Vitamin C Deficiency Inhibits Nonalcoholic Fatty Liver Disease Progression through Impaired de Novo Lipogenesis
2021; Elsevier BV; Volume: 191; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2021.05.020
ISSN1525-2191
AutoresSeoung-Woo Lee, Su-Min Baek, Kyung‐Ku Kang, A-Rang Lee, Tae–Un Kim, Seong‐Kyoon Choi, Yoon-Seok Roh, Il‐Hwa Hong, Sang‐Joon Park, Tae‐Hwan Kim, Kyu‐Shik Jeong, Jin‐Kyu Park,
Tópico(s)Lipid metabolism and disorders
ResumoDespite the increasing clinical importance of nonalcoholic fatty liver disease (NAFLD), little is known about its underlying pathogenesis or specific treatment. The senescence marker protein 30 (SMP30), which regulates the biosynthesis of vitamin C (VC) in many mammals, except primates and humans, was recently recognized as a gluconolactonase. However, the precise relation between VC and lipid metabolism in NAFLD is not completely understood. Therefore, this study aimed to clearly reveal the role of VC in NAFLD progression. SMP30 knockout (KO) mice were used as a VC-deficient mouse model. To investigate the precise role of VC on lipid metabolism, 13- to 15-week–old SMP30 KO mice and wild-type mice fed a 60% high-fat diet were exposed to tap water or VC-containing water (1.5 g/L) ad libitum for 11 weeks. Primary mouse hepatocytes isolated from the SMP30 KO and wild-type mice were used to demonstrate the relation between VC and lipid metabolism in hepatocytes. Long-term VC deficiency significantly suppressed the progression of simple steatosis. The high-fat diet–fed VC-deficient SMP30 KO mice exhibited impaired sterol regulatory element-binding protein-1c activation because of excessive cholesterol accumulation in hepatocytes. Long-term VC deficiency inhibits de novo lipogenesis through impaired sterol regulatory element-binding protein-1c activation. Despite the increasing clinical importance of nonalcoholic fatty liver disease (NAFLD), little is known about its underlying pathogenesis or specific treatment. The senescence marker protein 30 (SMP30), which regulates the biosynthesis of vitamin C (VC) in many mammals, except primates and humans, was recently recognized as a gluconolactonase. However, the precise relation between VC and lipid metabolism in NAFLD is not completely understood. Therefore, this study aimed to clearly reveal the role of VC in NAFLD progression. SMP30 knockout (KO) mice were used as a VC-deficient mouse model. To investigate the precise role of VC on lipid metabolism, 13- to 15-week–old SMP30 KO mice and wild-type mice fed a 60% high-fat diet were exposed to tap water or VC-containing water (1.5 g/L) ad libitum for 11 weeks. Primary mouse hepatocytes isolated from the SMP30 KO and wild-type mice were used to demonstrate the relation between VC and lipid metabolism in hepatocytes. Long-term VC deficiency significantly suppressed the progression of simple steatosis. The high-fat diet–fed VC-deficient SMP30 KO mice exhibited impaired sterol regulatory element-binding protein-1c activation because of excessive cholesterol accumulation in hepatocytes. Long-term VC deficiency inhibits de novo lipogenesis through impaired sterol regulatory element-binding protein-1c activation. Nonalcoholic fatty liver disease (NAFLD) is an umbrella term for a histologic spectrum in the liver without other secondary causes, such as alcohol consumption and viral hepatitis.1Baran B. Akyüz F. Non-alcoholic fatty liver disease: what has changed in the treatment since the beginning?.World J Gastroenterol. 