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• W2894528168 abstract "Hepatic vitamin D receptor (VDR) expression is increased in patients with nonalcoholic fatty liver (NAFL) and is required for liver steatosis in an NAFL mouse model. However, how hepatocyte VDR is involved in setting up steatosis remains unclear. The authors transduced human hepatocyte–derived cells with an adenoviral vector encoding human VDR and found that angiopoietin-like protein 8 (ANGPTL8) expression was increased upon VDR activation by vitamin D or lithocholic acid. The mRNA levels of hepatic VDR– and vitamin D–related genes [cytochrome P450 (CYP) 2R1, CYP27A1, and CYP3A4] were higher in NAFL patients compared with normal liver subjects. Noteworthy, hepatic ANGPTL8 mRNA and protein levels were elevated in NAFL patients, and its mRNA correlated with VDR mRNA and with the steatosis grade. Moreover, increases in serum conjugated bile acids, including the VDR agonist glycine-lithocholic acid, were observed in NAFL patients. Additionally, free fatty acids and insulin were able to up-regulate both VDR and ANGPTL8 mRNA in human hepatocytes, whereas ANGPTL8 gene knockdown attenuated free fatty acids–induced triglyceride accumulation in these cells. In conclusion, activated VDR up-regulates ANGPTL8 expression, contributing to triglyceride accumulation in human hepatocytes. Moreover, hepatic ANGPTL8 mRNA positively correlates with VDR mRNA content and the grade of steatosis in NAFL patients, suggesting that this novel pathway may play a key role in the pathogenesis of hepatosteatosis. Hepatic vitamin D receptor (VDR) expression is increased in patients with nonalcoholic fatty liver (NAFL) and is required for liver steatosis in an NAFL mouse model. However, how hepatocyte VDR is involved in setting up steatosis remains unclear. The authors transduced human hepatocyte–derived cells with an adenoviral vector encoding human VDR and found that angiopoietin-like protein 8 (ANGPTL8) expression was increased upon VDR activation by vitamin D or lithocholic acid. The mRNA levels of hepatic VDR– and vitamin D–related genes [cytochrome P450 (CYP) 2R1, CYP27A1, and CYP3A4] were higher in NAFL patients compared with normal liver subjects. Noteworthy, hepatic ANGPTL8 mRNA and protein levels were elevated in NAFL patients, and its mRNA correlated with VDR mRNA and with the steatosis grade. Moreover, increases in serum conjugated bile acids, including the VDR agonist glycine-lithocholic acid, were observed in NAFL patients. Additionally, free fatty acids and insulin were able to up-regulate both VDR and ANGPTL8 mRNA in human hepatocytes, whereas ANGPTL8 gene knockdown attenuated free fatty acids–induced triglyceride accumulation in these cells. In conclusion, activated VDR up-regulates ANGPTL8 expression, contributing to triglyceride accumulation in human hepatocytes. Moreover, hepatic ANGPTL8 mRNA positively correlates with VDR mRNA content and the grade of steatosis in NAFL patients, suggesting that this novel pathway may play a key role in the pathogenesis of hepatosteatosis. Nonalcoholic fatty liver disease (NAFLD) is becoming the most common form of chronic liver disorder worldwide1Garcia-Monzon C. Vargas-Castrillon J. Porrero J.L. Alonso M.T. Bonachia O. Castillo M.J. Marcos A. Quiros E. Ramos B. Sanchez-Cabezudo C. Villar S. Saez A. Rodriguez de Cia J. del Pozo E. Vega-Piris L. Soto-Fernandez S. Lo Iacono O. Miquilena-Colina M.E. Prevalence and risk factors for biopsy-proven non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in a prospective cohort of adult patients with gallstones.Liver Int. 2015; 35: 1983-1991Crossref PubMed Scopus (23) Google Scholar, 2Younossi Z.M. Koenig A.B. Abdelatif D. Fazel Y. Henry L. Wymer M. Global epidemiology of nonalcoholic fatty liver disease: meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology. 