Silencing of Hepatic Fatty Acid Transporter Protein 5 in Vivo Reverses Diet-induced Non-alcoholic Fatty Liver Disease and Improves Hyperglycemia

Non-alcoholic fatty liver disease is a serious health problem linked to obesity and type 2 diabetes. To investigate the biological outcome and therapeutic potential of hepatic fatty acid uptake inhibition, we utilized an adeno-associated virus-mediated RNA interference technique to knock down the expression of hepatic fatty acid transport protein 5 in vivo prior to or after establishing non-alcoholic fatty liver disease in mice. Using this approach, we demonstrate here the ability to achieve specific, non-toxic, and persistent knockdown of fatty acid transport protein 5 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. Importantly, knockdown of fatty acid transport protein 5 was also able to reverse already established non-alcoholic fatty liver disease, resulting in significantly improved whole-body glucose homeostasis. Thus, continued activity of hepatic fatty acid transport protein 5 is required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease. Non-alcoholic fatty liver disease is a serious health problem linked to obesity and type 2 diabetes. To investigate the biological outcome and therapeutic potential of hepatic fatty acid uptake inhibition, we utilized an adeno-associated virus-mediated RNA interference technique to knock down the expression of hepatic fatty acid transport protein 5 in vivo prior to or after establishing non-alcoholic fatty liver disease in mice. Using this approach, we demonstrate here the ability to achieve specific, non-toxic, and persistent knockdown of fatty acid transport protein 5 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. Importantly, knockdown of fatty acid transport protein 5 was also able to reverse already established non-alcoholic fatty liver disease, resulting in significantly improved whole-body glucose homeostasis. Thus, continued activity of hepatic fatty acid transport protein 5 is required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease. The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) 2The abbreviations used are: NAFLDnon-alcoholic fatty liver diseaseAAVadeno-associated virussdsAAVstabilized double-stranded AAVFATPfatty acid transport proteinFFAfree fatty acidTGtriglyceridesshRNAshort hairpin RNAPBSphosphate-buffered salinev.p.viral particlesa.i.after injectionSCRscrambled controlHFhigh fatNDnormal dietSREBP-1csterol regulatory element-binding protein 1cKOknockout. is presently estimated at 30% of the general population and affects a majority of patients with obesity and type 2 diabetes (1Clark J.M. Brancati F.L. Diehl A.M. Am. J. Gastroenterol. 2003; 98: 960-967Crossref PubMed Scopus (1096) Google Scholar, 2Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton J.D. Cohen J.C. Grundy S.M. Hobbs H.H. Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2955) Google Scholar). In obese individuals, chronically elevated serum free fatty acids (FFAs) and high insulin levels lead to both increased FFA uptake by the liver and increased synthesis of lipids, resulting in hepatic triglycerides (TG) accumulation, typically accompanied by hepatic insulin desensitization (1Clark J.M. Brancati F.L. Diehl A.M. Am. J. Gastroenterol. 2003; 98: 960-967Crossref PubMed Scopus (1096) Google Scholar, 3Samuel V.T. Liu Z.X. Wang A. Beddow S.A. Geisler J.G. Kahn M. Zhang X.M. Monia B.P. Bhanot S. Shulman G.I. J. Clin. Investig. 2007; 117: 739-745Crossref PubMed Scopus (394) Google Scholar) involving protein kinase C ϵ (3Samuel V.T. Liu Z.X. Wang A. Beddow S.A. Geisler J.G. Kahn M. Zhang X.M. Monia B.P. Bhanot S. Shulman G.I. J. Clin. Investig. 2007; 117: 739-745Crossref PubMed Scopus (394) Google Scholar). Current pharmacological treatment strategies for NAFLD focus principally on increasing hepatic fatty acid oxidation (4Laurin J. Lindor K.D. Crippin J.S. Gossard A. Gores G.J. Ludwig J. Rakela J. McGill D.B. Hepatology. 1996; 23: 1464-1467Crossref PubMed Scopus (543) Google Scholar) and improving extrahepatic insulin sensitivity (5Marchesini G. Brizi M. Bianchi G. Tomassetti S. Bugianesi E. Lenzi M. McCullough A.J. Natale S. Forlani G. Melchionda N. Diabetes. 