Intestine-specific expression of acyl CoA:diacylglycerol acyltransferase 1 reverses resistance to diet-induced hepatic steatosis and obesity in Dgat1 mice
2010; Elsevier BV; Volume: 51; Issue: 7 Linguagem: Inglês
10.1194/jlr.m002311
ISSN1539-7262
AutoresBonggi Lee, Angela M. Fast, Jiabin Zhu, Ji‐Xin Cheng, Kimberly K. Buhman,
Tópico(s)Diet, Metabolism, and Disease
ResumoMice deficient in acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in triacylglycerol (TG) biosynthesis, are resistant to high-fat (HF) diet-induced hepatic steatosis and obesity. DGAT1-deficient (Dgat1−/−) mice have no defect in quantitative absorption of dietary fat; however, they have abnormally high levels of TG stored in the cytoplasm of enterocytes, and they have a reduced postprandial triglyceridemic response. We generated mice expressing DGAT1 only in the intestine (Dgat1IntONLY) to determine whether this phenotype contributes to resistance to HF diet-induced hepatic steatosis and obesity in Dgat1−/− mice. Despite lacking DGAT1 in liver and adipose tissue, we found that Dgat1IntONLY mice are not resistant to HF diet-induced hepatic steatosis or obesity. The results presented demonstrate that intestinal DGAT1 stimulates dietary fat secretion out of enterocytes and that altering this cellular function alters the fate of dietary fat in specific tissues. Mice deficient in acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in triacylglycerol (TG) biosynthesis, are resistant to high-fat (HF) diet-induced hepatic steatosis and obesity. DGAT1-deficient (Dgat1−/−) mice have no defect in quantitative absorption of dietary fat; however, they have abnormally high levels of TG stored in the cytoplasm of enterocytes, and they have a reduced postprandial triglyceridemic response. We generated mice expressing DGAT1 only in the intestine (Dgat1IntONLY) to determine whether this phenotype contributes to resistance to HF diet-induced hepatic steatosis and obesity in Dgat1−/− mice. Despite lacking DGAT1 in liver and adipose tissue, we found that Dgat1IntONLY mice are not resistant to HF diet-induced hepatic steatosis or obesity. The results presented demonstrate that intestinal DGAT1 stimulates dietary fat secretion out of enterocytes and that altering this cellular function alters the fate of dietary fat in specific tissues. The absorption of dietary fat, the most energy-dense nutrient, by the small intestine is a highly efficient process. Greater than 95% of dietary fat consumed is absorbed whether a low- or high-fat (HF) diet is consumed (1.Tso P. Balint J.A. Bishop M.B. Rodgers J.B. Acute inhibition of intestinal lipid transport by Pluronic L-81 in the rat.Am. J. Physiol. 1981; 241: G487-G497Crossref PubMed Google Scholar), as evidenced by the small amount of fat that is excreted in feces. In the small intestine lumen, dietary fat in the form of triacylglycerol (TG) is hydrolyzed to generate free fatty acids and monoacylglycerol by pancreatic lipase. These products are then emulsified with the help of phospholipids and bile acids present in bile to form micelles. Free fatty acids and monoacylglycerol are then taken up by the absorptive cells of the small intestine, enterocytes, where they are resynthesized into TGs and incorporated into the core of chylomicrons, which are secreted via the lymphatic system into circulation (2.Mansbach C.M. Gorelick F. Development and physiological regulation of intestinal lipid absorption. II. Dietary lipid absorption, complex lipid synthesis, and the intracellular packaging and secretion of chylomicrons.Am. J. Physiol. Gastrointest. Liver Physiol. 