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All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver S

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All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver S Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/02/26/mol.116.103697.DC1 1521-0111/89/5/560–574$25.00 http://dx.doi.org/10.1124/mol.116.103697 MOLECULAR PHARMACOLOGY Mol Pharmacol 89:560–574, May 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver s Sasmita Tripathy, John D Chapman, Chang Y Han, Cathryn A Hogarth, Samuel L.M. Arnold, Jennifer Onken, Travis Kent, David R Goodlett, and Nina Isoherranen Departments of Pharmaceutics (S.T., S.L.M.A., N.I.), Medicinal Chemistry (J.D.C., D.R.G.), and Diabetes Obesity Center for Excellence and the Department of Medicine, Division of Metabolism, Endocrinology and Nutrition (C.Y.H.), University of Washington, Seattle, Washington; School of Molecular Biosciences and The Center for Reproductive Biology, Washington State University, Pullman, Washington (C.A.H., J.O., T.K.); and School of Pharmacy, University of Maryland, Baltimore, Maryland (D.R.G.) Downloaded from Received February 6, 2016; accepted February 25, 2016 ABSTRACT All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. mitochondrial DNA quantification. atRA also increased b-oxidation The liver is the main storage organ of vitamin A, but activation of and ATP production in HepG2 cells and in human hepatocytes. the retinoic acid receptors (RARs) in mouse liver and in human Knockdown studies of RARa,RARb, and PPARd revealed that molpharm.aspetjournals.org liver cell lines has also been shown. Although atRA treatment the enhancement of mitochondrial biogenesis and b-oxidation improves mitochondrial function in skeletal muscle in rodents, its by atRA requires peroxisome proliferator activated receptor role in modulating mitochondrial function in the liver is contro- delta. In vivo in mice, atRA treatment increased mitochondrial versial, and little data are available regarding the human liver. The biogenesis markers after an overnight fast. Inhibition of atRA aim of this study was to determine whether atRA regulates he- metabolism by talarozole, a cytochrome P450 (CYP) 26 specific patic mitochondrial activity. atRA treatment increased the mRNA inhibitor, increased the effects of atRA on mitochondrial bio- and protein expression of multiple components of mitochon- genesis markers in HepG2 cells and in vivo in mice. These drial b-oxidation, tricarboxylic acid (TCA) cycle, and respiratory studies show that atRA regulates mitochondrial function and chain. Additionally, atRA increased mitochondrial biogenesis in lipid metabolism and that increasing atRA concentrations in human hepatocytes and in HepG2 cells with and without lipid human liver via CYP26 inhibition may increase mitochondrial at ASPET Journals on September 26, 2021 loading based on peroxisome proliferator activated receptor gamma biogenesis and fatty acid b-oxidation and provide therapeutic coactivator 1a and 1b and nuclear respiratory factor 1 mRNA and benefit in diseases associated with mitochondrial dysfunction. Introduction genes responsible for bile acid metabolism and transport (Koh et al., 2014; Mamoon et al., 2014; Yang et al., 2014). The Vitamin A (retinol) is stored predominantly in the liver as activation of multiple pathways by atRA likely leads to diverse retinyl esters. However, the active metabolite of retinol, all- effects on liver physiology, including altered lipid homeostasis. trans-retinoic acid (atRA),appearstoalsoregulategene In humans both excessive intake of vitamin A or retinoids and transcription in the liver (O’Byrne and Blaner, 2013). Most actions of atRA are mediated via interactions with retinoic vitamin A deficiency lead to hypertriglyceridemia, elevated acid receptors (RARs) (Wolf, 2010; Yu et al., 2012), but atRA total cholesterol, and decreased high-density lipoprotein cho- also activates the peroxisome proliferator-activated receptor lesterol (Bershad et al., 1985; Lawrence et al., 2001; Staels, (PPAR)-b/d (Shaw et al., 2003; Berry and Noy, 2007; Wolf, 2001; Brelsford and Beute, 2008; Lilley et al., 2013). However, 2010). In addition, RETRACTEDatRA induces the expression of the tran- likely because of a dichotomy of the effects of vitamin A on the scriptional repressor small heterodimer partner in the liver, liver, the endogenous role of atRA in regulating lipid and fatty resulting in broad effects on gene transcription, especially of acid homeostasis and role of atRA in liver disease is not well understood. This research was supported by grants from National Institutes of Health In rodents atRA appears to regulate various processes in National Institute of General Medical Sciences [Grants R01 GM111772, R01 GM081569, and R01 GM081569-S1] and