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Ex Vivo Cell-Based Screening Platform for Modulators of Hepatosteatosis Shan Yu, Emily Chen, Lance Sherwood, Mitchell Hull, Ashley K. Woods, and Matthew S Tremblay ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 7, 2017
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Ex Vivo Cell-Based Screening Platform for Modulators of Hepatosteatosis Shan Yu, Emily Chen, Lance Sherwood, Mitchell Hull, Ashley K. Woods, Matthew S. Tremblay* California Institute for Biomedical Research, La Jolla, CA, 92037
* Corresponding author: Matthew S. Tremblay California Institute for Biomedical Research 11119 N. Torrey Pines Rd, Suite 100 La Jolla, CA, 92037
[email protected] Tel: (858)-242-1005 Fax: (858)-242-1001
Abstract: Non-alcoholic fatty liver disease (NAFLD) is the result of the ectopic accumulation of lipids in hepatic cells, and is the early stage of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. While some mechanisms of aberrant lipid storage are understood, unbiased phenotypic drug screening holds potential to identify new therapeutic small molecules mechanisms that reverse lipid accumulation in hepatic cells and prevent disease progression. Immortalized hepatocyte cell lines are often used as in vitro models of hepatocyte function, including to study lipid accumulation. However, mechanisms and therapeutic agents studied in these systems suffer from poor translation to primary cells and animal models of disease. Herein, we report an ex vivo high-throughput screening platform using primary mouse hepatocytes with a physiologically relevant lipid-laden phenotype isolated from mice that are administered a choline-methionine deficient diet. This screening platform using primary diseased hepatocytes may help to overcome a major hurdle in liver disease drug discovery and could lead to the development of new therapeutics for hepatosteatosis.
Introduction: NAFLD is one of the common metabolic diseases associated with obesity and diabetes. Up to 30% of the adult population and up to 80% of individuals with obesity and diabetes are currently 1 ACS Paragon Plus Environment
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affected by this chronic liver condition 1. The disease results from interactions between inherited genetic factors and environmental exposure, most notably a high-calorie diet rich in lipids. The hepatosteatosis that underlies NAFLD, if left untreated, may develop into non-alcoholic steatohepatitis (NASH), and related pathologies of fibrosis, cirrhosis and hepatocellular carcinoma. Combined, these pathologies represent the second leading cause of liver disease among adults awaiting liver transplantation in the United States and is projected to become the most common indication for liver transplantation in the next decade 2. The pathogenesis of non-alcoholic NAFLD is thought to be a result of multiple factors, including insulin resistance, lipotoxicity, oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, adipose tissue dysfunction and immune system dysfunction 3. Therefore, mediators of these events are potential therapeutic targets. However, no approved pharmacological treatments for NAFLD or NASH are currently available 4, pointing to an urgent need to develop effective therapeutic strategies for this spectrum of diseases. The current standard of care consists of lifestyle change aimed at decreasing insulin resistance, hyperlipidemia and body weight. Medications that impact these phenotypes are often also recommended, and many are undergoing clinical investigation for their putative secondary impact in ameliorating NAFLD and NASH. Candidate therapies that act in a primary fashion on NAFLD or NASH, typically through reducing de novo lipid synthesis, inflammation, apoptosis or fibrosis, are also being tested in the clinic. However, no drug candidates have been developed to specifically target the reduction of hepatic triglyceride levels 3. Herein, we report a novel screening platform with primary mouse lipid-laden hepatocytes isolated from choline-methionine deficient diet fed mice. Since these hepatocytes became lipidladen in vivo and are freshly isolated before testing, active compounds identified by this method are likely to be physiologically relevant.