2016; 64: 73-84Crossref PubMed Scopus (5388) Google Scholar and is closely linked to obesity, insulin resistance, diabetes, and cardiovascular complications,3Chalasani N. Younossi Z. Lavine J.E. Diehl A.M. Brunt E.M. Cusi K. Charlton M. Sanyal A.J. American Gastroenterological AssociationAmerican Association for the Study of Liver DiseasesAmerican College of GastroenterologyThe diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology.Gastroenterology. 2012; 142: 1592-1609Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar, 4Than N.N. Newsome P.N. A concise review of non-alcoholic fatty liver disease.Atherosclerosis. 2015; 239: 192-202Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar thus being currently considered as the hepatic component of the metabolic syndrome.5Salamone F. Bugianesi E. Nonalcoholic fatty liver disease: the hepatic trigger of the metabolic syndrome.J Hepatol. 2010; 53: 1146-1147Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar The potential for NAFLD to progress from a simple steatosis, also termed nonalcoholic fatty liver (NAFL), to more advanced forms of liver disease such as steatohepatitis, with variable stages of fibrosis, cirrhosis, and hepatocellular carcinoma, constitutes a major health problem worldwide6Loomba R. Cui J. Wolfson T. Haufe W. Hooker J. Szeverenyi N. Ang B. Bhatt A. Wang K. Aryafar H. Behling C. Valasek M.A. Lin G.Y. Gamst A. Brenner D.A. Yin M. Glaser K.J. Ehman R.L. Sirlin C.B. Novel 3D magnetic resonance elastography for the noninvasive diagnosis of advanced fibrosis in NAFLD: a prospective study.Am J Gastroenterol. 2016; 111: 986-994Crossref PubMed Scopus (137) Google Scholar and may become a significant burden for public health systems. However, NAFLD pathophysiology is still incompletely understood, thereby limiting the availability of effective diagnostic and therapeutic interventions. Though obesity and diabetes are well-known metabolic risk factors for NAFLD development,7Masuoka H.C. Chalasani N. Nonalcoholic fatty liver disease: an emerging threat to obese and diabetic individuals.Ann N Y Acad Sci. 2013; 1281: 106-122Crossref PubMed Scopus (195) Google Scholar another common endocrine disorder, the 25-hydroxy-vitamin D3 (25-OH-VD) deficiency, has been associated with NAFLD progression.8Eliades M. Spyrou E. Agrawal N. Lazo M. Brancati F.L. Potter J.J. Koteish A.A. Clark J.M. Guallar E. Hernaez R. Meta-analysis: vitamin D and non-alcoholic fatty liver disease.Aliment Pharmacol Ther. 2013; 38: 246-254Crossref PubMed Scopus (206) Google Scholar, 9Hazlehurst J.M. Tomlinson J.W. Non-alcoholic fatty liver disease in common endocrine disorders.Eur J Endocrinol. 2013; 169: R27-R37Crossref PubMed Scopus (64) Google Scholar, 10Nelson J.E. Roth C.L. Wilson L.A. Yates K.P. Aouizerat B. Morgan-Stevenson V. Whalen E. Hoofnagle A. Mason M. Gersuk V. Yeh M.M. Kowdley K.V. Vitamin D deficiency is associated with increased risk of non-alcoholic steatohepatitis in adults with non-alcoholic fatty liver disease: possible role for MAPK and NF-kappaB?.Am J Gastroenterol. 2016; 111: 852-863Crossref PubMed Scopus (92) Google Scholar, 11Targher G. Bertolini L. Scala L. Cigolini M. Zenari L. Falezza G. Arcaro G. Associations between serum 25-hydroxyvitamin D3 concentrations and liver histology in patients with non-alcoholic fatty liver disease.Nutr Metab Cardiovasc Dis. 2007; 17: 517-524Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar A number of clinical studies, however, have failed to confirm this association.12Bril F. Maximos M. Portillo-Sanchez P. Biernacki D. Lomonaco R. Subbarayan S. Correa M. Lo M. Suman A. Cusi K. Relationship of vitamin D with insulin resistance and disease severity in non-alcoholic steatohepatitis.J Hepatol. 