2007; 293: G645-G650Crossref PubMed Scopus (116) Google Scholar). TG is then delivered to cells throughout the body, where it serves diverse functions, including energy storage and generation depending on energy status. The postprandial triglyceridemic response (or levels of TG in blood after a meal) is thus dependent on both the appearance of TG in and the clearance of TG from circulation. Because of its high energy density, high efficiency of absorption, ability to be stored when energy is in excess, and ability to be oxidized to generate energy when needed, dietary fat and its absorption by the small intestine are important determinants of energy balance.TGs synthesized within enterocytes are secreted into circulation in a time- and amount-dependent manner. As the amount of dietary fat increases, postprandial triglyceridemia also increases (3.Dubois C. Beaumier G. Juhel C. Armand M. Portugal H. Pauli A.M. Borel P. Latge C. Lairon D. Effects of graded amounts (0–50 g) of dietary fat on postprandial lipemia and lipoproteins in normolipidemic adults.Am. J. Clin. Nutr. 1998; 67: 31-38Crossref PubMed Scopus (174) Google Scholar, 4.Lairon D. Macronutrient intake and modulation on chylomicron production and clearance.Atheroscler. Suppl. 2008; 9: 45-48Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In addition, under HF dietary challenges, TG is also found packaged in enterocytes in cytoplasmic lipid droplets (CLDs) (5.Zhu J. Lee B. Buhman K.K. Cheng J.X. A dynamic, cytoplasmic triacylglycerol pool in enterocytes revealed by ex vivo and in vivo coherent anti-Stokes Raman scattering imaging.J. Lipid Res. 2009; 50: 1080-1089Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). We recently demonstrated that this storage pool of TG in enterocytes expands and depletes relative to the fed-fasted state and is present whether mice are acutely or chronically challenged by high levels of dietary fat (5.Zhu J. Lee B. Buhman K.K. Cheng J.X. A dynamic, cytoplasmic triacylglycerol pool in enterocytes revealed by ex vivo and in vivo coherent anti-Stokes Raman scattering imaging.J. Lipid Res. 2009; 50: 1080-1089Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). These results suggest that TG stored in CLDs are eventually hydrolyzed, reesterified, and secreted on chylomicrons. Regulation of storage versus secretion of TG from enterocytes may be involved in determination of the postprandial triglyceridemic response. The postprandial triglyceridemic response, in terms of time and amount, is likely an important determinant of which cell types clear TG from circulation and the metabolic fate of the fatty acids within these cells (6.Fielding B.A. Frayn K.N. Lipoprotein lipase and the disposition of dietary fatty acids.Br. J. Nutr. 1998; 80: 495-502Crossref PubMed Google Scholar, 7.Yen C.L. Cheong M.L. Grueter C. Zhou P. Moriwaki J. Wong J.S. Hubbard B. Marmor S. Farese Jr, R.V. Deficiency of the intestinal enzyme acyl CoA:monoacylglycerol acyltransferase-2 protects mice from metabolic disorders induced by high-fat feeding.Nat. Med. 2009; 15: 442-446Crossref PubMed Scopus (131) Google Scholar). An exaggerated postprandial triglyceridemic response has been found to be associated with obesity and higher body fat content in humans (8.Blackburn P. Lamarche B. Couillard C. Pascot A. Tremblay A. Bergeron J. Lemieux I. Despres J.P. Contribution of visceral adiposity to the exaggerated postprandial lipemia of men with impaired glucose tolerance.Diabetes Care. 2003; 26: 3303-3309Crossref PubMed Scopus (49) Google Scholar, 9.Blackburn P. Lamarche B. Couillard C. Pascot A. Bergeron N. Prud'homme D. Tremblay A. Bergeron J. Lemieux I. Despres J.P. Postprandial hyperlipidemia: another correlate of the "hypertriglyceridemic waist" phenotype in men.Atherosclerosis. 2003; 171: 327-336Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 10.van Oostrom A.J. Castro C.M. Ribalta J. Masana L. Twickler T.B. Remijnse T.A. Erkelens D.W. Diurnal triglyceride profiles in healthy normolipidemic male subjects are associated to insulin sensitivity, body composition and diet.Eur. J. Clin. 