Hepatic FTO expression is increased in NASH and its silencing attenuates palmitic acid-induced lipotoxicity

Accepted Manuscript Hepatic FTO expression is increased in NASH and its silencing attenuates palmitic acid-induced lipotoxicity Andrea Lim, Jin Zhou, Rohit A. Sinha, Brijesh K. Singh, Sujoy Ghosh, Kiat-Hon Lim, Pierce Kah-Hoe Chow, Esther C.Y. Woon, Paul M. Yen PII:

S0006-291X(16)31546-7

DOI:

10.1016/j.bbrc.2016.09.086

Reference:

YBBRC 36465

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 1 September 2016 Accepted Date: 16 September 2016

Please cite this article as: A. Lim, J. Zhou, R.A. Sinha, B.K. Singh, S. Ghosh, K.-H. Lim, P. KahHoe Chow, E.C.Y. Woon, P.M. Yen, Hepatic FTO expression is increased in NASH and its silencing attenuates palmitic acid-induced lipotoxicity, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.09.086. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Hepatic FTO expression is increased in NASH and its silencing attenuates palmitic acidinduced lipotoxicity Andrea LIM*1,2, Jin Zhou*1, Rohit A. Sinha1, Brijesh K.Singh1, Sujoy Ghosh1, Kiat-Hon Lim3,

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Pierce Kah-Hoe Chow4,5, Esther C. Y. Woon2, Paul M. Yen1.

Program of Cardiovascular and Metabolic Disorders, Duke-NUS Medical School Singapore

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8 College Road, Singapore 169016 Deparment of Pharmacy, National University of Singapore

18 Science Drive 4, 117559

Department of Pathology, Singapore General Hospital, Singapore, Singapore,

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Professor, Clinical Science, Duke-NUS Graduate Medical School Singapore

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Senior Consultant Surgeon, Division of Surgical Oncology, National Cancer Centre Singapore

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* “These authors contributed equally to this work”

Authors:

Paul

M.

Yen

([email protected])

and

Jin

Zhou

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Corresponding

([email protected]), Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, 8 College Road, Level 8D, Singapore 169587 SINGAPORE

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Conflict of Interest: The authors declare no conflict of interest.

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Abstract Non-alcoholic steatohepatitis (NASH) is one of the most common causes of liver failure worldwide. It is characterized by excess fat accumulation, inflammation, and increased lipotoxicity in hepatocytes. Currently, there are limited treatment options for NASH due to lack

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of understanding of its molecular etiology. In the present study, we demonstrate that the expression of fat mass and obesity associated gene (FTO) is significantly increased in the livers of NASH patients and in a rodent model of NASH. Furthermore, using human hepatic cells, we show that genetic silencing of FTO protects against palmitate-induced oxidative stress,

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mitochondrial dysfunction, ER stress, and apoptosis in vitro. Taken together, our results show that FTO may have a deleterious role in hepatic cells during lipotoxic conditions, and strongly

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suggest that up-regulation of FTO may contribute to the increased liver damage in NASH.

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Research Highlights: • FTO expression is increased in NASH patients and rodent models of NASH. • Silencing of FTO protects against PA-induced ER-stress and apoptosis. • Silencing of FTO attenuates palmitate-induced mitochondrial dysfunction. Keywords: Non-alcoholic steatohepatitis (NASH), Fat and obesity associated (FTO) gene,

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Obesity, Lipotoxicity.

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Introduction

Genome-wide association studies (GWAS) have identified variants of the FTO gene that appear to be correlated with obesity in humans [1]. In animal models, transgenic overexpression of

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FTO leads to obesity [2] whereas loss of function mutations of FTO decreased body weight [3]. Furthermore, FTO is increased in muscle of type 2 diabetes patients, and its overexpression in myotubes alters insulin signaling, and induces mitochondrial dysfunction [4], suggesting that FTO may have an etiological role in metabolic disorders. FTO belongs to the AlkB family of

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enzymes and is an iron and 2-oxoglutarate-dependent dioxygenase that specifically demethylates N6-methyladenosine (m6A) in mRNA [5-7]. Recently, selective FTO inhibitors have been

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developed that block its enzymatic activity [8], and thus serve as potential treatments for metabolic disorders that have dysregulation of FTO.