2015; 62: 405-411Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Patel Y.A. Henao R. Moylan C.A. Guy C.D. Piercy D.L. Diehl A.M. Abdelmalek M.F. Vitamin D is not associated with severity in NAFLD: results of a paired clinical and gene expression profile analysis.Am J Gastroenterol. 2016; 111: 1591-1598Crossref PubMed Scopus (36) Google Scholar Similarly, there are apparent contradictions in animal models of NAFLD, where it has been reported that vitamin D3 (VD) deficiency exacerbates liver histology,14Nakano T. Cheng Y.F. Lai C.Y. Hsu L.W. Chang Y.C. Deng J.Y. Huang Y.Z. Honda H. Chen K.D. Wang C.C. Chiu K.W. Jawan B. Eng H.L. Goto S. Chen C.L. Impact of artificial sunlight therapy on the progress of non-alcoholic fatty liver disease in rats.J Hepatol. 2011; 55: 415-425Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar whereas it has also been observed that hepatocyte VD receptor (VDR) is required for liver steatosis in apolipoprotein E–deficient mice on a high fat diet.15Bozic M. Guzman C. Benet M. Sanchez-Campos S. Garcia-Monzon C. Gari E. Gatius S. Valdivielso J.M. Jover R. Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis.J Hepatol. 2016; 65: 748-757Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar VDR expression is significantly induced in the liver of NAFL patients, suggesting that hepatic VDR signaling might promote liver fat accumulation in early stages of NAFLD,15Bozic M. Guzman C. Benet M. Sanchez-Campos S. Garcia-Monzon C. Gari E. Gatius S. Valdivielso J.M. Jover R. Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis.J Hepatol. 2016; 65: 748-757Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar but further investigation is needed to unravel the role of hepatocyte VDR signaling in hepatosteatosis onset. On the other hand, the liver plays a major role in triglyceride (TG) homeostasis by releasing angiopoietin-like (ANGPTL) proteins, particularly ANGPTL3, -4, and -8.16Mattijssen F. Kersten S. Regulation of triglyceride metabolism by Angiopoietin-like proteins.Biochim Biophys Acta. 2012; 1821: 782-789Crossref PubMed Scopus (136) Google Scholar ANGPTL3 is expressed almost exclusively in liver, and its levels are modestly altered by fasting and refeeding, whereas ANGPTL4 is expressed at high levels in liver and adipose tissue, largely in the fasted state. ANGPTL8 (alias lipasin and betatrophin) is also expressed at the highest levels in liver and adipose tissue, but it is markedly up-regulated by feeding. These three ANGPTL members are lipoprotein lipase inhibitors, they act in concert to regulate trafficking of TG during the feeding–fasting cycle, and overexpression of any one of them results in hypertriglyceridemia.17Wang Y. Quagliarini F. Gusarova V. Gromada J. Valenzuela D.M. Cohen J.C. Hobbs H.H. Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis.Proc Natl Acad Sci U S A. 2013; 110: 16109-16114Crossref PubMed Scopus (257) Google Scholar, 18Zhang R. The ANGPTL3-4-8 model, a molecular mechanism for triglyceride trafficking.Open Biol. 2016; 6: 150272Crossref PubMed Scopus (148) Google Scholar It has also been recently shown that ANGPTL8 induces intracellular TG accumulation by down-regulating the adipose triglyceride lipase (ATGL).19Zhang Y. Li S. Donelan W. Xie C. Wang H. Wu Q. Purich D.L. Reeves W.H. Tang D. Yang L.J. Angiopoietin-like protein 8 (betatrophin) is a stress-response protein that down-regulates expression of adipocyte triglyceride lipase.Biochim Biophys Acta. 2016; 1861: 130-137Crossref PubMed Scopus (34) Google Scholar Moreover, several recent studies show a positive association of serum ANGPTL8 with NAFLD,20Lee Y.H. Lee S.G. Lee C.J. Kim S.H. Song Y.M. Yoon M.R. Jeon B.H. Lee J.H. Lee B.W. Kang E.S. Lee H.C. Cha B.S. Association between betatrophin/ANGPTL8 and non-alcoholic fatty liver disease: animal and human studies.Sci Rep. 2016; 6: 24013Crossref PubMed Scopus (68) Google Scholar, 21Mele C. Grugni G. Mai S. Vietti R. Aimaretti G. Scacchi M. Marzullo P. Circulating angiopoietin-like 8 (ANGPTL8) is a marker of liver steatosis and is negatively regulated by Prader-Willi Syndrome.Sci Rep. 2017; 7: 3186Crossref PubMed Scopus (12) Google Scholar, 22von Loeffelholz C. Pfeiffer A.F.H. Lock J.F. Lieske S. Docke S. Murahovschi V. Kriebel J. de Las Heras Gala T. Grallert H. Rudovich N. Stockmann M. Spranger J. Jahreis G. Bornstein S.R. Lau G. Xu A. Schulz-Menger J. Exner L. Haufe S. Jordan J. Engeli S. Birkenfeld A.L. ANGPTL8 (betatrophin) is expressed in visceral adipose tissue and relates to human hepatic steatosis in two independent clinical collectives.Horm Metab Res. 2017; 49: 343-349Crossref PubMed Scopus (22) Google Scholar but little is known about a potential pathogenic link between ANGPTL8 and NAFLD. The purpose of this work was to investigate how hepatocyte VDR signaling is involved in setting up steatosis. Herein, the possibility that ANGPTL8 is a VDR target gene is explored, and the feasibility of this novel VDR signaling pathway in cultured human hepatocytes and in patients with biopsy-proven NAFL is investigated. HepG2 cells (ATCC, Rockville, MD) were cultured in Ham's F-12/Leibovitz L-15 (1:1, v/v) medium (Gibco BRL/Invitrogen, Barcelona, Spain) supplemented with 7% newborn calf serum, 2 mmol/L l-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (Sigma Aldrich, Madrid, Spain). Second-generation human upcyte hepatocytes from three different donors and Hepatocyte Culture Medium, Hepatocyte High-Performance Medium, and Hepatocyte/LSECs Thawing Medium were all obtained from upcyte technologies GmbH (Hamburg, Germany). The recombinant adenoviral vector encoding human VDR (Ad-VDR) was developed in our laboratory with the kit Adeno-X Adenoviral System 3 (#632269; Clontech, Mountain View, CA). Full-length VDR cDNA was obtained from HeLa total cDNA with primers 5′-CACCCCTGGGCTCCACTTACC-3′ (forward) and 5′-CCGCCACAGGCTGTCCTAGTC-3′ (reverse). Recombinant adenovirus was plaque cloned and amplified in HEK293T and purified with the kit Vivapure AdenoPACK 20 (VS-AVPQ022; Sartorius, Madrid, Spain). Titration was performed by plaque-forming assay. For adenovirus infection, both upcyte hepatocytes and HepG2 cells were incubated with a noncytotoxic multiplicity of infection (plaque-forming units/cell) of Ad-VDR for 24 hours. Cells were then shifted to adenovirus-free medium and cultured for an additional period of time in the presence of VDR agonists. 