Tamoxifen induced hepatic steatosis in high-fat feeding rats through SIRT1-Foxo1 suppression and LXR-SREBP1c activation

Abstract

Background

Clinically, long-term use of tamoxifen (TAM) would lead to fatty liver disease in breast cancer patients, especially obese women. However, the exact mechanism of TAM-induced hepatic steatosis is still unclear. Meanwhile, there is no drug to prevent and treat it.

Aims and Methods

In view of silent information regulator 1 (SIRT1) playing a key role in hepatic lipid metabolism regulation, this study was conducted to investigate whether SIRT1 is a potential therapeutic target for TAM-induced hepatic steatosis. In this study, obese female Wistar rats fed with high-fat diet (HFD) for 15 weeks were given TAM (4, 8 mg/kg, intragastric) for 14 days. In vitro, human hepatocarcinoma cell line HepG2 was used to establish a high-fat model with 50 μM oleic acid and TAM (10 μM) was treated simultaneously for 72 h.

Results

The results showed that TAM was more likely to upregulate the expression of lipid synthetase that caused the increase of lipid content in HepG2 cells and rat liver. The expression of SIRT1 was downregulated both in vitro and in vivo. SIRT1 agonist SRT1720 (15 mg/kg, 30 mg/kg, i.p.) could resist TAM-induced hepatic lipid synthetase overexpression to relieve TAM-induced hepatic steatosis. Meanwhile, the upregulation of p-forkhead box O1 and LXRα induced by TAM was reversed by SRT1720.

Conclusions

These results indicated that TAM-induced hepatic steatosis was based on SIRT1-p-FoxO/LXRα-sterol regulatory element binding protein 1c pathway under HFD condition. SIRT1 agonist might be a potential therapeutic drug to relieve this side effect.

Highlights

• Tamoxifen increased lipid synthesis and regulated lipid transport in HFD rat liver.

• p-FoxO1/LXRα-SREBP1c signaling was upregulated through the inhibition of SIRT1 in tamoxifen-induced hepatic steatosis under HFD condition.

• SIRT1 agonist SRT1720 could relieve tamoxifen-induced hepatic steatosis.

Introduction

Tamoxifen (TAM), as a nonsteroidal estrogen receptor antagonist, is an important therapeutic drug for estrogen receptor positive breast cancer patients, especially postmenopausal ones.¹ However, long-term use of TAM will cause a series of side effects, such as vaginal bleeding, deep vein thrombosis, and even endometrial cancer.^2,3 But the more common side effect of TAM is the nonalcoholic fatty liver disease (NAFLD).

Studies have shown that 43% of breast cancer patients treated with TAM reached NAFLD within the first 2 years of treatment, especially some overweight women even developed nonalcoholic steatohepatitis and cirrhosis.⁴ A propensity score-matched cohort study among 328 matched subjects showed that the incidence of fatty liver in TMX group was 128.7 per 1,000 person-years, and it was related to the increased risk of new fatty liver within 5 years.⁵ Besides, a meta-analysis of 6,962 patients who received TAM and 975 patients who did not received TAM showed that the incidence of fatty liver in TAM group was much higher (incidence rate ratio: 3.12, 95% confidence interval: 2.05–4.75, I2 = 61%) regardless of region.⁶ Studies also indicate that elevated body mass index (BMI) and decreased high-density lipoprotein (HDL) are predictors of NAFLD induced by TAM.⁷ Among them, BMI ≥ 22 kg/m² is a potential risk factor for TAM-induced fatty liver.⁸

The primary cause of hepatic lipid accumulation is the imbalance between lipid deposition and clearance, which is directly related to the increase of liver fat production, the decrease of fatty acid β-oxidation, the increase of lipid intake, and the decrease of triglyceride (TG) output.⁹ Nowadays, there are several different views on the mechanism of TAM-induced fatty liver. It has been reported that the increase of lipogenesis caused by the upregulation of sterol regulatory element binding protein 1c (SREBP1c) and lipid synthase is the main reason of TAM-induced liver steatosis.^4,10 Others have proved that TAM-induced liver steatosis is related to the inhibition of fatty acid oxidation.^11,12 Besides, studies also suggested that TAM treatment may affect lipid uptake or secretion.^13,14 Nevertheless, the exact mechanism still remains to be discussed.

