Insulin resistance dysregulates CYP7B1 leading to oxysterol accumulation: a pathway for NAFL to NASH transition
2020; Elsevier BV; Volume: 61; Issue: 12 Linguagem: Inglês
10.1194/jlr.ra120000924
ISSN1539-7262
AutoresGenta Kakiyama, Dalila Marques, Rebecca Martin, Hajime Takei, Daniel Rodrı́guez-Agudo, Sandra A. LaSalle, Taishi Hashiguchi, Xiaoying Liu, Richard Green, Sandra K. Erickson, Gregorio Gil, Michael Fuchs, Mitsuyoshi Suzuki, Tsuyoshi Murai, Hiroshi Nittono, Phillip B. Hylemon, Huiping Zhou, William M. Pandak,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoNAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD. NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD. NAFLD has emerged as the most common chronic liver disease and is soon to become a major cause for liver cancer and transplantation (1Younossi Z.M. Marchesini G. Pinto-Cortez H. Petta S. Epidemiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: implications for liver transplantation.Transplantation. 2019; 103: 22-27Crossref PubMed Scopus (201) Google Scholar). With one-fourth of the population affected, it will have a dramatic global economic impact on healthcare systems and patient's well-being (2Younossi Z. Tacke F. Arrese M. Chander Sharma B. Mostafa I. Bugianesi E. Wai-Sun Wong V. Yilmaz Y. George J. Fan J. et al.Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.Hepatology. 2019; 69: 2672-2682Crossref PubMed Scopus (787) Google Scholar). NAFLD is a disease spectrum ranging from simple steatosis [nonalcoholic fatty liver (NAFL)] to steatohepatitis (NASH) with or without liver fibrosis or cirrhosis. The natural history of disease progression is complex, supported by findings of rapid fibrosis progression on one hand and spontaneous disease regression on the other hand (3Reddy Y.K. Marella H.K. Jiang Y. Ganguli S. Snell P. Podila P.S.B. Maliakkal B. Satapathy S.K. Natural history of non-alcoholic fatty liver disease: a study with paired liver biopsies.J. Clin. Exp. Hepatol. 2020; 10: 245-254Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 4De A. Duseja A. Natural history of simple steatosis or nonalcoholic fatty liver.J. Clin. Exp. Hepatol. 2020; 10: 255-262Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Unfortunately, we do not have a pathophysiology-driven explanation for the observed phenotypic variability of this liver disease. NAFLD is considered as the hepatic manifestation of the metabolic syndrome, as it is strongly associated with conditions of dietary excess such as obesity, dyslipidemia, type 2 diabetes mellitus (T2DM), and hypertension (5Eslam M. Newsome P.N. Sarin S.K. Anstee Q.M. Targher G. Romero-Gomez M. Zelber-Sagi S. Wai-Sun Wong V. Dufour J.F. Schattenberg J.M. et al.A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement.J. Hepatol. 2020; 73: 202-209Abstract Full Text Full Text PDF PubMed Scopus (1284) Google Scholar). Insulin resistance and lipid excess within the liver are considered as the earliest markers or evidence of progression of NAFLD followed by inflammation and subsequent fibrosis (6Fon Tacer K. Rozman D. Nonalcoholic fatty liver disease: focus on lipoprotein and lipid deregulation.J. Lipids. 2011; 2011783976 Crossref PubMed Google Scholar). Interestingly, excess liver lipid is contributed to by increased de novo fatty acid synthesis within the liver, further exacerbating dietary induced liver lipid excess (7Browning J.D. Horton J.D. Molecular mediators of hepatic steatosis and liver injury.J. Clin. Invest. 