1α, 25-dihydroxyvitamin D3 (calcitriol or VD) and lithocholic acid (LCA; Sigma Aldrich) stock solutions were prepared in dimethyl sulfoxide (Sigma Aldrich), and added at a final concentration of 10 nmol/L and 100 μmol/L, respectively, to cultured cells for a variable period of time as indicated. Control cultures were treated with the same amount of solvent. Human hepatoma Huh7 cells (ATTC) were cultured in DMEM (Gibco BRL/Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma Aldrich) at 37°C with 5% CO2. Confluent HepG2 and Huh7 cells were treated with 600 and 1000 μmol/L free fatty acid (FFA) mix (oleate:palmitate ratio 2:1) or with 100 nmol/L insulin (all from Sigma Aldrich) for 16 hours. Total RNA was isolated from snap-frozen liver biopsy samples available of 34 NAFL patients and 28 normal liver (NL) subjects, representatives of the study population shown in Table 1, as well as from cultured liver cells after the corresponding treatment with Trizol reagent (Invitrogen). DNase-treated RNA was reverse transcribed into cDNA with d(N)6 random hexamer primers and real-time quantitative RT-PCR was performed with an ABI 7900HT sequence detector (Applied Biosystems, Foster City, CA) using the SyBr Green method. RNA expression was corrected by β-actin expression.Table 1Characteristics of the Study PopulationCharacteristicNL (n = 89)NAFL (n = 80)P-valueAge, years42.6 ± 14.652.8 ± 14.20.003Women74 (83.1)50 (62.5)0.015Race Caucasian66 (74.2)51 (63.8)NS Latin American18 (20.2)23 (28.8)NS Others5 (5.6)6 (7.4)NSBody mass index, kg/m227.0 ± 4.930.5 ± 8.70.031Waist perimeter, cm92.5 ± 11.5101.6 ± 15.5<0.001Hip perimeter, cm104.0 ± 11.1109.7 ± 13.70.019Glucose, mg/dL91.8 ± 11.2100.8 ± 17.50.029Insulin levels, μU/L7.6 ± 4.210.1 ± 4.90.013HOMA score1.7 ± 1.12.5 ± 1.40.021Triglycerides, mg/dL110.1 ± 57.7137.9 ± 64.40.003Total cholesterol193.7 ± 85.8204.6 ± 98.4NSHDL-cholesterol, mg/dL49.5 ± 11.848.6 ± 12.2NSALT, IU/L17.1 ± 5.320.7 ± 7.3<0.001AST, IU/L17.3 ± 9.125.4 ± 15.70.029γ-GT, IU/L31.4 ± 30.050.2 ± 58.90.019Ferritin, ng/mL62.6 ± 66.898.4 ± 70.3<0.001Alkaline phosphatase, IU/L69.9 ± 23.376.1 ± 33.2NS25-OH vitamin D levels, ng/mL18.0 ± 6.917.4 ± 8.6NSSteatosis Grade 089 (100)0 Grade 1054 (67.5) Grade 2020 (25) Grade 306 (7.5)Hepatocyte ballooning Grade 089 (100)80 (100)Fibrosis Stage 089 (100)80 (100)Data are shown as mean ± SD or as number of cases (%).γ-GT, gamma-glutamyltransferase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL, high-density lipoprotein; HOMA, homeostatic model assessment; NAFL, nonalcoholic fatty liver; NL, normal liver; NS, not significant. Open table in a new tab Data are shown as mean ± SD or as number of cases (%). γ-GT, gamma-glutamyltransferase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL, high-density lipoprotein; HOMA, homeostatic model assessment; NAFL, nonalcoholic fatty liver; NL, normal liver; NS, not significant. The following primer sequences were used: VDR, 5′-CCCTGAGTCACCAGGAATGT-3′ (forward) and 5′-GGGTTTTCAGGGAACTACG-3′ (reverse); ANGPTL8, 5′-GCCTGTTGGAGACTCAGATGG-3′ (forward) and 5′-CCATAGGATGTGGCTCTGCTT-3′ (reverse); and CYP3A4, 5′-CCTTACATATACACACCCTTTGGAAGT-3′ (forward) and 5′-AGCTCAATGCATGTACAGAATCCCCGGTTA-3′ (reverse). Cell lysates were obtained by scraping off cultured dishes with M-PER reagent from Pierce (Fisher Scientific, Madrid, Spain) and a protease inhibitor cocktail. After protein content determination with the Protein Assay Kit (Bio-Rad Laboratories, Madrid, Spain), total protein was boiled in Laemmli sample buffer and was resolved by 12% SDS-PAGE. Proteins were transferred to Immobilion membranes (Millipore Iberica, Madrid, Spain), and after blocking with 5% nonfat dry milk, membranes were incubated overnight with antibodies as indicated. Blots were developed with horseradish peroxidase–labeled IgG (Dako, Glostrup, Denmark) using an ECL kit (Bio-Rad Laboratories). Equal loading was verified by Coomassie Brilliant Blue or Ponceau S staining. The anti-ANGPTL8 antibody (SAB3501080) was purchased from Sigma Aldrich and the anti-GAPDH (SC-365062) from Santa Cruz Biotechnology (Dallas, TX). Human scrambled (control) or ANGPTL8 shRNA lentiviral particles (Sigma Aldrich) were used to produce stable ANGPTL8 gene knockdown in HepG2 and Huh7 human hepatic cells. Proliferating cells were coincubated with lentiviral transducing particles in culture media containing polybrene (hexadimethrine bromide; Santa Cruz Biotechnology) for 24 hours and then cultured with 5 to 10 μg/mL of puromycin (Santa Cruz Biotechnology). Resistant cells were expanded and examined for ANGPTL8 mRNA levels. Lipids were extracted as described.23Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42799) Google Scholar After purification, lipids were resuspended in isopropyl alcohol, and triglycerides were analyzed with a colorimetric kit (Spinreact, Barcelona, Spain). The values of TG content were obtained in μg/mg protein and expressed as fold change. This study comprises 80 patients with biopsy-proven NAFL. Eighty-nine subjects with NL on whom liver biopsy was performed during programed laparoscopic cholecystectomy were further studied. Characteristics of the entire study population are detailed in Table 1. All NAFL patients and NL subjects studied drank <20 g/day of alcohol, were not having potentially hepatotoxic drugs, had no analytical evidence of iron overload, and were seronegative for autoantibodies and for hepatitis B, hepatitis C, and human immunodeficiency virus. This study was performed in agreement with the Declaration of Helsinki, and with local and national laws. The human ethics committee of the Santa Cristina University Hospital approved the study procedures, and all participants signed an informed written consent before inclusion in the study. Venous blood samples of each participant were obtained at the time of liver biopsy to determine serum levels of liver enzymes, metabolic parameters, and serologic tests using routine laboratory methods. In addition, plasma insulin was determined by a chemiluminescent microparticle immunoassay (ARCHITECT insulin; Abbott Laboratories, Abbott Park, IL). Insulin resistance was calculated by the homeostasis model assessment method.24Matthews D.R. Hosker J.P. Rudenski A.S. Naylor B.A. Treacher D.F. Turner R.C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.Diabetologia. 1985; 28: 412-419Crossref PubMed Scopus (25575) Google Scholar Metabolic syndrome was defined according to the ATP III criteria.25National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III)Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report.Circulation. 2002; 106: 3143-3421Crossref PubMed Scopus (11286) Google Scholar 25-OH-VD was tested by a validated chemiluminescent immunoassay (LIAISON 25 OH Vitamin D TOTAL Assay; DiaSorin Inc., Stillwater, MN). As previously reported,26Kwok R.M. Torres D.M. Harrison S.A. Vitamin D and nonalcoholic fatty liver disease (NAFLD): is it more than just an association?.Hepatology. 2013; 58: 1166-1174Crossref PubMed Scopus (109) Google Scholar, 27Rosen C.J. Clinical practice. Vitamin D insufficiency.N Engl J Med. 2011; 364: 248-254Crossref PubMed Scopus (671) Google Scholar VD status was defined according to serum VD concentrations as sufficiency (≥20 ng/mL) or deficiency (<20 ng/mL). Hematoxylin-eosin and Masson's trichrome–stained paraffin-embedded liver biopsy sections were examined and interpreted by the same experienced hepatopathologist (J.V.-C.), who was unaware of the clinical data. Steatosis was assessed as outlined by Brunt et al,28Brunt E.M. Janney C.G. Di Bisceglie A.M. Neuschwander-Tetri B.A. Bacon B.R. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions.Am J Gastroenterol. 