Therapeutic FGF19 promotes HDL biogenesis and transhepatic cholesterol efflux to prevent atherosclerosis
2019; Elsevier BV; Volume: 60; Issue: 3 Linguagem: Inglês
10.1194/jlr.m089961
ISSN1539-7262
AutoresMei Zhou, R. Marc Learned, Stephen J. Rossi, Hui Tian, Alex M. DePaoli, Lei Ling,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoFibroblast growth factor (FGF)19, an endocrine hormone produced in the gut, acts in the liver to control bile acid synthesis. NGM282, an engineered FGF19 analog, is currently in clinical development for treating nonalcoholic steatohepatitis. However, the molecular mechanisms that integrate FGF19 with cholesterol metabolic pathways are incompletely understood. Here, we report that FGF19 and NGM282 promote HDL biogenesis and cholesterol efflux from the liver by selectively modulating LXR signaling while ameliorating hepatic steatosis. We further identify ABCA1 and FGF receptor 4 as mediators of this effect, and that administration of a HMG-CoA reductase inhibitor or a blocking antibody against proprotein convertase subtilisin/kexin type 9 abolished FGF19-associated elevations in total cholesterol, HDL cholesterol (HDL-C), and LDL cholesterol in db/db mice. Moreover, we show that a constitutively active MEK1, but not a constitutively active STAT3, mimics the effect of FGF19 and NGM282 on cholesterol change. In dyslipidemic Apoe−/− mice fed a Western diet, treatment with NGM282 dramatically reduced atherosclerotic lesion area in aortas. Administration of NGM282 to healthy volunteers for 7 days resulted in a 26% increase in HDL-C levels compared with placebo. These findings outline a previously unrecognized role for FGF19 in the homeostatic control of cholesterol and may have direct impact on the clinical development of FGF19 analogs. Fibroblast growth factor (FGF)19, an endocrine hormone produced in the gut, acts in the liver to control bile acid synthesis. NGM282, an engineered FGF19 analog, is currently in clinical development for treating nonalcoholic steatohepatitis. However, the molecular mechanisms that integrate FGF19 with cholesterol metabolic pathways are incompletely understood. Here, we report that FGF19 and NGM282 promote HDL biogenesis and cholesterol efflux from the liver by selectively modulating LXR signaling while ameliorating hepatic steatosis. We further identify ABCA1 and FGF receptor 4 as mediators of this effect, and that administration of a HMG-CoA reductase inhibitor or a blocking antibody against proprotein convertase subtilisin/kexin type 9 abolished FGF19-associated elevations in total cholesterol, HDL cholesterol (HDL-C), and LDL cholesterol in db/db mice. Moreover, we show that a constitutively active MEK1, but not a constitutively active STAT3, mimics the effect of FGF19 and NGM282 on cholesterol change. In dyslipidemic Apoe−/− mice fed a Western diet, treatment with NGM282 dramatically reduced atherosclerotic lesion area in aortas. Administration of NGM282 to healthy volunteers for 7 days resulted in a 26% increase in HDL-C levels compared with placebo. These findings outline a previously unrecognized role for FGF19 in the homeostatic control of cholesterol and may have direct impact on the clinical development of FGF19 analogs. Homeostasis of cholesterol, an important risk factor for coronary artery disease, is maintained by the coordinated regulation of de novo synthesis, catabolism, nutritional intake, intestinal absorption, reverse transport, and biliary excretion (1.Goldstein J.L. Brown M.S. A century of cholesterol and coronaries: from plaques to genes to statins.Cell. 2015; 161: 161-172Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Genome-wide association studies have uncovered a plethora of genetic loci associated with alterations in plasma cholesterol levels that have provided valuable mechanistic insights (2.Teslovich T.M.K. Musunuru A.V. Smith A.C. Edmondson I.M. Stylianou M. Koseki J.P. Biological, clinical and population relevance of 95 loci for blood lipids.Nature. 2010; 466: 707-713Crossref PubMed Scopus (2787) Google Scholar). Despite notable successes in developing medical therapies to reduce plasma levels of LDL cholesterol (LDL-C), cardiovascular disease remains the leading cause of death and years-of-life-lost worldwide (3.GBD 2016 Causes of Death Collaborators Global, regional, and national age-sex specific mortality for 264 causes of death, 1980–2016: a systematic analysis for the Global Burden of Disease Study 2016.Lancet. 