2004; 114: 147-152Crossref PubMed Scopus (1757) Google Scholar). What complicates this relatively straightforward sequence of metabolic events is that only a small percentage of those with fatty liver progress to NASH. The amount of lipid excess does not seem to correlate with disease activity or progression, i.e., the presentation of liver lipid excess that occurs in lean NASH patients (8Satapathy S.K. Sanyal A.J. Epidemiology and natural history of nonalcoholic fatty liver disease.Semin. Liver Dis. 2015; 35: 221-235Crossref PubMed Scopus (247) Google Scholar, 9Younes R. Bugianesi E. NASH in lean individuals.Semin. Liver Dis. 2019; 39: 86-95Crossref PubMed Scopus (111) Google Scholar). Furthermore, the coexistence of NASH in type 1 diabetes mellitus suggests other causes than insulin resistance promoting fatty liver to NASH (10Bhatt H.B. Smith R.J. Fatty liver disease in diabetes mellitus.Hepatobiliary Surg. Nutr. 2015; 4: 101-108PubMed Google Scholar). In the absence of a common thread, NAFLD has become described as a complex metabolic entity of liver lipid excess. It is obvious that only a better understanding of the pathophysiologic derangement promoting progression from fatty liver to NASH will allow us to develop effective treatment approaches other than weight loss. Our laboratory hypothesized that liver lipid excess in NAFLD and NASH might be linked to dysregulation of cholesterol metabolism through the alternative pathway of bile acid synthesis (Fig. 1A) (11Pandak W.M. Kakiyama G. The acidic pathway of bile acid synthesis: not just an alternative pathway.Liver Res. 2019; 3: 88-98Crossref PubMed Scopus (51) Google Scholar). As previously hypothesized by Javitt and colleagues and our own group, this pathway was the initial pathway of bile acid synthesis; which was likely not originally a pathway to form bile acids, but a pathway that controlled the levels of the regulatory oxysterols regarded as key metabolic regulators of both cholesterol and lipid biosynthesis (12Javitt N.B. 25R,26-Hydroxycholesterol revisited: synthesis, metabolism, and biologic roles.J. Lipid Res. 2002; 43: 665-670Abstract Full Text Full Text PDF PubMed Google Scholar, 13Arnon R. Yoshimura T. Reiss A. Budai K. Lefkowitch J.H. Javitt N.B. Cholesterol 7-hydroxylase knockout mouse: a model for monohydroxy bile acid-related neonatal cholestasis.Gastroenterology. 1998; 115: 1223-1228Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 14Kakiyama G. Marques D. Takei H. Nittono H. Erickson S. Fuchs M. Rodriguez-Agudo D. Gil G. Hylemon P.B. Zhou H. et al.Mitochondrial oxysterol biosynthetic pathway gives evidence for CYP7B1 as controller of regulatory oxysterols.J. Steroid Biochem. Mol. Biol. 2019; 189: 36-47Crossref PubMed Scopus (18) Google Scholar). Moreover, the inner mitochondrial membrane has low levels of cholesterol and the alternative pathway may have also evolved to remove excess cholesterol via side-chain oxidation by sterol 27-hydroxylase (CYP27A1) and further metabolism by oxysterol 7α-hydroxylase (CYP7B1). As a vital metabolic pathway, it is pervasive within body tissues. The ultimate ability to form bile acids likely developed within hepatocytes as a way to aid the absorption of dietary lipids. However, an inability to increase rates of bile acids to aid in the absorption of larger amounts of dietary lipids without generating toxic amounts of oxysterols and their metabolic products likely led to the neutral pathway of bile acid synthesis; a pathway where 7α-hydroxylation of cholesterol is the initial metabolic step allowing for the synthesis of nonregulatory less toxic metabolites (14Kakiyama G. Marques D. Takei H. Nittono H. Erickson S. Fuchs M. Rodriguez-Agudo D. Gil G. Hylemon P.B. Zhou H. et al.Mitochondrial oxysterol biosynthetic pathway gives evidence for CYP7B1 as controller of regulatory oxysterols.J. Steroid Biochem. Mol. Biol. 2019; 189: 36-47Crossref PubMed Scopus (18) Google Scholar). In this regard, Setchell et al. (15Setchell K.D. Schwarz M. O'Connell N.C. Lund E.G. Davis D.L. Lathe R. Thompson H.R. Weslie Tyson R. Sokol R.J. Russell D.W. Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease.J. Clin. Invest. 