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Whether or not an exaggerated postprandial triglyceridemic response is due to altered regulation of TG storage versus secretion in enterocytes and thus appearance of TG in circulation deserves further attention.Specific pathways for TG synthesis may determine whether the TGs are stored or secreted by the enterocyte. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final reaction in the synthesis of TG from diacylglycerol and fatty acyl-CoA in many cell types (13.Buhman K.K. Chen H.C. Farese R.V. The enzymes of neutral lipid synthesis.J. Biol. Chem. 2001; 276: 40369-40372Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 14.Cases S. Smith S.J. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. et al.Identification of a gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.Proc. Natl. Acad. Sci. USA. 1998; 95: 13018-13023Crossref PubMed Scopus (858) Google Scholar). DGAT activity has been described on the cytoplasmic and luminal sides of the endoplasmic reticulum and proposed to synthesize TG with fates of storage and secretion, respectively (15.Owen M. Zammit V.A. Evidence for overt and latent forms of DGAT in rat liver microsomes. Implications for the pathways of triacylglycerol incorporation into VLDL.Biochem. Soc. Trans. 1997; 25: 21SCrossref PubMed Scopus (21) Google Scholar, 16.Owen M.R. Corstorphine C.C. Zammit V.A. Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.Biochem. J. 1997; 323: 17-21Crossref PubMed Scopus (110) Google Scholar). More recently, two unrelated gene families encoding DGAT enzymes have been identified. DGAT1 and DGAT2 are expressed ubiquitously in cell types and tissues including enterocytes (14.Cases S. Smith S.J. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. et al.Identification of a gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.Proc. Natl. Acad. Sci. USA. 1998; 95: 13018-13023Crossref PubMed Scopus (858) Google Scholar, 17.Buhman K.K. Smith S.J. Stone S.J. Repa J.J. Wong J.S. Knapp F.F. Burri B.J. Hamilton R.L. Abumrad N.A. Farese R.V. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis.J. Biol. Chem. 2002; 277: 25474-25479Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 18.Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members.J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). Studies of their intracellular localization suggest that DGAT1 and DGAT2 do not have redundant functions within cells. DGAT1 and DGAT2 localize to specific regions of the ER in seeds of the tung tree (19.Shockey J.M. Gidda S.K. Chapital D.C. Kuan J.C. Dhanoa P.K. Bland J.M. Rothstein S.J. Mullen R.T. Dyer J.M. Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum.Plant Cell. 2006; 18: 2294-2313Crossref PubMed Scopus (417) Google Scholar). In addition, DGAT2 has been found in and associated with other cellular organelles such as lipid droplets (20.Kuerschner L. Moessinger C. Thiele C. Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets.Traffic. 2008; 9: 338-352Crossref PubMed Scopus (300) Google Scholar, 21.Lardizabal K.D. Mai J.T. Wagner N.W. Wyrick A. Voelker T. Hawkins D.J. DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity.J. Biol. Chem. 2001; 276: 38862-38869Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), mitochondria-associated membranes, and mitochondria (22.Stone S.J. Levin M.C. Zhou P. Han J. Walther T.C. Farese Jr, R.V. The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal that promotes its association with mitochondria.J. Biol. Chem. 2009; 284: 5352-5361Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). DGAT1, on the other hand, is not associated with lipid droplets in COS-7 cells after oleate loading (22.Stone S.J. Levin M.C. Zhou P. Han J. Walther T.C. Farese Jr, R.V. The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal that promotes its association with mitochondria.J. Biol. Chem. 2009; 284: 5352-5361Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar).Investigation of mice with targeted deletion or tissue-specific overexpression of either of these enzymes has shed some light on the in vivo functions of these enzymes; however, multiple questions remain. DGAT1-deficient (Dgat1−/−) mice are resistant to HF diet-induced obesity due in part to an increase in systemic energy expenditure (23.Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (725) Google Scholar). They have reduced tissue TG concentration in all tissues except the small intestine after a HF dietary challenge. Enterocytes from HF-fed Dgat1−/− mice abnormally accumulate multiple, large CLDs that are TG rich (17.Buhman K.K. Smith S.J. Stone S.J. Repa J.J. Wong J.S. Knapp F.F. Burri B.J. Hamilton R.L. Abumrad N.A. Farese R.V. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis.J. Biol. Chem. 2002; 277: 25474-25479Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar); however, these mice have no gross defects in dietary TG absorption, as determined by fecal fat analysis (23.Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (725) Google Scholar). However, they have a reduced postprandial triglyceridemic response compared with wild-type (WT) mice (17.Buhman K.K. Smith S.J. Stone S.J. Repa J.J. Wong J.S. Knapp F.F. Burri B.J. Hamilton R.L. Abumrad N.A. Farese R.V. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis.J. Biol. Chem. 2002; 277: 25474-25479Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). DGAT2-deficient mice die shortly after birth and thus the in vivo role of DGAT2 deficiency in TG metabolism remains less clear (24.Stone S.J. Myers H.M. Watkins S.M. Brown B.E. Feingold K.R. Elias P.M. Farese Jr, R.V. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice.J. Biol. Chem. 2004; 279: 11767-11776Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar).In this study, we tested the hypothesis that the intestine phenotype of Dgat1−/− mice (excess enterocyte TG storage and reduced postprandial triglyceridemic response) contributes to their resistance to HF diet-induced hepatic steatosis and obesity. We generated transgenic mice that overexpress mouse DGAT1 specifically in the intestine (Dgat1Int) and crossed them with Dgat1−/− mice to generate mice with expression of DGAT1 only in the intestine (Dgat1IntONLY). Here, we report the phenotype of Dgat1IntONLY mice related to their susceptibility to HF-diet induced hepatic steatosis and obesity.MATERIALS AND METHODSDiets and miceAll procedures were approved by the Purdue Animal Care and Use Committee. The VILL-Dgat1 transgene was made with a 12.4 kb VILL promoter/enhancer from the pUC12.4 kb-villin plasmid (Dr. Deborah L. Gumucio, University of Michigan, Ann Arbor, Michigan) and a 1.6 kb amino terminal flag tagged Dgat1 cDNA sequence from the pBSSK/moDGATflag plasmid (Dr. Robert V. Farese Jr., Gladstone Institutes, San Francisco, California). The final transgene clone containing the villin promoter/enhancer and mouse Dgat1 cDNA was verified by restriction mapping and sequence analysis. The transgene was prepared by digestion with PmeI and purification before introduction into C57BL/6 fertilized eggs by microinjection (Purdue Transgenic Core Facility, Purdue University, West Lafayette, Indiana). Founder animals were backcrossed with C57BL/6 mice to generate Dgat1Int mice. Dgat1Int mice were crossed with Dgat1−/− mice to generate Dgat1IntONLYmice. All mice in the studies described were female unless otherwise noted and were maintained in a specific pathogen-free barrier facility with a 12 h-light/-dark cycle (6 AM/6 PM). Mice were fed either a low-fat, rodent chow (PicoLab 5053, Lab Diets, Richmond, IN) or a HF (D12492, Research Diets, Inc., New Brunswick, NJ) diet for the indicated times. For chow, 62.1% of calories came from carbohydrate (starch), 24.7% from protein, and 13.2% from fat. For HF, 20% of calories came from carbohydrate (35% sucrose, 65% starch), 20% from