2017; 390: 1151-1210Abstract Full Text Full Text PDF PubMed Scopus (2981) Google Scholar). This high unmet medical need necessitates going beyond the established paradigm to explore alternative approaches to lower the risk of cardiovascular disease, especially those of an atherosclerotic nature. Plasma HDL cholesterol (HDL-C) concentrations inversely associate with the risk of cardiovascular disease (4.Gordon D.J. Probstfield J.L. Garrison R.J. Neaton J.D. Castelli W.P. Knoke J.D. Jacobs Jr., D.R. Bangdiwala S. Tyroler H.A. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies.Circulation. 1989; 79: 8-15Crossref PubMed Scopus (2660) Google Scholar), a finding that has led to the hypothesis that HDL-C may protect against cardiovascular disease. Indeed, the development of novel therapies to exploit the atheroprotective property of HDL has been an area of intense investigation in recent years, but has proven to be particularly challenging, as high-profile clinical trials of drugs targeting the cholesteryl ester transfer protein ended in disappointment (5.Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J. Komajda M. Lopez-Sendon J. Mosca L. Tardif J.C. Waters D.D. et al.Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2610) Google Scholar, 6.HPS3/TIMI55–REVEAL Collaborative Group Bowman L. Hopewell J.C. Chen F. Wallendszus K. Stevens W. Collins R. Wiviott S.D. Cannon C.P. Braunwald E. et al.Effects of anacetrapib in patients with atherosclerotic vascular disease.N. Engl. J. Med. 2017; 377: 1217-1227Crossref PubMed Scopus (612) Google Scholar). Fibroblast growth factor (FGF)19 is an endocrine hormone that has a crucial role in controlling bile acid, carbohydrate, protein, and energy homeostasis (7.Kliewer S.A. Mangelsdorf D.J. Bile acids as hormones: the FXR-FGF15/19 pathway.Dig. Dis. 2015; 33: 327-331Crossref PubMed Scopus (240) Google Scholar, 8.Degirolamo C. Sabba C. Moschetta A. Therapeutic potential of the endocrine fibroblast growth factors FGF19, FGF21 and FGF23.Nat. Rev. Drug Discov. 2016; 15: 51-69Crossref PubMed Scopus (277) Google Scholar). Through the FGF receptor (FGFR)4-βKlotho receptor complex, FGF19 potently suppresses mRNA levels of CYP7A1, the gene encoding cholesterol-7α-hydroxylase, to inhibit de novo bile acid synthesis (7.Kliewer S.A. Mangelsdorf D.J. Bile acids as hormones: the FXR-FGF15/19 pathway.Dig. Dis. 2015; 33: 327-331Crossref PubMed Scopus (240) Google Scholar, 8.Degirolamo C. Sabba C. Moschetta A. Therapeutic potential of the endocrine fibroblast growth factors FGF19, FGF21 and FGF23.Nat. Rev. Drug Discov. 2016; 15: 51-69Crossref PubMed Scopus (277) Google Scholar). FGF19 also increases metabolic rate and reduces adiposity (9.Tomlinson E. Fu L. John L. Hultgren B. Huang X. Renz M. Stephan J.P. Tsai S.P. Powell-Braxton L. French D. et al.Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity.Endocrinology. 2002; 143: 1741-1747Crossref PubMed Scopus (287) Google Scholar), acts as a postprandial insulin-independent activator of hepatic glycogen and protein synthesis (10.Kir S. Beddow S.A. Samuel V.T. Miller P. Previs S.F. Suino-Powell K. Xu H.E. Shulman G.I. Kliewer S.A. Mangelsdorf D.J. FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis.Science. 2011; 331: 1621-1624Crossref PubMed Scopus (445) Google Scholar), and regulates hepatic glucose metabolism by inhibiting the CREB-PGC1α pathway (11.Potthoff M.J. Boney-Montoya J. Choi M. He T. Sunny N.E. Satapati S. Suino-Powell K. Xu H.E. Gerard R.D. Finck B.N. et al.FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway.Cell Metab. 