Binding to Peroxisome Proliferator-Activated Receptor Gamma

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Molecular mechanism of betaine on hepatic lipid metabolism: Inhibition of FoxO1 binding to PPARg Dae Hyun Kim, Bonggi Lee, Min Hi Park, Min Jo Kim, Hye Jin An, Eun Kyeong Lee, Ki Wung Chung, June Whoun Park, Byung Pal Yu, Jae Sue Choi, and Hae Young Chung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02644 • Publication Date (Web): 21 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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Journal of Agricultural and Food Chemistry

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Molecular mechanism of betaine on hepatic lipid metabolism: Inhibition of

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FoxO1 binding to PPARγγ

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Dae Hyun Kim† , Bonggi Lee† , Min Jo Kim†, Min Hi Park†, Hye Jin An†, Eun Kyeong

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Lee†, Ki Wung Chung†, June Whoun Park†, Byung Pal Yu§, Jae Sue Choi∥, Hae Young

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Chung†*

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†

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Pharmacy, Pusan National University, Busan 609-735, Republic of Korea;

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Molecular Inflammation Research Center for Aging Intervention (MRCA), College of

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§

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∥

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Republic of Korea

Department of Physiology, The University of Texas Health Science Center at San Antonio;

Department of Food and Life Science, Pukyong National University, Busan, 608-737,

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#

These authors contributed equally to this work.

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*To whom all correspondence should be addressed:

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Prof. Dr. Hae Young Chung

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Department of Pharmacy, College of Pharmacy, Pusan National University

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Gumjung-gu, Busan 609-735, Korea

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Telephone: 82-51-510-2814; Fax: 82-51-518-2821;E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Betaine is a major water-soluble component of Lycium chinensis. Although

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there are reports about the protective effects of betaine on hepatic steatosis, the underlying

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mechanisms are unclear. We used db/db mice and HepG2 cells to examine mechanism

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underlying betaine-mediated protection against hepatic steatosis. Here, we showed increased

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hepatic lipid accumulation in db/db mice which is associated with increased activation of

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lipogenic transcription factors including FoxO1 and PPARγ, whereas betaine administration

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by oral gavage reversed these characteristics. We investigated whether betaine ameliorates

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hepatic steatosis by inhibiting FoxO1/PPARγ signaling in HepG2 cells. Although adenovirus-

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mediated FoxO1 overexpression notably increased mRNA expression levels of PPARγ and its

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target genes including FAS and ACC, betaine treatment reversed them. Furthermore, betaine

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inhibited FoxO1 binding to the PPARγ promoter and PPARγ transcriptional activity in

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HepG2 cells, which was previously shown to induce hepatic steatosis. We concluded that

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betaine ameliorates hepatic steatosis, at least in part, by inhibiting the FoxO1 binding to

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PPARγ and their downstream lipogenic signaling cascade.

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Keywords: Betaine; FoxO1; PPARγ; lipogenesis; steatosis

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INTRODUCTION

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Betaine, an oxidative metabolite of choline, is related to the synthesis of methionine from

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homocysteine in the liver. Betaine is rich in various foods including wheat bran (13.4mg/g),

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wheat germ (12.4mg/g), spinich (6mg/g), beets (2.97mg/g), and goji berry (10.1mg/g) (1).

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Previous studies showed that betaine administration protected the liver from hepatotoxicants,

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such as ethanol and lipopolysaccharide (LPS), possibly by decreasing oxidative stress (2, 3).

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A recent study showed that betaine administration reduced fatty liver and increased hepatic

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insulin signaling in high fat-fed mice (4). However, mechanisms underlying betaine-mediated

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protection against fatty liver are not fully understood. Thus, it is necessary to examine

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specific signaling pathways regulated by betaine in order to ameliorate hepatic steatosis.

