Betaine suppresses hepatic steatosis: Inhibition of FoxO6 and PPARγ interaction

doi:10.21203/rs.3.rs-2219821/v1

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Betaine suppresses hepatic steatosis: Inhibition of FoxO6 and PPARγ interaction

https://doi.org/10.21203/rs.3.rs-2219821/v1

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Betaine is the major water-soluble component of Lycium chinensis. Although there are reports of a protective effect of betaine on fatty liver disease, the underlying mechanisms are unclear. We investigated the effects of betaine on forkhead box O6 (FoxO6) and peroxisome proliferator-activated receptor gamma (PPARγ) expression, which are associated with hepatic lipid accumulation. In this study, we attempted to elucidate the molecular regulation of betaine on hyperglycemia-induced lipid accumulation via FoxO6 activation. HepG2 cells and liver tissue isolated from db/db mice treated with betaine at a dose of 50 mg/kg/day for 3 weeks were used. In the present study, we investigated whether betaine ameliorates hepatic steatosis by inhibiting FoxO6/PPARγ signaling in liver cells. Interestingly, betaine notably decreased lipid accumulation in FoxO6-induced mRNA expression of lipogenesis-related genes. In addition, hepatic insulin signaling was decreased; and activation of FoxO6, which is negatively regulated by Akt, was reduced by betaine treatment. Furthermore, betaine inhibited the FoxO6 interaction with PPARγ and cellular triglycerides in high-glucose- or FoxO6-overexpression-treated liver cells. In addition, we confirmed that betaine administration via oral gavage significantly ameliorated hepatic steatosis in db/db mice. The protein level of PPARγ, a lipogenic transcription factor, was decreased in the livers of db/db mice. Therefore, it has previously been shown to induce hepatic steatosis. We conclude that betaine ameliorates hepatic steatosis, at least in part, by inhibiting the interaction between FoxO6 and PPARγ, thereby suppressing lipogenic gene transcription.

Betaine

FoxO6

PPARγ

hepatic steatosis

interaction

Betaine, an oxidative metabolite of choline, is involved in the synthesis of methionine from homocysteine in the liver. Previous studies have shown that betaine administration protects the liver from hepatotoxicants such as ethanol and lipopolysaccharide [1, 2]. A recent study showed that betaine administration reduces fatty liver and increases hepatic insulin signaling in high-fat diet (HFD) mice [3]. However, the mechanisms underlying betaine-mediated protection against fatty liver disease are not fully understood. Thus, it is necessary to examine the specific signaling pathways regulated by betaine to ameliorate hepatic steatosis.

Forkhead box O (FoxO) proteins are evolutionarily conserved transcription factors in mammals that include FoxO1, FoxO3a, FoxO4, and FoxO6 [4]. FoxOs are regulated by various nutritional and molecular factors. Phosphorylation is a key regulatory mechanism of FoxO. For instance, Akt-mediated phosphorylation inactivates FoxO by inducing its translocation from the nucleus to cytoplasm [5–7]. Although there is consensus regarding the role of FoxO1 in controlling gluconeogenic gene expression, its role in regulating hepatic lipid metabolism remains unclear. Some studies have reported that expression of a constitutively active FoxO1 in the liver induce lipogenic sterol regulatory element binding protein 1c (SREBP-1c) gene expression [8] and hepatic TG accumulation [9]; however, others have not found this response. A study in mice reported that FoxO1 is necessary and sufficient to promote hepatic very-low-density lipoprotein-associated TG (VLDL-TG) production and hypertriglyceridemia via the regulation of microsomal TG transfer protein (MTP) [10]. FoxO6 stimulates the hepatic production of MTP, a molecular chaperone that catalyzes the rate-limiting step in VLDL-TG assembly and secretion [11]. FoxO6 depletion attenuates hepatic gluconeogenesis in HFD mice [12]. However, FoxO6 activity induces peroxisome proliferator-activated receptor gamma (PPARγ) in the liver, leading to lipid accumulation [13]. Further studies are necessary to examine the physiological role of betaine in controlling hepatic lipid metabolism via FoxO6.