2011; 13: 729-738Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The effect on weight reduction and glycemic regulation by FGF19 is largely dependent on the FGFR1c-βKlotho receptor complex, through its actions on the nervous system (12.Wu A.L. Kolumam G. Stawicki S. Chen Y. Li J. Zavala-Solorio J. Phamluong K. Feng B. Li L. Marsters S. et al.Amelioration of type 2 diabetes by antibody-mediated activation of fibroblast growth factor receptor 1.Sci. Transl. Med. 2011; 3: 113ra126Crossref PubMed Scopus (136) Google Scholar, 13.Foltz I.N. Hu S. King C. Wu X. Yang C. Wang W. Weiszmann J. Stevens J. Chen J.S. Nuanmanee N. et al.Treating diabetes and obesity with an FGF21-mimetic antibody activating the betaKlotho/FGFR1c receptor complex.Sci. Transl. Med. 2012; 4: 162ra153Crossref PubMed Scopus (165) Google Scholar, 14.Lan T. Morgan D.A. Rahmouni K. Sonoda J. Fu X. Burgess S.C. Holland W.L. Kliewer S.A. Mangelsdorf D.J. FGF19, FGF21, and an FGFR1/β-Klotho-activating antibody act on the nervous system to regulate body weight and glycemia.Cell Metab. 2017; 26: 709-718.e3Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In recent years, FGF19 has emerged as an attractive target for treating chronic liver diseases where bile acids play pivotal roles, and to this end, an engineered nontumorigenic analog of FGF19, NGM282 (also known as M70) (15.Zhou M. Wang X. Phung V. Lindhout D.A. Mondal K. Hsu J.Y. Yang H. Humphrey M. Ding X. Arora T. et al.Separating tumorigenicity from bile acid regulatory activity for endocrine hormone FGF19.Cancer Res. 2014; 74: 3306-3316Crossref PubMed Scopus (130) Google Scholar), is currently being evaluated as a potential treatment for patients with nonalcoholic steatohepatitis, primary sclerosing cholangitis, or primary biliary cholangitis (16.Harrison S.A. Rinella M.E. Abdelmalek M.F. Trotter J.F. Paredes A.H. Arnold H.L. Kugelmas M. Bashir M.R. Jaros M.J. Ling L. et al.NGM282 for treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.Lancet. 2018; 391: 1174-1185Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 17.Mayo M.J. Wigg A.J. Leggett B.A. Arnold H. Thompson A.J. Weltman M. Carey E.J. Muir A.J. Ling L. Rossi S.J. et al.NGM282 for treatment of patients with primary biliary cholangitis: a multicenter, randomized, double-blind, placebo-controlled trial.Hepatol. Commun. 2018; 2: 1037-1050Crossref PubMed Scopus (69) Google Scholar). In NGM282, the deletion of five amino acids (P24–S28) coupled with the substitution of three amino acids at critical positions (A30S, G31S, H33L) within the N terminus enables biased FGFR4 signaling so that NGM282 retains the ability to repress CYP7A1 expression, but is unable to activate signal transducer and activator of transcription 3 (STAT3) signaling to trigger hepatic tumorigenesis (15.Zhou M. Wang X. Phung V. Lindhout D.A. Mondal K. Hsu J.Y. Yang H. Humphrey M. Ding X. Arora T. et al.Separating tumorigenicity from bile acid regulatory activity for endocrine hormone FGF19.Cancer Res. 2014; 74: 3306-3316Crossref PubMed Scopus (130) Google Scholar, 18.Luo J. Ko B. Elliott M. Zhou M. Lindhout D.A. Phung V. To C. Learned R.M. Tian H. DePaoli A.M. et al.A nontumorigenic variant of FGF19 treats cholestatic liver diseases.Sci. Transl. Med. 2014; 6: 247ra100Crossref PubMed Scopus (124) Google Scholar). In this study, we describe a previously unrecognized role of FGF19 in selectively modulating hepatic LXR signaling to promote transhepatic cholesterol efflux and to increase HDL biogenesis, and that the effects of FGF19 on cholesterol metabolism include a complex interplay between multiple regulatory pathways not limited to the inhibition of bile acid synthesis. We also evaluated the effects of an FGF19 analog on aortic plaque and lesion formation in mouse models of atherosclerosis and on plasma cholesterol levels in healthy human volunteers. All animal experiments were approved by the Institutional Animal Care and Use Committee at NGM and conducted in compliance with ethical regulations. Mice were housed in a pathogen-free animal facility at 22°C under controlled 12 h light and12 h dark cycles. All mice were maintained in filter-topped cages on standard chow diet (Teklad 2918), or special diets when indicated, and autoclaved water ad libitum. Male mice were used unless otherwise specified. Sample sizes were determined on the basis of prior experience in the selected models to allow detection of statistically significant differences in metabolic parameters between groups. Mice were randomized into the treatment groups based on body weight and blood glucose. Studies were replicated in two to three independent cohorts of animals. All injections and tests were performed during the light cycle. Technicians treating the animals and measuring