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Forkhead box O (FoxO) proteins are evolutionally conserved transcription factors in

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mammals and include FoxO1, FoxO3a, FoxO4, and FoxO6 (5). Of these, FoxO1 is well-

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known for its regulatory effects on hepatic lipid metabolism. Studies showed that increased

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FoxO1 is closely associated with hepatic steatosis. It was reported in an animal study that

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expression of a constitutively active FoxO1 in liver induced hepatic lipid accumulation

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arising from elevated lipogenic signaling and reduced fatty acid oxidation (6). Furthermore,

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a human study indicated that FoxO1 nuclear localization and expression were markedly

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increased in patients with steatohepatitis (7). In addition, FoxO1 mRAN levels were

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positively associated with nonalcoholic fatty liver score and were controlled by drug

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targeting hepatic lipogenesis (7). Thus, FoxO1 may be a key transcription factor that

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regulates hepatic lipid accumulation.

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In the current study, we investigated the mechanisms underlying betaine-mediated

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amelioration of hepatic steatosis in db/db mice. We focused on the inhibitory effects of 3



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betaine on FoxO1 and related lipogenic signaling.

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MATERIALS AND METHODS

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Materials

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2',7'-Dichlorodihydrofluorescein diacetate (DCFDA) was obtained from Molecular

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Probes, Inc. (Carlsbad, CA, USA). Western blotting detection reagents were obtained from

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Amersham (Chalfont St. Giles, UK). All antibodies examined in our study were purchased

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from Santa Cruz Biotechnology (Dallas, TX, USA) except p-FoxO1 (Ser 256) (Cell Signaling,

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Beverly, MA, USA). Anti-rabbit and anti-mouse secondary antibodies were purchased from

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Amersham Pharmacia Biotech. Anti-sheep/goat secondary antibodies were obtained from

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Serotec (Oxford, UK).

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Mouse studies

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C57BLKS/J-db/db mice and their normal littermate C57BLKS/J-db/+ mice (8 weeks old,

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male) were purchased from Japan SLC (Hamamatsu, Japan). Betaine or control solution was

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administered to the mice by oral gavage (50 mg/kg/day) for 3 weeks. Body weight and food

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intake were measured every other day. The mice were housed in cages under a 12 h light and

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dark cycle at ~24°C and ~50% relative humidity. The mice were sacrificed after 3 weeks.

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Tissue samples were collected separately and frozen in the nitrogen tank. For long-term

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storage, the tissue samples were moved to -80°C deep freezer located at Aging Tissue Bank

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in Pusan National University. For further analysis, tissue samples were sliced on the dry ice.

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For a glucose tolerance test, mice were fasted overnight. Glucose (2g/kg) was administered 4


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through intraperitoneal cavity and blood was obtained from the tail vein. Glucose

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concentration was measured by a glucometer (Accu-Chek Active, Roche). All animal studies

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were designed by the Aging Tissue Bank and approved by the Institutional Animal Care

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Committee of Pusan National University. We followed the guidelines for animal experiments

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issued by Pusan National University (Approval Number PNU-2012-0088).

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Cell culture system

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HepG2 cells were purchased from the ATCC (Rockville, MD, USA) and cultured in the

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DMEM media (Nissui Co., Tokyo, Japan) supplemented with 10% FBS (Gibco, Grand Island,

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NY, USA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

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Cell viability after treatments was measured by the 3-(4,5-dimethylthiazol-2-Yl)-2,5-

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diphenyltetrazolium bromide (MTT) assay. For palmitate treatment, palmitate was dissolved

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in 0.1M NaOH at 70°C. Then, the dissolved palmitate was mixed with 5% BSA solution at

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60°C.

