Hepatic FOXA3 overexpression prevents Western diet–induced obesity and MASH through TGR5

Forkhead transcription factor 3 (FOXA3) has been shown to regulate metabolism and development. Hepatic FOXA3 is reduced in obesity and fatty liver disease. However, the role of hepatic FOXA3 in regulating obesity or steatohepatitis remains to be investigated. In this work, C57BL/6 mice were i.v. injected with AAV8-ALB-FOXA3 or the control virus. The mice were then fed a chow or Western diet for 16 weeks. The role of hepatic FOXA3 in energy metabolism and steatohepatitis was investigated. Plasma bile acid composition and the role of Takeda G protein–coupled receptor 5 (TGR5) in mediating the metabolic effects of FOXA3 were determined. Overexpression of hepatic FOXA3 reduced hepatic steatosis in chow-fed mice and attenuated Western diet–induced obesity and steatohepatitis. FOXA3 induced lipolysis and inhibited hepatic genes involved in bile acid uptake, resulting in elevated plasma bile acids. The beneficial effects of hepatic FOXA3 overexpression on Western diet–induced obesity and steatohepatitis were abolished in Tgr5−/− mice. Our data demonstrate that overexpression of hepatic FOXA3 prevents Western diet–induced obesity and steatohepatitis via activation of TGR5.

Hepatic triglyceride (TG) levels are regulated by de novo lipogenesis (DNL), fatty acid uptake, lipolysis, fatty acid oxidation (FAO), and VLDL secretion.Excessive TG deposition in the liver is the hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), which is an emerging healthcare issue and a major chronic liver disease worldwide.Disruption of hepatic fatty acid/TG homeostasis may cause lipotoxicity and the development of metabolic dysfunction-associated steatohepatitis (MASH) (1,2).MASH is an advanced form of MASLD and is characterized by steatosis, hepatocyte ballooning, lobular inflammation, and fibrosis.So far, no Food and Drug Administration-approved drugs are available for the treatment of MASH.
Bile acids (BAs) play an important role in regulating intestinal fat absorption and metabolic homeostasis mainly via activating farnesoid X receptor and Takeda G protein-coupled receptor 5 (TGR5) (3,4).Primary BAs are produced from cholesterol by cholesterol 7αhydroxylase (CYP7A1) in the classic pathway and converted to secondary BAs by gut microbiota.Elevated total BAs and altered BA composition are observed in the plasma of MASLD and MASH patients (5).BAactivated TGR5 promotes thermogenesis and improves glucose homeostasis (6)(7)(8).TGR5 activation also inhibits the development of MAFLD/MASH (9)(10)(11)(12).
Forkhead box A3 (FOXA3) is a member of the winged-helix transcription factors.FOXA3 is expressed in the liver, intestine, pancreas, etc., and regulates glucose homeostasis, lipoprotein metabolism, and liver and pancreas development (13)(14)(15)(16)(17). Overexpression of hepatic FOXA3 is shown to protect against atherosclerosis by promoting reverse cholesterol transport (14).However, the role of hepatocyte FOXA3 in regulating diet-induced MASH has not been reported.
In the current study, we investigated the role of overexpression of hepatic FOXA3 in obesity and MASH development and the underlying mechanisms.Our data showed that overexpression of hepatic FOXA3 raised plasma BA levels and attenuated Western diet-induced obesity and MASH in WT mice but not in Tgr5 −/− mice.In addition, overexpression of hepatic FOXA3 also induced hepatic lipolysis and reduced hepatic TG levels in chow-fed mice.

Mice and diets
C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME).Tgr5 −/− mice have been described previously (18).The Western diet (21% fat/0.2%cholesterol) was purchased from Envigo (cat# TD.88137).Unless otherwise stated, 2-month-old male mice were used and fed a Western diet for 16 weeks.The mice were fasted for 5-6 h during the light cycle before euthanasia.All the animal experiments were approved by the Institutional Animal Care and Use Committee at Northeast Ohio Medical University.

Adeno-associated viruses
Human FOXA3 was cloned into an adeno-associated virus (AAV) vector under the control of a mouse albumin promoter to generate AAV-ALB-FOXA3 (14,19).AAV8-ALB-Null (control) or AAV8-ALB-FOXA3 was produced and titrated by Vector BioLabs.Each mouse was i.v.injected with 3 × 10 11 genome copies of AAVs.

Quantitative real-time PCR
Total RNA was extracted from liver or brown adipose tissues (BATs) using Trizol reagent (Thermo Fisher Scientific), and mRNA levels were quantified by quantitative real-time PCR using Powerup SYBR Green Master mix (Thermo Fisher Scientific) on a 7500 real-time PCR machine (Applied Biosystems).mRNA levels were normalized to 36b4.