PPARs belong to the nuclear hormone receptor superfamily and are comprised of ligand-modulated transcription factors. PPARs heterodimerize with retinoid X receptors (RXRs) and bind to PPAR response elements (PPRE) in the promoter region of specific target genes, regulating their transcription. Currently, three PPAR subtypes have been identified: PPARα, PPARβ, and PPARγ. PPARγ promotes adipogenesis, controls lipid accumulation in adipocytes, and regulates the expression of adipocyte-secreted proteins and adipocytokines (leptin and adiponectin) to reduce lipotoxicity [14] and hepatic lipid metabolism [15, 16]. Kim et al. [11] reported that in diabetic db/db mice, hepatic FoxO6 significantly induced hepatic PPARγ expression in insulin-resistant livers, and consequently induced hepatic lipogenesis as well as increased hepatic fat content.

Elderly individuals exhibit an increased incidence of diabetes and obesity, which are closely associated with insulin resistance [17]. Insulin resistance is a pathological condition in which cells fail to respond to normal insulin signals to store glucose in the tissues. As a result, hyperglycemia and hyperinsulinemia occur because of the reduced glucose uptake from tissues in response to insulin and the consequent increase in insulin secretion by pancreatic beta cells in an attempt to control glucose homeostasis. In addition, diabetes overproduces glucose and triglycerides (TG), contributing to twin abnormalities of this disease, such as hyperglycemia and hypertriglyceridemia [18]. However, medicinal plants such as Alisma orientalis have been proposed to have therapeutic effects on metabolic syndromes [19], including insulin resistance, hyperlipidemia, obesity, and non-alcoholic fatty liver disease (NAFLD) [20]. In the current study, we investigated the mechanisms underlying betaine-mediated amelioration of hepatic steatosis, which has been widely used for studying obesity-associated lipid accumulation, as well as FoxO6 interaction with PPARγ in both db/db mouse liver and HepG2 cells.

Materials

Betaine was obtained from Sigma-Aldrich (St. Louis, MO, USA). Western blotting detection reagents were obtained from Amersham (Chalfont St. Giles, UK). Antibodies against β-actin, p-Akt, Akt, PPARγ, and ACC were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against FoxO6 and p-FoxO6 (Ser184) were obtained from Dr. H. Henry Dong (University of Pittsburgh, Pittsburgh, PA, USA). Anti-rabbit IgG-horseradish peroxidase-conjugated antibody and anti-mouse IgG-horseradish peroxidase-conjugated antibody were obtained from Amersham Pharmacia Biotech. Horseradish peroxidase-conjugated donkey anti-sheep/goat IgG was purchased from Serotec (Oxford, UK). Polyvinylidene difluoride (PVDF) membranes were obtained from the Millipore Corporation (Bedford, MA, USA). Dokdo-MARK™ protein size marker was obtained from ElpisBiotech (Daejeon, Korea).

Mouse Models

C57BLKS/J-lean and C57BLKS/J-db/db mice (8 weeks old, male) were purchased from Japan SLC (Hamamatsu, Japan). The mice were maintained under a 12 h light/dark cycle at 23 ± 1°C and 50 ± 5% relative humidity under specific pathogen-free conditions. Betaine was injected to db/db mice by oral gavage (50 mg/kg/day) for 3 weeks. Body weight and food intake were measured every other day. The mice were sacrificed after 3 weeks. All experiments were approved and performed in accordance with the regulations of the Chosun Univeristy Care and Used Committee (IACUC). All mice were housed at the Chosun University Specific pathogen free (SPF) Animal Care Unit.

Cell Culture System

HepG2 (human hepatocellular carcinoma) cells were obtained from the ATCC (American Type Culture Collection, Rockville, MD, USA). HepG2 cells were cultured in Dulbecco’s Modified Eagle Media (DMEM) (Nissui Co., Tokyo, Japan) supplemented with 10% heat-inactivated (56°C for 30 min) fetal bovine serum (Gibco, Grand Island, NY, USA), 233.6 mg/mL glutamine, 72 µg/mL penicillin streptomycin, and 0.25 µg/mL amphotericin B, and adjusted to pH 7.4–7.6 with NaHCO₃ in a 5% CO₂ atmosphere. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Western Blotting

Homogenized samples were boiled for 5 min in gel-loading buffer (125 mM Tris-Cl, 4% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol, pH 6.8, 0.2% bromophenol blue) at a volume ratio of 1:1. Total protein-equivalents for each sample were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes at 15 V for 1 h using a semi-dry transfer system. Membranes were immediately placed into a blocking buffer (1% non-fat milk) in 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween-20. Blots were allowed to block at room temperature for 1 h. Membranes were then incubated with specific primary antibodies at 4ºC overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at 25ºC for 1 h. Antibody labeling was detected by enhanced chemiluminescence according to the manufacturer's instructions. Pre-stained protein markers were used for measuring molecular weight.