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Protein fractionation

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The cultured cells were washed with cold PBS. The cytosolic fraction buffer (10 mM Tris

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at pH 8.0, 1.5 mM MgCl2, 1 mM dithiothreitol, 0.1% nonidet P-40, and protease inhibitors)

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was used for isolating the cytosolic fraction of proteins. The nuclear fraction buffer (10 mM

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Tris at pH 8.0, 50 mM KCl, 100 mM NaCl, and protease inhibitors) was used to isolate the

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nuclear fraction from the pellet obtained after the cytosolic protein isolation. The protein

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fractionation was performed by centrifugation at 13,420 g at 4ºC for 15 min. 5



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FoxO1 adenoviral overexpression

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FoxO1-expressing adenovirus is a kind gift from Dr. H. Henry Dong at University of

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Pittsburgh School of Medicine. HepG2 cells were transduced with adenoviral vectors at a

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defined dose of 100 plaque-forming units (pfu) per cell. FoxO1-expressing adenovirus

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(1.0×1011 pfu/ml) was transduced to HepG2 cells for 24h and the null adenovirus (1.25×1011

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pfu/ml) was used as a control (8).

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Western Blotting

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Homogenized samples were boiled for 5 min in gel-loading buffer (125 mM Tris-Cl, 4%

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sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol, pH 6.8, 0.2% bromophenol blue) at a

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volume ratio of 1:1. Total protein-equivalents for each sample were separated by SDS-

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polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes.

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Membranes were then incubated with specific primary antibodies at 4ºC overnight, followed

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by incubation with horseradish peroxidase-conjugated secondary antibodies at 25ºC for 1 h.

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Western images were visualized by enhanced chemiluminescence according to the

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manufacturer's instructions. Western blotting images were semi-quantified using the image J

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software.

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ROS measurements

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To quantify intracellular ROS generation (9), HepG2 cells were grown in a 96-well plate.

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After 24 h, the medium was changed to fresh serum-free medium. Cells were pretreated with

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or without betaine for 3 h followed by treatment with palmitate for 7 h. Then, the medium 6


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was replaced with fresh medium including DCFDA (f.c. 2.5 µM). The fluorescence intensity

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of DCF was measured every 5 min for 30 min using a microplate fluorescence reader (Tecan,

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Salzburg, Austria) using excitation and emission wavelengths at 485 and 535 nm, respectively.

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RNA isolation and real time RT-PCR

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RNA was isolated form 20mg of liver slice or HepG2 cells at ~2x106 concentration using

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the RNeasy Mini Kit (Qiagen, Hilden, Germany). Real-time PCR was conducted using the

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Roche LightCycler-RNA amplification kit (Roche Diagnostics, Indianapolis, IN, USA). All

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the primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). The

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primer sequences are shown in Supplementary Table 1.

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Chromatin immunoprecipitation (ChIP) and luciferase assays

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ChIP assays were performed using an EZ ChIP Kit (Millipore) based on the manufacturer's

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instructions. Luciferase assays were performed using the One-Glo luciferase assay system

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(Promega, Madison, WI, USA). Detailed methods for ChIP and luciferase assays were

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shown in our previous publication (10) .

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Statistical Analysis

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Data are expressed as the mean ± standard error means (SEM). GraphPad Prism 5.0

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software (GraphPad Software, San Diego, CA) was used for one-way ANOVA followed by

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Bonferroni post-tests, where p < 0.05 was considered statistically significant. Area under the 7



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curve, a plot of concentration of glucose in blood against time was calculated based on the

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glucose tolerance test.

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RESULTS

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Ameliorated glucose tolerance and blood lipid profile in betaine-treated db/db mice

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Genetically obese db/db mice were used to investigate the effects of betaine on glucose

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metabolism and the blood lipid profile. As expected, db/db mice exhibited markedly

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increased body weight due to increased food intake compared to the db/+ control group

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(Table 1). Furthermore, db/db mice showed highly elevated levels of glucose, insulin,

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triacylglycerol, and free fatty acids in serum (Fig.1a-b and table 1). A glucose tolerance test

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showed that serum glucose concentration in db/db mice was maintained much higher than

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db/+ mice over the whole experimental periods (Fig.1c). These data confirmed that the db/db

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mice used in our study had obesity-related metabolic syndrome. On the other hand, although

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body weight and food intake were unaltered (table 1), oral administration of betaine to db/db

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mice decreased serum glucose level by ~45% (p=0.025), serum insulin level by ~20%

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(p=0.0098), serum free fatty acid level by ~35% (p=0.032), and serum triacylglycerol level

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by ~22% (p=0.0087). In addition, betaine treatment decreased glucose intolerance in db/db

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mice by ~23% based on area under the curve (AUC) of glucose tolerance test (p=0.034).