2001; 50: 1844-1850Crossref PubMed Scopus (1989) Google Scholar). However, none of these treatment methods reduce hepatic uptake of dietary fats, and novel therapeutics that specifically aim at reversing NAFLD in the context of obesity would be highly desirable. non-alcoholic fatty liver disease adeno-associated virus stabilized double-stranded AAV fatty acid transport protein free fatty acid triglycerides short hairpin RNA phosphate-buffered saline viral particles after injection scrambled control high fat normal diet sterol regulatory element-binding protein 1c knockout. Based on the premises that obesity-associated NAFLD is primarily driven by the continuous protein-mediated uptake of fatty acids by the liver and that NAFLD is a contributing factor to whole-body insulin desensitization, we argued that blocking proteins responsible for hepatic fatty acid uptake should prevent or reverse hepatic steatosis, thus improving insulin sensitivity and glucose homeostasis. Two members of the fatty acid transport protein (FATP) family, FATP2 and FATP5, are robustly expressed in liver (6Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar) and are thought to be involved in the early steps of long-chain fatty acid uptake/activation (7Doege H. Stahl A. Physiol. (Bethesda). 2006; 21: 259-268Crossref PubMed Scopus (173) Google Scholar, 8Stahl A. Gimeno R.E. Tartaglia L.A. Lodish H.F. Trends Endocrinol. Metab. 2001; 12: 266-273Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). We recently demonstrated the importance of FATP5 in hepatic lipid metabolism by showing that deletion of FATP5 partially protected mice from developing high fat diet-induced obesity and improved insulin-sensitivity (9Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 10Hubbard B. Doege H. Punreddy S. Wu H. Huang X. Kaushik V.K. Mozell R.L. Byrnes J.J. Stricker-Krongrad A. Chou C.J. Tartaglia L.A. Lodish H.F. Stahl A. Gimeno R.E. Gastroenterology. 2006; 130: 1259-1269Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To explore the consequences of hepatic FATP5 ablation in the context of NAFLD for hepatic fatty acid uptake and whole-body lipid fluxes, we took advantage of a recently developed stabilized double-stranded (sds) adeno-associated virus (AAV) 8-mediated delivery method for liver-directed small hairpin (sh) RNA expression (11Grimm D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. Kay M.A. Nature. 2006; 441: 537-541Crossref PubMed Scopus (1384) Google Scholar). Using this approach, we could achieve specific silencing of endogenous FATP5 in mice after a single delivery of sdsAAV-shRNA expression constructs, resulting in the protection/reversal of NAFLD and hyperglycemia in diet-induced obesity mice. Antibodies and Reagents—BODIPY fatty acid (C1-BODIPY-C12) was obtained from Molecular Probes (Eugene, OR). 14COleic acid was purchased from ARC, Inc. (St. Louis, MO). Polyclonal antisera against the C termini of FATP2, -4, and -5 were raised as described previously (9Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12Stahl A. Hirsch D.J. Gimeno R.E. Punreddy S. Ge P. Watson N. Patel S. Kotler M. Raimondi A. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Anti-β-tubulin and anti-insulin-degrading enzyme antibodies were purchased from BD Biosciences and BD Transduction Laboratories, respectively. Immunoblot analysis was performed as reported previously (13Stahl A. Evans J.G. Pattel S. Hirsch D. Lodish H.F. Dev. Cell. 2002; 2: 477-488Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). All other chemicals were obtained from Sigma. AAV-shRNA Constructs—Oligonucleotides against FATP5 were designed as suggested (14Reynolds A. Leake D. Boese Q. Scaringe S. Marshall W.S. Khvorova A. Nat. Biotechnol. 2004; 22: 326-330Crossref PubMed Scopus (1648) Google Scholar) and did not share any significant homology with other genes in the mouse genome. All shRNAs used in vitro were expressed from the human H1 promoter (pSUPERIOR-based expression construct; OligoEngine, Seattle, WA). Loop sequence was 5′-TTCAAGAGA-3′. Stabilized double-stranded (sds) AAV vectors for persistent and efficient expression of shRNAs in the liver were derived from elements from AAV serotypes 2, 4, and 8 (11Grimm D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. Kay M.A. Nature. 2006; 441: 537-541Crossref PubMed Scopus (1384) Google Scholar). Viral particles were generated, purified, and titered as described by Grimm et al. (15Grimm D. Kay M.A. Kleinschmidt J.A. Mol. Ther. 2003; 7: 839-850Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Fatty Acid Uptake Assay in HEK293 Cells—Fatty acid uptake assays were performed as described previously (12Stahl A. Hirsch D.J. Gimeno R.E. Punreddy S. Ge P. Watson N. Patel S. Kotler M. Raimondi A. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). General Animal Procedures—C57BL/6 or Swiss Webster mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and acclimated for 1 week after arrival before they were used for experiments. Animals were maintained on regular lab chow (5P75, LabDiet, Richmond, IN) or on a special diet (see below), receiving food and water ad libitum, and kept at 22 °C on a 12-h/12-h light/dark cycle. FATP5 knock-out mice were generated and maintained as described previously (9Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). All study groups had comparable initial ages and weights. Intravenous sdsAAV8 deliveries (5 × 1010 or 3 × 1011 viral particles (v.p.), total volume 250 μl in PBS) were performed by tail vein injections using established methods (16Nakai H. Fuess S. Storm T.A. Muramatsu S. Nara Y. Kay M.A. J. Virol. 2005; 79: 214-224Crossref PubMed Scopus (256) Google Scholar). For diet studies, separately housed 8-week-old male mice were fed ad libitum a high fat diet containing 60% fat (D12492, Research Diets, NJ) or a low fat diet (D12450) containing 10% fat. Weight was measured weekly, and food intake was measured twice a week. Standard glucose tolerance tests and insulin tolerance tests as well as lipid gavages were performed as described previously (17Wu Q. Ortegon A.M. Tsang B. Doege H. Feingold K.R. Stahl A. Mol. Cell. Biol. 2006; 26: 3455-3467Crossref PubMed Scopus (215) Google Scholar). All procedures were approved by the Animal Care Committee at the Palo Alto Medical Foundation Research Institute and the University of California Berkeley Institutional Animal Care and Use Committee. Hepatocyte Preparation—Mouse livers were cannulated through the portal vein and an incision was made in the inferior vena cava. Liver perfusion with digestion and perfusion media and isolation of hepatocytes was carried out according to the manufacturer's instructions (Invitrogen) followed by long-chain fatty acid uptake assays as described (9Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Tissue Lipid Analysis—Samples from various tissues were powdered in liquid nitrogen, and total lipids were extracted by the method of Folch et al. (18Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Total triglycerides were assayed using a colorimetric kit (Sigma Diagnostics). Liver Morphology—Cryosections obtained from livers of FATP knockdown or knock-out animals and the respective controls were stained with either Masson's trichrome reagent or hematoxylin and eosin. Neutral lipid were stained with analysis of FATP5 knockdown or FATP5 knock-out scrambled control or controls was by or analysis of as are was of Fatty Acid in generated (14Reynolds A. Leake D. Boese Q. Scaringe S. Marshall W.S. Khvorova A. Nat. Biotechnol. 2004; 22: 326-330Crossref PubMed Scopus (1648) Google Scholar) FATP5 expression and an with to any sequence in the mouse and the of the on FATP expression and FFA uptake by into HEK293 with FATP5 expression Two FFA uptake and FATP protein levels were using assays (12Stahl A. Hirsch D.J. Gimeno R.E. Punreddy S. Ge P. Watson N. Patel S. Kotler M. Raimondi A. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) and respectively. assays of a RNA interference We did not any for the other or the control with with the respective FATP expression vectors and of Fatty Acid 5 in in vitro to an in vivo of diet-induced we used viral (11Grimm D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. Kay M.A. Nature. 2006; 441: 537-541Crossref PubMed Scopus (1384) Google Scholar) to and in vitro for FATP5 and the resulting in the vectors and Two mouse and Swiss Webster were the tail vein with the viral at (5 × 1010 and 3 × 1011 after injection hepatic protein expression of FATP5 was by FATP5 expression was in the not in the animals is to the that in vivo of The be to from as FATP5 knockdown was at the viral (5 × 1010 is in using 1 × (11Grimm D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. Kay M.A. Nature. 