Materials and methods: Animal studies. All procedures were carried out in accordance with protocols approved by the California Institute for Biomedical Research (Calibr) Animal Care and Use Committee. 8 to 10 week-old C57BL/6 mice were purchased from the Jackson Laboratory and housed in Calibr’s animal facility. For induction of fatty liver disease, mice were given a choline-methionine deficient diet (MP biomedical, Cat No. 02960439) for 2 to 5 weeks, or a high fat diet (Research Diet, Cat No. D12492) for 20 weeks. Isolation of primary mouse hepatocytes. C57Bl6 wild type mice were given an intraperitoneal injection of 10 mg/mL Inactin hydrate (Sigma) at a volume of 10 mL/kg to induce anesthesia. The liver was perfused using 50 mL pre-warmed Liver Perfusion Medium (Gibco) followed by 50 mL pre-warmed Liver Digest Medium (Gibco) at a rate of 5 mL/min with a butterfly needle inserted into the portal vein. An incision in inferior vena cava was made to allow blood and buffer to flow out of circulation. Liver was carefully removed and placed in the ice cold sterile transport medium consisting of Hepatozyme (Gibco) with 1% BSA. The liver cells were gently scraped off from the liver and filtered through a 40 micron nylon cell strainer. The cells were 2 ACS Paragon Plus Environment
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then centrifuged twice at 800Xg for 3 mins at room temperature to pellet hepatocytes. The cell pellets were then washed with Hepatocyte Wash Medium (Gibco) twice, and re-suspended in complete hepatocyte culture medium, consisting of HepatoZYME-SFM (Gibco), 10% FBS and 10 mM penicillin-streptomycin and 10 mM L-glutamine. Cell were then plated in tissue culture plates with the number of cells as indicated in the specific experiments. Immunocytochemistry. Hepatocytes (104 cells per well) were seeded in collagen-coated 384well black clear bottom plate. Cells were stained with anti-Albumin (Abcam, Cat No. ab8940) primary antibody followed by FITC conjugated donkey anti-sheep secondary antibody, and the nuclei was stained with DAPI. The cells were then imaged with a Zeiss Axio Observer microscope. Triglyceride colorimetry assay. Hepatocytes (104 cells per well) were seeded in collagencoated 384-well black clear bottom plate with compounds pre-spotted in the wells. The compound solvent DMSO final concentration was 0.1%. After 48-hour incubation, cell culture supernatant was flicked off, and the rest of the medium was pat dry on an absorbent paper. 15 uL lysis buffer (DPBS supplemented with 0.1% Triton-X 100 and 250 mM sucrose) was added into each well, and the plate was placed on an orbital plate shaker and shaken for 1 hour at room temperature. Triglyceride enzyme mixture (Cayman chemical, Cat No. 10010511) and triglyceride standards (Cayman chemical, Cat No. 10010509) were prepared according to manufacturer’s recommendation. 10 uL of triglyceride enzyme mixture per well was added into the sample wells and standard wells. The reaction was carried out while shaking the plate on an orbital plate shaker for 15 minutes. The absorbance at 530 – 550 nm was then read using an Envision plate reader. HCS LipidTOX TM neutral lipid stain assay. Hepatocytes (5000 cells per well) were plated in 384 well plate. The cells were treated with compounds at 1 uM concentration for different time points as indicated, followed by fixing with 3% formaldehyde and staining with HCS LipidTOX TM neutral lipid stain (Invitrogen, Cat No. H34475) according to the manufacturer’s protocol. The lipid content was quantified and analyzed by Cellomics high-content imaging system and software. Oil Red O staining. Hepatocytes (105 cells per well) were plated in collagen-coated 24 well plate and cultured overnight, and the floating cells were then washed off. The cells were treated with compounds for 48 hours before Oil Red O staining (Abcam, Cat No. ab150678) according to the manufacturer’s instruction. The cells were then imaged with Zeiss Axio Observer microscope. RT-qPCR assay. Cells were lysed with 400 µL RLT buffer provided in the RNeasy Mini kit (Qiagen), followed by adding 400 µL of 70% ethanol and purifying RNA as instructed by the user’s manual. RNA was reverse transcribed into cDNA using qScript cDNA synthesis kit (Quanta). qPCR was performed with PerfeCTa qPCR ToughMix (Quanta) on 50 ng cDNA template/reaction using Taqman probes (Life Technologies). Lipase activity assay. Hepatocytes (106 cells per well) were plated in collagen-coated 96-well plate and were allowed for overnight adherence. Cells were treated with compounds at indicated 3 ACS Paragon Plus Environment
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concentrations for 48 hours before cells were collected in ice-cold lipase assay buffer provide in the Lipase Activity Assay Kit (Sigma-aldrich, Cat No. MAK046). Free fatty acid assay. Hepatocytes (106 cells per well) were plated in collagen-coated 96-well plate and were allowed for overnight adherence. Cells were treated with compounds at indicated concentration for 48 hours followed by analysis of free fatty acid level according to the instructions of Free Fatty Acid Quantification Kit (Sigma-aldrich, Cat No. MAK044). β-oxidation assay. Hepatocytes (0.5 X 106 cells per well) were plated in collagen-coated 96-well plate and were allowed for overnight adherence. Cells were treated with compounds at indicated concentrations for 48 hours before cells were homogenized in FAD assay buffer according to the instructions of FAD Assay Kit (Sigma-aldrich, Cat No. MAK035). Cytotoxicity assay. After cells were incubated for certain time period as indicated for each experiment, CellTiter-Glo Luminescent cell viability reagent (Promega) was added at a ratio of 1:5 in volume to cell culture directly. The reagent was mixed with cell culture on a plate shaker. The signal was read on a PerkinElmer EnVision Multilabel Reader after 10 minutes incubation. Statistics and calculations. Statistics analysis was conducted using T-test or one-way ANOVA with Tukey’s post hoc test on Prism 5 software with significance of * p