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However, no marked differences were observed in adipose tissue weight and liver weight as

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percentage among groups although kidney weight as percentage was reduced in db/db mice

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and db/db mice treated with betaine (Table 1). These data suggest that betaine may be helpful

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for ameliorating obesity-induced dyslipidemia and glucose intolerance and the beneficial

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effects appear to be independent of changes in energy balance. 8


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Betaine ameliorates hepatic steatosis in db/db mice

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To examine whether betaine protects liver from obesity-induced lipid accumulation, we

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measured triacylglycerol content in liver homogenates. Triacylglycerol concentration was

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increased by ~135% in the liver of db/db mice compared to db/+ mice (p=0.0049), and

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betaine treatment decreased it by ~38% compared to db/db mice (p=0.00047) (Fig. 2a). ROS

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generation is closely associated with hepatic lipid accumulation (11). Thus, ROS level was

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measured in liver homogenates. ROS level was increased by ~260% in the liver of db/db

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mice compared to that of db/+ mice (p=0.014), and betaine treatment decreased it by ~36%

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compared to that of db/db mice (p=0.044) (Fig.2b). These data indicate that betaine

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ameliorated obesity-related hepatic steatosis and oxidative stress.

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Betaine increases insulin signaling and inhibits FoxO1 in liver of db/db mice

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To examine whether betaine improves insulin signaling in the liver of db/db mice,

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western blotting was performed to examine the insulin signaling pathway. Compared to the

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liver of the db/+ control mice, phosphorylation of IRS at Ser307 was increased and

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phosphorylation of IRS at Tyr 632 was decreased in the liver of db/db mice (Fig. 3a and 3c-

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3d). However, betaine treatment reversed them to the level comparable to the control group

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(Fig. 3a and 3c-3d). Consistent with this finding, betaine treatment reversed the reduced

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phosphorylation of AKT in db/db mice (Fig. 3a and 3e), indicating that betaine increased

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insulin signaling in the liver of db/db mice. FoxO1, negatively regulated by AKT, has been

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shown to stimulate lipogenesis, thereby contributing to hepatic steatosis (6, 12). To

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investigate whether betaine-mediated amelioration of hepatic steatosis is associated with

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inhibition of FoxO1, we measured the protein levels of FoxO1 and p-FoxO1 and semi9



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quantified and calculated p-FoxO1/FoxO1. Compared to the liver of the control group, p-

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FoxO1/FoxO1 was decreased in db/db mice, whereas it was significantly increased by

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betaine treatment (Fig.3b and 3f), indicating that betaine inhibits FoxO1 activation. PPARγ is

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another lipogenic protein which is involved in hepatic steatosis (13-15). We investigated

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whether PPARγ is activated in the liver of db/db mice. The protein level of PPARγ was

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significantly increased in the nucleus fraction of the liver of db/db mice, whereas betaine

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treatment reversed it (Fig.3b and 3g), indicating that betaine suppresses PPARγ activation.

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Together, these data suggest that betaine ameliorates hepatic steatosis in db/db mice at least

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partially trough inhibiting FoxO1 and PPARγ activation.

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Betaine inhibits FoxO1 binding to PPARγ

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We used a HepG2 liver cell line to investigate further the mechanism underlying

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betaine-mediated reduction in hepatic steatosis. We first examined whether FoxO1

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overexpression increases lipid accumulation in HepG2 cells and found that FoxO1 adenoviral

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overexpression markedly increased triacylglycerol concentration in HepG2 cells (Fig. 4a).

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Furthermore, FoxO1 overexpression with palmitate treatment additively increased cellular

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triacylglycerol levels, which was reversed by betaine treatment (Fig.4a). Because palmitate

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treatment notably increased FoxO1 mRNA (Fig.4b), which was reversed by betaine, we

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assumed that the protective effect of betaine against lipid accumulation in HepG2 cells is, at

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least in part, due to inhibition of FoxO1, which was consistent with the in vivo result.

