Niemann-Pick C1-Like 1 deletion in mice prevents high-fat diet-induced fatty liver by reducing lipogenesis

• DOI: 10.1194/jlr.m006353
• Publication Date: 2010-08-11T05:45:28Z
• Authors: Lin Jia; Yinyan Ma; Shunxing Rong; Jenna L. Betters; Ping Xie; Soonkyu Chung; Nanping Wang; Weiqing Tang; Liqing Yu
• Keywords: Lipogenesis; Steatosis; Hyperinsulinemia; Ezetimibe; Fatty acid synthesis

Niemann-Pick C1-Like 1 (NPC1L1) mediates intestinal absorption of dietary and biliary cholesterol. Ezetimibe, by inhibiting NPC1L1 function, is widely used to treat hypercholesterolemia in humans. Interestingly, ezetimibe treatment appears attenuate hepatic steatosis rodents humans without a defined mechanism. Overconsumption high-fat diet (HFD) represents major cause metabolic disorders including fatty liver. To determine whether how deficiency prevents HFD-induced steatosis, this study, we fed knockout (L1-KO) mice their wild-type (WT) controls an HFD, found that 24 weeks HFD feeding causes no liver L1-KO mice. Hepatic acid synthesis levels mRNAs for lipogenic genes are substantially reduced but lipoprotein-triglyceride production, oxidation, triglyceride hydrolysis remain unaltered versus WT Strikingly, completely protected against hyperinsulinemia under both fasted states during glucose challenge. Despite similar tolerance, relative more insulin sensitive the overnight-fasted state display significantly lower plasma concentrations. In conclusion, reducing lipogenesis, at least part, through attenuating resistance, known drive lipogenesis elevated circulating levels. polytopic transmembrane protein essential cholesterol (1Davies J.P. Levy B. Ioannou Y.A. Evidence Niemann-pick C (NPC) gene family: identification characterization NPC1L1.Genomics. 2000; 65: 137-145Crossref PubMed Scopus (189) Google Scholar, 2Altmann S.W. Davis Jr., H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng et al.Niemann-Pick C1 Like critical absorption.Science. 2004; 303: 1201-1204Crossref (1441) Scholar). The tissue distribution expression differs among species. humans, abundantly expressed small intestine (2Altmann 3Davies Scott C. Oishi K. Liapis Inactivation multiple lipid transport defects protects diet-induced hypercholesterolemia.J. Biol. Chem. 2005; 280: 12710-12720Abstract Full Text PDF (247) 4Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Davies Nilsson Yu L. C1-like regulates concentration target ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref (306) rodents, almost exclusively where it resides apical membrane absorptive enterocytes resistant high-cholesterol due substantial reduction 5Davis Liu J. Lam M.H. Lund E.G. phytosterol transporter key modulator whole-body homeostasis.J. 279: 33586-33592Abstract (594) 6Davis Graziano M.P. Altmann Deficiency atherosclerosis ApoE−/− mice.Arterioscler. Thromb. Vasc. 27: 841-849Crossref (112) (7Garcia-Calvo Lisnock Bull H.G. Hawes B.E. Burnett D.A. Braun Crona J.H. Dean D.C. Detmers P.A. al.The (NPC1L1).Proc. Natl. Acad. Sci. USA. 102: 8132-8137Crossref (656) 8Weinglass A.B. Kohler Schulte U. Nketiah E.O. Thomas Schmalhofer Williams Bildl McMasters D.R. al.Extracellular loop important binding ezetimibe.Proc. 2008; 105: 11140-11145Crossref (94) Scholar), potent inhibitor blood low density lipoprotein (LDL)-cholesterol (9Davis Veltri E.P. Zetia: inhibition reduce hyperlipidemia.J. Atheroscler. 14: 99-108Crossref (145) Unexpectedly, or was recently shown improve many besides obese (10Chan Watts G.F. Gan S.K. Ooi E.M. Barrett P.H. Effect on fat, inflammatory markers, apolipoprotein B-100 kinetics insulin-resistant subjects weight loss diet.Diabetes Care. 2010; 33: 1134-1139Crossref (142) 11Yagi S. Akaike Aihara Iwase T. Ishikawa Yoshida Sumitomo-Ueda Kusunose Niki Yamaguchi al.Ezetimibe ameliorates microalbuminuria patients with 17: 173-180Crossref (40) For instance, lessened improved sensitivity leptin receptor-deficient Zucker rats (12Deushi Nomura Kawakami Haraguchi Ito Okazaki Ishii H. Ezetimibe improves resistance rat model syndrome.FEBS Lett. 581: 5664-5670Crossref (147) 13Nomura Inhibition Neiman-Pick resistance.Am. Physiol. Endocrinol. Metab. 2009; (Epub ahead print. August 4, 2009)Crossref (62) also methionine choline-deficient (14Assy N. Grozovski Bersudsky I. Szvalb Hussein O. insulin-sensitizing agents combination ezetimibe, valsartan non-alcoholic disease.World Gastroenterol. 