Compensatory Increase in Hepatic Lipogenesis in Mice with Conditional Intestine-specific Mttp Deficiency
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m510622200
ISSN1083-351X
AutoresYan Xie, Elizabeth P. Newberry, Stephen G. Young, Sylvie Robine, Robert L. Hamilton, Jinny S. Wong, Jianyang Luo, Susan Kennedy, Nicholas O. Davidson,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoMicrosomal TG transfer protein (MTTP) is required for the assembly and secretion of TG (TG)-rich lipoproteins from both enterocytes and hepatocytes. Liver-specific deletion of Mttp produced a dramatic reduction in plasma very low density lipoprotein-TG and virtually eliminated apolipoprotein B100 (apoB100) secretion yet caused only modest reductions in plasma apoB48 and apoB48 secretion from primary hepatocytes. These observations prompted us to examine the phenotype following intestine-specific Mttp deletion because murine, like human enterocytes, secrete virtually exclusively apoB48. We generated mice with conditional Mttp deletion in villus enterocytes (Mttp-IKO), using a tamoxifen-inducible, intestine-specific Cre transgene. Villus enterocytes from chow-fed Mttp-IKO mice contained large cytoplasmic TG droplets and no chylomicron-sized particles within the secretory pathway. Chow-fed, Mttp-IKO mice manifested steatorrhea, growth arrest, and decreased cholesterol absorption, features that collectively recapitulate the phenotype associated with abetalipoproteinemia. Chylomicron secretion was reduced dramatically in vivo, in conjunction with an ∼80% decrease in apoB48 secretion from primary enterocytes. Additionally, although plasma and hepatic cholesterol and TG content were decreased, Mttp-IKO mice demonstrated a paradoxical increase in both hepatic lipogenesis and very low density lipoprotein secretion. These findings establish distinctive features for MTTP involvement in intestinal chylomicron assembly and secretion and suggest that hepatic lipogenesis undergoes compensatory induction in the face of defective intestinal TG secretion. Microsomal TG transfer protein (MTTP) is required for the assembly and secretion of TG (TG)-rich lipoproteins from both enterocytes and hepatocytes. Liver-specific deletion of Mttp produced a dramatic reduction in plasma very low density lipoprotein-TG and virtually eliminated apolipoprotein B100 (apoB100) secretion yet caused only modest reductions in plasma apoB48 and apoB48 secretion from primary hepatocytes. These observations prompted us to examine the phenotype following intestine-specific Mttp deletion because murine, like human enterocytes, secrete virtually exclusively apoB48. We generated mice with conditional Mttp deletion in villus enterocytes (Mttp-IKO), using a tamoxifen-inducible, intestine-specific Cre transgene. Villus enterocytes from chow-fed Mttp-IKO mice contained large cytoplasmic TG droplets and no chylomicron-sized particles within the secretory pathway. Chow-fed, Mttp-IKO mice manifested steatorrhea, growth arrest, and decreased cholesterol absorption, features that collectively recapitulate the phenotype associated with abetalipoproteinemia. Chylomicron secretion was reduced dramatically in vivo, in conjunction with an ∼80% decrease in apoB48 secretion from primary enterocytes. Additionally, although plasma and hepatic cholesterol and TG content were decreased, Mttp-IKO mice demonstrated a paradoxical increase in both hepatic lipogenesis and very low density lipoprotein secretion. These findings establish distinctive features for MTTP involvement in intestinal chylomicron assembly and secretion and suggest that hepatic