protein, and 60% from fat (mostly lard). Mice were enrolled in body weight studies at 11–13 weeks of age and fed the indicated diets for 9 weeks. Mice not enrolled in body weight studies were 3–5 months of age and fed the indicated diets. Mice were euthanized at 9 AM after fasting for 2 h unless specifically noted. For analysis of the small intestine, we divided the small intestine into three equal-length segments and labeled them S1–S3 (proximal to distal). For specific experiments, we divided each of these segments additionally into two equal-length segments and labeled these a and b (proximal to distal). We scraped mucosa from each region of intestine for RT-PCR or quantitative PCR (QPCR) and intestinal TG quantification.GenotypingGenotyping was performed on genomic DNA extracted from mouse tails to determine both transgene and endogenous gene presence. To determine transgene presence, a FLAG primer set was used with forward primer F 5′-ATGGGAGATTACAAAGATGATGATGATG-3′ and reverse primer R 5′-AGAATCTTGCAGACGATGGCA-3′ amplifying a 433 bp product in transgenic mice and no product in WT mice. To determine endogenous gene presence or absence, a previously described genotyping reaction for Dgat1−/− mice was used (23.Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (725) Google Scholar).RT-PCR and QPCRTotal RNA was extracted from tissues with RNA STAT60 (Tel-Test, Friendswood, TX) and then DNase treated with Turbo DNA-free (Ambion, Austin, TX). cDNA was synthesized from 1µg DNase-treated RNA by AffinityScript QPCR cDNA using oligo dT and random hexamer primers (Stratagene, La Jolla, CA). SYBR green QPCR and RT-PCR were performed using a Mx3000P QPCR System (Stratagene) and Brilliant SYBR green master mix (Stratagene) or GoTaq Green master mix (Promega), respectively. Thermocycling parameters for QPCR included 1 cycle of 95°C for 10 min; 40 cycles at 95°C for 30 s, 55°C for 60 s, and 72°C for 30 s; and 1 cycle at 95°C for 60 s, 55°C for 30 s, and 95°C for 30 s. Thermocycling parameters for RT-PCR included 1 cycle of 95°C for 2 min; 30 cycles at 95°C for 30 s, 55°C for 60 s, and 72°C for 60 s; and 1 cycle at 72°C for 5 min. Post-PCR products were subjected to 1.5% agarose gel electrophoresis for imaging product size. The expression of each gene was normalized to 18s rRNA and calculated with the comparative CT method. Primers used for this study were all validated for efficiency and correct product size in cDNA from mouse intestinal mucosa. The sequences are as follows: DGAT1, F 5′-ACCGCGAGTTCTACAGAGATTGGT-3′ and R 5′-ACAGCTGCATTGCCATAGTTCCCT-3′; DGAT2, F 5′-TGGGTCCAGAAGAA GTTCCAGAAGTA-3′ and R 5′-ACCTCAGTCTCTG GAAGG CCAAAT-3′; 18s rRNA, F 5′-TTAGAGTGTTCAAA GCAGGCCCGA-3′ and R 5′-TCTTGGCAAATGCTTTCGCTCTGG-3.' For RT-PCR, the FLAG primer described under genotyping and the DGAT1 primer were used to amplify products of 433 and 301 bp, respectively.Immunoblot analysisIntestinal mucosa from region S1a (defined under Diets and Mice) was homogenized using a 26G needle in TNET-C buffer [1% Triton, 150 mM NaCl, 50 mM Tris (pH 7.4), 2 mM EDTA, 0.5% cholate] including protease inhibitor cocktail (Complete Mini, Roche, Indianapolis, IN). Proteins (50 μg) were separated by 12% Tris-glycine gel (Invitrogen, Carlsbad, CA) and transferred to PVDF membrane (Hybond-P PVDF membrane, Amersham Bioscience, Piscataway, NJ). Ponceau S staining was done to confirm equal protein loading and transfer efficiency. The membrane was incubated in 5% skim milk buffer [5% skim milk in 0.1% PBS-T (Tween 20) solution] for 1 h with shaking to block nonspecific binding. The membrane was treated with primary goat polyclonal antibodies for DGAT1 (Santa Cruz Biotechnology, Santa Cruz, CA) and primary mouse monoclonal antibody for β-actin (Santa Cruz Biotechnology) for 1 h with shaking. After washing with 0.1% PBS-T three times, donkey anti-goat IgG-HRP for DGAT1 and donkey anti-mouse IgG-HRP for β-actin (Santa Cruz Biotechnology) were incubated for 1 h with shaking and then an enhanced chemiluminescence kit (ECL Western Blotting Substrate, Pierce, Rockford, IL) was used for detection.Determination of TG and glucose concentrationsLipids in intestinal mucosa (S2b, representing jejunum) and liver were extracted by the hexane/isopropanol (3:2) method (25.Hara A. Radin N.S. Lipid extraction of tissues with a low-toxicity solvent.Anal. Biochem. 