1999; 94: 2467-2474Crossref PubMed Scopus (3139) Google Scholar grading the percentage involvement by steatotic hepatocytes as follows: grade 0, <5%; grade 1, 5% to 33%; grade 2, >33% to 66%; and grade 3, >66%. Histologic diagnosis of all liver biopsies from NAFLD patients studied was simple steatosis, also termed as NAFL. Paraffin-embedded liver biopsy sections from a representative sample of the study population were immunostained with a primary rabbit antibody against ANGPTL8 (SAB3501080; Sigma Aldrich), diluted to 1:50 by using the Dako EnVision+ System (Dako) as described by the manufacturer. Liver tissue area occupied by ANGPTL8-positive cells was measured by using a high-resolution digital video camera (Nikon DXM 1200; Nikon, Tokyo, Japan) connected to a light microscope (Nikon Eclipse E400) equipped with a Plan Apochromat 20× objective (Nikon). Image analysis procedures were performed with the FIJI software version Java 8 (NIH, Bethesda, MD; https://imagej.net/Fiji/Downloads). Values were obtained in six different lobular areas where hepatocytes are the predominant cell type. The average value was considered as the ANGPTL8 expression index for each liver biopsy sample and expressed as arbitrary units that reflect the intensity of the immunostaining. Serum bile acids (BA) were profiled by a ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method as described previously29Barchetta I. Carotti S. Labbadia G. Gentilucci U.V. Muda A.O. Angelico F. Silecchia G. Leonetti F. Fraioli A. Picardi A. Morini S. Cavallo M.G. Liver vitamin D receptor, CYP2R1, and CYP27A1 expression: relationship with liver histology and vitamin D3 levels in patients with nonalcoholic steatohepatitis or hepatitis C virus.Hepatology. 2012; 56: 2180-2187Crossref PubMed Scopus (170) Google Scholar in 37 NAFL patients and 40 NL subjects, representatives of the study population shown in Table 1. This method, validated according to Food and Drug Administration guidelines, allows the quantification of 12 nonconjugated, 8 glycine-conjugated, and 11 taurine-conjugated BA, using 5 additional deuterated BA as internal standards in a single analytical run and was performed in the Analytical Unit, Core Facility, IIS Hospital La Fe in Valencia, Spain. Categorical variables are presented as frequency and percentage. Continuous variables are shown as means ± SD or standard error of the mean (SEM), as indicated. The baseline characteristics of the patients studied were compared by the Pearson χ2 test for categorical variables and the unpaired t-test or U-test for continuous variables. Data from gene and protein expression levels as well as from serum VD and BA concentrations were compared by using the Kruskal-Wallis analysis of variance test. The Spearman's r-test was used to evaluate correlations. These statistical analyses were performed using SPSS statistical software version 24.0 (IBM SPSS Statistics, Armonk, NY) with 2-sided tests; P < 0.05 was considered as statistically significant. In an attempt to find new VDR targets able to explain the steatogenic role of VDR signaling in the liver,15Bozic M. Guzman C. Benet M. Sanchez-Campos S. Garcia-Monzon C. Gari E. Gatius S. Valdivielso J.M. Jover R. Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis.J Hepatol. 