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Based on the above finding showing betaine-mediated inhibition of FoxO1 and

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PPARγ in the liver of db/db mice, we investigated whether FoxO1 regulates PPARγ in

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HepG2 cells and whether betaine affects this process to reduce hepatic steatosis. FoxO1 10


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adenoviral overexpression notably increased mRNA expression of PPARγ and its target genes,

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such as FAS, acetyl-CoA carboxylase (ACC), and SREBP-1C, which was additively

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increased by palmitate (Fig. 4b). However, betaine notably decreased mRNA expression of

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these genes (Fig.4b), indicating that FoxO1 upregulated PPARγ signaling in HepG2 cells,

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which was inhibited by betaine treatment. To examine whether FoxO1 directly regulates

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PPARγ transcriptional activity and whether betaine inhibits this process, we performed a

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PPARγ luciferase assay after palmitate or/and FoxO1 adenovirus treatment. FoxO1

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significantly increased PPARγ transcriptional activity, which was additively increased by

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palmitate treatment, and betaine treatment reversed it in HepG2 cells (Fig.4c). In order to

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examine whether FoxO1 directly binds to PPARγ and whether betaine affects this process in

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vivo, a ChIP assay was performed using liver homogenates from db/db mice. Interestingly,

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FoxO1 binding to the PPARγ promoter region was increased in the liver of db/db mice and

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betaine treatment reversed it to the level comparable to the db/+ control group (Fig. 4d).

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These data suggest that betaine inhibits the FoxO1 binding to PPARγ, leading to the reduced

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lipogenic gene expression.

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DISCUSSION

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It has been reported that betaine improves hepatic insulin signaling and fatty liver,

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although the underlying mechanisms remain to be elucidated (4, 16, 17). Here, we examined

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the molecular mechanism by which betaine ameliorates hepatic steatosis using obese db/db

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mice and HepG2 cells. Our data showed that betaine improved obesity-induced glucose

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intolerance and hepatic steatosis without altering energy balance. We found that the decreased

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protein levels of pAKT/AKT and pFoxO1/FoxO1 in db/db mice were recovered by betaine 11



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treatment. In addition, the obesity-induced increase in protein or mRNA expression levels of

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PPARγ and its downstream genes related to lipogenesis were markedly reduced by betaine

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treatment.

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this binding was increased in liver of db/db mice but decreased in the liver of betaine-treated

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db/db mice. Our in vitro studies showed that FoxO1 directly increased PPARγ promoter

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activity as well as its target gene expression, and betaine treatment decreased them. Together,

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our study suggests that the inhibition of FoxO1 binding to PPARγ followed by decreased

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lipogenic gene expression contributes to betaine-mediated amelioration of hepatic steatosis.

As a potential mechanism, FoxO1 can directly bind to the PPARγ promoter, and

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It is well documented that PPARγ stimulates hepatic lipid accumulation (13-15, 18).

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Increased PPARγ expression is closely associated with hepatic steatosis in genetically and

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diet-induced obese mice by increasing lipogenic gene expression (13, 14, 18). On the other

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hand, liver-specific deletion of PPARγ in db/db mice markedly ameliorated hepatic steatosis

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by downregulating FAS, SCD1, and ACC (15). Although various studies reported that both

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FoxO1 and PPARγ stimulated hepatic steatosis, the relationship between PPARγ and FoxO1

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in liver is unclear. Our data showed that FoxO1 directly binds to the PPARγ promoter, and

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this binding was highly increased in hepatic steatosis, which is possibly associated with

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increased PPARγ expression after FoxO1 overexpression. Furthermore, FoxO1 increased

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PPARγ transcriptional activity based on the peroxisome proliferator responsive element

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(PPRE) luciferase assay, indicating that FoxO1 not only increases PPARγ transcription

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possibly by direct binding to the promoter, but also increases PPARγ activity. These data also

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indicate that the FoxO1-mediated increase in PPARγ activity at least partially contributes to

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the increase in hepatic lipid accumulation.