aortic lesions were blinded to the identity of the test articles. No animals were excluded from analysis at study completion. The db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J, #000642), Abca1 fl/flAbcg1 fl/fl mice (B6.Cg-Abca1tm1Jp Abcg1tm1Tall/J, #21067), Apoe−/− mice (B6.129P2-Apoetm1Unc/J, #002052), Ldlr−/− mice (B6.129S7-Ldlrtm1Her/J, #002207), and C57BL6/J mice (#000664) were obtained from Jackson Laboratory. Fgfr4−/− mice were kindly provided by Dr. Grace Guo (Rutgers University). The diabetic db/db mouse model was chosen because it allowed us to simultaneously study lipid and glucose regulations by FGF19. The db/db mice (10–12 weeks old) received a single 200 μl intravenous injection of 1 × 1011 vector genomes (vg) of adeno-associated virus (AAV)-FGF19, AAV-NGM282, or a control virus encoding green fluorescent protein via tail vein. Animals were euthanized and livers were collected 2 weeks after injection of the AAV vectors for gene expression analysis. For experiments investigating constitutively active MAPK/ERK kinase 1 (caMEK1) or constitutively active STAT3 (caSTAT3), mice were administered with AAV-caMEK1 (1 × 1011 vg), AAV-caSTAT3 (1 × 1011 vg), AAV-FGF19 (1 × 1011 vg), or a control virus, and blood was collected 4 weeks after AAV injection for measurements of serum levels of cholesterol, HDL-C, and LDL-C. For experiments investigating various inhibitors, db/db mice were administered with AAV-FGF19 (1 × 1011 vg) via tail vein. Three weeks later, mice were treated with rosuvastatin (0.005% in diet; BioServ), ezetimibe (0.01% in diet; BioServ), or anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralizing antibody (10 mg kg−1 ip qw) for an additional 4 weeks. Blood was collected for measurements of serum levels of cholesterol, HDL-C, and LDL-C. For studies in hepatocyte-specific Abca1g1-deficient mice, 10- to 14-week-old Abca1 fl/flAbcg1 fl/fl mice received a single intravenous dose of 1 × 1011 vg of AAV-NGM282 in combination with 3 × 1011 vg of AAV-thyroxine-binding globulin (TBG)-Cre recombinase or a control virus encoding green fluorescent protein through the tail vein. Abca1 fl/flAbcg1 fl/fl mice served as WT controls. AAV-TBG-Cre drives Cre recombinase expression under TBG promoter, which allows hepatocyte-specific expression. Four weeks after AAV administration, serum levels of cholesterol, HDL-C, and LDL-C were measured. For studies in Fgfr4-deficient mice, Fgfr4−/− mice were backcrossed to C57BL/6 mice for at least 10 generations, and age- and gender-matched WT C57BL6 mice were used as Fgfr4+/+ controls. AAV-FGF19 (3 × 1011 vg) or a control virus encoding green fluorescent protein was injected intravenously in a volume of 200 μl to 6- to 8-week-old female Fgfr4+/+ or Fgfr4−/− mice. Mice were euthanized 12 months post AAV administration for cholesterol, HDL-C, LDL-C, and body weight measurements. For liver tumor assessment, the maximum diameter of liver tumor nodules in each mouse was measured with a caliper and total numbers of tumor nodules per liver were recorded. Livers were weighed and collected for histological examination. For studies in Apoe-deficient mice, 12- to 14-week-old Apoe−/− mice received a single 200 μl intravenous injection of 1 × 1011 vg AAV-NGM282 or a control virus encoding green fluorescent protein via tail vein. Mice were placed on a high-fat high-cholesterol Western diet (Teklad TD88137) immediately following AAV injection, and this diet was continued ad libitum throughout the study. Mice were euthanized 18 weeks after AAV administration for en face and histology analysis. For studies in Ldlr-deficient mice, 10-week-old Ldlr−/− mice received a single 200 μl intravenous injection of 1 × 1011 vg AAV-NGM282 or a control virus encoding green fluorescent protein via tail vein. Mice were placed on a standard chow diet ad libitum throughout the study. Serum cholesterol, HDL-C, and LDL-C were analyzed 4 weeks after AAV administration. Human FGF19 cDNA (NM005117) was subcloned into pAAV-EF1α vector using SpeI and NotI sites with primers 5′-CCGACTAGTCACCATGCGGAGCGGGTGTGTGG-3′ (sense) and 5′-ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC-3′ (antisense). Construct for NGM282 was generated by Quick-Change site-directed mutagenesis kit (Agilent