Hepatic lipids, hydroxyproline, and plasma ALT and AST
Approximately 100 mg of liver tissue was homogenized in methanol, and lipids were extracted using chloroform/ methanol (2:1 v/v) as previously described (20).Hepatic TGs and total cholesterol (TC) were determined using Infinity reagents from ThermoFisher Scientific.Free cholesterol (FC) and FFAs were measured using Fujifilm Wako Chemicals.Hepatic hydroxyproline levels were measured using a kit from Cell Biolabs (cat # STA675).Plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels were determined using Infinity reagents.

Triglyceride hydrolase activity and FAO
Hepatic total cell lysates were isolated for measuring triglyceride hydrolase (TGH) activity using 3 H-triolein as substrate (21).Primary hepatocytes were isolated and then cultured in Dulbecco's modified Eagle's medium containing 10% FBS in 12-well plates.FAO was analyzed using 3 H-palmitic acid as substrate as previously described (21).

Intestinal fat absorption
To determine intestinal fat absorption, mice were fasted for 6 h and then i.v.injected with tyloxapol (500 μg/kg) prior to gavage with olive oil (15 μl/g body weight) as described (19,22).Plasma TG levels were determined at indicated time points.
BA levels, BA pool size, and BA composition BA levels were determined using a BA kit from Diazyme (cat # DZ042A-KY1).BAs in the liver, gall bladder, and intestine were extracted using ethanol (23).Total BAs in the liver, gall bladder, and intestine were determined and used to calculate the total BA pool size.The BA composition was determined by LC-MS/MS.

Glucose tolerance test
The glucose tolerance test was performed as previously described (24).Briefly, mice were fasted overnight, followed by i.p. injection of D-glucose (Sigma) at a dose of 1.8 mg/kg body weight.Plasma glucose levels at indicated time points were measured.

Fatty acid composition and DNL
Hepatic fatty acid composition and DNL were analyzed using GC-MS as described previously (25,26).For DNL, mice were given water containing 8% 2 H 2 O for the last 7 days.Four hours prior to anesthesia, mice were i.p. injected with 30 μl/g 2 H 2 O.

Oil Red O, H&E, and picrosirius red staining
Liver tissues were fixed in 10% formalin and then embedded in paraffin or OCT.Liver sections were stained with Oil Red O, H&E, or picrosirius red.Images were acquired using an Olympus microscope.

Body fat measurement, food intake, and energy expenditure
Body fat was measured using EchoMRI (EchoMRI, Houston, TX).Oxygen consumption, carbon dioxide production, and heat production were determined using Comprehensive Lab Monitoring System (Columbus Instruments) as described previously (27).In brief, mice underwent an acclimation period, and a 48-h measurement of energy expenditure was analyzed using an eight-chamber system.Each run included both the control and overexpression groups, with four mice per group.The energy expenditure data were analyzed using the web-based CalR program (28).

Statistical analysis
All the data were expressed as mean ± SEM.Statistical significance was analyzed using an unpaired Student's t test or ANOVA (GraphPad Prism, San Diego, CA).Differences were considered as statistically significant at P < 0.05.

Hepatic overexpression of FOXA3 lowers hepatic TG accumulation in chow-fed mice
Previous studies show that hepatic FOXA3 is markedly reduced in genetic or high-fat diet-induced obesity and MASH patients (14).Since FOXA3 was expressed in mouse primary hepatocytes, AML12 cells, or HepG2 cells at a moderate level (supplemental Fig. S1A-C), we evaluated the role of hepatocyte FOXA3 in the regulation of hepatic lipid metabolism.C57BL/6 mice were i.v.injected with either AAV8-ALB-Null or AAV8-ALB-FOXA3, and then fed a chow diet for 4 weeks.Overexpression of FOXA3 had no impact on body weight (supplemental Fig. S2A, B) but caused a 66% reduction of hepatic TG levels (Fig. 1A), whereas hepatic FFA, TC, or FC levels remained unchanged (supplemental Fig. S2C, D).Interestingly, analysis of hepatic fatty acid composition by GC-MS showed that C14:0, C16:0, C18:0, C18:1, C18:2, and C20:4 fatty acid levels were significantly reduced in mice-overexpressing FOXA3 (Fig. 1B).Since hepatic fatty acid levels are determined by DNL, fatty acid uptake, FAO, fatty acid incorporation of TG, and TG hydrolysis (lipolysis), we analyzed hepatic DNL using GC-MS.There was no change in de novo synthesis of hepatic palmitate levels (Fig. 1C), suggesting that FOXA3 does not regulate DNL.Gene expression analysis showed that overexpression of hepatic FOXA3 significantly induced carboxylesterases 1g (Ces1g), Ces2c, Ces2g, and Ces2e without affecting adipose TG lipase or Ces1b (Fig. 1D).We have previously shown that Ces1g/ Ces1 and Ces2c/Ces2 have TG lipase (TGH) activity (21,25), suggesting that FOXA3 may regulate lipolysis.In addition, hepatic overexpression of FOXA3 inhibited the expression of Pparγ, microsomal TG transfer protein, Apob, and inflammatory and fibrogenic genes (interleukin 1β and α-smooth muscle actin) (supplemental Fig. S2E).Together, these data indicate that hepatic FOXA3 prevents hepatic TG accumulation in chow-fed mice likely via inducing lipolysis.