Immunoprecipitation (Ip) Assay

Nuclear extracts were immunoprecipitated in a buffer containing 40 mM Tris-HCl (pH 7.6), 120 mM NaCl, 20 mM β-glycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate, 5 mM EDTA, 1 mM PMSF, 0.1% NP40, leupeptin (2 µg/ml), aprotinin (1 µg/ml), and pepstatin A (1 µg/ml). Aliquots of cell extracts were centrifuged at 12,000 g at 4ºC for 15 min, incubated overnight at 4ºC with the correspondent antibody, and then incubated overnight at 4ºC with 50% protein A-agarose slurry. After washing the immunoprecipitates three times with immunoprecipitation buffer, the immunoprecipitated proteins were analyzed by SDS-PAGE and western blotting, as described above.

Serum Biochemical Analyses

Blood samples were collected from animals in each group after sacrifice. Serum samples were prepared by centrifugation (4℃, 2000 ⅹ g for 15min). ALT and AST levels were analyzed by using kits from Asan pharm (Asan, South Korea).

Rna Isolation And Real Time Rt-pcr

RNA isolation from liver (20 mg) or HepG2 cells (~ 2x10⁶ cells) was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Real-time polymerase chain reaction (RT-PCR) was performed to measure relative mRNA concentrations using the Roche LightCycler-RNA amplification kit (Roche Diagnostics, Indianapolis, IN, USA). All primers were obtained commercially from Integrated DNA Technologies (Coralville, IA, USA). The primer sequences are listed in Supplementary Table 1.

Luciferase Assays

PPARγ activities were estimated using an PPARγ-luciferase vector. Transfection was carried out using Lipofectamine 2000 (Invitrogen). Briefly, 1 × 10⁴ cells per well were seeded in 48-well plates. When the cultured cells reached about 40% confluence, they were treated with DNA/Lipofectamine 2000 complexes (1 µg/µl) in 500 µl normal media (with 10% serum) for 24 h, then treated with the vector containing Akt (100 MOI), or FoxO6-siRNA (100 MOI) at 24 h after transfection. Subsequently, glucose (30 mM) was added for 8 h. Then, the cells were washed with PBS, and subjected to the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA). Luciferase activity was measured by a luminometer (GENious, TECAN, Salzburg, Austria).

Immunofluorescence

HepG2 cells were seeded at 1 × 10³ cells per well in a 6-well plate, incubated for 24 h, fixed in 4% paraformaldehyde solution (15 min at room temperature), washed with PBS buffer, blocked with 3% normal goat serum (Gibco, Grand Island, USA), and immunostained using rabbit anti-PPARγ antibody (1:1000 dilution, Santa Cruz, CA) at 4°C overnight. Cells were then washed with TBS and incubated for 3 h in the presence of anti-rabbit IgG labeled with Alexa Fluor 488 (1:200; Invitrogen, CA, USA). Cell nuclei were visualized by immunostaining with Hoechst 33342 (1:1000; Invitrogen), and PPARγ was determined by confocal laser scanning microscopy (TCS SP2, Leica, Wetzler, Germany).

Lipid Content

Liver tissue and cell samples were homogenized in 400 µL of HPLC-grade acetone. After an overnight incubation with agitation at room temperature, 50 µL aliquots of acetone-extracted lipid suspensions were used to determine triglyceride concentrations via the Infinity triglyceride reagent (Thermo Electron). Liver lipid content was defined as mg of triglyceride per gram of total liver tissue or cellular proteins, as described earlier.