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Betaine has been shown to increase insulin signaling and ameliorate fatty liver (4). 12


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However, it is unclear how betaine-mediated protection of insulin signaling is associated with

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the amelioration of fatty liver. Because FoxO1 is phosphorylated and inactivated by AKT, we

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investigated whether betaine-mediated protection of hepatic steatosis is associated with

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FoxO1 regulation. In the liver of db/db mice, betaine treatment increased insulin signaling,

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which is consistent with a previous report (4). In parallel with this, FoxO1 was deactivated by

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betaine in the liver of db/db mice. Interestingly, betaine notably decreased FoxO1 adenovirus-

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mediated increase in mRNA expression of lipogenic genes, such as PPARγ, ACC, and FAS in

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HepG2 cells, suggesting that betaine may inhibit FoxO1-mediated lipogenesis in liver.

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Because adenoviral FoxO1 overexpression increased mRNA expression of PPARγ and its

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target genes, which is closely related to hepatic steatosis, we examined whether betaine

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directly inhibits FoxO1 binding to PPARγ. Data showed that betaine markedly inhibited

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FoxO1 binding to the PPARγ promotor in the liver of db/db mice. In addition,

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FoxO1/palmitate-induced PPARγ-PPRE luciferase activity was decreased by betaine

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treatment in HepG2 cells. Taken together, these data suggest that betaine inhibits FoxO1-

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mediated up-regulation of PPARγ expression and activity, which partially contributes to

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betaine-mediated amelioration of hepatic steatosis.

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Although the betaine-mediated inhibition of FoxO1 binding to PPARγ contributes to the

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reduction in hepatic steatosis by betaine, it is not the only mechanism underlying the decrease

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in hepatic steatosis because hyperglycemia and hyperinsulinemia are ameliorated by betaine.

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It has been shown that hyperglycemia and hyperinsulinemia contributes to hepatic steatosis

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directly increasing de novo lipogenesis and indirectly by increasing free fatty acids flux to the

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liver through reduced inhibition of lipolysis (19). Therefore, it is also possible that betaine-

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mediated improvement of hyperglycemia and hyperinsulinemia also contributes to the

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decrease in the hepatic steatosis in db/db mice. Because FoxO1 is regulated by insulin 13



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signaling, further studies are necessary to investigate whether increased insulin signaling is

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an upstream factor to reduce FoxO1 binding to PPARγ.

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Together, our study revealed that FoxO1 is able to bind to and increase PPARγ activity,

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which was markedly inhibited by betaine treatment. Thus, we conclude that inhibiting FoxO1

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binding to PPARγ may be a partial mechanism underlying betaine-mediated protection

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against hepatic steatosis.

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AUTHOR INFORMATION

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Corresponding Author: Hae Young Chung

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Telephone: 82-51-510-2814; Fax: 82-51-518-2821;E-mail: [email protected]

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NOTE

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The authors have no conflict of interest to disclose

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ACKNOWLEDGMENTS

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This work was supported by a 2-Year Research Grant of Pusan National University. We also thank the Aging Tissue Bank (Busan, Korea) for supplying research materials.

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leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes. J Clin Invest

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effects on one-carbon metabolism. Amino Acids 2015, 47, 839-46.

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Ables, G. P., Update on ppargamma and nonalcoholic Fatty liver disease. PPAR Res

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Utzschneider, K. M.; Kahn, S. E., Review: The role of insulin resistance in

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387 388 389 390 391

Table 1. Ameliorated serum lipid profile without changes in energy balance in db/db mice

392

treated with betaine

393

db/+

db/db

db/db+betaine

Body Weight (kg)

25.10 ± 2.17

44.98 ± 3.78***

45.66 ± 3.41

Epididymal fat (%)

6.48 ± 0.31

6.97 ± 0.69

6.00 ± 0.34

Liver (%)