Overexpression of hepatic FOXA3 increases energy expenditure
To understand how overexpression of hepatic FOXA3 prevented Western diet-induced obesity, we analyzed energy expenditure using Comprehensive Lab Animal Monitoring System.Overexpression of hepatic FOXA3 significantly increased energy expenditure during the day and night time (Fig. 4A, B).Consistent with the increased energy expenditure, hepatic FOXA3-induced oxygen consumption (Fig. 4C) without affecting respiratory exchange ratio (Fig. 4D).Surprisingly, mice-overexpressing hepatic FOXA3 had increased food intake (Fig. 4E, F).Thus, hepatic FOXA3 overexpression attenuates Western diet-induced obesity via increasing energy expenditure.
Gene expression data show that overexpression of hepatic FOXA3 induced uncoupled protein 1 (Ucp1), Ucp2, and Ucp3 expression in BAT of WT mice, but not in Tgr5 −/− mice (Fig. 6M).Hepatic FOXA3 overexpression had no impact on Dio2 or Cpt1a expression in BAT (supplemental Fig. S4C, D), but induced Cpt1b or Cd36 expression in BAT in an Tgr5-dependent manner (supplemental Fig. S4E, F).Taken together, these studies indicate that activation of the TGR5 signaling is required for hepatic FOXA3 to ameliorate Western diet-induced obesity and hepatic steatosis.

DISCUSSION
Previous studies have shown that hepatic FOXA3 expression is markedly reduced in obesity or MASH (14).However, whether hepatic FOXA3 regulates dietinduced obesity or MASH has not been investigated.In this project, we show that overexpression of hepatic FOXA3 reduces hepatic TG accumulation in chow-fed mice and ameliorates Western diet-induced obesity and MASLD/MASH.Mechanistically, hepatic FOXA3 induces lipolysis and thermogenesis and prevents Western diet-induced metabolic disorders via TGR5.
One important finding of this study is that overexpression of hepatic FOXA3 raises plasma BA levels by inhibition of hepatic NTCP and OATP1 expression.NTCP is expressed exclusively in the liver (30).Previous studies by Donkers et al. (31) show that loss of NTCP prevents diet-induced obesity and hepatic steatosis by inducing energy expenditure and inhibiting intestinal fat absorption.Our data show that hepatic FOXA3 improves diet-induced obesity and MASH in WT mice, but not in Tgr5 −/− mice, suggesting that activation of TGR5 plays a key role in mediating the beneficial effects of FOXA3.BAs activate TGR5 in the order of potency of LCA>DCA>CDCA>CA with an EC50 of 0.53 μM, 1.0 μM, 4.4 μM, and 7.7 μM, respectively (32,33).Activated TGR5 induces thermogenesis and improves diet-induced MAFLD/MASH (6,(8)(9)(10)(11)(12).Interestingly, Donkers et al. (31) show that loss of NTCP or both NTCP and TGR5 has similar effects on obesity or hepatic steatosis, suggesting that loss of NTCP exerts beneficial effects independent of activation of TGR5.It is unclear whether OATP1 is compensatively induced in Ntcpdeficient mice.In our studies, FOXA3 inhibits both NTCP and OATP1, which may allow a drastic increase in plasma BA levels and thus activation of TGR5.It remains unclear how the loss of NTCP leads to increased energy expenditure (31).
In MASH patients, plasma BA levels are generally increased by ∼2to 3-fold (5,34,35).Interestingly, the increase in plasma BAs does not or is not sufficient to protect against MASH development.Grzych et al. (35) show that plasma BA levels are elevated only in MASH patients with pronounced insulin resistance, suggesting that the increase in plasma BAs in MASH may result from liver damage.In contrast, FOXA3 overexpression raises plasma BA levels by 6.5-fold via the inhibition of Hepatic FOXA3 inhibits obesity and steatohepatitis NTCP and OATP1, which appears to be sufficient to activate TGR5 to exert beneficial metabolic effects.
In addition to raising plasma BA levels by inhibition of NTCP and OATP1, FOXA3 overexpression also induces hepatic TGH activity likely through increasing the expression of CESs, such as CES1 and CES2, which have been demonstrated to have TGH activity (21,25).The increased lipolytic activity may account for reduced hepatic TG levels in chow-fed mice-overexpressing hepatic FOXA3, as body weight is not changed in these mice.FOXA3 also induces FAO and inhibits SREBP2, which, together with reduced obesity, may account for reduced hepatic FFA and FC levels in Western diet-fed mice.A reduction in hepatic FFA and FC levels may attenuate hepatic lipotoxicity, resulting in reduced hepatic inflammation and fibrosis (1,2).
In summary, we have identified a novel role of hepatic FOXA3 in regulating lipid and BA metabolism, obesity, and MASH.Since hepatic FOXA3 is markedly reduced in obesity and MASH, targeting hepatic FOXA3 may be a useful strategy for the treatment of obesity and MASH.