Non-alcoholic fatty liver disease (NAFLD) is a common co-morbidity associated with obesity, and is represented by a clinical spectrum that includes hepatosteatosis, non-alcoholic steatohepatitis (NASH), and cirrhosis. The severity of NASH is characterized by histological

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criteria such as hepatocyte ballooning, inflammation, focal fibrosis, and steatosis [9]. Advanced NASH is a serious of liver condition since the hepatocyte apoptosis and ensuing fibrosis usually are not reversible. The prevalence of NASH varies depending on the population and geographical location, but it is estimated to occur in more than 15% of obese individuals with

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NASH.

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other features of the metabolic syndrome [9]. Currently, there is no FDA-approved drug for

Lipotoxicity, from saturated free fatty acids such as palmitic acid (PA), is a major pathological feature of NASH. It is caused by the accumulation of toxic lipid intermediates in the liver that lead to cellular dysfunction and death [9]. At a cellular level, PA induces reactive oxygen species (ROS) that cause lipid peroxidation and stress in mitochondria and endoplasmic reticulum (ER). PA also serves as a precursor for the generation of ceramides that can alter the membrane compositions of organelles to induce intracellular stress pathways [10], leading to mitochondrial dysfunction, activation of the caspase cascade, and cell death [11].

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In this study, we have demonstrated that the hepatic FTO levels are significantly increased in both human NASH patients and a diet-induced rodent model of NASH. Furthermore, we demonstrated that depletion of FTO decreased PA-induced lipotoxicity in hepatic cells by

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restoring mitochondrial function and reducing ER stress.

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Materials and Methods

Microarray analysis For analysis of FTO expression in human NASH and steatosis a publically available database

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was used [12] . The data set is accessible at the ArrayExpress public repository for microarray data under the accession number E-MEXP-3291 (http://www.webcitation.org/5zyojNu7T). The distribution of FTO gene expression in Control, NASH and steatotic samples was ascertained via boxplots, and the statistical significance of expression differences across the three groups was

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determined via ANOVA. All calculations were performed in the statistical package, R 3.2.3.

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Immunohistochemistry

FTO protein expression in the liver specimens was examined by immunohistochemistry (IHC). Samples used in the IHC were archived tissues. All patients gave written informed consent, and all tissue samples were collected with approvals from respective ethics committees. Standard IHC techniques were used to stain the FFPE histological samples using the Leica Bond III Automated Stainers. Staining for FTO was noted to be nuclear and results were scored using

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0,1-3 + intensity with 3+ being strongest and 1+ the weakest, a percentage of cells for each sample was also given with a range from 0-100%.

Animal Model

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C57BL/6J mice (6 to 8 weeks old) were fed on either normal chow or methionine/cholinedeficient (MCD) diet for 6 weeks. The study was carried out in strict accordance to the standards

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as described in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animal and approved by National University of Singapore Institutional Animal Care and Use Committee (NUS IACUC) at Duke-NUS Medical School.

Cell culture

Human hepatocellular carcinoma cell line, HepG2 (HB-8065) were cultured at 37oC in Dulbecco’s modified Eagle’s medium, DMEM (Life Technologies) containing 4.5 g/L D-glucose, L-glutamine and 10% fetal bovine serum (FBS) (Sigma-Aldrich), in a humidified incubator containing 5% CO2 environment. 5

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siRNA transfection was performed using Lipofectamine RNAiMAX (Life Technologies) according to manufacturer’s instructions. Forty-eight hours after transfection, cells were treated

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overnight with or without BSA-conjugated PA (0.75mM).

Cell viability assay

Cell number was evaluated by the crystal violet staining as described previously [13]. Briefly, cells were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde and

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stained with 0.1% crystal violet in 2% methanol. Dye was extracted with 10% acetic acid (Merck

Western blotting

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Millipore) and absorbance was measured at 595 nm with a microplate reader (Tecan).

Western blotting was done as described previously [14] . Cells or tissue samples were lyzed using mammalian lysis buffer (Sigma-Aldrich), and immunoblotting was performed as per the manufacturer’s guidelines (Bio-Rad, Hercules, CA). Densitometry analysis was performed using

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ImageJ software (NIH, Bethesda, MD).

Cellular oxygen consumption rate (OCR) analysis Oxygen consumption was measured at 37 degree using an XF24 extracellular analyzer (Seahorse Bioscience Inc., North Billerica, MA). HepG2 cells (40,000) were seeded in 24-well plates and

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transfected with either negative or FTO siRNA. Forty-eight hours after transfection, palmitic acid (0.75 mM) was added overnight. Mitochondrial metabolic parameters were assessed using

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Mitostress test kit (Seahorse Biosciences, Cat. 103015-100) according to manufacturer’s instructions. Every point represents an average from five different wells.