2014; 20: 14219Crossref PubMed Scopus (29) Google Scholar NAFLD is broadly categorized by the two major histologic classifications of simple steatosis [nonalcoholic fatty liver (NAFL)], which is an accumulation of fatty lipids in hepatocytes with mild or no inflammatory lesions; and nonalcoholic steatohepatitis (NASH), which is a more exacerbated form of fatty liver that includes hepatocyte necrosis, inflammatory cell infiltration, and fibrosis.2Arulanandan A. Loomba R. Noninvasive testing for NASH and NASH with advanced fibrosis: are we there yet?.Curr Hepatol Rep. 2015; 14: 109-118Crossref PubMed Scopus (18) Google Scholar NASH also has a potential to progress to irreversible lesions, such as cirrhosis and even hepatocellular carcinoma.3Michelotti G.A. Machado M.V. Diehl A.M. NAFLD, NASH and liver cancer.Nat Rev Gastroenterol. 2013; 10: 656-665Crossref PubMed Scopus (643) Google Scholar The progression from simple steatosis to NASH is highly affected by oxidative stress and occurs following lipid peroxidation.4Leclercq I.A. Farrell G.C. Field J. Bell D.R. Gonzalez F.J. Robertson G.R. CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis.J Clin Invest. 2000; 105: 1067-1075Crossref PubMed Scopus (658) Google Scholar However, the precise pathogenesis of NAFLD, including lipid metabolism, oxidative stress, and inflammatory responses, is not clearly understood.5Trauner M. Arrese M. Wagner M. Fatty liver and lipotoxicity.Biochim Biophys Acta Mol Cell Biol Lipids. 2010; 1801: 299-310Crossref PubMed Scopus (218) Google Scholar Generally, NAFLD is highly correlated with other lipid metabolic disorders, such as obesity, diabetes, and hyperlipidemia.6Henao-Mejia J. Elinav E. Jin C. Hao L. Mehal W.Z. Strowig T. Thaiss C.A. Kau A.L. Eisenbarth S.C. Jurczak M.J. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity.Nature. 2012; 482: 179-185Crossref PubMed Scopus (1611) Google Scholar,7Anstee Q.M. Targher G. Day C.P. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis.Nat Rev Gastroenterol. 2013; 10: 330Crossref PubMed Scopus (1017) Google Scholar Because of the emerging epidemic of these diseases in various developed countries, such as in Asia, Europe, and the United States, NAFLD has become one of the most common liver diseases worldwide, with an estimated prevalence ranging from 25% to 45%.8Lazo M. Clark J.M. The epidemiology of nonalcoholic fatty liver disease: a global perspective.Semin Liver Dis. 2008; 28: 339-350Crossref PubMed Scopus (555) Google Scholar,9Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton J.D. Cohen J.C. Grundy S.M. Hobbs H.H. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2859) Google Scholar Although the global burden of NAFLD is increasing, specific treatments for NAFLD have not yet been approved, and NAFLD treatments mainly depend on prevention.10Benedict M. Zhang X. Non-alcoholic fatty liver disease: an expanded review.World J Hepatol. 2017; 9: 715Crossref PubMed Scopus (380) Google Scholar Therefore, additional research is essential to understand the underlying mechanisms of NAFLD. The senescence marker protein 30 (SMP30) is the first identified age-associated protein with androgen-independent decreased patterns in aged rat livers.11Fujita T. Uchida K. Maruyama N. Purification of senescence marker protein-30 (SMP30) and its androgen-independent decrease with age in the rat liver.Biochim Biophys Acta. 1992; 1116: 122-128Crossref PubMed Scopus (162) Google Scholar The general structure of SMP30 is well preserved in many vertebrates.12Scott S.H. Bahnson B.J. Senescence marker protein 30: functional and structural insights to its unknown physiological function.Biomol Concepts. 