lipogenesis undergoes compensatory induction in the face of defective intestinal TG secretion. The mobilization and secretion of triglyceride (TG) 3The abbreviations used are: TG, triglyceride; apoB, apolipoprotein B; FPLC, fast protein liquid chromatography; HDL, high density lipoprotein; HMG-CoA, hydroxymethylglutaryl coenzyme A; LDL, low density lipoprotein; MTTP, microsomal TG transfer protein; Q-PCR, quantitative PCR; VLDL, very low density lipoprotein.3The abbreviations used are: TG, triglyceride; apoB, apolipoprotein B; FPLC, fast protein liquid chromatography; HDL, high density lipoprotein; HMG-CoA, hydroxymethylglutaryl coenzyme A; LDL, low density lipoprotein; MTTP, microsomal TG transfer protein; Q-PCR, quantitative PCR; VLDL, very low density lipoprotein.-rich lipoproteins from mammalian enterocytes and hepatocytes are critically dependent on the integrated function of at least two dominant genes. These include the microsomal triglyceride transfer protein (MTTP), a resident endoplasmic reticulum protein that facilitates the transfer of neutral lipid to a large hydrophobic acceptor protein, apolipoprotein B (apoB) (1.Davidson N.O. Shelness G.S. Annu. Rev. Nutr. 2000; 20: 169-193Crossref PubMed Scopus (228) Google Scholar). ApoB in turn functions as a requisite structural component of the surface of TG-rich lipoproteins and plays a vital role in plasma lipoprotein metabolism (2.Young S.G. Circulation. 1990; 82: 1574-1594Crossref PubMed Scopus (322) Google Scholar). Their physiological importance is exemplified through the phenotypes associated with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia, where structural defects in either the MTTP or APOB gene, respectively, lead to a syndrome of hypocholesterolemia (including low levels of high density lipoprotein (HDL)), mild fat malabsorption with fasting lipid accumulation in the small intestine along with hepatic steatosis (3.Linton M.F. Farese Jr., R.V. Young S.G. J. Lipid Res. 1993; 34: 521-541Abstract Full Text PDF PubMed Google Scholar, 4.Scanu A.M. Aggerbeck L.P. Kruski A.W. Lim C.T. Kayden H.J. J. Clin. Invest. 1974; 53: 440-453Crossref PubMed Scopus (60) Google Scholar). Exploration of the mechanisms underlying these phenotypes has been advanced through studies in murine genetic models in which either the Apob or Mttp gene was deleted. Germ line deletion of either the Apob or Mttp gene, however, resulted in embryonic lethality (5.Farese Jr., R.V. Ruland S.L. Flynn L.M. Stokowski R.P. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1774-1778Crossref PubMed Scopus (210) Google Scholar, 6.Huang L.S. Voyiaziakis E. Markenson D.F. Sokol K.A. Hayek T. Breslow J.L. J. Clin. Invest. 1995; 96: 2152-2161Crossref PubMed Scopus (100) Google Scholar, 7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar), the result of defective lipid delivery to the developing embryo, necessitating alternative approaches to the study of their genetic disruption in the adult animal. In regard to the effects of Mttp deletion, this approach was particularly informative. Heterozygous Mttp knock-out mice grow normally, demonstrate no apparent intestinal phenotype, no effects on plasma TG levels, and only marginally elevated hepatic TG content (5.Farese Jr., R.V. Ruland S.L. Flynn L.M. Stokowski R.P. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1774-1778Crossref PubMed Scopus (210) Google Scholar, 8.Leung G.K. Veniant M.M. Kim S.K. Zlot C.H. Raabe M. Bjorkegren J. Neese R.A. Hellerstein M.K. Young S.G. J. Biol. Chem. 2000; 275: 7515-7520Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), suggesting that half-normal levels of MTTP are well tolerated. Liver-specific deletion of MTTP gene expression, by contrast, produced a much more dramatic phenotype, with almost complete elimination of hepatic TG secretion, reduction in plasma TG and cholesterol levels, and the accumulation of large hepatocyte lipid droplets, with a ∼3-fold increase in hepatic TG content (7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar, 9.Bjorkegren J. Beigneux A. Bergo M.O. Maher J.J. Young S.G. J. Biol. Chem. 2002; 277: 5476-5483Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10.Lieu H.D. Withycombe S.K. Walker Q. Rong J.X. Walzem R.L. Wong J.S. Hamilton R.L. Fisher E.A. Young S.G. Circulation. 2003; 107: 1315-1321Crossref PubMed Scopus (106) Google Scholar). An additional feature of the liver-specific Mttp deletor mice reported by Young and colleagues was the divergent responses observed in hepatocyte secretion of the apoB isoforms. Specifically, these workers (7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar) demonstrated that serum apoB100 levels were decreased by ∼90%, and the secretion of apoB100 from primary murine hepatocytes was virtually eliminated. By contrast, the secretion of apoB48 was essentially unchanged, and there were no significant changes in plasma apoB48 levels (7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar). These findings raised the question of whether the effects of MTTP ablation would be intrinsically less dramatic in murine small intestine, where apoB48 is overwhelmingly the dominant isoform expressed (11.Blanc V. Davidson N.O. J. Biol. Chem. 2003; 278: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). A corollary question posed in the current study was the extent to which intestinal apoB48 secretion requires normal MTTP abundance/activity because evidence from either genetic deletion (7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar) or pharmacologic inhibition of MTTP in murine hepatocytes (12.Kulinski A. Rustaeus S. Vance J.E. J. Biol. Chem. 2002; 277: 31516-31525Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13.Larsson S.L. Skogsberg J. Bjorkegren J. J. Biol. Chem. 2004; 279: 831-836Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) indicated only marginal effects of MTTP deletion on apoB48 secretion. The role of intestinal apoB gene expression in chylomicron assembly and secretion has been inferred from the study of apoB knock-out mice (which otherwise die during embryonic development) rescued by crossing into a hemizygous human apoB transgenic line (HuBTg+/0, ApoB–/–). The offspring of these crosses express apoB in the liver, but not in the small intestine (14.Young S.G. Cham C.M. Pitas R.E. Burri B.J. Connolly A. Flynn L. Pappu A.S. Wong J.S. Hamilton R.L. Farese Jr., R.V. J. Clin. Invest. 1995; 96: 2932-2946Crossref PubMed Scopus (105) Google Scholar). Neonatal HuBTg+/0, Apob–/– mice developed lipid malabsorption, fat-filled enterocytes with large cytoplasmic lipid droplets, and one-half to two-thirds of the animals died before 3 weeks of age (14.Young S.G. Cham C.M. Pitas R.E. Burri B.J. Connolly A. Flynn L. Pappu A.S. Wong J.S. Hamilton R.L. Farese Jr., R.V. J. Clin. Invest. 1995; 96: 2932-2946Crossref PubMed Scopus (105) Google Scholar). Nevertheless, the surviving mice appeared to grow normally while consuming a chow diet and eventually weighed the same as their littermates. These latter findings suggested that intestinal TG absorption may be compensated at levels of low dietary fat intake, despite the absence of intestinal apoB. Although cholesterol absorption in these surviving HuBTg+/0, Apob–/– animals was completely eliminated and serum cholesterol levels slightly lower, serum TG levels were unchanged compared with HuBTg+/0 mice (14.Young S.G. Cham C.M. Pitas R.E. Burri B.J. Connolly A. Flynn L. Pappu A.S. Wong J.S. Hamilton R.L. Farese Jr., R.V. J. Clin. Invest. 