Technologies). cDNAs for TBG promoter and Cre recombinase (AF298789) were chemically synthesized (DNA2.1) and subcloned into promoterless pAAV vector. cDNAs for caMEK1 (with EE mutations in the activation loop) and caSTAT3 were chemically synthesized (DNA2.1) and subcloned into pAAV-EF1α vector. AAV293 cells (Agilent Technologies) were cultured in DMEM (Mediatech) supplemented with 10% FBS and 1× antibiotic antimycotic solution (Mediatech). Cells were cultured in a humidified incubator with 5% CO2 and 95% air at 37°C, confirmed to be mycoplasma free, and authenticated by short tandem repeat DNA profiling. The cells were transfected with three plasmids [AAV transgene, pHelper (Agilent Technologies), and AAV2/9] for viral production. Viral particles were purified using a discontinuous iodixanol (Sigma) gradient and resuspended in PBS with 10% glycerol and stored at −80°C. Viral titer or vector genome number was determined by quantitative PCR using custom TaqMan assays specific for woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequences. Standard curves for WPRE were obtained from serial dilutions over a 6 log range of the corresponding plasmids. AAV-mediated gene delivery provides a means to achieve long-lasting transgene expression without the inflammatory responses that are commonly associated with other viral vectors. When introduced into adult mice, sustained expression of up to 1 year has been observed. The major site of transgene expression is the hepatocytes. RNA was isolated from the livers of db/db mice 2 weeks after administration of AAV-FGF19 or a control virus and treated with DNase I (Thermo Fisher Scientific). RNA integrity and purity were confirmed by Bioanalyzer (Agilent Technologies) with RIN numbers >8.0. The raw expression data from Affymetrix mouse gene 1.0 ST whole-transcript arrays (Thermo Fisher) were normalized using the robust multi-array average method. The metadata and matrix tables have been deposited to the Gene Expression Omnibus (GEO) repository (accession number GSE117855). Ingenuity Pathway Analysis (IPA) (Qiagen), including canonical pathways, upstream analysis, diseases, and functions, was conducted on genes differentially represented in FGF19-treated versus control livers. The top canonical pathways were ranked by −Log (P value) with a threshold P value of 0.05. The highest ranking categories were sorted in a decreasing order of significance. The Gene Tissue Expression (GTEx) project collected tissue samples from 554 human donors and carried out RNA-seq and other genomic profiling on these tissue samples. All GTEx datasets used in the analyses described here are available through the GTEx portal (http://gtexportal.org). The donor samples with gene expression data are summarized in supplemental Table S3. Cardiovascular disease-related datasets (supplemental Table S4) were extracted from OmicSoft DiseaseLand database (Qiagen), which contains datasets retrieved from a variety of public projects including GEO, Sequence Read Archive, ArrayExpress, and the Database of Genotypes and Phenotypes. Bioinformatics analysis, including gene expression correlation and disease versus normal comparison related to cardiovascular diseases, was conducted using ArrayStudio software version 10.0 from OmicSoft (Qiagen). Mouse livers or ileum were snap-frozen in liquid nitrogen upon euthanization of animals. Total RNA was extracted using RNeasy Mini kit (Qiagen) and treated with DNase I (Thermo Fisher Scientific). Quantitative PCR with reverse transcription (qPCR) assays were performed using QuantiTect multiplex qRT-PCR master mix (Qiagen) and premade TaqMan gene expression assays (Life Technologies). Samples were loaded into an optical 384-well plate and qPCR was performed in duplicates on QuantStudio 7 Flex real-time PCR system (Applied Biosystems). After an initial hold at 50°C for 30 min to allow reverse transcription to complete, HotStart Taq DNA polymerase was activated at 95°C for 15 min. Forty cycles of a three-step PCR (94°C for 45 s, 56°C for 45 s, and 76°C for 45 s) were applied, and the fluorescence intensity was measured at each change of temperature to monitor amplification. Target gene expression was determined using the comparative threshold cycle (ΔΔCt) method and normalized to the