2011; 2: 469Crossref PubMed Scopus (23) Google Scholar SMP30 is mainly expressed in parenchymal organs, such as the liver, kidney, lungs, and brain.13Maruyama N. Ishigami A. Kondo Y. Pathophysiological significance of senescence marker protein-30.Gerontol Geriatr Med. 2010; 10: S88-S98Google Scholar SMP30 was identified as a gluconolactonase that catalyzes l-gulonic acid to l-gulono-γ-lactone in the vitamin C (VC) biosynthesis pathway.13Maruyama N. Ishigami A. Kondo Y. Pathophysiological significance of senescence marker protein-30.Gerontol Geriatr Med. 2010; 10: S88-S98Google Scholar Therefore, SMP30 performs a crucial role in the production of VC, and SMP30 knockout (KO) mice are generally used as a VC-deficient mouse model.14Kondo Y. Inai Y. Sato Y. Handa S. Kubo S. Shimokado K. Goto S. Nishikimi M. Maruyama N. Ishigami A. Senescence marker protein 30 functions as gluconolactonase in L-ascorbic acid biosynthesis, and its knockout mice are prone to scurvy.Proc Natl Acad Sci U S A. 2006; 103: 5723-5728Crossref PubMed Scopus (202) Google Scholar Vitamin C (alias an ascorbic acid) is a water-soluble, six-carbon lactone that acts as an electron donor by donating electrons to other substances.15Padayatty S.J. Katz A. Wang Y. Eck P. Kwon O. Lee J.-H. Chen S. Corpe C. Dutta A. Dutta S.K. Vitamin C as an antioxidant: evaluation of its role in disease prevention.J Am Coll Nutr. 2003; 22: 18-35Crossref PubMed Scopus (1229) Google Scholar Vitamin C is synthesized from d-glucose in most mammalian species; however, a few species, including humans, primates, and guinea pigs, cannot synthesize vitamin C because of a mutation in gluconolactone oxidase.16Burns J. Missing step in man, monkey and guinea pig required for the biosynthesis of L-ascorbic acid.Nature. 1957; 180: 553Crossref PubMed Scopus (110) Google Scholar Vitamin C, a strong antioxidant, reduces oxidative stress by scavenging hydroxyl, peroxyl, and superoxide radicals.17Rezazadeh A. Yazdanparast R. Molaei M. Amelioration of diet-induced nonalcoholic steatohepatitis in rats by Mn-salen complexes via reduction of oxidative stress.J Biomed Sci. 2012; 19: 1-8Crossref PubMed Scopus (34) Google Scholar Moreover, vitamin C works as a cofactor in various enzymatic responses that regulate essential biological functions in animals.18Carr A.C. Maggini S. Vitamin C and immune function.Nutrients. 2017; 9: 1211Crossref PubMed Scopus (631) Google Scholar Previous studies have reported that prolonged exposure of the liver to excessive free fatty acid generates reactive oxygen species and endoplasmic reticulum stress, and these inflammatory responses play a key role in the progression of simple steatosis to NASH.19Buzzetti E. Pinzani M. Tsochatzis E.A. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD).Metabolism. 2016; 65: 1038-1048Abstract Full Text Full Text PDF PubMed Scopus (1267) Google Scholar,20Kim J.Y. Garcia-Carbonell R. Yamachika S. Zhao P. Dhar D. Loomba R. Kaufman R.J. Saltiel A.R. Karin M. ER stress drives lipogenesis and steatohepatitis via caspase-2 activation of S1P.Cell. 2018; 175: 133-145.e15Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar Thus, vitamin C treatment is a promising strategy for treating NAFLD progression. However, there are many conflicting findings regarding the relation between vitamin C and NAFLD progression.21Ipsen D.H. Tveden-Nyborg P. Lykkesfeldt J. Does vitamin C deficiency promote fatty liver disease development?.Nutrients. 2014; 6: 5473-5499Crossref PubMed Scopus (35) Google Scholar Moreover, recent studies about the effects of vitamin C on NAFLD not only focused on the antioxidant effect of vitamin C but have also used a treatment cocktail including vitamins C and E rather than vitamin C only.21Ipsen D.H. Tveden-Nyborg P. Lykkesfeldt J. Does vitamin C deficiency promote fatty liver disease development?.Nutrients. 