Histological Analysis

Oil Red O staining is used to identify cellular neutral lipid droplet accumulation. The cells were stained by the Oil Red O method. After treatments, the cells were washed three times with ice-cold PBS and fixed with 4% formalin for 30 min, and then dipped in 60% isopropanol for 5 min. After fixing, the cells were washed and stained with Oil Red O solution for 60 min at room temperature. After staining, the tissue or cells were washed three times with PBS to remove the unbound stain.

Livers were fixed in 10% neutral formalin and paraffin-embedded sections were stained with hematoxylin and eosin (H&E staining). The Oil Red O (Sigma, O1391) staining was done by described previously methods with optimal cutting temperature of frozen samples and consequent staining.

Cytotoxicity Assays

Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Aldrich Chemical Co. (Madison, WI). Cells were seeded into 96-well plates, incubated overnight for adherence, and subsequently exposed to 30 mM glucose with betaine for various periods of conc. At the end of the treatment, MTT reagent dissolved in PBS was added to the medium (final concentration of 0.5 mg/ml) and the plates were incubated in the dark for 1 h. After incubation, the supernatant was removed and formazan crystals were dissolved in 100 µl of dimethyl sulfoxide (DMSO) with gentle agitation. The absorbance in each well was measured at 450 nm by using a spectrophotometer.

Statistical Analysis

Data are expressed as the mean ± standard error means (SEM). GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA) was used for one-way ANOVA followed by Bonferroni post-tests, where p < 0.05 was considered statistically significant.

Effect of betaine on lipid accumulation by high-glucose-mediated FoxO6 activation

To this end, serum-starved liver cells were treated with 30 mM of glucose. HepG2 cells treated with betaine showed higher levels of phosphorylated FoxO6 than did glucose-treated HepG2 cells (Fig. 1A). The treatment enhanced the levels of the lipogenic marker PPARγ (Fig. 1A) and induced cellular TG (Fig. 1B). However, betaine inhibited the expression of lipogenic markers and cellular TG levels (Fig. 1A, B). Hepatic mRNA levels of lipogenic factors, including FAS, ACC, and PPARγ were significantly reduced in response to betaine with glucose in HepG2 cells. Betaine treatment increased the expression of β-oxidation genes including ACOX, CPT, and PPARα (Fig. 1C). These results were confirmed in liver cells by Oil Red O staining, showing that fat accumulated in glucose-treated HepG2 cells. As a control, we determined that cellular TG levels were significantly decreased in the betaine-treated HepG2 cells (Fig. 1D). In addition, we investigated the effect of betaine on cell viability using an MTT assay. HepG2 cells were pre-incubated with 30 mM betaine and then treated with glucose. Treatment with 40 µ or 80 µM betaine resulted in cell viabilities of 68% and 79%, respectively (Supp. Figure 1). Based on the above data, betaine did not appear to cause toxicity in HepG2 cells (Supp. Figure 1). These data indicate that betaine blocks glucose-induced lipogenesis and FoxO6 activation. We used HepG2 liver cells to further investigate the functional role of FoxO6 in hepatic steatosis. We hypothesized that FoxO6-induced lipid accumulation in the liver occurs, at least in part, via PPARγ activation. Immunoprecipitation experiments showed that the interaction between FoxO6 and PPARγ was induced in glucose-treated HepG2 cells. Betaine inhibited the interaction between FoxO6 and PPARγ (Fig. 1E). Moreover, glucose caused the transcriptional activation of PPARγ, as determined by the PPARγ luciferase assay. Thus, betaine suppressed PPARγ activation (Fig. 1F).

The binding of insulin and growth factors to specific receptor tyrosine kinases, activates PI3K and serine-threonine kinase Akt. Akt promotes cell survival and proliferation, in part by directly phosphorylating and inhibiting members of the FoxO subfamily of forkhead transcription factors [7]. Therefore, we examined the signaling molecules that lead to Akt activation via oxidative stress. Palmitate-induced oxidative stress activates PI3K and its downstream target Akt. Therefore, to determine whether the activation of the PI3K/Akt pathway causes a change in FoxO6 phosphorylation, we investigated the modulation of FoxO by phosphorylated Akt (the active form of Akt). Although the amount of total Akt was unchanged by glucose, the levels of phosphorylated Akt (Ser473) decreased. Conversely, pretreatment with betaine prevented glucose-induced Akt activation (Fig. 1G).