1995; 96: 2932-2946Crossref PubMed Scopus (105) Google Scholar). Thus, given the modest effects of intestinal apoB deletion on TG absorption in the surviving adults (on a chow diet), it seemed reasonable to raise the question of whether TG absorption would be affected to a similar extent in young adult animals with intestine-specific Mttp deletion. A corollary question posed in the current study was the extent to which alterations in intestinal Mttp expression might influence systemic cholesterol and TG metabolism. To address these questions, we have generated mice with conditional deletion of Mttp in the small intestinal epithelium. The findings reveal subtle distinctions between the effects of intestinal Mttp and Apob deletion and demonstrate an unexpected compensatory adaptation in hepatic lipogenesis in the setting of defective chylomicron formation and secretion. Animals—Mttpflox/flox mice (7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar) (50% C57BL/6, ∼50% 129/SvJ) were crossed with transgenic villin-Cre-ERT2 deletor mice (15.el Marjou F. Janssen K.P. Chang B.H. Li M. Hindie V. Chan L. Louvard D. Chambon P. Metzger D. Robine S. Genesis. 2004; 39: 186-193Crossref PubMed Scopus (678) Google Scholar) (C57BL/6) mice to generate a compound line, Mttpflox/flox villin-Cre-ERT2 (Mttpf/f VilCre), in a background of (∼75% C57BL/6 and ∼ 25% 129/SvJ). Cre recombinase expression in villus enterocytes was induced by intraperitoneal injection of 1 mg/100 μl tamoxifen (Sigma) 1 mg/day for 5 days, as described previously (15.el Marjou F. Janssen K.P. Chang B.H. Li M. Hindie V. Chan L. Louvard D. Chambon P. Metzger D. Robine S. Genesis. 2004; 39: 186-193Crossref PubMed Scopus (678) Google Scholar). Experiments were performed on mice between 8 and 15 weeks of age, unless otherwise noted, and were undertaken 3 weeks after the tamoxifen injection. Growth analysis was undertaken in groups of mice starting at 4 weeks of age, as noted in the relevant figure legend. Genotyping was performed by PCR using the following primers: for MTTPf/f, sense, 5′-GCTCTCAAGAGGAGGTTAAGG-3′; antisense, 5′-CGTCTTTCAAGAGAATGCCC-3′; for villin-Cre-ERT2, sense, 5′-CAAGCCTGGCTCGACGGCC-3′; antisense, 5′-CGCGAACATCTTCAGGTTCT-3′. In some experiments, where noted, wild type and Apobec-1–/– mice (C57BL/6) were used as a source of primary intestinal enterocytes. Histologic and Ultrastructural Analysis of Tissues—Frozen sections of intestine were stained by oil Red O and examined by light microscopy. Formalin-fixed tissue was also examined by routine hematoxylin staining. For electron microscopic examination, intestinal samples from the proximal jejunum were prepared after perfusion fixation with freshly prepared 1.5% glutaraldehyde and 4% polyvinylpyrrolidone in 0.05% calcium chloride, 0.1 m sodium cacodylate, pH 7.4, for 10 min. Tissues were stained in 2% aqueous uranyl acetate and sections viewed by electron microscopy exactly as detailed previously (16.Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar). For immunochemical detection of Cre staining, the proximal intestine was fixed in 10% neutral formalin, sectioned, and stained with a 1:1,500 dilution of rabbit anti-Cre IgG (Novagen, Madison, WI). Enterocyte Isolation, Lipid Determination, and Metabolic Labeling— Enterocytes were isolated from the entire small intestine as described previously (17.Xie Y. Nassir F. Luo J. Buhman K. Davidson N.O. Am. J. Physiol. 