National Institute of Diabetes and lipid homeostasis in a species-specific manner. Data from Digestive and Kidney Diseases [Grant P30 DK035816]. several mouse studies suggest that atRA signaling via RAR dx.doi.org/10.1124/mol.116.103697. s This article has supplemental material available at molpharm. is needed to maintain liver mitochondrial fatty acid oxida- aspetjournals.org. tion and lipid homeostasis. Hepatocyte-specific RAR knockout ABBREVIATIONS: ACSL,07 acyl CoA synthase January ligase; ATGL, adipose triglyceride lipase; atRA, all-trans- retinoic2020 acid; CPT1a, carnitine palmitoyltransferase 1a; CYP, cytochrome P450; DMEM, Dulbecco’s modified essential medium; DMSO, dimethylsulfoxide; ECH1, enoyl CoA hydratase 1; FASN, fatty acid synthase; FGF21, fibroblast growth factor 21; IDH2, isocitrate dehydrogenase; NAFLD, nonalcoholic fatty liver disease; NRF1, nuclear respiratory factor 1; PGC1a, peroxisome proliferator activated receptor gamma coactivator 1a;PGC1b, peroxisome proliferator activated receptor gamma coactivator 1b; PPAR, peroxisome proliferator activated receptor; RA, retinoic acid; RAR, retinoic acid receptor; SDHA, succinate dehydrogenase subunit A; SREBP1, sterol regulatory element binding protein 1. 560 Retinoic Acid Increases Fatty Acid b-Oxidation 561 mice develop microvesicular steatosis, hepatocellular car- Minneapolis, MN. Talarozole (R115866) was obtained from Med- cinoma, decreased mitochondrial fatty acid oxidation, and Chem Express, Princeton, NJ. siRNAs for RARa, RARb,and increased peroxisomal and microsomal fatty acid oxidation PPARd were obtained from Ambion, Life Science Technologies, presumably due to deficient atRA signaling (Yanagitani et al., Grand Island, NY. ATP assay kit was purchased from Biovision 2004). Similarly, in normal mice atRA enhances fatty acid Inc., Milpitas, CA. Primer and probe pairs for human peroxisome proliferator activated receptor gamma coactivator 1a (PGC1a) oxidation and ketogenesis via RAR activation and FGF21 (Hs01016719_m1, FAM), PGC1b (Hs00991677_m1, FAM), nuclear induction (Amengual et al., 2012; Li et al., 2013). In agreement respiratory factor 1(NRF1) (Hs00192316_m1, FAM), carnitine with a role of atRA in maintaining lipid homeostasis in the palmitoyltransferase 1a (CPT1a; (Hs00912671_m1, FAM), PPARa liver, decrease of hepatic atRA concentrations via inhibition of (Hs00947536_m1, FAM), PPARd (Hs04187066_g1, FAM), PPARg atRA synthesis in mice led to microvesicular vacuolation but (Hs01115513_m1, FAM), GAPDH (Hs99999905_m1, VIC), RARa without a change in liver triglycerides (Paik et al., 2014). (Hs00940446_m1, FAM), RARb (Hs00233407_m1, FAM), adipose However, in diet-induced obese mice atRA treatment de- triglyceride lipase (ATGL; Hs00386101_m1, FAM), diacylglycerol creased hepatic triglyceride content (Berry and Noy, 2009) O-acyl transferase 2 (Hs01045913_m1, FAM), microsomal triglyc- and hepatic lipid accumulation (Kim et al., 2014). In contrast, eride transfer protein (Hs00165177_m1, FAM), and sterol regula- in rats vitamin A deficiency led to increased expression of tory element binding protein 1(SREBP1; Hs01088691_m1, FAM) were obtained from Applied Biosystems, Carlsbad, CA. Primer and genes involved in fatty acid metabolism in the liver (McClintick probe pairs for mouse Rarb (Mm01319677_m1, FAM), Cyp26a1 Downloaded from et al., 2006) and decreased liver total phospholipid content and (Mm00514486_m1, FAM), Pgc1a (Mm01208835_m1, FAM), phosphatidylcholine synthesis (Oliveros et al., 2007), demon- Cpt1a (Mm01231183_m1, FAM), Atgl (Mm00503040_m1, FAM), strating opposite effects to those observed in mice. Low retinol Pgc1b (Mm00504720_m1), Nrf1 (Mm01135606_m1), and b-Actin diet also significantly decreased stellate cell free fatty acids and (Mm00607939_s1, FAM) were obtained from Applied Biosystems. total lipids, whereas high retinol diet increased triglycerides, Mitochondrial genes and nuclear gene primer set #7246 (for human) cholesteryl esters, free fatty acids, and total lipids in rat stellate and #RR290 (for mouse) were purchased from Takara Clontech, cells (Moriwaki et al., 1988). How well these observations in Mountain View, CA. molpharm.aspetjournals.org mice or rats reflect atRA signaling in healthy or diseased HepG2 Cell Culture. HepG2 cells were cultured in 6-well plates  6 human liver is not known. (Corning Life Sciences, Corning, NY) with a cell density 1 10 cells per well in DMEM with 25 mM glucose media with 10% fetal calf Hepatic vitamin A stores are depleted in alcoholic liver serum in a 5% carbon dioxide environment in a humidified incubator disease, and vitamin A deficiency is believed to play a role in at 37°C. All media contained penicillin and streptomycin, and all the the development and progression of the disease
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