1978; 90: 420-426Crossref PubMed Scopus (2038) Google Scholar). Briefly, after homogenization of the mucosa with 1 M Tris-HCL (pH 7.4), hexane/isopropanol (3:2) and water were added and then the mucosa sample was incubated for 30 min with occasional mixing. The upper part containing lipids was removed to a new tube. After evaporating the organic phase under nitrogen, lipids were dissolved in isopropanol. The amounts of TG were then determined by Wako L-Type TG determination kit (Wako Chemical USA, Richmond, VA) and were normalized to the protein concentration (Pierce). To measure fasting plasma TG and glucose concentrations, mice were overnight fasted and blood samples obtained from the submandibular vein. TG concentration was determined by a Wako L-Type TG determination kit (Wako Chemicals USA), and glucose concentration was determined by OneTouch glucometer (LifeScan, Milpitas, CA).Determination of fecal fatFeces were collected for 4 days during the sixth week of the HF diet feeding period for fecal fat analysis. The feces were dried for 2 h using an ANKOMRD dryer (ANKOM technology, Macedon, NY) and then lipids were extracted by automated Soxhlet extraction (petroleum ether) using the ANKOMXT15 extraction system (ANKOM technology, Macedon, NY, American Oil Chemist Society Official Procedure Am 5-04). The analysis is achieved by measuring the loss of mass due to the extraction of fat from the dried fecal samples.Coherent anti-Stokes Raman scattering microscopyFor intact tissue imaging, ex vivo fresh tissues (5 mm) from small intestine (S2a, representing upper jejunum) were placed in 3 ml Dulbecco's Modified Eagle's Medium (Gibco, Carlsbad, CA) supplemented with 20 mM HEPES, 100 U/ml penicillin-streptomycin (Gibco), and 10% fetal bovine serum. Tissues kept at 4°C maintained good morphology over 5 h. Small intestine tissue was cut longitudinally and laid flat for luminal imaging. All tissues were imaged within 3 h after euthanasia. Coherent anti-Stokes Raman scattering (CARS) imaging were performed at a multimodal microscope (26.Wang H.W. Le T.T. Cheng J.X. Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope.Opt. Commun. 2008; 281: 1813-1822Crossref PubMed Scopus (125) Google Scholar). Pump and Stokes lasers were generated from two synchronized Ti:sapphire lasers (Tsunami, Spectra-Physics, Mountain View, CA), with a pulse width of 5 ps. These two lasers were tightly synchronized (Lock-to-Clock, Spectra-Physics), colinearly combined, and directed into a laser scanning confocal microscope (FV300/IX71, Olympus America, Center Valley, PA). A 60× water immersion objective (numerical aperture = 1.2) or a 20× air objective (numerical aperture = 0.75) were used to focus the laser beams into the sample. The average powers of the pump and Stokes beams at the sample were 40 and 30 mW, respectively. For imaging TGs, the pump laser and the Stokes laser were tuned to 14,140 cm−1 and 11,300 cm−1, respectively, to generate a Raman shift of ∼2,840 cm−1 that excites the symmetric CH2 vibration (27.Nan X. Cheng J.X. Xie X.S. Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy.J. Lipid Res. 2003; 44: 2202-2208Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 28.Cheng J.X. Xie X.S. Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications.J. Phys. Chem. B. 2004; 108: 827-840Crossref Scopus (843) Google Scholar). The forward-detected CARS signals were collected using an air condenser (N.A. = 0.55). A external photomultiplier tube (R3896, Hamamatsu, Japan) detector was used to receive the forward-detected CARS signals.Data and statisticsAll the data are shown as mean ± SEM. Statistical differences were evaluated with one-way ANOVA, with Tukey's Studentized grouping test between groups or a t-test where appropriate (P < 0.05) using SAS 9.1 program.RESULTSGeneration of Dgat1Int miceDgat1Int mice were generated with a DNA construct containing the villin promoter/enhancer driving expression of mouse DGAT1 containing a FLAG epitope at the N terminus (Fig. 1A). The villin promoter/enhancer drives expression in epithelial cells of the small and large intestine as well as from crypt to tip on the villus beginning as early as 12.5 days post coitum (29.Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J. Biol. Chem. 2002; 277: 33275-33283Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). Of five founder lines, we further investigated three lines with 2- (Dgat1Int2×), 5- (Dgat1Int5×), and 20- (Dgat1Int20×) fold increased levels of Dgat1 mRNA (transgene plus endogenous gene) compared with WT mice fed a low fat, chow diet (Fig. 1B) determined by QPCR. Using RT-PCR, we found that all three lines had Dgat1 transgene present along the length of the small intestine and colon as well as in the kidney consistent with other mouse models generated using this promoter (29.Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J. Biol. Chem. 2002; 277: 33275-33283Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 30.Xue Y. Fleet J.C. Intestinal vitamin D receptor is required for normal calcium and bone metabolism in mice.Gastroenterology. 2009; 136: 1317-1327Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), but not in muscle, heart, liver, or adipose tissue (Fig. 1C). We refer to the mice throughout this manuscript as intestine specific or intestine only; however, we acknowledge that expression is also present in the kidney and all results should be viewed in light of this fact. DGAT1 protein levels were found to be significantly higher in intestinal mucosa from Dgat1Int20× mice compared with WT mice (Fig. 3C). As a marker of DGAT1 function in enterocytes, we examined the ability of DGAT1 overexpression in the small intestine of Dgat1−/− mice and found that it complements the intestine phenotype of Dgat1−/− mice in terms of TG storage and postprandial triglyceridemic response (Fig. 4, Fig. 5, described below). Dgat1Int5× and Dgat1Int20× mice fed a low-fat (data not shown) or a HF diet for 9 weeks (Fig. 2 and 6A, respectively) had similar body weights and dietary fat absorption (Table 1) as WT mice. Further analysis of the Dgat1Int20× mouse line is described below.Fig. 3Generation of Dgat1IntONLY mice. A: RT-PCR analysis was performed with tissues from WT, Dgat1−/−, and Dgat1IntONLY mice using primers specific for the FLAG epitope, Dgat1 primers for both endogenous and transgene Dgat1, and 18S rRNA. Tissues included: small intestine (S1, S2, and S3, divided in three equal-length regions), kidney (K), muscle (M), adipose tissue (A), and liver (L). B: Dgat1 and Dgat2 relative mRNA levels. mRNA levels of Dgat1 and Dgat2 were determined by QPCR analysis of small intestine tissue representing upper jejunum (S1b) from WT, Dgat1−/−, Dgat1Int20×, and Dgat1IntONLY mice. Data are represented as mean ± SEM. Bars with asterisks significantly differ from each other, P < 0.05, n = 5 mice. Gene expression levels were normalized to 18S rRNA. C: DGAT1 protein levels. DGAT1 protein levels were determined by immunoblot analysis of small intestinal mucosa representing duodenum (S1a) from WT, Dgat1−/−, Dgat1Int20×, and Dgat1IntONLY mice fed a HF diet for 9 weeks. Abbreviations: NTC, no template control, NoRT, no reverse transcriptase control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Abr
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