2014; 6: 5473-5499Crossref PubMed Scopus (35) Google Scholar,22Curcio A. Romano A. Cuozzo S. Nicola A.D. Grassi O. Schiaroli D. Nocera G.F. Pironti M. Silymarin in combination with vitamin C, vitamin E, coenzyme Q10 and selenomethionine to improve liver enzymes and blood lipid profile in NAFLD patients.Medicina. 2020; 56: 544Crossref Scopus (3) Google Scholar Thus, the precise relation between vitamin C intake and NAFLD remains controversial. Therefore, we designed the present study using SMP30 KO mice as a vitamin C–deficient mouse model to clarify the precise role of vitamin C in high-fat diet (HFD)–induced NAFLD and lipid metabolism. Male 12- to 15-week–old C57BL/6 SMP30 KO mice (n = 20) and wild-type (WT) mice (n = 24), weighing 26 to 30 g, were used in this study. During the experimental period (11 weeks), the WT and SMP30 KO mice were housed in a room at 22°C ± 3°C with a relative humidity of 50% ± 10%, a 12-hour light-dark cycle (lights on at 8:00 am), and ad libitum access to food and water. The mice were subdivided into eight groups based on the diet type, vitamin C supplementation, and genetic types (WT or SMP30). The mice received either a chow diet (SAFE D40; Safe Diet, Rosenberg, Germany) or an HFD (D12492; Research Diet, New Brunswick, NJ). All groups were given a vitamin C–free diet, and vitamin C was provided in the drinking water (1.5 g/L). All of the animal experiments and protocols using animal tissues used in this study were approved by the Kyungpook National University Institutional Animal Care and Use Committee (approval numbers 2017-0112 and 2019-0070). The tissue samples, which were collected in 10% neutral-buffered formalin, were processed routinely and embedded in paraffin wax. The sections were cut into 4-μm–thick sections for hematoxylin and eosin staining. A histologic analysis of NAFLD was performed using the NASH Clinical Research Network grading system (Table 1).23Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (6754) Google Scholar The NAFLD Activity Score was defined as the sum of the scores for steatosis, lobular inflammation, and ballooning degeneration. The histologic grades were averaged on the basis of at least five random fields at ×200 magnification for each liver sample. Oil Red O staining was performed to visualize hepatic triglyceride accumulation. For the Oil Red O staining, the liver tissue was fixed in 4% paraformaldehyde for frozen section. The frozen liver sections were then stained with freshly prepared Oil Red O working solution.Table 1Grading and Staging System for NAFLDFeaturesGrade/scoreNASH clinical research network grading system23Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (6754) Google ScholarSteatosis0 67%Lobular inflammation0No foci1 4 Foci per ×20 fieldBallooning degeneration0None1Few2ManyNAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. Open table in a new tab NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. The following antibodies were used: anti–β-actin (A1978; Sigma, St. Louis, MO; and sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA), anti-SMP30 (SML-RO1001-EX; COSMOBIO CO, LTD, Tokyo, Japan), anti–phosphorylated AMP-activated protein kinase (AMPK; 2535S; Cell Signaling Technology, Danvers, MA), anti-AMPK (2532S; Cell Signaling Technology), anti–peroxisome proliferator-activated receptor-α (PPAR-α; sc-398394; Santa Cruz Biotechnology), anti–sterol regulatory element-binding protein-1c (SREBP-1c; ab28481; Abcam, Cambridge, UK), and anti–fatty acid synthase (FAS; sc-48357; Santa Cruz Biotechnology). The following chemicals were used: oleic acid (OA; O1008; Sigma, Steinheim, Germany), bovine serum albumin (10735078001; Roche, Basel, Switzerland), vitamin C (832; DUKSAN, Ansan, Republic of Korea), cholesterol (C8667; Sigma, St. Louis, MO), fructose (F0366; SANCHUN, Yeosu, Republic of Korea), and glucose (64220S0601; JUNSEI, Tokyo, Japan). For the immunohistochemistry analysis, mouse liver tissues in paraffin-embedded slides were deparaffinized in toluene and rehydrated in a graded alcohol series. After deparaffinization, antigen retrieval was performed in a solution of 3% hydrogen peroxide in methanol at room temperature for 35 minutes and steamed for 30 minutes in 10 mmol/L citrate buffer for antigen retrieval. After being cooled at room temperatures (25°C) for 2 hours, the sections were incubated with blocking solution (Life Technologies, Frederick, MD) at room temperature for 1 hour. Subsequently, the sections were incubated with the primary antibody overnight at 4°C. After the sections were washed in phosphate-buffered saline, the sections were incubated with a broad-spectrum secondary antibody (Life Technologies) and horseradish peroxidase–conjugated streptavidin (Life Technologies) at room temperature for 10 minutes. Then, the antigen-antibody complex was visualized with an avidin-biotin peroxidase complex solution using an ABC kit (Vector Laboratories, Burlingame, CA). The sections were rinsed in distilled water, counterstained with hematoxylin, and dehydrated with an alcohol series and toluene. For immunofluorescence, the frozen liver tissue sections were washed with phosphate buffer saline and incubated overnight at 4°C in blocking buffer with the primary antibody (primary antibodies and 0.1% Triton X-100 were diluted in 5% donkey serum). Alexa Fluor 555–donkey anti-rabbit IgG (ab150066; Abcam) was used for detection. The slides were covered by a drop of ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology) for nuclear staining. ToupView (ToupTek Photonics, Hangzhou, China) was used to assess the immunofluorescence results. Confocal images were acquired with an LSM700 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). For the immunoblotting of the liver, the snap-frozen liver tissues were homogenized in lysis buffer that contained 0.1 mmol/L Na3VO4, protease inhibitor cocktail tablet (Roche, Mannheim, Germany), Pefabloc SC (Roche, Mannheim, Germany), sodium fluoride, and sodium pyrophosphate. The protein concentration was measured using a DC Protein Assay Kit (Bio Rad, Hercules, CA). Equal amounts of proteins were loaded and separated by SDS-PAGE. Proteins transferred to polyvinylidene difluoride membranes (IPVH00010; Millipore, Billerica, MA) were analyzed by immunoblotting with primary antibodies. After washing with Tris-buffered saline containing 0.1% Tween-20, membranes were incubated with a horseradish peroxidase–conjugated goat–anti-rabbit (401393; Calbiochem, San Diego, CA) or goat–anti-mouse (401253; Calbiochem) antibody for 1 hour at room temperature. Proteins were visualized using the ProNA ECL Ottimo (Translab, Seoul, Republic of Korea), and the images were acquired using the Amersham Imager 680 (GE Healthcare, Bjorkgatan, Sweden). β-Actin was used as a loading control. Whole blood was collected from all mice and centrifuged at 900 × g for 15 minutes at 4°C. Then, the supernatant was separated. Serum triglycerides were measured using the spectrophotometer method and a Triglyceride L-Type Kit (Wako, Osaka, Japan) in accordance with the manufacturer's instructions, and the reconstituted lipid calibrator was used as a standard. Serum free fatty acid concentrations were measured with an automated analyzer (TBA-120FR; Toshiba Corp., Tokyo, Japan). Serum vitamin C and alanine aminotransferase (ALT) activity were measured using ALT activity assay kit (K752-100; BioVision, Milpitas, CA) and vitamin C assay kit (K661-100; BioVision) in accordance with the manufacturer's instructions. Snap-frozen liver tissues were placed in isopropanol solution (278475; Sigma, St. Louis, MO) and incubated overnight at 4°C. Then, the samples were centrifuged at 9600 × g for 15 minutes at 4°C to aspirate the supernatants. The supernatants were measured with a Triglyceride L-Type Kit, according to the same method as that used for the serum triglyceride measurements. The hepatic cholesterol and total bile acid values were mused using an EZ-total cholesterol assay kit (DG-TSC100; DoGen, Seoul, Republic of Korea) and Total Bile Acid Assay kit (STA-631; CELL BIOLABS, San Diego, CA) in accordance with the manufacturer's instructions. Total liver RNAs were extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA), and the RNA concentrations were measured by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The extracted RNA was used as a template for cDNA synthesis using the RT Prime kit (EBT-1520; ELPIS Biotech, Daejeon, Republic of Korea) with M-MLV Reverse Transcriptase, random hexamers, and oligo dT. Synthesized cDNAs were mixed with the TOPreal qPCR 2× PreMIX (RT500M; Enzynomics, Daejeon, Republic of Korea) with 5 pmol of primers. The 18S ribosomal RNA was used as an internal control to normalize mRNA expression. The following mouse primers were used: 18s, carbohydrate-responsive element-binding protein (ChREBP), SREBP-1c, FAS, and Sodium-dependent vitamin C transporter 1 (SVCT-1). The sequence and source of primer used in the present study are listed in Table 2.24Tomita Y. Cakir B. Liu C.-H. Fu Z. Huang S. Cho S.S. Britton W.R. Sun Y. Puder M. Hellström A. Free fatty acid receptor 4 activation protects against choroidal neovascularization in mice.Angiogenesis. 2020; 23: 385-394Crossref PubMed Scopus (15) Google Scholar, 25Dubuquoy C. Robichon C. Lasnier F. Langlois C. Dugail I. Foufelle F. Girard J. Burnol A.-F. Postic C. Moldes M. Distinct regulation of adiponutrin/PNPLA3 gene expression by the transcription factors ChREBP and SREBP1c in mouse and human hepatocytes.J Hepatol. 2011; 55: 145-153Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 26Li G. Hernandez-Ono A. Crooke R.M. Graham M.J. Ginsberg H.N. Antisense reduction of 11β-hydroxysteroid dehydrogenase type 1 enhances energy expenditure and insulin sensitivity independent of food intake in C57BL/6J mice on a western-type diet.Metabolism. 2012; 61: 823-835Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 27Gui Y.-z. Yan H. Gao F. Xi C. Li H.-h. Wang Y.-p. Betulin attenuates atherosclerosis in apoE−/− mice by up-regulating ABCA1 and ABCG1.Acta Pharmacol Sin. 2016; 37: 1337-1348Crossref PubMed Scopus (23) Google ScholarTable 2Mouse Primer Sequences for Quantitative Real-Time PCRTarget geneReferencePrimer sequences (5′-3′)18s24Tomita Y. Cakir B. Liu C.-H. Fu Z. Huang S. Cho S.S. Britton W.R. Sun Y. Puder M. Hellström A. Free fatty acid receptor 4 activation protects against choroidal neovascularization in mice.Angiogenesis. 2020; 23: 385-394Crossref PubMed Scopus (15) Google ScholarForward5′-ACGGAAGGGCACCACCAGGA-3′Reverse5′-CACCACCACCCACGGAATCG-3′ChREBP25Dubuquoy C. Robichon C. Lasnier F. Langlois C. Dugail I. Foufelle F. Girard J. Burnol A.-F. Postic C. Moldes M. Distinct regulation of adiponutrin/PNPLA3 gene expression by the transcription factors ChREBP and SREBP1c in mouse and human hepatocytes.J Hepatol. 2011; 55: 145-153Abstract Full Text Full Text PDF PubMed Scopus (107) Google ScholarForward5′-ATGACCCCTCACTCAGGGAAT-3′Reverse5′-GATCCAAGGGTCCAGAGCAG-3'SREBP-1c26Li G. Hernandez-Ono A. Crooke R.M. Graham M.J. Ginsberg H.N. Antisense reduction of 11β-hydroxysteroid dehydrogenase type 1 enhances energy expenditure and insulin sensitivity independent of food intake in C57BL/6J mice on a western-type diet.Metabolism. 