2016; 65: 748-757Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar mRNA expression analysis was performed by real-time quantitative RT-PCR in HepG2 cells and human upcyte hepatocytes, transduced with Ad-VDR, and either left alone or incubated with 10 nmol/L calcitriol (VD) for 4 hours. Among the lipid metabolism genes examined, ANGPTL8, which encodes a hepatocyte-derived protein that inhibits lipolysis and regulates plasma triglycerides, was differentially expressed. More precisely, activation of VDR by VD increased the mRNA level of ANGPTL8 between six- and eightfold in both human HepG2 cells (Figure 1A) and human upcyte hepatocytes from three different donors (Figure 1B). The response of ANGPTL8 to VD was fast and reached more than sixfold induction in only 2 hours after VD (Figure 1C). Moreover, the expression of ANGPTL8 showed a dose-dependent response to increasing multiplicity of infection of Ad-VDR (Figure 1D). Besides ANGPTL8 expression, VDR-transfected cells also up-regulated the well-characterized hepatic VDR-target gene CYP3A4 upon VD exposure (Supplemental Figure S1). Given that VDR can be activated by specific BA, it was investigated whether LCA might regulate hepatic ANGPTL8 expression in a VDR-dependent manner. Results showed that LCA increased ANGPTL8 mRNA content in both VDR-transfected HepG2 cells and human upcyte hepatocytes (Figure 1, E and F). The response of ANGPTL8 to both VD and LCA was next compared in VDR-transfected human upcyte hepatocytes. Although VD-dependent up-regulation of ANGPTL8 expression was higher at early time points (15-fold by 4 hours), the LCA-dependent induction of ANGPTL8 expression was the highest at later time points (25-fold by 24 hours) (Figure 1G). More importantly, the combined effect of VD and LCA resulted in an additive 38-fold induction of ANGPTL8 gene expression. Finally, immunoblotting analysis of ANGPTL8 demonstrated that the VDR-dependent induction observed at the mRNA level translated to the protein level (Figure 1H). In line with our previously reported findings,15Bozic M. Guzman C. Benet M. Sanchez-Campos S. Garcia-Monzon C. Gari E. Gatius S. Valdivielso J.M. Jover R. Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis.J Hepatol. 2016; 65: 748-757Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar liver VDR mRNA levels were significantly higher in NAFL patients (2.74-fold increase; P = 0.0057) than in NL subjects (Figure 2A). Because cytochrome P450 (CY0P) enzymes such as CYP2R1, CYP27A1, and CYP3A4 are essential for the 25-hydroxylation of VD in the liver, the hepatic mRNA levels of these three CYPs were measured; they were found to be up-regulated in NAFL patients (1.4-fold for CYP3A4; P = 0.115; 2-fold for CYP27A1; P < 0.0001; and 3.2-fold for CYP2R1; P < 0.0001) (Figure 2B). Of interest, mRNA levels of the VDR-target gene CYP3A4 and, also, both CYP27A1 and CYP2R1 mRNA contents showed a positive significant correlation with VDR mRNA levels in the liver of NAFL patients (Figure 2C), indicating that an active intrahepatic VDR signaling exists in human fatty livers. ANGPTL8 gene expression was significantly increased in NAFL patients with respect to NL subjects (3.3-fold; P < 0.0001) (Figure 3A). Noteworthy, a significant positive correlation between the hepatic mRNA levels of VDR and ANGPTL8, and between hepatic ANGPTL8 mRNA levels and the grade of steatosis in the entire study population was also observed (Figure 3B). However, hepatic ANGPTL8 mRNA levels did not correlate with either body mass index (r = 0.1628; P = 0.2140) or homeostasis model assessment of insulin resistance (r = −0.119; P = 0.3805). In addition, the hepatic ANGPTL8 protein content was found to be significantly higher in NAFL patients than in NL individuals by immunohistochemistry (Figure 3C). Serum BA was tested in the study pop" @default.
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• W2894528168 title "Angiopoietin-Like Protein 8 Is a Novel Vitamin D Receptor Target Gene Involved in Nonalcoholic Fatty Liver Pathogenesis" @default.
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