expression of the housekeeping gene, GAPDH. Blood was collected from the tail vein in unanesthetized animals using Microvette serum gel tubes (Sarstedt) for measurements of total cholesterol, HDL-C, LDL-C, aspartate aminotransferase (AST), and FGF19 concentrations. Serum samples were prepared by centrifugation at 4°C for 10 min at 2,000 g after clotting at room temperature for 30 min. Concentrations of total cholesterol, HDL-C, and LDL-C were measured by enzymatic methods using COBAS INTEGRA enzyme kit and an automated analyzer (COBAS INTEGRA 400 Plus clinical analyzer; Roche Diagnostics). In these enzymatic methods, esterified cholesterol is converted to free cholesterol by cholesterol esterase. The resulting cholesterol is then oxidized by cholesterol oxidase to produce Δ4-cholestenone and hydrogen peroxide. The hydrogen peroxide then reacts with 4-aminoantipyrine in the presence of peroxidase to produce a colored product that is measured at 583 nm. Direct determination of HDL-C is achieved through the combination of polyethylene glycol-modified enzymes, α-cyclodextrin sulfate, and magnesium chloride, which provides selectivity for HDL-C. The results of this method correlate with those obtained by precipitation-based or ultracentrifugation methods (19.Kimberly M.M. Leary E.T. Cole T.G. Waymack P.P. Selection, validation, standardization, and performance of a designated comparison method for HDL-cholesterol for use in the cholesterol reference method laboratory network.Clin. Chem. 1999; 45: 1803-1812PubMed Google Scholar). Direct determination of LDL-C is achieved through the selective micellar solubilization of LDL-C by a nonionic detergent and the interaction of a sugar compound and lipoproteins. The combination of a sugar compound with detergent enables the selective determination of LDL-C in serum (Roche Diagnostics). These reagents are compatible for quantification of total cholesterol, HDL-C, and LDL-C levels in samples of human, mouse, or rat origin. For fractionation of serum lipoproteins, pooled mouse serum samples (50 μl) were injected on two Superose 6 HR 10/30 columns connected in series on an AKTA Explorer fast protein LC system (GE Healthcare). Lipoproteins were eluted at a constant 0.3 ml min−1 flow rate with PBS (pH 7.4) containing 0.02% EDTA. Individual fractions were collected for total cholesterol measurements. FGF19 and NGM282 concentrations were determined by FGF19 ELISA (Biovendor; RD191107200R). All assays were performed according to the manufacturers' instructions. Blood concentrations of ad libitum-fed glucose were measured in conscious animals from a hand-held glucometer (Accu-check; Roche Diagnostics) using tail vein blood. Frozen liver samples (∼100 mg each) were homogenized in chloroform/methanol (2:1, v/v) using the Folch method. Aliquots of extracts were washed with isotonic saline, and the chloroform layers were dried under nitrogen gas. The lipids were reconstituted in isopropanol, and concentrations of total triglyceride and cholesterol were measured using Infinity liquid stable reagents (Thermo Fisher). Values were expressed as milligrams of triglyceride or cholesterol per gram wet weight of liver (mg g−1). To determine levels of free cholesterol and hydroxycholesterol in the liver, frozen liver samples in glass tubes combined with internal standard were extracted with chloroform/methanol (2:1, v/v) (Creative Dynamics). Phospholipids were removed by SPE columns (Waters). The chromatographic system was a Michrom Paradigm HPLC equipped with a C18AQ analytical column (2.0 × 150 mm, 4 μm), an autosampler, a helium degassing system, and a column oven. Column eluent was introduced to a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher) with a heated electrospray ionization source. Retention times for each sterol were compared with reference standards (Steroloids Inc.). Mouse livers or hearts were fixed in 10% neutral-buffered formalin and embedded in paraffin. Five micron sections were deparaffinized in xylenes (5 min) and rehydrated sequentially in graded ethanol (100, 95, 80, 70, and 50%; 2 min each) and PBS (2 min). Stains with H&E, hematoxylin-phloxine-saffron (HPS), Masson's trichrome, or von Kossa were performed using standard methods. For osmium tetroxide staining of