1998; 102: 1690-1703Crossref PubMed Scopus (293) Google Scholar) reported that the absence of CYP7B1 in children led to oxysterol-induced liver inflammation and cirrhosis within the first year of life. Dai et al. (16Dai D. Mills P.B. Footitt E. Gissen P. McClean P. Stahlschmidt J. Coupry I. Lavie J. Mochel F. Goizet C. et al.Liver disease in infancy caused by oxysterol 7 alpha-hydroxylase deficiency: successful treatment with chenodeoxycholic acid.J. Inherit. Metab. Dis. 2014; 37: 851-861Crossref PubMed Scopus (40) Google Scholar) subsequently showed that the absence of CYP7B1 led first to the presence of fat within the liver with subsequent liver inflammation in a pattern found in the transition from fatty liver to NASH. Interestingly, Tang, Pettersson, and Norlin (17Tang W. Pettersson H. Norlin M. Involvement of the PI3K/Akt pathway in estrogen-mediated regulation of human CYP7B1: identification of CYP7B1 as a novel target for PI3K/Akt and MAPK signalling.J. Steroid Biochem. Mol. Biol. 2008; 112: 63-73Crossref PubMed Scopus (12) Google Scholar) showed that upregulation of the insulin signal pathway seemed able to upregulate Cyp7b1. Biddinger et al. (18Biddinger S.B. Haas J.T. Yu B.B. Bezy O. Jing E. Zhang W. Unterman T.G. Carey M.C. Kahn C.R. Hepatic insulin resistance directly promotes formation of cholesterol gallstones.Nat. Med. 2008; 14: 778-782Crossref PubMed Scopus (223) Google Scholar), in a cholesterol gallstone mouse model, reported that Cyp7b1 mRNA levels were markedly suppressed in liver selective insulin receptor knockout mice. However, the potential importance of regulation of CYP7B1 by insulin has never been truly addressed in the context of NAFLD and NASH. NAFLD is a rheostat of disease. Within the existing literature, the timing of the transition of fatty liver to NASH and to fibrosis is not clearly delineated, i.e., lacking a clear starting point from which to initiate investigation. As a result, the lines of initiation appear to have become blurred within the multitude of ensuing metabolic consequences of cellar toxicity, inflammatory cell infiltrate, cell to cell interactions, collagen synthesis, liver architectural changes, and increased cell metabolic contents such as bile acids associated with cholestasis. Thus, establishing inciting events appeared unlikely in the absence of coupling several early-event in vivo models. In the current study, we chose to pursue five overlapping mouse models of NAFL, coupled to human data, and describe evidence for a new model explaining how the dysregulation in cellular oxysterol levels initiated by the chronic suppression of CYP7B1 plays a role in the transition from NAFLD to NASH. All chemicals were of the highest purity commercially available. HPLC grade solvents were purchased from Fisher Scientific (New Lawn, NJ), and all other reagents were from Sigma-Aldrich, Inc. (St. Louis, MO), unless indicated otherwise: oxysterol standards, 7α-hydroxycholesterol (7α-HC), 24(S)-hydroxycholesterol (24-HC), 25-hydroxycholesterol (25-HC), (25R)-26-hydroxycholesterol (26-HC), 7α,24(S)-dihydroxycholesterol (7,24-diHC), 7α,25-dihydroxycholesterol (7α,25-diHC), and (25R)-7α,26-dihydroxycholesterol (7α,26-diHC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). d6-25-HC (26,26,26,27,27,27-[2H6]25-HC) was purchased from Cayman Chemical (Ann Arbor, MI). Tauro (T) α-Muricholic acid (MCA), αMCA, TβMCA, βMCA, TωMCA, ωMCA, cholic acid (CA)-3-sulfate (S), chenodeoxycholic acid (CDCA)-3S, ursodeoxycholic acid (UDCA)-3S, deoxycholic acid (DCA)-3S, lithocholic acid (LCA)-3S, glyco (G)CA-3S, GCDCA-3S, GUDCA-3S, GDCA-3S, GLCA-3S, TCA-3S, TCDCA-3S, TUDCA-3S, TDCA-3S, and TLCA-3S were kind gifts from Professor Takashi Iida (Tokyo, Japan). [2,2,4,4-d4]CA [d4-CA, internal standard (IS) for unconjugated bile acids], [2,2,4,4-d4]GCA (d4-GCA, IS for glycine-conjugated bile acids), and [2,2,4,4-d4]TCA (d4-TCA, and IS for taurine-conjugated and double-conjugated bile