2012; 61: 823-835Abstract Full Text Full Text PDF PubMed Scopus (10) Google ScholarForward5′-GGCACTAAGTGCCCTCAACCT-3′Reverse5′-GCCACATAGATCTCTGCCAGTGT-3′FAS27Gui Y.-z. Yan H. Gao F. Xi C. Li H.-h. Wang Y.-p. Betulin attenuates atherosclerosis in apoE−/− mice by up-regulating ABCA1 and ABCG1.Acta Pharmacol Sin. 2016; 37: 1337-1348Crossref PubMed Scopus (23) Google ScholarForward5′-GCTGCGGAAACTTCAGGAAAT-3′Reverse5′-AGAGACGTGTCACTCCTGGACTT-3′SVCT1NM_011397Forward5′-GGCATCATTGAGTCCATCGG-3′Reverse5′-GTAGCCCAGCGATAATGCAG-3′Data are available at https://www.ncbi.nlm.nih.gov/nuccore (last accessed June 22, 2021). Open table in a new tab Data are available at https://www.ncbi.nlm.nih.gov/nuccore (last accessed June 22, 2021). Primary mouse hepatocytes (PMHs) were isolated from the WT mice and SMP30 KO mice by the collagenase liver perfusion methods using 0.075% collagenase diluted in Hanks' balanced salt solutions (LB-003-01; WELGENE, Gyeongsan, Republic of Korea). The extracted cells were centrifuged with 50% 1× Percoll solution (1-0891-02; GE Healthcare, Waukesha, WI). The purified primary hepatocytes were placed in 6-well collagen (354236; Corning, Tewksbury, MA) coated plates at a density of 1 × 105 cells per well and cultured in William's media (12551-032; Gibco, Carlsbad, CA) containing 5% fetal bovine serum (16000044; Gibco, Rockville, MD), 1 mol/L HEPES, l-glutamine, gentamycin, streptomycin–penicillin–amphotericin B, dexamethasone, and insulin for 8 hours. The medium was then replaced by maintenance media, which is a William's media containing gentamycin, streptomycin–penicillin–amphotericin B, dexamethasone, and insulin. For facilitated de novo lipogenesis, PMHs were incubated in maintenance media containing high sugar (25 mmol/L fructose, 25 mmol/L glucose, and 0.2 mmol/L OA). The 25 μmol/L of cholesterol was administered in WT PMHs to mimic the in vivo experiments. The 75 μmol/L of vitamin C was also administered in WT PMHs every 8 hours for 24 hours in accordance with a previous report.28Frikke-Schmidt H. Lykkesfeldt J. Keeping the intracellular vitamin C at a physiologically relevant level in endothelial cell culture.Anal Biochem. 2010; 397: 135-137Crossref PubMed Scopus (18) Google Scholar After the cholesterol and vitamin C treatments, the WT PMHs were incubated in maintenance media containing high sugar for 24 hours at 37°C, 5% CO2. The cells were then harvested by lysis buffer that contained protease inhibitors. All obtained data from the experiments were expressed as means ± SD, and statistical significance among the groups was determined on the basis of unpaired test, U-test, or Kruskal-Wallis one-way analysis of variance on ranks. To investigate the exact role of vitamin C in NAFLD, the WT and SMP30 KO mice were fed an HFD for 11 weeks. Interestingly, the vitamin C–deficient SMP30 KO mice showed a significantly decreased body weight and a reduced increase in body weight ratio (percentage) compared with the WT mice and the vitamin C–supplemented SMP30 KO mice (Figure 1, A and B ). As expected, the vitamin C–deficient SMP30 KO mice exhibited significantly decreased serum vitamin C levels compared with the WT mice and the vitamin C–supplemented SMP30 KO mice (Figure 1C). Similarly, in a gross observation, the vitamin C–deficient SMP30 KO mice exhibited a significantly reduced liver weight and size compared with the WT and vitamin C–supplemented mice groups (Figure 1, D and E). In addition, the HFD-fed vitamin C–deficient SMP30 KO mice exhibited