lipids in the liver, frozen livers were embedded in optimal cutting temperature compound and directly processed for staining. For immunohistochemistry, specimens were subjected to antigen retrieval in a citrate-based antigen unmasking solution (Vector Laboratories; #H-3300) and incubated for 30 min with 3% H2O2 at room temperature to block endogenous peroxidase activity. Sections were blocked in PBST (PBS + 0.1% Tween-20) containing 10% goat serum and stained with primary antibodies against glutamine synthetase (Abcam), CD68, or smooth muscle actin diluted in blocking solution at 4°C overnight. Specimens were then washed three times for 5 min each in PBST and incubated with biotinylated secondary antibodies in blocking solution for 1 h at room temperature. ImmPRESS-alkaline phosphatase reagent (Vector Laboratories) and ImmPACT Vector Red substrate or ABC reagent with 3,3-diaminobenzidine substrate were used for detection. Digital imaging microscopy was performed using a Leica DM4000 microscope equipped with a DFC500 camera and a high-precision motorized scanning platform (Leica). Images for the entire liver section were acquired by Turboscan and real-time imaging stitching at camera frame rates using the Surveyor program. For morphometric analysis of tumor area, glutamine synthetase-positive tumor areas were quantified using the measure/count/area tool from ImagePro software. Apoe−/− mice on a high-fat high-cholesterol Western diet, an established model for atherosclerosis with features of aortic plaque and lesion formation, were used for the evaluation of the effects of NGM282 on atherosclerosis development. At 18 weeks on Western diet, mice were euthanized and atherosclerosis development in aortas and aortic roots was measured. En face analysis was conducted at Wake Forest School of Medicine Metabolic Core (Winston-Salem, NC). Briefly, the aortas were cleaned of any adventitial and fat tissue, cut open longitudinally, and pinned flat on a black wax surface. Aorta images were captured through a stereomicroscope (Leica S8 APO) with a digital camera (Leica MC-120). Lesion area was quantified using ImageJ software according to published protocols and expressed as percent stained area relative to total aortic area. All quantifications were carried out by an observer blinded to the sample identity. No animals were excluded from atherosclerotic lesion analysis. Aortic contents of total cholesterol, free cholesterol, and cholesterol ester were quantified at Wake Forest School of Medicine Metabolic Core Facility. Hearts from Apoe−/− mice treated with NGM282 or control mice were isolated, fixed in formalin, and embedded in paraffin. Mouse hearts were sectioned perpendicular to the axis of the aorta, and once the aortic root was identified by the appearance of aortic valve leaflets and smooth muscle cells, cross-sections (5 μm thick) were taken and mounted on AAS-coated slides. The sections were stained with HPS for necrotic area and Masson's trichome for collagen content. The images were recorded using Aperio software. A phase 1, first-in-human, randomized, placebo-controlled, double-blind clinical trial was performed to assess the safety, tolerability, and pharmacodynamics of NGM282 administered subcutaneously to healthy volunteers. The study was conducted in accordance with the Declaration of Helsinki and with Good Clinical Practice guidelines. The Human Research Ethics Committee approved the study protocol. Written informed consent was obtained from all subjects prior to participation. Treatment allocation and concealment were conducted by a computerized random-number generator and numbered containers with active and placebo syringes of identical appearance. The randomization list was centrally held by an independent contract research organization. All randomized subjects, the sponsor, the study center, and contract research organization personnel were blinded to treatment allocation until data were locked and analyzed. After a screening period, participants were randomized to receive 3 mg NGM282 protein (n = 9) or placebo (n = 17) subcutaneous treatment once daily for 7 days. The sample size chosen was based on precedents set by other first-in-human studies o
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