1998; 273: 22052-22058Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), so we hypothesized that the levels of mature SREBP-1 would be reduced in livers of Mttp Δ/Δ mice. Indeed, this was the case. SREBP-1 (but not SREBP-2) levels were reduced by ∼50% in the livers of Mttp Δ/Δ mice (Fig.2 C). SREBP-1c expression is reduced by low levels of insulin and induced by insulin replacement (28Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (628) Google Scholar, 29Shimano H. Prog. Lipid Res. 2001; 40: 439-452Crossref PubMed Scopus (587) Google Scholar). To determine whether the inactivation ofMttp affected glucose or insulin levels, plasma triglycerides, glucose, and insulin levels were measured inMttp flox/flox mice (n = 20),Mttp flox/flox mice treated with subcutaneous injections of water (n = 10), andMttp Δ/Δ mice (i.e. Mttp flox/flox mice treated with subcutaneous injections of pI-pC; n = 10). Consistent with the results in Table I, plasma triglyceride levels were significantly reduced in Mttp Δ/Δ mice (p< 0.001). Plasma glucose levels were reduced by ∼20% inMttp Δ/Δ mice (14.06 ± 0.88 mmol/liter in Mttp flox/flox mice versus11.44 ± 0.50 in Mttp Δ/Δ mice;p < 0.05). Plasma insulin levels were reduced by ∼45% in Mttp Δ/Δ mice (0.39 ± 0.30 ng/ml in Mttp flox/flox mice versus0.21 ± 0.06 in Mttp Δ/Δ mice;p < 0.001). Thus, the lower plasma insulin levels inMttp Δ/Δ mice might well contribute to the lower SREBP-1 levels. Scd1 expression is also down-regulated by polyunsaturated fatty acids (30Ntambi J.M. J. Lipid Res. 1999; 40: 1549-1558Abstract Full Text Full Text PDF PubMed Google Scholar, 31Hannah V.C., Ou, J. Luong A. Goldstein J.L. Brown M.S. J. Biol. Chem. 2001; 276: 4365-4372Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 32Xu J. Teran-Garcia M. Park J.H.Y. Nakamura M.T. Clarke S.D. J. Biol. Chem. 2001; 276: 9800-9807Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), so we sought to determine whether levels of polyunsaturated fatty acids were increased inMttp Δ/Δ mice. Interestingly, the predominant polyunsaturated fatty acid, linoleic acid, was increased significantly in the livers of Mttp Δ/Δ mice. The amount of linoleic acid (as a percentage of the total fatty acids) in liver triglycerides was 34.2 ± 4.2 inMttp flox/flox mice (n = 8) and 40.8 ± 2.7 in Mttp Δ/Δ mice (n = 7) (p = 0.0037); the percentage of linoleic acid in liver free fatty acids was 15.8 ± 2.7 inMttp flox/flox mice and 22.7 ± 2.7 inMttp Δ/Δ mice (p = 0.0003). These differences could not be accounted for by differences in the fatty acid composition of the plasma. The amount of linoleic acid (as a percentage of the total fatty acids) in plasma triglycerides was 27.2 ± 5.9 in Mttp flox/flox mice (n = 8) and 24.2 ± 16.3 inMttp Δ/Δ mice (n = 7) (p = 0.62). Because hepatic steatosis in some mouse models leads to hepatic inflammation (26Leclercq I.A. Farrell G.C. Field J. Bell D.R. Gonzalez F.J. Robertson G.R. J. Clin. Invest. 2000; 105: 1067-1075Crossref PubMed Scopus (670) Google Scholar), we suspected that the accumulation of lipids inMttp Δ/Δ mice might affect the expression of many genes, including those involved in inflammatory responses. However, the microarray experiments did not uncover evidence for an active inflammatory response in Mttp Δ/Δlivers. Expression levels for inflammation-related genes (e.g. macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, interferon-α, interferon-β, interferon-γ; and TNF-α) and apoptosis-related genes (bax, bcl-2, caspases 1, 2, 3 and 7, c-jun, c-myc, cytochromec, and fas) were either equally low inMttp Δ/Δ andMttp flox/flox livers or below the threshold of detection. To determine whetherMttp Δ/Δ mice were particularly sensitive to hepatic injury, Mttp Δ/Δ andMttp flox/flox mice were challenged with three toxins known to cause acute liver inflammation (LPS, ConA, and PEA). The inflammatory response triggered by these toxins is characterized by the release of pro-inflammatory cytokines (e.g. TNF-α, interferon-γ, IL-2, and IL-6), which leads to hepatocyte injury and increased plasma levels of AST, ALT, and LDH (33Parrillo J.E. N. Engl. J. Med. 1993; 328: 1471-1478Crossref PubMed Scopus (1507) Google Scholar, 34Tiegs G. Hentschel J. Wendel A. J. Clin. Invest. 1992; 90: 196-203Crossref PubMed Scopus (973) Google Scholar, 35Schümann J. Angermüller S. Bang R. Lohoff M. Tiegs G. J. Immunol. 1998; 161: 5745-5754PubMed Google Scholar). LPS stimulates monocytes and macrophages (33Parrillo J.E. N. Engl. J. Med. 1993; 328: 1471-1478Crossref PubMed Scopus (1507) Google Scholar), whereas ConA primarily stimulates T lymphocytes (34Tiegs G. Hentschel J. Wendel A. J. Clin. Invest. 1992; 90: 196-203Crossref PubMed Scopus (973) Google Scholar). PEA inhibits pro" @default.
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• W2032779971 title "Blocking the Secretion of Hepatic Very Low Density Lipoproteins Renders the Liver More Susceptible to Toxin-induced Injury" @default.
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