acids) were obtained from C/D/N Isotopes, Inc. (Pointe-Claire, Quebec, Canada). All other oxysterol standards and bile acids were from Steraloids, Inc. (Newport, RI). SV Total RNA Isolation System was obtained from Promega Co. (Madison, WI). Cyp7b1 antibody was available from Proteintech Group Inc. (Rosemont, IL). Anti-GRP78 (BiP) and anti-GAPDH antibodies were from Abcam (Cambridge, MA). Monoclonal anti-CCAAT-enhancer-binding homologous protein (CHOP) antibody was from Thermo Fisher Scientific (Waltham, MA). Human liver tissues were obtained from the National Institutes of Health (NIH)-sponsored Liver Tissue Distribution Center at the University of Minnesota. An F2 intercross of the mouse strains 129S1/SvlmJ and C57Bl/6J (B6/129) that develop classic fatty liver with progression to NASH on a supplemental WD chow, were obtained from Dr. Sandra Erickson (University of California, San Francisco). The mice were bred at the animal facility of McGuire Veterans Affairs Medical Center with a 12 h light cycle (6:00 AM to 6:00 PM). All mice had free access to water. Unless specified differently, mice were fed an ad libitum WD with 42% of calories from fat and 43% from carbohydrates (Harlan-Teklad TD.88137; Envigo, Frederick, MD) for 2–6 weeks. One group of mice was prolonged on the WD feeding for 32 weeks for reaffirming the B6/129 mouse as previously characterized as a viable NASH model. Controls were age-matched B6/129 mice fed a regular chow (RC; irradiated Teklad LM-485). All mice were 12 weeks old at the time they were euthanized unless otherwise noted. For the study of in vivo steroidogenic acute regulatory protein (StarD1) overexpression (Fig. 1, Fig. 2, Fig. 3), Ad-StarD1 or Ad-β-galactosidase (β-Gal; for control) recombinant virus (1 × 109 pfu) was injected into the tail vein of B6/129 mice on the seventh day from start of WD feeding. These adenovirus constructs were prepared as previously described (19Pandak W.M. Schwarz C. Hylemon P.B. Mallonee D. Valerie K. Heuman D.M. Fisher R.A. Redford K. Vlahcevic Z.R. Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis.Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 281: G878-G889Crossref PubMed Google Scholar). WD feeding was continued for another week, and the mice were euthanized on the fourteenth day. Blood was centrifuged at 1,620 g for 15 min at 4°C, and serum in the supernatant was collected. All specimens were snap-frozen in liquid nitrogen and stored at −78°C until further analysis. A portion of liver was also fixed in 10% neutral buffered formalin and sent to the Department of Pathology at Virginia Commonwealth University to be processed and stained with H&E. Images were taken with a Nikon Ti-U microscope and NIS Elements software.Fig. 3Sterile inflammation is associated with increased oxysterol formation in mice. StarD1 was overexpressed in B6/129 male mice to drive endogenous oxysterol formation as outlined in the Methods. Mouse livers were evaluated at day 14, which was 7 days following StarD1 overexpression, by both flow cytometry and qPCR. All cells were gated off of live singlets. A: Measurement of total percent hematopoietic cells (CD45+), percent B cells (CD45+B220+MHCII+), percent eosinophils (CD45+CD11b+CCR3+), percent neutrophils (CD45+CD11bhiF4/80lowLy6G+Ly6C+), percent dendritic cells (CD45+CD11c+MHCII+), and percent Kupffer cells (CD45+CD11b+F4/80+). B: Normalized relative mRNA expression of inflammation markers related to NAFLD, normalized to the housekeeping gene (Tbp). Statistics were performed as a Student's t-test between groups in A and a Mann-Whitney nonparametric comparison in B. Error bars depict SEM. *P ≤ 0.05 versus β-Gal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The STAM™ mice were prepared and characterized at SMC Laboratories Inc. (Tokyo, Japan) as previously described (20Fujii M. Shibazaki Y. Wakamatsu K. Honda Y. Kawauchi Y. Suzuki K. Arumugam S. Watanabe K. Ichida T. Asakura H. et al.A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma.Med. Mol. Morphol. 