Modulation Of Hepatic Triglyceride In Ca-akt Transfected Cells

To further establish the importance of FoxO6 in lipid accumulation, we examined the effect of Akt pathway on FoxO6 phosphorylation by utilizing constitutively active Akt (CA-Akt) and evaluated Akt-dependent phosphorylation of FoxO6 in HepG2 cells (Fig. 2A). Akt overexpression additively decreased cellular triglyceride levels, which were suppressed by betaine treatment (Fig. 2B). To examine whether Akt directly or indirectly regulates PPARγ transcriptional activity, and whether betaine inhibits this process, we performed a PPARγ-luciferase assay after Akt adenovirus treatment. Akt significantly decreased PPARγ transcriptional activity, and betaine treatment decreased this increase in HepG2 cells (Fig. 2C). Cellular mRNA levels of lipogenic factors, including FAS, ACC, and PPAR, γ were significantly suppressed in response to betaine with CA-Akt in HepG2 cells (Fig. 2D).

Effect Of Betaine On Foxo6 Overexpression-mediated Lipid Accumulation In Liver Cells

We used a HepG2 liver cell line to further investigate the mechanism underlying betaine-mediated reduction in hepatic steatosis. We hypothesized that betaine partially decreased lipid accumulation in the liver by inhibiting FoxO6. We examined whether FoxO6 overexpression increased lipid accumulation in HepG2 cells and found that HepG2 cells treated with betaine showed lower levels of FoxO6 and ACC protein than FoxO6 overexpressiontreated HepG2 cells (Fig. 3A). FoxO6 overexpression additively increased cellular TG levels, which was reversed by betaine treatment (Fig. 3B). These results were confirmed in HepG2 cells using Oil Red O staining (Fig. 3C).

PPARγ plays an important role in stimulating hepatic steatosis by inducing lipogenesis-related gene expression [21–23]. It has been reported that FoxO6 regulates PPARγ in adipocytes; therefore, we investigated whether FoxO6 regulates PPARγ in HepG2 cells and whether betaine affects this process to reduce hepatic steatosis. FoxO6 adenoviral overexpression notably increased the mRNA expression of PPARγ and its target genes FAS and ACC. However, betaine notably decreased the mRNA expression of these genes (Fig. 3D), indicating that FoxO6 upregulated PPARγ signaling in HepG2 cells, which was inhibited by betaine treatment. Betaine treatment increased the expression of β-oxidation genes, including ACOX, CPT, and PPARα, compared to FoxO6-CA-treated groups (Fig. 3D). However, its expression increased PPARγ localization in the nucleus, as assessed by immunostaining, which was reversed by betaine treatment (Fig. 3E).

Effect Of Betaine On Foxo6-sirna Transduced Cells

We investigated the role of betaine and the relationship between glucose and lipid accumulation under FoxO6 knockdown conditions. Thus, betaine showed lower protein levels of FoxO6 and PPARγ than glucose in FoxO6-siRNA-treated HepG2 cells (Fig. 4A). We performed a PPARγ-luciferase assay after treatment with FoxO6-siRNA adenovirus, betaine, and glucose. Glucose with FoxO6-siRNA significantly decreased PPARγ transcriptional activity, and betaine with glucose and FoxO6-siRNA-treatment more suppressed this decrease in HepG2 cells (Fig. 4B). Glucose with FoxO6-siRNA treatment additively decreased cellular TG levels, which were decreased by betaine treatment (Fig. 4C).

Betaine Inhibits Foxo6 And Pparγ Interaction In Liver Of Db/db Mice

To provide further physiological underpinning for betaine regulation of hepatic lipid metabolism, we fed betaine to 3-weeks the livers of obese and diabetic db/db mice. Diabetic db/db mice were intraperitoneally injected with betaine, followed by the determination of lipid metabolism.