2006; 12: 4369-4376Crossref (49) Scholar) diets containing high amounts (15Zheng Cook van Heek Hwa J.J. fat disease mice.Eur. Pharmacol. 584: 118-124Crossref study focusing obesity diabetes, gain (HFD), though remains be determined intervention influences development (16Labonte E.D. Camarota Rojas J.C. Jandacek R.J. Gilham D.E. Tso P. Hui D.Y. Howles P.N. Reduced saturated acids diabetes ezetimibe-treated Npc1l1−/− mice.Am. Gastrointest. Liver 295: G776-G783Crossref (97) another role trafficking hypercholesterolemia, were observed induced Paigen diet, lithogenic contains bile (3Davies Additionally, decreased content these interesting observations, mechanisms which alleviates largely unexplored. steatohepatitis, fibrosis, cirrhosis collectively as nonalcoholic (NAFLD) (17Browning J.D. Horton Molecular mediators injury.J. 114: 147-152Crossref (1768) general population, estimated prevalence NAFLD could 24% (18Clark J.M. epidemiology adults.J. 40: S5-S10PubMed may occur 17–33% Americans (19Browning Szczepaniak L.S. Dobbins R. Nuremberg Cohen Grundy S.M. Hobbs H.H. Prevalence urban population United States: impact ethnicity.Hepatology. 1387-1395Crossref (2955) representing burgeoning health problem worldwide. Fat overconsumption plays etiology steatosis. Effects have not been examined. Earlier studies performed animals either deficient pathway challenged nonphysiological 14Assy Different likely distinct mechanisms. conceivable cholesterol-dependent X receptor (LXR) activation (20Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling mediated nuclear LXR alpha.Nature. 1996; 383: 728-731Crossref (1476) activation-induced (21Schultz Tu Luk Repa Medina Li Schwendner Wang Thoolen al.Role LXRs control lipogenesis.Genes Dev. 2831-2838Crossref (1404) limiting absorption. Consistent this, accumulate less after T0901317 (22Tang Jia required agonist raise HDL 28: 448-454Crossref (43) synthetic promotes During feeding, develop pathological associated state, selectively insulin-induced suppression gluconeogenesis, stays insulin-stimulated sterol regulatory element-binding (SREBP)-1c (23Chen Liang Ou Goldstein J.L. Brown M.S. Central insulin-mediated Srebp-1c transcription stimulation liver.Proc. 101: 11245-11250Crossref (439) membrane-bound factor whose (24Horton SREBPs: activators complete program liver.J. 2002; 109: 1125-1131Crossref (3835) thereby developing (25Brown Selective total resistance: pathogenic paradox.Cell 7: 95-96Abstract (692) itself turn dampen signaling therefore aggravate systemic (26Shulman G.I. Cellular resistance.J. 106: 171-176Crossref (2201) This vicious cycle mechanism underlying We hypothesized prevent mainly stopping cycle. hypothesis, showed does level ∼85% mice, all Thus, HFD-driven resistance-hepatic blocked NPC1L1-deficient Our findings promote clinical effects pure C57BL/6 background reported previously All housed specific pathogen-free animal facility plastic cages 22°C daylight from 6AM 6PM. provided water standard chow (Prolab RMH 3000) ad libitum unless stated otherwise. procedures approved Institutional Animal Care Use Committee Wake Forest University Health Sciences. Male (TD.93075; Harlan Teklad, Madison, WI) starting 6 age. derives 54.8% calories 21.2% protein, carbohydrates. It only trace amount (∼0.007%). composition 28% saturated-, 30% monounsaturated-(trans), monounsaturated-(cis), 14% polyunsaturated-(cis) (27Biddinger S.B. Almind Miyazaki Kokkotou E. Ntambi Kahn C.R. genetic protein-1c, stearoyl-CoA desaturase 1, syndrome.Diabetes. 54: 1314-1323Crossref (202) After 18 determination fecal neutral excretion described (28Tang Genetic inactivation sitosterolemia lacking ABCG5/ABCG8.J. Lipid Res. 50: 293-300Abstract (45) 13 individually 7 days test except 5% replaced nonabsorbable marker, sucrose poly-behenate (29Jandacek Heubi J.E. A novel, noninvasive method measurement absorption.Gastroenterology. 