Protein Oxidation Detection Protein carbonyl formation was measured using an Oxyblot system (Millipore, S7150) according to the manufacturer’s instructions [15]. In brief, the protein lysate was denatured with 12% SDS, and the carbonyl groups in the protein side chains were subsequently derivatized to 2,4dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH).

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The DNP-derivatized protein samples were separated by polyacrylamide gel electrophoresis followed by Western blotting using an anti-DNP primary antibody provided in the kit.

Measurement of cellular ceramide

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To quantify levels of ceramide, cells were washed with PBS and trypsinized. The cell pellet was collected and re-suspended homogeneously in 100 µL cold PBS. 10 µL was used for protein measurement, and lipids were extracted using ice-cold chloroform/methanol (1:2). The organic phase was separated with chloroform and Milli-Q water. The organic phase was collected into a

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fresh tube, dried with a gentle nitrogen stream, and stored at -80 degree freezer for liquid

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chromatography/mass spectroscopy analysis as described previously [14].

Statistical analysis

Cell cultures were performed in triplicates. All results were expressed in terms of mean ± standard deviation (SD) values, unless otherwise indicated. Differences between groups were

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compared using ANOVA analysis; p < 0.05 was considered statistically significant.

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Results and Discussion

FTO expression is significantly increased in livers from NASH patients and a rodent model of NASH

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Hepatic FTO expression was analyzed in a cohort of Caucasian patients with hepatosteatosis or NASH that were compared to healthy subjects. Hepatic FTO mRNA expression was increased in patients with hepatosteatosis and NASH compared to healthy subjects. However, a significant increase in FTO mRNA was observed in NASH patients when compared to healthy subjects

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(Figure 1A). In order to validate FTO expression in NASH at the protein level, we performed IHC staining using liver biopsies obtained from healthy subjects and NASH patients from

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Singapore General Hospital. As shown in Figure 1B and Supplementary Table 1, hepatic nuclear FTO staining was increased in NASH patients and associated with increased fat accumulation and NAFLD Activity Score (NAS) score [16]. The latter is based upon current histopathology guidelines [17] that characterize and grade hallmarks of NASH such as inflammation, ballooning and degree of steatosis), and glycogenated nuclear staining.

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We then examined FTO protein expression in an experimental rodent NASH model in which mice were fed a methionine/choline-deficient (MCD) diet. Unlike fatty liver models of benign steatosis that are induced by high fat diet (HFD), this animal model exhibits several phenotypic characteristics of human NASH, including large areas of mixed inflammatory cell infiltration,

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hepatocyte necrosis, and discrete perivenular and pericellular fibrosis [18]. We found that MCD diet induced liver damage, as evidenced by increased expression of the apoptotic marker-cleaved

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caspase 3, and ER stress marker-CHOP, respectively (Figure 2A). Interestingly, FTO protein level also was significantly increased in mice fed MCD diet, suggesting that hepatic FTO may have a significant role in NASH progression. Several previous studies have suggested that hepatic expression of FTO in rodents is sensitive to nutritional status since FTO mRNA level is reduced by fasting [6] and increased by HFD [19, 20]. However, unlike the dietary models of fatty liver disease such as HFD feeding, the MCD mice did not gain weight and/or increase dietary fat intake. The development of fatty liver and steatosis in this model is due to the inability of the liver to secrete VLDL which leads to triglyceride accumulation and inflammation. Of note, the histological characteristics of liver damage resemble human NASH. Based on these results, it 8

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is possible that the increase in FTO expression in NASH may not be driven by fatty acid accumulation in hepatocytes per se but may be induced by inflammation and/or its sequelae. This notion is further supported by our in vitro experiments in which FTO expression was not up-regulated by PA treatment in hepatic cell culture (Figure 3). These results notwithstanding,

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the precise cause of FTO up-regulation in humans and the rodent model of NASH still needs to be determined.

Genetic ablation of FTO protects against hepatic cell apoptosis by PA

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To understand the biological effect of FTO activity in influencing hepatocyte survival in NASH, we next examined a cell culture-based model of hepatic lipotoxicity. To induce lipotoxicity in

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hepatocytes, PA was added to cultured HepG2 human hepatoma cells. Similar to Figure 2, FTO knockdown cells had higher viability than control cells after PA treatment (Figure 3A). PA induced the expression of the apoptotic and ER stress markers, cleaved caspase 3 and CHOP respectively, in control cells and these effects were significantly attenuated in FTO knockdown cells (Figure 3B). These effects were confirmed using another individual siRNA targeting human FTO (Supplementary Figure 1). Our finding that FTO depletion in hepatic cells in vitro

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protected against PA-induced ER stress demonstrated that FTO had a direct and cell-autonomous role in mediating lipotoxicity in hepatic cells.

