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. 2008 Aug 8;283(32):22186-92.
doi: 10.1074/jbc.M803510200. Epub 2008 Jun 3.

Silencing of hepatic fatty acid transporter protein 5 in vivo reverses diet-induced non-alcoholic fatty liver disease and improves hyperglycemia

Holger Doege  1 , Dirk Grimm, Alaric Falcon, Bernice Tsang, Theresa A Storm, Hui Xu, Angelica M Ortegon, Melissa Kazantzis, Mark A Kay, Andreas Stahl
Affiliations

Silencing of hepatic fatty acid transporter protein 5 in vivo reverses diet-induced non-alcoholic fatty liver disease and improves hyperglycemia

Holger Doege et al. J Biol Chem. .

Abstract

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.

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Figures

FIGURE 1.
FIGURE 1.
In vitro and in vivo knockdown of FATP2 and FATP5. A, HEK293 cells were co-transfected with empty expression vector (black bar) or FATP5 expression plasmids alone (white bar) or in combination with the indicated shRNA constructs. Uptake of fluorescent fatty acid (FA) (C1-BODIPY-C12) was determined 2 days after transfection by flow cytometry. Mean fluorescence normalized to empty vector. Error bars indicate standard deviation. B, Western blot analysis of cell lysates from co-transfections. α-Tubulin served as loading control. IB, immunoblot. C, Western blot analysis of sdsAAV-shRNA-mediated knockdown of FATP5 protein in vivo. Proteins were extracted from liver tissue homogenates of different mouse strains (C57BL/6 and Swiss Webster) 4 weeks after injection with the indicated constructs and viral titers and probed with antisera specific for FATP5, FATP2, FATP4, and insulin-degrading enzyme. AB, antibody. D, fatty acid uptake by hepatocytes isolated 4 weeks after virus injection. FFA uptake was determined ex vivo using a fluorescent fatty acid uptake assay.
FIGURE 2.
FIGURE 2.
Effects of FATP knockdown on postprandial lipid flows. Four weeks after viral transduction, mice were gavaged with 250 μl of olive oil spiked with 3.5 μCi of [14C]oleic acid. A, 14C counts were determined in serum samples drawn at 0, 30, 60, 120, and 240 min after gavage. B, 240 min after gavage, mice were euthanized, and 14C counts normalized to protein content were determined for liver, heart, skeletal muscle (Sk.M.), white adipose tissue (WAT), and kidney lysates. Error bars indicate standard deviation, and asterisks indicate p < 0.05 in Student's t test.
FIGURE 3.
FIGURE 3.
Comparision of AAVFATP5 knockdown and FATP5 gene knock-out phenotypes. A and B, weight gain (left panels) and caloric consumption (middle panels) of AAVFATP5 (solid line) versus AAVSCR (broken line) animals (A) and FATP5KO (broken line) versus wild-type (WT, solid line) animals (B) (all in a C57BL/6 background) fed HF diet (60% fat) for 5 weeks. Insets show Western blots for FATP5 and a loading control (tubulin (Tub)). Serum TG levels (right panels) were determined at the end of the 5-week study for controls (black bars) and hypomorphs (white bars). C, Masson's trichrome staining of liver sections from PBS-, AAVSCR-, and AAVFATP5-injected mice at the end of the 5-week HF feeding study. Error bars indicate standard deviation, and asterisks indicate p < 0.05 in Student's t test.
FIGURE 4.
FIGURE 4.
Reversal of obesity-induced NAFLD in vivo. Six-week-old C57BL/6J mice were fed HF or ND chows for 6 weeks followed by tail vein injections of PBS or the indicated AAV-shRNA constructs and then maintained for an additional 7 weeks on their respective diets. A, Western blot showing expression of FATP5, FATP2, FATP4, or tubulin in liver lysates from C57BL/6 mice transduced with the indicated viruses. IB, immunoblot; AB, antibody. B, total hepatic TG content normalized to protein of mice fed low or high fat diet for a total of 13 weeks determined 7 weeks a.i. of the indicated AAVs. C, Masson's trichrome staining. D, BODIPY493/503 fluorophore (green)/4′,6-dia-midino-2-phenylindole (blue) staining for neutral lipids and nuclei of liver sections at the end of the diet study.
FIGURE 5.
FIGURE 5.
Metabolic consequences of FATP5 knockdown in animals with NAFLD. A-C, mice from Fig. 3 were analyzed throughout the study for weight gain (A), food consumption (B), and ND-fed serum glucose (C). D and E, glucose tolerance test (D) and insulin tolerance test (E) were performed at the end of the study with fasted animals. Circles, AAVFATP5; squares, AAVSCR. Solid lines, HF diet; dotted lines, nutrient-matched low fat diet. Error bars indicate standard deviation, and asterisks indicate p < 0.05 in Student's t test.
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