127: 139-144Abstract (93) prepared our core primate center last 3 samples collected. feces gas chromatography fractional individual calculated Plasma adiponectin measured Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem, Inc.) Quantikine Adiponectin/Acrp30 (R & D Systems), respectively. Fed Glucose Assay (Cayman Chemical Co.). β-hydroxybutyrate (LiquiColor, Stanbio) nonesterified [HR Series NEFA-HR Wako] following manufacturer's instructions. concentrations cholesterol, free analyzed using Cholesterol/HP (Roche), Free Cholesterol (Wako), Triglycerides/GB (Roche) enzymatic assay kits, 4 h fast cycle, euthanized bleeding livers removed snap-frozen liquid nitrogen analysis (4Temel Livers fixed 10% buffered formalin processed hematoxylin eosin (H E) staining laboratory department. These measurements Mulvihill, al. (30Mulvihill E.E. Allister Sutherland B.G. Telford Sawyez C.G. Edwards J.Y. Markle Hegele R.A. Huff M.W. Naringenin dyslipidemia, B overproduction, LDL receptor-null resistance.Diabetes. 58: 2198-2210Crossref (255) followed intraperitoneal injection 10 µCi [1-14C]acetic (PerkinElmer, Boston, MA). One hour later, euthanized. tissues (∼250 mg) collected, saponified 2.2 ml mixture 50% potassium hydroxide 95% ethanol (1:10), then extracted hexane. sterol-containing hexane extract dried down solubilized scintillation cocktail counting de novo synthesis. remaining aqueous phase acidified 0.8 glacial acetic (pH 6), hexane, Fatty oxidation activity (31Linden D. William-Olsson Ahnmark Ekroos Hallberg Sjogren H.P. Becker Svensson Clapham Oscarsson al.Liver-directed overexpression mitochondrial glycerol-3-phosphate acyltransferase results increased triacylglycerol secretion oxidation.FASEB 20: 434-443Crossref h. Fresh (∼200 homogenized four volumes 0.25 M mM EDTA. About 5 mg homogenate incubated 30 min 25°C buffer 150 KCl, HEPES 7.2), 0.1 EDTA, phosphate malonate, MgCl2, carnitine, 0.15% free-BSA, ATP, 50 μM palmitic μCi [9,10(n)-3H] (PerkinElmer). Reactions stopped addition 200 μl 0.6 N perchloric acid. Unreacted extraction. Radioactive degradation products counted data presented DPM/min/mg protein. production (32Millar J.S. Cromley McCoy M.G. Rader Billheimer J.T. Determining mice: comparison poloxamer 407 Triton WR-1339.J. 46: 2023-2028Abstract (203) fast, anesthetized injected Tyloxapol (Triton WR-1339) (Sigma) 500 mg/kg body (BW) via retro-orbital vein block lipolysis. Each mouse received ∼100 saline solution 15 g/dl Tyloxapol. Blood collected 0, 30, 60, 90, 120, 180 injection. above. Linear regression line drawn corresponding time points GraphPad Prism 5. rate derived slope line. hydrolase cytosolic extracts droplets isolated HepG2 cells substrates (33Schweiger Schoiswohl Lass Radner F.P. Haemmerle Malli Graier Cornaciu Oberer Salvayre C-terminal region human adipose lipase affects enzyme droplet binding.J. 283: 17211-17220Abstract (119) 34Radner Streith I.E. Schweiger Kumari Eichmann T.O. Rechberger Koefeler H.C. Eder Schauer al.Growth retardation, impaired catabolism, lethal skin barrier defect comparative identification-58 (CGI-58).J. 285: 7300-7311Abstract (153) radioactive labeling droplets, treated 2.5 [3H-9,10]oleic acid/ml + cold oleic complexed BSA 16 Cells washed ice-cold PBS centrifuged (1000 g 4°C min). supernatant resuspended hypotonic lysis medium (HLM) (20 Tris-HCl, pH 7.4, sodium fluoride, protease cocktail). isolate transferred Potter-Elvehjem homogenizer gently ice. centrifugation, floating layer adjusted final 20% HLM. layered into 13.2 ultracentrifuge tube overlaid HLM sucrose. remainder filled 28,000 SW40 rotor. white (TG) contents enzymatically. activity, homogenizing (0.25 sucrose, DTT, Homogenate 20,000 4°C. infranatant ultracentrifuged 100,000 hundred micrograms triglyceride-free 25 nmol [3H-9,10]oleate labeled substrate volume 37°C. reaction adding 3.25 methanol:chloroform:heptane (10:9:7) carbonate, boric 10.5). radioactivity upper counting. tolerance tests, measuring baseline tail nick Glucometer (Bayer Contour), intraperitoneally 1.5 mg/g BW. 15, 120 level, recombinant (Novo Nordisk, 1.2 mU/g BW, post administration. Glucose-stimulated (35Norris A.W. Chen Fisher S.J. Szanto Ristow Jozsi A.C. Hirshman M.F. Rosen Goodyear Gonzalez F.J. al.Muscle-specific PPARgamma-deficient adiposity respond thiazolidinediones.J. 2003; 112: 608-618Crossref (367) Seven-week-old 6-week-old bolus cavity tails kit Inc.). quantitative real-time PCR (qPCR) (36Yang Hammer Moon Decreased disrupted Site-1 gene.Proc. 2001; 98: 13607-13612Crossref (192) primers qPCR available upon request. peroxidation product malondialdehyde thiobarbituric reactive (TBARS) TBARS redox environment glutathione (GSH)/oxidize