2003; 285: G735-G746Crossref PubMed Scopus (0) Google Scholar) and pooled. Fresh cells were used for pulse-chase experiments; alternatively, cell pellets were frozen at –80 °C for further experiments. For determination of enterocyte lipid content, ∼107 enterocytes were lysed in 1 ml of phosphate-buffered saline by sonication, whereupon 600 μl of lysate was extracted into 5 ml of hexane/isopropyl alcohol, 3:2. The hexane phase was collected, dried under nitrogen, and resuspended in 1% Triton X-100 for enzymatic assay. Determinations of TG, cholesterol, free fatty acid, and phospholipid content were undertaken enzymatically using an L-type triglyceride H kit, Cholesterol E kit, NEFA C kit, or phospholipid B kit, respectively (Wako Chemicals, Neuss, Germany). Aliquots of enterocyte lysate were used for protein content determination by the Bio-Rad protein assay reagent (Bio-Rad Laboratories). Pulse-chase experiments were conducted as described previously (17.Xie Y. Nassir F. Luo J. Buhman K. Davidson N.O. Am. J. Physiol. 2003; 285: G735-G746Crossref PubMed Scopus (0) Google Scholar). In brief, freshly isolated enterocytes (107/incubation) were pulse labeled for 30 min with 250 μCi of 35S-protein labeling mix (PerkinElmer Life Sciences) and chased for 120 min in Dulbecco's modified Eagle's medium supplemented with 10 mm methionine and 5 mm cysteine. A mixed micellar lipid solution (0.4 mm sodium taurocholate, 0.54 mm sodium taurodeoxycholate, 0.3 mm phosphatidylcholine, 0.45 mm oleic acid, 0.26 mm monoolein) was added to both pulse and chase medium for all of the experiments. At the end of the chase, medium and cells were collected and aliquots of lysate and medium immunoprecipitated with rabbit anti-mouse apoB IgG. Quantitation of 35S incorporation into apoB48 was conducted using 4–15% PAGE separation of the immunoprecipitation reactions, which were then quantified using PhosphorImager analysis (Molecular Dynamics, Sunnydale, CA). In some experiments, where noted, isolated enterocytes were labeled as above with 250 μCi of 35S-protein labeling mix/ml for 2 h with or without 10 μm MTTP inhibitor BMS197636 (a generous gift from Dr. John Wetterau at Bristol-Myers Squibb Company). 35S-apoB synthesis and secretion were analyzed in cell lysates and media as described above. Real Time Quantitative Reverse Transcription-PCR (Real Time Q-PCR)—RNA was extracted from isolated enterocytes or liver tissue using TRIzol (Invitrogen) and treated with DNase. Reverse transcription was performed using the SuperScript II First-strand Synthesis System (Invitrogen), with 3 μg of total RNA and random hexamers, to generate cDNA. For Cre detection, cDNA products were amplified with Cre-specific primers: 5′-ATGGAAAATAGCGATCGCTGCCAG-3′ (forward), 5′-ACCAGGCCAGGTATCTCTGACCAG-3′ (reverse). Real time Q-PCR assays were performed in triplicate on an ABI Prim 7000 Sequence Detection System using SYBR Green PCR Master Mix. The mRNA level of individual genes was quantified and normalized against a control reaction for mouse 18 S mRNA. Relative mRNA abundance of individual genes was calculated as -fold change compared with its mRNA level in pooled preparations of isolated enterocytes or livers from 5 Mttpf/f mice. The primers used for real time Q-PCR are listed in Table 1.TABLE 1Oligodeoxyribonucleotide primer sequences for real time Q-PCRGeneForward primerReverse