2013; 46: 141-152Crossref PubMed Scopus (216) Google Scholar). Briefly, 200 μg of streptozotocin (STZ) were subcutaneously injected into C57Bl/6J male mice at 2 days after birth. The mice were fed a low-fat RC diet (CE-2; CLEA Japan Inc.) until 4 weeks of age, and then fed an ad libitum HFD (HFD32; CLEA Japan Inc) until 8 weeks (NASH stage). STZ injection led to approximately 60% of β cells being impaired when they were evaluated at 9 weeks of age (supplemental Fig. S1). One group of mice was continued on their RC diet for an additional 4 weeks (fed RC for 8 weeks). All mice were fasted 4 h prior to being euthanized unless otherwise noted. At the time of euthanasia, mice were subjected to isoflurane inhalation. All animal protocols were approved by the IACUC of the McGuire Veterans Affairs Medical Center and the Virginia Commonwealth University; both are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and comply with the Guide for Care and Use of Laboratory Animals published by the NIH. Serum total cholesterol (TC), HDL-C, LDL-C, TGs, glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase levels were measured by the enzymatic procedures run on Siemens Vista 1500 instrumentation. Serum insulin was measured by the sensitive ELISA sandwich assay method using Crystal Chem ultra-sensitive mouse insulin ELISA kit (Elk Grove Village, IL) according to manufacturer's instructions. For plasma insulin measurement of the STZ-treated mice, Ultra-Sensitive Mouse Insulin ELISA kit (Morinaga Institute of Biological Science, Inc., Yokohama, Japan) was used. Plasma glucagon of C57Bl/6J mice was measured using Glucagon Quantikine ELISA kit (R&D Systems, Inc., Minneapolis, MN). Liver TC and free cholesterol (FC) were measured by enzymatic assay using the Cholesterol E and Free Cholesterol E kits (FUJIFILM Wako Chemicals U.S.A. Co, Richmond, VA). Infinity Triglycerides kit (Thermo Fisher Scientific) was used for liver TG measurement. The serum lipoprotein profile was obtained by the FPLC using an Agilent 1100 series HPLC system as follows: 100 μl of serum was injected onto a Superose 6, 10/300 GL column (GE Healthcare, Chicago, IL). NaCl (154 mM) containing 1 mM EDTA (pH 8.0) was used for the elution buffer at a flow rate of 0.2 ml/min. Effluents were monitored at 280 nm (21Jiao S. Cole T.G. Kitchens R.T. Pfleger B. Schonfeld G. Genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel-permeation chromatography.Metabolism. 1990; 39: 155-160Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Lipoproteins were identified by coelution with VLDL, LDL and HDL standards (Sigma-Aldrich). Starting at 20 min, fractions were collected every 1.2 min (240 μl/fraction) until 100 min. Cholesterol content was measured by adapting the Cholesterol E kit, adding 20 μl of a 10× reagent buffer to 180 μl of each fraction in a 96-well plate, heating at 37°C, and measuring absorbance at 595 nm. Primary hepatocytes were removed from the livers of the StarD1-overexpressed B6/129 mice using the collagenase-perfusion technique as previously described (22Pandak W.M. Bohdan P. Franklund C. Mallonee D.H. Eggertsen G. Bjorkhem I. Gil G. Vlahcevic Z.R. Hylemon P.B. Expression of sterol 12alpha-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo.Gastroenterology. 