Hepatic steatosis is closely associated with impaired insulin signaling in the liver. To examine whether betaine improves insulin signaling in the livers of db/db mice, western blotting was performed to examine the insulin signaling pathway. To investigate whether betaine-mediated amelioration of hepatic steatosis was due to inhibition of FoxO6, serum alanine aminotransferase (ALT) and aspartate transaminase (AST) levels were measured. However, the serum ALT and AST levels did not change in db/db and betaine-fed db/db mice (Fig. 5A, B). We measured the protein levels of the active forms of the FoxO6 and PPARγ target gene, ACC. Active FoxO6 and ACC levels were decreased by betaine treatment in the liver of db/db mice (Fig. 5C), indicating that betaine inhibits FoxO6 activation, thereby leading to suppression of obesity-induced hepatic steatosis. To address the underlying mechanism, we determined the effect of betaine on hepatic expression of key enzymes involved in lipogenesis vs. fatty acid oxidation. The hepatic mRNA levels of lipogenic factors, including FAS, ACC, and PPARγ were significantly reduced in db/db mice treated with betaine (Fig. 5D). In addition, we detected a significant increase in the mRNA levels of β-oxidation genes (ACOX, CPT, and PPARα) in the liver of betaine-treated db/db mice (Fig. 5D). Next, we analyzed lipid parameters in the liver. Oil red O staining showed that lipid droplets accumulated in the liver of db/db mice, and lipid accumulation was further decreased in the livers of betaine-fed db/db mice (Fig. 5E). To examine whether betaine affected the interaction between FoxO6 and PPARγ in vivo, IP was performed using liver homogenates from db/db mice. Interestingly, FoxO6 interaction with PPARγ was increased in the liver of db/db mice compared to control mice, and betaine treatment reduced this interaction to levels comparable to those in db/db mice (Fig. 5F). These data suggest that betaine inhibits the interaction between FoxO6 and PPARγ, thereby decreasing lipogenic gene expression.

Betaine has been used in traditional oriental medicine to treat hepatic toxicity and disorders [2, 24], and recent studies have shown that betaine increases insulin signaling and ameliorates fatty liver [25]. Betaine has been reported to improve hepatic insulin signaling and fatty liver; however, the underlying mechanisms remain to be elucidated [25–27]. Moreover, it is unclear how betaine-mediated protection of insulin signaling is associated with amelioration of fatty liver. As FoxO6 is inactivated (increased phosphorylation) by activated Akt in the insulin signaling pathway, we investigated whether the betaine-mediated protection of hepatic steatosis is associated with FoxO6 regulation. Considerable data suggests that betaine plays an important role in the regulation of FoxO6-mediated hepatic steatosis (Fig. 3). However, their importance in hepatic lipid metabolism remains unclear. A previous study indicated that liver-specific overexpression of active FoxO1 results in notably increased lipogenesis, hepatic steatosis, and impaired glucose tolerance in db/db mice [28]. Furthermore, overexpression of FoxO1 in the liver causes hepatic steatosis owing to increased lipogenesis and decreased fatty acid oxidation [29], suggesting that FoxO6 increases lipogenesis, thereby stimulating hepatic steatosis [30]. A positive trend was noted, showing that hepatic triglyceride content was higher in livers deficient in FoxO1 after siRNA-mediated knockdown of FoxO1 [10]. Therefore, the role of FoxO6 in hepatic lipid accumulation remains unclear. Our data indicated that adenoviral overexpression of FoxO6 increased the mRNA expression of lipogenic genes such as ACC and FAS in HepG2 cells (Fig. 3). Consistently, betaine notably inhibited increased triglyceride accumulation by FoxO6 overexpression, supporting the role of FoxO6 in the stimulation of hepatic lipid accumulation through increased lipogenesis marker PPARγ (Fig. 3).