primerABCA15′-AGGGTTTCTTTGCTCAGATTGTC-3′5′-TGCCAAAGGGTGGCACA-3′ABCG55′-TTGCGATACACAGCGATGCT-3′5′-TGACTGCCTCTACCTTCTTGTTGT-3′ABCG85′-CCGTCGTCAGATTTCCAATGA-3′5′-GGCTTCCGACCCATGAATG-3′ACAT25′-GCCTATACTGCCAGGAGTGGTATG-3′5′-ACCATCCCCCAGAATGTTGTC-3′ApoA15′-CCACACCCTTCAGGATGAAAG-3′5′-TGGCTCCCTGTCAGGAAGAC-3′ApoA-IV5′-CCCGGGCTGAGGTCACTT-3′5′-GCATTGTTGCTTAGCTGGGTAA-3′ApoB5′-TGAATGCACGGGCAATGA-3′5′-GGCATTACTTGTTCCATGGTTCT-3′CD365′-GCCAAGCTATTGCGACATGA-3′5′-ATCTCAATGTCCGAGACTTTTCAAC-3′DGAT15′-TCCGCCTCTGGGCATTC-3′5′-GAATCGGCCCACAATCCA-3′DGAT25′-AGAACCGCAAAGGCTTTGTG-3′5′-AGGAATAAGTGGGAACCAGATCAG-3′FANS5′-GGCATCATTGGGCACTCCTT-3′5′-GCTGCAAGCACAGCCTCTCT-3′FATP45′-TTGCAAGTCCCATCAGCAACT-3′5′-GCATACAGAGGCAGCTCCTTT-3′HMGS5′-TGGTGGATGGGAAGCTGTCTA-3′5′-TTCTTGCGGTAGGCTGCATAG-3′HMGR5′-ATTCTGGCAGTCAGTGGGAACT-3′5′-CCTCGTCCTTCGATCCAATTTA-3′I-FABP5′-ACTAATCCAGACCTACACATATGAAGGA-3′5′-GCTCCAGGCTCTGAGAAGTTGA-3′LDLR5′-GCTCCATAGGCTATCTGCTCTTCA-3′5′-CTGCGGTCCAGGGTCATC-3′L-FABP5′CCAGGAGAACTTTGAGCCATTC-3′5′-TGTCCTTCCCTTTCTGGATGA-3′LXRa5′-GCTCTGCTCATTGCCATCAG-3′5′-TGTTGCAGCCTCTCTACTTGGA-3′MGAT25′-GGTGAGTGCCGATCACATTCT-3′5′-CAACGATGATGGCAAGCAAGT-3′MTTP5′-ATGATCCTCTTGGCAGTGCTT-3′5′-TGAGAGGCCAGTTGTGTGAC-3′NPC1L15′-CCACAGACCCTGTGGAACTG-3′5′GCTCGTCATGGAAAGCCTTT-3′PPARa5′-TATTCGGCTGAAGCTGGTGTAC-3′5′-CTGGCATTTGTTCCGGTTCT-3′SAR1a5′-GGGCCGTTGTAAGCATCAATA-3′5′-TTCCAGATTTCTTGTAGAGTCCTAGGA-3′SAR1b5′-GCTAAAAAGGCAGGGCTATGG-3′5′-GGCCGCTGCTAATCGATGTA-3′SR-B15′-TCAGAAGCTGTTCTTGGTCTGAAC-3′5′-GTTCATGGGGATCCCAGAGA-3′SREBP1c5′-GGAGCCATGGATTGCACATT-3′5′-CCTGTCTCACCCCCAGCATA-3′SREBP25′-ACAGCCGCCCTTCAAGTG-3′5′-TCACAGGCATTGTGGTCAGAA-3′18 S5′-CGGCTACCACATCCAAGGAA-3′5′-GCTGGAATTACCGCGGCT-3′ Open table in a new tab Detection of Enterocyte MTTP and ApoB Protein by Western Blotting— Enterocyte lysates were prepared as described previously (17.Xie Y. Nassir F. Luo J. Buhman K. Davidson N.O. Am. J. Physiol. 2003; 285: G735-G746Crossref PubMed Scopus (0) Google Scholar). Aliquots of lysates representing 100 μg of total protein were separated by 4–15% PAGE. Western blots were performed with rabbit antiserum against mouse apoB (1:4,000) or Goat anti-serum against MTTP (1:8,000), and ECL detection reagents. Quantitation was performed by Kodak 440CF imager system. Reactivity against Hsp40 was used as an internal control (StressGen). Cholesterol Absorption and Fecal Lipid Content—Cholesterol absorption was measured by a fecal dual-isotope ratio method, essentially as described previously (18.Wang D.Q. Paigen B. Carey M.C. J. Lipid Res. 2001; 42: 1820-1830Abstract Full Text Full Text PDF PubMed Google Scholar). Mice were gavaged with 150 μl of corn oil mixed with 1 μCi of [14C] cholesterol (PerkinElmer Life Sciences) and 2 μCi of β-[3H]sitostanol (American Radiolabeled Chemicals, Inc.). Mice were then housed individually in fresh metabolic cages, and feces were collected over the next 2 days. The ratio of 14Cto 3Hin each fecal sample was determined, corrected for the ratio in the dosing mixture, and the percent of cholesterol absorption calculated as described previously (18.Wang D.Q. Paigen B. Carey M.C. J. Lipid Res. 2001; 42: 1820-1830Abstract Full Text Full Text PDF PubMed Google Scholar). The lipid content of feces was determined gravimetrically after chloroform/methanol extraction. Determination of Intestinal Apolipoprotein and Triglyceride Secretion in Vivo—Mice were fasted for 16 h, weighed, and injected intravenously with 500 mg/kg body weight Tyloxapol (Sigma). Immediately after tyloxapol administration, the mice received an intragastric bolus of 0.5 ml of lipid emulsion containing 400 μl of 20% Intralipid, 90 μl of corn oil, and 10 μCi of [3H]triolein (American Radiolabel chemicals). Blood samples were collected at 0, 1, 2, 3, and 4 h after injection and plasma TG measured enzymatically. Aliquots of plasma