8.79 ± 0.38

9.16 ± 0.64

7.69 ± 0.46

Kidney (%)

0.69 ± 0.02

0.44 ± 0.01**

0.43 ± 0.04**

Food Intake (g/day)

2.96 ± 0.13

5.74 ± 0.31***

5.37 ± 0.19

FFA (mEq/l)

232.00 ± 26.08

400.00 ± 47.27**

259.50 ± 21.36#

Triacylglycerol (ug/ml)

56.80 ± 11.08

88.20 ± 2.16*

69.40 ± 5.46##

394 395 396

Each value is the mean ± SEM (n=5/each group) *

p< 0.05,

**

p< 0.01,

***

p< 0.001, vs. db+ mice; #p< 0.05, ##p< 0.01 vs. db/db mice.

397 398 399 400

18


Page 19 of 26


401 402 403 404 405

Figure legends

406

Figure 1. Ameliorated glucose tolerance and blood lipid profile in betaine-treated db/db

407

mice. Betaine (50 mg/kg/day) was administered into db/db mice by oral gavage for 3 weeks.

408

Serum (a) glucose and (b) insulin levels were measured after 3 weeks of betaine

409

administration. (c) Oral glucose tolerance test was performed after 2 weeks of betaine

410

administration. (d) Area under the curve, a plot of concentration of glucose in blood against

411

time was calculated based on the Fig.1c. The results are shown as a bar graph. The data are

412

expressed as a mean ± SEM (n=5).

413

db/db mice.

***

p < 0.001 vs. db+ mice. #p < 0.05 and ##p < 0.01 vs.

414 415

Figure 2. Betaine ameliorates hepatic steatosis and ROS level in db/db mice.

416

(a) ROS level and (b) triacylglycerol concentration in liver were quantified using the

417

DCFHDA method and colorimetric assay kit, respectively after administrating betaine to

418

mice for 3 weeks. The data are expressed as a mean ± SEM (n=5). *p < 0.05 and **p < 0.01

419

vs. db/+ mice. #p < 0.05 and ###p < 0.001 vs. db/db mice.

420 421

Figure 3. Betaine increases insulin signaling and inhibits forkhead box O1 (FoxO1) in

422

liver of db/db mice. Western blotting was performed to examine protein levels of (a) p-

423

insulin receptor substrate (IRS) (Ser307), p-IRS (Tyr623), IRS, p-AKT, and AKT in the

424

insulin signaling pathway in the cytosol fraction of liver of db/db mice treated with betaine.

425

β-actin was used as a loading control for the cytosolic fraction. (b) Protein levels of p-FoxO1,

426

FoxO1, and peroxisome proliferator-activated receptor gamma (PPARγ) related to lipid 19



Page 20 of 26

427

accumulation in the nucleus fraction of liver of db/db mice treated with betaine. Histone H1

428

was used as a loading control for the nucleus fraction. (c-g) Western blotting images were

429

semi-quantified using the image J software. Three independent experiments were performed

430

and similar results were obtained.

431 432

Figure 4. Betaine inhibits FoxO1 binding to PPARγ

433

HepG2 cells were pre-treated with betaine for 3 h followed by FoxO1 adenovirus or/and

434

palmitate treatment for 24 h. (a) Cellular triacylglycerol concentration was measured by a

435

colorimetiric assay. (b) Real-time PCR analysis was performed for measuring mRNA levels

436

of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS),

437

acetyl-CoA carboxylase (ACC), PPARγ, and FoxO1. (c) PPARγ- peroxisome proliferator

438

responsive element (PPRE) luciferase assay was performed to examine whether betaine

439

inhibits PPARγ transcriptional activity. (d) Chromatin immunoprecipitation (ChIP) assay was

440

performed using liver samples of db/db mice to investigate whether betaine inhibits binding

441

of FoxO1 to the PPARγ promoter. The data are expressed as a mean ± SEM. Three

442

independent experiments were performed and similar results were obtained. *p < 0.05, **p

Binding to Peroxisome Proliferator-Activated Receptor Gamma

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