PA induction of ER stress and apoptosis after mitochondrial oxidative stress [23] plays an

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important role in the pathogenesis and progression of NASH [24]. Therefore, we next measured reactive oxygen species (ROS) production and mitochondrial function to better understand the

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protective mechanism(s) mediated by FTO knockdown. Using immunoblot-based detection of carbonyl groups introduced into proteins by oxidative reactions, we found that PA treatment significantly increased intracellular ROS, and knockdown of FTO decreased PA-induced oxidative stress (Figure 4A). We next tested mitochondrial function by measuring cellular oxygen consumption rate to further explore the metabolic consequences of FTO signaling in a lipotoxic environment. Basal oxygen consumption, ATP turnover, maximal respiration and spare respiratory capacity all were impaired by PA treatment in control hepatic cells, but they were restored in FTO knockdown cells treated with PA (Figure 4B). Thus, FTO knockdown protected against ROS production and mitochondrial dysfunction caused by PA. Our results indicate that 9

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the increased FTO expression is likely to be accounted for the increased ROS and mitochondrial dysfunction that is commonly found in NASH.

Generation of lipotoxic lipid species such as ceramides from PA has been associated with

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mitochondrial dysfunction [22, 27] and decreased mitochondrial oxygen consumption in hepatocytes [27]. Therefore, we next measured cellular ceramide content by metabolomics. C-16 ceramide, the most abundant and toxic ceramide, was significantly increased after PA treatment, whereas this induction was attenuated after FTO knockdown (Figure 4C). Altered ceramide

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content in the mitochondrial membrane perturbs the membrane structure and inhibits Complex I and III of the electron transport chain [28, 29]. Thus, attenuation of PA-induced C-16 ceramide

from PA-induced lipotoxicity.

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formation may be a novel mechanism by which decreased FTO led to mitochondrial protection

In conclusion, our study shows that hepatic FTO mRNA and protein levels are increased in rodent and human NASH, and its silencing reduces PA-induced lipotoxicity in human hepatic

Acknowledgement

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cells.

This work was supported by NMRC/CSA/ 0054/2013 (PMY), NMRC/CIRG/1340/2012 (PMY), and from Singapore Ministry of Health NMRC/BNIG/2025/2014 (RAS). The authors would like

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to thank Dr Benjamin L. Farah from Duke-NUS Medical School for his valuable advice in the

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Figure legends

Figure 1. FTO expression is significantly increased in liver of NASH patients. (A) FTO mRNA level is significantly increased in the liver of NASH patient. (B) FTO protein level is increased in NASH patient. Representative image of immunohistochemistry staining of FTO in liver biopsy

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from NASH patient or healthy subject.

Figure 2. FTO protein level is significantly increased in liver of rodent model of NASH. (A)

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Immunoblots and (B) densitometric analysis of FTO, cleaved caspase 3 and CHOP in mouse livers harvested from mice on normal chow or methionine/choline-deficient (MCD) diets for 6

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weeks.

Figure 3. Knockdown of FTO protects against PA-induced ER-stress and apoptosis in HepG2 cells. HepG2 cells were transfected with negative control or FTO siRNA. 48 hours after transfection, cells were treated with or without BSA-conjugated PA (0.75mM) overnight. (A) Knockdown of FTO decreased PA-induced apoptosis. Cell viability was measured by crystal violet staining. (B) Knockdown of FTO decreased PA-induced cleaved caspase 3, and CHOP.

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Figure 4. Knockdown of FTO protects against PA-induced mitochondrial dysfunction. HepG2 cells were transfected with negative control or FTO siRNA. 48 hours after transfection, cells were treated with or without BSA-conjugated PA (0.75mM) overnight. (A) Oxyblot analysis showing that knockdown of FTO decreased PA-induced oxidative stress. (B)Knockdown of FTO

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partially restored mitochondrial metabolic parameters upon PA treatment. Cellular oxygen consumption rate (OCR) was measured using Mitostress test kit. (C) Metabolomics analysis

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showing that knockdown of FTO decreased C-16 ceramide upon PA treatment.

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Research Highlights: FTO expression is increased in NASH patients and rodent models of NASH.

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Silencing of FTO protects against PA-induced ER-stress and apoptosis.

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Silencing of FTO attenuates palmitate-induced mitochondrial dysfunction.

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