were separated by 4–15% SDS-PAGE and apoB and apoE were detected by Western blot. To ensure a linear response, 1 μl of serum at time zero was run alongside 0.5 μl from the 3 h time point and the images quantitated using a Kodak 440CF Imager System. The increase of plasma apoB48 at 3 h was expressed as -fold increase of apoB48 compared with time zero. In other experiments, where indicated, 200 μl of plasma obtained 4 h after tyloxapol injection and intragastric lipid bolus was size fractionated by fast performance liquid chromatography (FPLC). 20 fractions were collected and used for Western blot analysis of apoB48 distribution. Lipids were extracted from each fraction, separated by TLC, and radiolabeled TG quantitated by scintillation counting. Determination of Hepatic Lipid Content and Secretion and ApoB Secretion in Vivo—Approximately 50 mg of liver was homogenized and used to prepare lipid extracts for enzymatic assay as described previously (19.Newberry E.P. Xie Y. Kennedy S. Han X. Buhman K.K. Luo J. Gross R.W. Davidson N.O. J. Biol. Chem. 2003; 278: 51664-51672Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Hepatic TG and apoB secretion were determined as described previously (20.Nassir F. Xie Y. Patterson B.W. Luo J. Davidson N.O. J. Lipid Res. 2004; 45: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Mice were fasted for 4 h and then injected via tail vein with 500 mg/kg tyloxapol and 500 μCi of 35S-protein labeling mix. Blood samples were collected before injection and every 30 min for 2 h. Plasma TGs and cholesterol were measured enzymatically, as above. Newly synthesized apoB and albumin were estimated by PhosphorImager analysis after 4–15% SDS-PAGE of 2 μl of plasma, as described previously (20.Nassir F. Xie Y. Patterson B.W. Luo J. Davidson N.O. J. Lipid Res. 2004; 45: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). To determine VLDL TG secretion, 50 μl of plasma obtained 2 h after injection was overlaid with 850 μl of 0.15 m NaCl and d < 1.006 (VLDL) and d > 1.006 fractions subsequently separated by ultracentrifugation at 40,000 rpm, 16 h at 14 °C in a Beckman MLA-130 rotor model. TG content was determined enzymatically in the isolated fractions. Lipid Synthesis and Secretion from Isolated Primary Hepatocytes— Primary mouse hepatocytes were isolated by liver perfusion (20.Nassir F. Xie Y. Patterson B.W. Luo J. Davidson N.O. J. Lipid Res. 2004; 45: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and seeded at 3 × 106 cells/T25 collagen-coated flask in hepatocyte wash medium with 10% lipoprotein-deficient serum (21.Gianturco S.H. Bradley W.A. Methods Enzymol. 1986; 129: 319-344Crossref PubMed Scopus (32) Google Scholar). Cells were allowed to attach for 4 h. After a wash with hepatocyte wash medium, cells were radiolabeled for 18 h with 5 μCi/flask [14C]acetate in hepatocyte wash medium with 10% lipoprotein-deficient serum. Media were collected and cells lysed in phosphate-buffered saline and 1% Triton X-100 by sonication. Lipids were extracted from cell lysate and medium as described above. Lipid extracts were dried under nitrogen, solubilized in chloroform, and separated on TLC plates using a solvent system of hexane:ethyl ether:acetic acid (70:30:1). Specific bands were identified by their comigration with authentic standards, scraped, and quantitated by scintillation spectroscopy. Floxed Mttp mice were crossed into the ER-T2 villin-Cre deletor line and recombination induced by tamoxifen administration. Reverse transcription-PCR analysis of intestinal and hepatic RNA (Fig. 1A) confirmed intestine-restricted Cre expression, and nuclear