The fact that PPARγ stimulates hepatic lipid accumulation is well-documented [21–23, 31]. Increased PPARγ expression is closely associated with hepatic steatosis in genetically- and diet-induced obese mice by increasing lipogenic gene expression [21, 22, 31]. In contrast, liver-specific deletion of PPARγ in db/db mice markedly ameliorated hepatic steatosis by downregulating FAS, SCD1, and ACC [23]. Although various studies have reported that both FoxO1 and PPARγ stimulate hepatic steatosis [32], the relationship between PPARγ and FoxO6 in the liver is unclear. In the livers of db/db mice, betaine treatment increased insulin signaling, which is consistent with a previous report [25]. In parallel, FoxO6 was inactivated by betaine in the liver of db/db mice (Fig. 5). Interestingly, betaine notably decreased the glucose-mediated increase in the mRNA expression of lipogenic genes, such as PPARγ, ACC, and FAS, in HepG2 cells, suggesting that betaine may inhibit FoxO6-mediated lipogenesis in the liver. As glucose increased the mRNA expression of PPARγ and its target genes, which are closely related to hepatic steatosis, we examined whether betaine directly inhibited the interaction between FoxO6 and PPARγ (Fig. 5). Additionally, glucose-induced PPARγ-luciferase activity was decreased by betaine treatment in HepG2 cells (Fig. 1). Taken together, these data suggest that betaine inhibits FoxO6-mediated PPARγ gene expression and activity, which at least partially contributes to betaine-mediated amelioration of fatty liver disease (Figs. 1 and 5). In this study, we examined the molecular mechanism by which betaine ameliorated hepatic steatosis in obese db/db mice. We found that the protein level of Akt under insulin signaling was increased and inactive FoxO6 phosphorylated by Akt was increased by betaine treatment (Fig. 1). Furthermore, FoxO6 overexpression increased PPARγ transcriptional activity, indicating that FoxO6 not only increased PPARγ expression but also increased PPARγ activity (Fig. 3). These data also indicate that betaine regulates the FoxO6-mediated increase in PPARγ activity, at least partially contributing to the increase in hepatic lipid accumulation. Since PPARγ and FoxO1 are closely associated with hepatic steatosis [21, 28, 29], we examined whether betaine ameliorates hepatic steatosis by downregulating FoxO6 and PPARγ. Together, our study suggests that the inhibition of FoxO6 and PPARγ interaction, followed by decreased lipogenic gene expression, contributes to the betaine-mediated amelioration of hepatic steatosis (Fig. 6).

In conclusion, FoxO6 increased PPARγ activity, which was markedly inhibited by betaine treatment. Thus, we concluded that inhibiting the interaction between FoxO6 and PPARγ may be a partial mechanism underlying betaine-mediated protection against hepatic steatosis.

Acknowledgements

This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2021R1I1A1A01052055).

Conflict of Interest

The authors declare no conflict of interest.

Author contributions

MEK, MHP, and DHK collected the samples and performed the experiments. JSL and DHK wrote the manuscript. All authors contributed to the interpretation of the results. All authors revised and approved the manuscript.

Date availability statement

The data sets supporting the findings of the current study are avail-able from the corresponding author upon reasonable request.

Ethics approval All experiments were approved and performed in accordance with the regulations of the Care and Used Committee (IACUC). All mice were housed at the Chosun University Specific pathogen free (SPF) Animal Care Unit.

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Supp. Fig. 1 The cells were pretreated with various concentrations of betaine for 2 h and followed by treatment with or without 30 mM glucose for 24 h. The MTT assay was performed as described in Material and Methods. One-factor ANOVA: ^###p< 0.001 vs. Normal; **p< 0.01 vs. glucose-treated cells.
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Editorial decision: Major Revisions Needed
16 Apr, 2023
Reviewers agreed at journal
15 Nov, 2022
Reviewers invited by journal
15 Nov, 2022
Editor assigned by journal
03 Nov, 2022
First submitted to journal
02 Nov, 2022

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Betaine suppresses hepatic steatosis: Inhibition of FoxO6 and PPARγ interaction

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials

Mouse Models

Cell Culture System

Western Blotting

Immunoprecipitation (Ip) Assay

Serum Biochemical Analyses

Rna Isolation And Real Time Rt-pcr

Luciferase Assays

Immunofluorescence

Lipid Content

Histological Analysis

Cytotoxicity Assays

Statistical Analysis

Results

Effect of betaine on lipid accumulation by high-glucose-mediated FoxO6 activation

Modulation Of Hepatic Triglyceride In Ca-akt Transfected Cells

Effect Of Betaine On Foxo6 Overexpression-mediated Lipid Accumulation In Liver Cells

Effect Of Betaine On Foxo6-sirna Transduced Cells

Betaine Inhibits Foxo6 And Pparγ Interaction In Liver Of Db/db Mice

Discussions

Declarations

References

Supplementary Files

Status:

Version 1