2006; 441: 537-541Crossref PubMed Scopus (1384) Google Scholar). Importantly, we did not any of FATP5 knockdown on the expression levels of other liver proteins the highly FATP2 and with Based on serum and levels and liver expression and FATP knockdown after viral not Thus, using this in vivo RNA interference approach, we were able to achieve specific and protein knockdown of endogenous To the by of FATP5 would reduce FFA uptake, we performed vivo uptake assays with hepatocytes from ad libitum mice after viral as described before (9Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). was in the fatty acid uptake hepatocytes from the and demonstrated a reduction in long-chain fatty acid uptake after knockdown of FATP5 The uptake activity either may be to other hepatic as FATP2 and may of Lipid in the of lipids in and we performed gavages containing after an using mice fed a normal chow of in serum was comparable groups of normal in However, as well as serum and FFA not were elevated for in the FATP5 knockdown at lipid from the to of hepatic FATP after we by the livers of FATP5 knockdown animals with increased lipid in and fat are with the of liver total high fat feeding (see and that of hepatic a of lipids from the liver to tissues on other FATP as and these the that FATP5 a in hepatic fatty acid uptake and in the of of NAFLD in mice were fed or for followed by tail vein injections of or the AAV-shRNA and maintained for an on respective showing expression of FATP5, FATP2, or in liver from mice with the total hepatic to protein of mice fed low or high fat diet for a total of of the Masson's trichrome for lipids and of liver at the of the diet of FATP5 and we the of mice with mice in a on a Weight and food by and animals to significantly from the and controls 2 to by the of the study in a reduction and significantly improved serum levels for both groups with respective controls and livers of and control mice of steatosis, both mice and mice (9Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 10Hubbard B. Doege H. Punreddy S. Wu H. Huang X. Kaushik V.K. Mozell R.L. Byrnes J.J. Stricker-Krongrad A. Chou C.J. Tartaglia L.A. Lodish H.F. Stahl A. Gimeno R.E. 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Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). in were by significantly fat in FATP5 knockdown animals with in both FATP5, and livers FATP5, were significantly from animals from the control the the initial of mice developed of FATP5 to a reduction in serum glucose by week 5 in glucose was also in glucose tolerance and insulin tolerance showing for FATP5 knockdown mice tolerance FATP5 that were from animals and prior study of vectors that in and against that out of in liver by of D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. Kay M.A. Nature. 2006; 441: 537-541Crossref PubMed Scopus (1384) Google Scholar). Importantly, and that to be were also (11Grimm D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. 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We argued that obesity-associated NAFLD is primarily driven and maintained by the continuous protein-mediated uptake of fatty acids by the of FATP5 should at partially hepatic we that knockdown of FATP5 reduced established hepatic and hyperglycemia of mice fed a high fat We that may these of FATP5 hepatic fatty acid uptake as with and hepatic fatty acid hepatic lipid is to insulin sensitivity in this (3Samuel V.T. Liu Z.X. Wang A. Beddow S.A. Geisler J.G. Kahn M. Zhang X.M. Monia B.P. Bhanot S. Shulman G.I. J. Clin. Investig. 2007; 117: 739-745Crossref PubMed Scopus (394) Google Scholar). the lipid was in with FATP expressed by extrahepatic tissues showing that of activity to a in fat and and in liver lipids (17Wu Q. Ortegon A.M. Tsang B. Doege H. Feingold K.R. Stahl A. Mol. Cell. Biol. 2006; 26: 3455-3467Crossref PubMed Scopus (215) Google Scholar). 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Gimeno R.E. Stahl A. Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 10Hubbard B. Doege H. Punreddy S. Wu H. Huang X. Kaushik V.K. Mozell R.L. Byrnes J.J. Stricker-Krongrad A. Chou C.J. Tartaglia L.A. Lodish H.F. Stahl A. Gimeno R.E. Gastroenterology. 2006; 130: 1259-1269Abstract Full Text Full Text PDF PubMed Scopus (101) Google could be that the elevated serum fatty acid levels as a G.J. Nat. 2005; PubMed Scopus Google Scholar). we made we demonstrate that expression is an efficient for the of hepatic genes in normal and is an as this is the of non-toxic, and RNA interference stabilized double-stranded 8 AAV particles and should for to the hepatic lipid and we that knockdown of FATP5 from high fat diet-induced hepatic and lipid from the of FATP5 is to reverse established NAFLD in diet-induced obesity and to glucose homeostasis. Thus, reduction of hepatic either or small is a novel to lipid and may novel for the treatment of NAFLD and insulin We and at We the from the Palo Alto Medical Foundation Research Institute with