a significantly reduced average adipocyte size compared with those of the other HFD-fed mice groups (Figure 1, F and G). Interestingly, the increasing body weight ratio exhibited a significant positive correlation with serum vitamin C levels despite the same caloric intake per body weight ratio in all HFD-fed mice groups (Figure 1, H and I). These results indicate that vitamin C deficiency might inhibit HFD-induced liver weight increase. Taken together, these studies indicate that vitamin C might be associated with liver lipid metabolism. The NAFLD lesions among all HFD-fed mice groups were evaluated next. Vitamin C–deficient SMP30 KO mice have notably decreased hepatic triglyceride accumulation compared with that of vitamin C–supplemented mice groups, as indicated by the hematoxylin and eosin and Oil Red O staining (Figure 2, A and B ). Vitamin C deficiency reduced the average hepatocyte size the SMP30 KO mice, which was reversed by vitamin C supplementation (Supplemental Figure S1). Next, NAFLD progression was assessed using NASH Clinical Research Network grading system. Notably, histopathologic examination of liver sections demonstrated a significantly decreased steatosis grade in vitamin C–deficient mice compared with vitamin C–supplemented mice, suggesting the inhibitory effect of vitamin C deficiency on NAFLD progression (Figure 2C). However, the inflammatory lesions of NAFLD were attenuated by vitamin C supplements (Figure 2, D–F). Vitamin C–deficient SMP30 KO mice displayed reduced hepatic triglyceride accumulation compared with WT control mice, as indicated by the biochemical assays (Figure 2G). In the serum biochemistry analysis, the HFD-fed vitamin C–deficient SMP30 KO mice exhibited significantly decreased ALT activity compared with that of the HFD-fed WT mice (Figure 2H). Similarly, the serum triglyceride levels were also significantly decreased in the HFD-fed vitamin C–deficient SMP30 KO mice, and vitamin C supplementation elevated serum triglyceride levels to the same levels as those in the HFD-fed WT mice groups, despite having the same serum-free fatty acid levels (Figure 2, I and J). These data indicate that vitamin C deficiency decreases hepatic steatosis independently of its antioxidant effects on the inflammatory responses. To clarify the potential effects of SMP30 in HFD-induced NAFLD progression, immunohistochemistry and immunoblot analyses were done to assess hepatic SMP30 expression. The SMP30 expression was similar in all WT mice; however, SMP30 expression was not observed in the SMP30 KO mice (Figure 3). Taken together, the HFD-mediated NAFLD is generally was not associated with the expression of SMP30, and vitamin C deficiency decreased the NAFLD progression via SMP30-independent pathways. Decreased NAFLD progression in the vitamin C–deficient SMP30 KO mice compared with the WT and vitamin C–supplemented SMP30 KO mice corroborated results from prior studies. To clarify how vitamin C–deficient SMP30 KO mice suppressed NAFLD progression compared with the WT and vitamin C–supplemented SMP30 KO mice, the phosphorylation levels of AMPK, a crucial energy sensor of cells, were evaluated. The HFD-fed vitamin C–deficient SMP30 KO mice exhibited increased AMPK phosphorylation compared with the HFD-fed WT mice (Figure 4, A and B ). However, the PPAR-α protein expression levels were almost equal in all HFD-fed groups (Figure 4, C and D). Given that PPAR-α is a crucial factor of lipid degradation, we inferred that fatty acid oxidation was not affected by vitamin C deficiency, despite suppressed NAFLD progression in the vitamin C–deficient SMP30 KO mice. Vita
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