translocation was confirmed by immunochemical localization in heterozygous mice (Fig. 1B, compare left two panels). Biallelic inactivation of Mttp was associated with gross lipid accumulation (Fig. 1B, right panel), described in detail below. Enterocyte MTTP mRNA and protein abundance were decreased after tamoxifen administration, with a range of expression ranging from ∼50% of wild type levels down to undetectable (Fig. 1C). As detailed below, a phenotype associated with Mttp-IKO mice (MTTPΔ/Δ, Fig. 1C) was demonstrated with enterocyte MTTP mRNA and protein expression of less than 20% of wild type levels. Starting at approximately 1 week after tamoxifen administration, Mttp-IKO mice demonstrated a plateau in weight gain (Fig. 2A) associated with the onset of steatorrhea (Fig. 2, B and D). Regression analysis of 53 animals (encompassing control, floxed, and Mttp-IKO mice) revealed a significant negative correlation between MTTP mRNA abundance and fecal fat, with an evident shift when relative MTTP mRNA abundance fell below 20% control (Fig. 2B). The presence of steatorrhea (>7%) on a chow diet excluded animals with residual MTTP expression greater than 20% of control levels, providing a convenient functional screen to identify Mttp-IKO (or MTTPΔ/Δ) mice. Cholesterol absorption, measured by dual isotope ratio methodology, revealed a positive correlation with enterocyte MTTP mRNA abundance (Fig. 2C). The findings demonstrate an ∼60–70% reduction in cholesterol absorption in Mttp-IKO mice (Fig. 2E), suggesting that cholesterol absorption, although decreased, was not eliminated. Even in a subgroup of 11 animals with less than 3% residual MTTP expression, there was still ∼10.5% cholesterol absorption detectable (comparable with the Mttp-IKO group as a whole (Fig. 2E)), which represents values approximately one-third of controls. These findings contrast with results from mice producing apoB in the liver but not the small intestine (14.Young S.G. Cham C.M. Pitas R.E. Burri B.J. Connolly A. Flynn L. Pappu A.S. Wong J.S. Hamilton R.L. Farese Jr., R.V. J. Clin. Invest. 1995; 96: 2932-2946Crossref PubMed Scopus (105) Google Scholar), where cholesterol absorption was undetectable. After a 4-h fast, the entire small intestine of Mttp-IKO mice was visibly engorged with lipid (Fig. 3A), and oil Red O staining of frozen sections revealed confluent lipid droplets from the lower villus to the tip (Fig. 3B). These findings contrast with the findings in Mx1-Cre transgenic mice crossed into the Mttpflox/flox mice, where fat-filled enterocytes were confined to the lower half of the villus (7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar). We next analyzed the proximal small intestine of Mttp-IKO mice using electron microscopy. The apical portions of Mttp-IKO mice contained numerous large lipid droplets, although virtually no VLDL or chylomicron-sized lipoprotein particles were seen within the endoplasmic reticulum or Golgi (Fig. 3, C–E). Occasional, small, electron-dense particles were observed within the Golgi apparatus of Mttp-IKO mice, the functional significance of which is unknown. These findings are reminiscent of the findings of Raabe and co-workers (7.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (354) Google Scholar) and suggest an important element of conservation in the effects of Mttp disruption in murine hepatocytes and enterocytes. Accompanying these morphologic changes, isolated enterocytes from Mttp-IKO mice demonstrated an ∼12-fold increase in TG content and an ∼2-fold increase in free fatty acid con
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