2001; 120: 1801-1809Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly, the liver was digested with 0.025% collagenase in William's E culture medium (40 ml) at 37°C for 10 min. The digest was filtered through a double thickness of sterile gauze. Hepatocytes were removed by centrifugation (60 g at 4°C for 5 min), and nonparenchymal cells (approximately 9.0 × 106 cells) in the supernatant were collected. The live-dead staining was conducted using Zombie Aqua (#423102; BioLegend, San Diego, CA) according to manufacturer's protocol. Cells were washed with FACS buffer (5% FBS in PBS with 2 mM EDTA). Fc receptors were blocked with 5 μg 2.4G2 for 10 min at 4°C. Antibodies were added for 45 min at 4°C. Cells were washed twice with FACS buffer and fixed in fixation buffer (BioLegend, #420801) for 10 min at room temperature. Flow cytometry data were collected on a BD LSR Fortessa™ X-20 (BD, Franklin Lakes, NJ) and analyzed in FlowJo (BD) software, all as previously described (23Lownik J.C. Conrad D.H. Martin R.K. A disintegrin and metalloproteinase 17 is required for ILC2 responses to IL-33.Biochem. Biophys. Res. Commun. 2019; 512: 723-728Crossref PubMed Scopus (5) Google Scholar). Antibodies used in this study were as follows: APC-conjugated anti-mouse CD45, PE/Cy7-conjugated anti-mouse F4/80, PE-conjugated anti-mouse CCR3, BV421-conjugated anti-mouse MHCII, FITC-conjugated anti-mouse CD11c, PE-conjugated anti-mouse B220, APCFIRE750-conjugated anti-mouse Ly6C, FITC-conjugated anti-mouse Ly6G (all from BioLegend), and BUV395-conjugated anti-mouse CD11b (BD). Total RNA from the liver tissue was isolated with the Promega Total RNA Isolation system according to manufacturer's protocol. qPCR was performed with 2 μg of RNA using the Affimetrix (Santa Clara, CA) VeriQuest SYBR Green PCR Master Mix in an Applied Biosystems 7500 PCR system (Foster City, CA). Primers are listed in supplemental Table S2. All data were produced in triplicates for each mRNA. The comparative CT method was used to calculate relative quantification of gene expression. The mRNA expression levels obtained for each gene were normalized to the expression of the Gapdh housekeeping gene by using the following equation: relative mRNA expression = 2−(Ct of each gene−Ct of Gapdh) (where Ct is the threshold cycle). For Fig. 3, total RNA was extracted from the nonhepatocyte-enriched fraction using Invitrogen™ TRIzol™ reagent per the manufacturer's instructions. RNA was quantified using a NanoDrop ND-100 spectrophotometer. One microgram of total RNA was reverse transcribed using SuperScript IV (Thermo Fisher Scientific) with oligo(dT20). The mRNA expression levels obtained for each gene were normalized to the expression level of the housekeeping gene, tbp: Normalized relative expression = 2−ΔΔCt (24Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (124148) Google Scholar). Protein samples from B6/129 mouse livers were separated on 12% SDS-PAGE gel and then transferred onto a PVDF membrane using a Bio-Rad semidry transfer cell apparatus. The membrane was incubated in TBST (TBS and Polysorbate 20) with 5% nonfat dry milk for 2 h at room temperature. The membrane was then incubated overnight in TBST with 2.5% nonfat dry milk containing a dilution of a primary antibody at 4°C, as indicated. The membrane was then washed three times in TBST for 30 min at room temperature. After washing, the membrane was incubated in a 1:2,000 dilution of the corresponding HRP-conjugated IgG in TBST with 2.5% nonfat dry milk for 1.5 h at room temperature. Finally, the membrane was washed three more times in TBST. Protein bands were visualized using SuperSignal West Pico chemiluminescent substrate and developed on a Bio-Rad ChemiDoc Touch imaging system. Quantification of protein expression levels of Cyp7b1, BiP, and GAPDH were performed using ImageJ software. Relative levels of Cyp7b1 and BiP expression were normalized by GAPDH. Quantification of oxysterols was performed by LC/ESI-MS/MS according to the reported method (25Sidhu R. Jiang H. Farhat N.Y. Carrillo-Carrasco N. Woolery M. Ottinger E. Porter F.D. Schaffer J.E. Ory D.S. Jiang X. A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid.J. Lipid Res. 2015; 56: 1222-1233Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) with modification: Liver tissue (100 mg) was