Vitamin C Deficiency Inhibits Nonalcoholic Fatty Liver Disease Progression through Impaired de Novo Lipogenesis

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 biosynthesis vitamin C (VC) in many mammals, except primates and humans, was recently recognized as a gluconolactonase. However, precise relation between VC lipid metabolism NAFLD not completely understood. Therefore, this study aimed to clearly reveal role progression. SMP30 knockout (KO) mice were used VC-deficient mouse model. To investigate on metabolism, 13- 15-week–old KO wild-type fed 60% high-fat diet exposed tap water VC-containing (1.5 g/L) ad libitum for 11 weeks. Primary hepatocytes isolated from demonstrate hepatocytes. 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During experimental period (11 weeks), WT housed room at 22°C ± 3°C relative humidity 50% 10%, 12-hour light-dark cycle (lights 8:00 am), access food water. subdivided into eight groups based type, supplementation, genetic types (WT SMP30). received either chow (SAFE D40; Safe Diet, Rosenberg, Germany) HFD (D12492; Research New Brunswick, NJ). All given C–free diet, provided drinking g/L). animal experiments protocols tissues approved Kyungpook National University Institutional Animal Care Use Committee (approval numbers 2017-0112 2019-0070). tissue samples, collected 10% neutral-buffered formalin, processed routinely embedded paraffin wax. sections cut 4-μm–thick hematoxylin eosin staining. A analysis performed Clinical Network grading system (Table 1).23Kleiner D.E. Brunt E.M. Van Natta Behling Contos Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida Design validation histological scoring disease.Hepatology. 2005; 41: 1313-1321Crossref (6754) Activity Score defined sum scores steatosis, lobular inflammation, ballooning degeneration. grades averaged basis least five random fields ×200 magnification each sample. Oil Red O staining visualize triglyceride accumulation. For staining, fixed 4% paraformaldehyde frozen section. then stained freshly prepared working solution.Table 1Grading Staging System NAFLDFeaturesGrade/scoreNASH network system23Kleiner ScholarSteatosis0<5%15%–33%233%–67%3>67%Lobular inflammation0No foci1<2 Foci per ×20 field22–4 field3>4 fieldBallooning degeneration0None1Few2ManyNAFLD, disease; NASH, steatohepatitis. Open table new tab antibodies used: anti–β-actin (A1978; Sigma, St. Louis, MO; sc-47778; Santa Cruz Biotechnology, Cruz, CA), anti-SMP30 (SML-RO1001-EX; COSMOBIO CO, LTD, Tokyo, Japan), anti–phosphorylated AMP-activated kinase (AMPK; 2535S; Signaling Technology, Danvers, MA), anti-AMPK (2532S; Technology), anti–peroxisome proliferator-activated receptor-α (PPAR-α; sc-398394; Biotechnology), anti–sterol (SREBP-1c; ab28481; Abcam, Cambridge, UK), anti–fatty synthase (FAS; sc-48357; Biotechnology). chemicals oleic (OA; O1008; Steinheim, Germany), bovine serum albumin (10735078001; Roche, Basel, Switzerland), (832; DUKSAN, Ansan, Republic Korea), (C8667; MO), fructose (F0366; SANCHUN, Yeosu, glucose (64220S0601; JUNSEI, Japan). immunohistochemistry analysis, paraffin-embedded slides deparaffinized toluene rehydrated graded series. After deparaffinization, antigen retrieval solution 3% hydrogen peroxide methanol temperature 35 minutes steamed 10 mmol/L citrate buffer retrieval. being cooled temperatures (25°C) 2 hours, incubated blocking (Life Technologies, Frederick, MD) 1 hour. Subsequently, primary antibody overnight 4°C. washed phosphate-buffered saline, broad-spectrum Technologies) horseradish peroxidase–conjugated streptavidin minutes. Then, antigen-antibody complex visualized avidin-biotin peroxidase ABC kit (Vector Laboratories, Burlingame, CA). rinsed distilled water, counterstained hematoxylin, dehydrated series toluene. immunofluorescence, phosphate saline 4°C (primary 0.1% Triton X-100 diluted 5% donkey serum). Alexa Fluor 555–donkey anti-rabbit IgG (ab150066; Abcam) detection. covered drop ProLong Gold Antifade Reagent DAPI (Cell Technology) nuclear ToupView (ToupTek Photonics, Hangzhou, China) assess immunofluorescence results. Confocal images acquired LSM700 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). immunoblotting snap-frozen homogenized lysis contained 0.1 Na3VO4, protease inhibitor tablet (Roche, Mannheim, Pefabloc SC sodium fluoride, pyrophosphate. concentration measured DC Protein Assay Kit (Bio Rad, Hercules, Equal amounts proteins loaded separated SDS-PAGE. Proteins transferred polyvinylidene difluoride membranes (IPVH00010; Millipore, Billerica, MA) analyzed antibodies. washing Tris-buffered containing Tween-20, goat–anti-rabbit (401393; Calbiochem, San Diego, CA) goat–anti-mouse (401253; Calbiochem) hour temperature. ProNA ECL Ottimo (Translab, Seoul, Amersham Imager 680 (GE Healthcare, Bjorkgatan, Sweden). β-Actin loading control. Whole all centrifuged 900 × g 15 supernatant separated. Serum triglycerides spectrophotometer method Triglyceride L-Type (Wako, Osaka, Japan) accordance manufacturer's instructions, reconstituted calibrator standard. concentrations automated analyzer (TBA-120FR; Toshiba Corp., alanine aminotransferase (ALT) activity ALT assay (K752-100; BioVision, Milpitas, (K661-100; BioVision) instructions. Snap-frozen placed isopropanol (278475; MO) samples 9600 aspirate supernatants. supernatants Kit, according same measurements. total bile values mused EZ-total (DG-TSC100; DoGen, Korea) Total Bile Acid (STA-631; CELL BIOLABS, RNAs extracted TRIzol (Invitrogen, Carlsbad, RNA NanoDrop ND-1000 (NanoDrop Wilmington, DE). template cDNA synthesis RT Prime (EBT-1520; ELPIS Biotech, Daejeon, M-MLV Reverse Transcriptase, hexamers, oligo dT. Synthesized cDNAs mixed TOPreal qPCR 2× PreMIX (RT500M; Enzynomics, 5 pmol primers. 18S ribosomal internal control normalize mRNA expression. primers 18s, carbohydrate-responsive (ChREBP), SREBP-1c, FAS, Sodium-dependent transporter (SVCT-1). sequence source primer listed Table 2.24Tomita Cakir C.-H. Fu Z. Huang Cho S.S. Britton W.R. Sun Puder Hellström Free receptor 4 protects against choroidal neovascularization mice.Angiogenesis. 23: 385-394Crossref (15) Scholar, 25Dubuquoy Robichon Lasnier Langlois Dugail I. Foufelle Girard Burnol A.-F. Postic Moldes Distinct regulation adiponutrin/PNPLA3 gene expression transcription factors ChREBP SREBP1c human hepatocytes.J 55: 145-153Abstract (107) 26Li Hernandez-Ono Crooke R.M. Graham Ginsberg H.N. Antisense 11β-hydroxysteroid dehydrogenase type enhances energy expenditure insulin sensitivity independent C57BL/6J western-type diet.Metabolism. 61: 823-835Abstract (10) 27Gui Y.-z. Yan H. Gao Xi Li H.-h. Y.-p. Betulin attenuates atherosclerosis apoE−/− up-regulating ABCA1 ABCG1.Acta Pharmacol Sin. 37: 1337-1348Crossref ScholarTable 2Mouse Primer Sequences Quantitative Real-Time PCRTarget geneReferencePrimer sequences (5′-3′)18s24Tomita ScholarForward5′-ACGGAAGGGCACCACCAGGA-3′Reverse5′-CACCACCACCCACGGAATCG-3′ChREBP25Dubuquoy ScholarForward5′-ATGACCCCTCACTCAGGGAAT-3′Reverse5′-GATCCAAGGGTCCAGAGCAG-3’SREBP-1c26Li ScholarForward5′-GGCACTAAGTGCCCTCAACCT-3′Reverse5′-GCCACATAGATCTCTGCCAGTGT-3′FAS27Gui ScholarForward5′-GCTGCGGAAACTTCAGGAAAT-3′Reverse5′-AGAGACGTGTCACTCCTGGACTT-3′SVCT1NM_011397Forward5′-GGCATCATTGAGTCCATCGG-3′Reverse5′-GTAGCCCAGCGATAATGCAG-3′Data available https://www.ncbi.nlm.nih.gov/nuccore (last accessed June 22, 2021). Data (PMHs) collagenase perfusion methods 0.075% Hanks' balanced salt solutions (LB-003-01; WELGENE, Gyeongsan, Korea). cells 1× Percoll (1-0891-02; GE Waukesha, WI). purified 6-well collagen (354236; Corning, Tewksbury, coated plates density 105 cultured William's media (12551-032; Gibco, fetal (16000044; Rockville, MD), mol/L HEPES, l-glutamine, gentamycin, streptomycin–penicillin–amphotericin B, dexamethasone, 8 hours. medium replaced maintenance media, William’s insulin. facilitated lipogenesis, PMHs high sugar (25 fructose, 25 glucose, 0.2 OA). μmol/L administered mimic vivo experiments. 75 every hours 24 previous report.28Frikke-Schmidt Keeping intracellular physiologically relevant level endothelial culture.Anal Biochem. 397: 135-137Crossref treatments, 37°C, CO2. harvested inhibitors. obtained data means SD, statistical among determined unpaired test, U-test, Kruskal-Wallis one-way variance ranks. exact Interestingly, showed body weight reduced increase ratio (percentage) compared C–supplemented (Figure 1, B ). As expected, levels 1C). Similarly, gross observation, size D E). In addition, HFD-fed average adipocyte those F G). significant positive correlation despite caloric H I). These results indicate might inhibit HFD-induced increase. Taken together, be associated lesions evaluated next. notably groups, indicated 2, mice, reversed supplementation (Supplemental Figure S1). Next, assessed system. Notably, histopathologic examination demonstrated grade suggesting inhibitory 2C). attenuated supplements D–F). displayed biochemical assays 2G). biochemistry 2H). elevated having serum-free I J). decreases independently responses. progression, immunoblot analyses done similar mice; observed 3). HFD-mediated SMP30, SMP30-independent pathways. Decreased corroborated prior studies. how phosphorylation AMPK, sensor cells, evaluated. increased AMPK 4, PPAR-α almost equal D). Given factor degradation, inferred oxidation deficiency, mice. Vita