digested in 1.0 mg/ml Proteinase K (0.5 ml in water) at 55°C for 6 h. Chloroform/methanol (1:2, v/v, 2 ml) was added to the digest, and the mixture was ultra-sonicated in a Branson type B-220 ultra-sonic bath (Danbury, CT) for 30 min. After centrifugation at 1,620 g for 5 min, the supernatant was collected in a clean glass tube. This procedure was repeated twice, and the combined extract was evaporated to dryness under a nitrogen stream at 40°C. The residue was redissolved in ethanol (1 ml). Aliquots (100 μl each) of this solution were used for oxysterol and bile acid analysis (see next section). For oxysterol analysis, a 100 μl aliquot of this solution was mixed with 10 μl of d6-25-HC (100 pmol/ml in methanol) and 10 μl of butylated hydroxytoluene (5 mg/ml in ethanol). After adding water (0.5 ml) and n-hexane (1.0 ml), the mixture was thoroughly vortexed (approximately 2 min), centrifuged at 180 g for 5 min, and the organic upper layer was collected in a glass tube. The bottom layer was washed again with n-hexane (1.0 ml) by the same procedure, and the combined extract was evaporated under reduced pressure. The residue was redissolved in n-hexane (0.5 ml) and loaded onto an InertSep NH2 cartridge (100 mg/1 ml; GL Sciences Inc., Tokyo, Japan) to remove cholesterol. Prior to loading the sample, the column was rinsed with 1.0 ml of chloroform/methanol (1:1) and conditioned with 3 ml of n-hexane. After loading the sample, the column was washed with n-hexane (1.0 ml), and the desired oxysterols were eluted with 1.0 ml of chloroform/methanol (20:1). Oxysterols were then derivatized to nicotinyl ester form according to the method by Sidhu et al. (25Sidhu R. Jiang H. Farhat N.Y. Carrillo-Carrasco N. Woolery M. Ottinger E. Porter F.D. Schaffer J.E. Ory D.S. Jiang X. A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid.J. Lipid Res. 2015; 56: 1222-1233Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) with modifications. Derivatization reagent (100 μl), which was a mixture of nicotinic acid (80 mg), N,N′-dimethyl-4-aminopyridine (30 mg), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; 100 mg) in N,N′-dimethylformamide (1.0 ml), was added to the oxysterol sample, and the mixture was incubated at 60°C for 1 h. Water (0.5 ml) and n-hexane (1.0 ml) were added, and the solution was thoroughly vortexed (approximately 2 min). After centrifugation at 180 g for 5 min, the n-hexane layer was collected and evaporated under a reduced pressure. The residue was dissolved in acetonitrile (100 μl), and an aliquot (10 μl) was injected into a Shimadzu LCMS-8050 system equipped with an ESI probe. A Nexera X2 HPLC pump with SunShell C30 column (100 × 2.1 mm inner diameter, 2.6 μm particle size; ChromaNik Tecnology Inc., Osaka, Japan) was used for the chromatographic separation. The column was heated at 35°C. Mobile phase A (5 mM ammonium acetate with 0.1% formic acid) and mobile phase B [isopropanol-acetonitrile (3:2) containing 0.1% formic acid] were used for gradient elution as follows: 0–2 min 70% B, 2–6 min 70–76% B, 6–16 min 78% B, 16–18 min 96% B, 18–20 min 96% B, 20–20.1 min 96–70% B, and 20.1–25 min 70% B. Flow rate was kept constant at 0.25 ml/min. The nebulizer gas flow was set at 3.0 liters per minute, and the heating gas and the drying gas were set at 10 liters per minute. The interface temperature was 300°C; the desolvation line temperature was 250°C; and the heat block temperature was set at 400°C with an interface voltage of 4,000 V. The collision gas (argon) pressure was 270 kPa. Quantification of all analytes was achieved in positive multiple reaction monitoring mode (supplemental Table S1). Oxysterols were quantified according to the calibration curve constructed by IS against known concentration of authentic standards. A 100 μl aliquot of the liver extract sus
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