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent class III histone deacetylase,¹⁵ which has been proved to play a crucial role in lipid metabolism. A previous study reported that SIRT1 could deacetylate forkhead box O1 (FoxO1),¹⁶ which inhibits the transcriptional activation of SREBP1c, thus suppressing fat synthesis.¹⁷ SIRT1 also directly modulates the expression of SREBP1c, carbohydrate response element-binding protein (CHREBP), and liver X receptor (LXR), which are involved in glucose and lipid metabolism.^18,19 SRT1720, a chemical agonist of SIRT1, has been proved to inhibit the expression of SREBP1c target gene in vivo and in vitro.²⁰ However, whether SIRT1 is a promising therapeutic target for TAM-induced hepatic steatosis has not been reported.

Clarifying the molecular mechanism of NAFLD induced by TAM is urgent to be explained for the clinical application of TAM. In this study, we aimed to deeply and systematically investigate the molecular mechanism of hepatic steatosis caused by TAM. Besides, the role of SIRT1 in TAM-induced hepatic steatosis was investigated. It has important scientific significance and theoretical value for reducing the clinical risk of TAM and developing effective NAFLD therapeutic drugs.

Materials and methods

Reagents and antibodies

TAM (Citrate) was purchased from Medchem Express Co., Ltd (Princeton, NJ, United States). SRT1720 was purchased from Shanghai Yongcan Chemical Co. (Shanghai, China). High-fat purified feed (MD12032) (45% fat Kcal%) was purchased from Jiangsu meidisen biology medicine co., Ltd (Yangzhou, China). Oleic acid (OA) was obtained from Sigma-Aldrich (MO, United States). Albumin, from Bovine Serum, Fatty Acid Free (BSA) was obtained from FUJIFILM Wako Pure Chemical Corporation(FUJIFILM, Japan). RIPA lysis buffer and BCA protein assay reagent kit were purchased from Beyotime Biotechnology (Shanghai, China). The phospho-acetyl-CoA carboxylase (ACC) (11818T), ACC (3676T), phospho-adenosinemonophosphate (AMP)-activated protein kinase (AMPK) (2535S), AMPK (2532), and SIRT1 (9475s) primary antibodies were from CellSignaling Technology (Danvers, MA, United States). The SREBP1c (sc-8984), LXRα (sc-1202), and FAS (sc-715) primary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The microsomal triglyceride transporter (MTTP) (A1746), cluster of differentiation 36 (CD36) (A1470), and CHREBP (A7630) primary antibodies were from ABclonal Technology (Wuhan, China). The stearyl coenzyme A desaturase 1 (SCD1) (ab19862), low-density lipoprotein receptor (LDLR) (ab30532) and elongation long chain fatty acids family member 6 (ELOVL6) (ab69857) primary antibodies were purchased from Abcam (Cambridge, UK).

Animal experiments

Wistar rats (female, 180–220 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All rats were housed in ventilated cages under a 12-h light–dark cycle with free access to water, and the temperature was kept at 25 ± 2 °C.

After 1 week of adaptive feeding, the rats were randomly divided into 4 groups (n = 6/group): (i) chow diet (CD)-control, (ii) High-fat diet (HFD)-control, (iii) HFD-TAM-L (4 mg/kg/day), and (iv) HFD-TAM-H (8 mg/kg/day). After feeding chow diet or HFD for 15 weeks, rats were given control solution (5% ethanol solution) or TAM for 14 consecutive days in an intragastric manner.

For SRT1720 treatments, the Wistar rats (female, 180–220 g) were randomly divided into 4 groups (n = 6/group): (i) control, (ii) TAM (8 mg/kg/day), (iii) TAM + SRT1720 (15 mg/kg/day), and (iv) TAM + SRT1720 (30 mg/kg/day). After 15 weeks of HFD, the rats were administered intragastrically with TAM and injected intraperitoneally with SRT1720 or control solution (10% ethyl alcohol and 40% polyethylene glycol 400) for 14 consecutive days.

All experiments and procedures involved were approved by the Ethical Committee of China Pharmaceutical University and were conducted in accordance with the institutional guidelines of Chinese experimental animal care and use institutions.

Cell culture

The human hepatocarcinoma cell line HepG2 (American Type Culture Collection, Manassas, VA, United States) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere composed of 95% air and 5% CO₂.

MTT assay for cell viability

TAM was dissolved in DMSO, then diluted with DMEM containing 10% FBS. HepG2 cells were grown in 96-well plates at a density of 1 × 10⁴ cells. Cell culture medium was replaced with media containing TAM (0.5, 1, 5, 10, 15, and 20 μM) or 0.2% DMSO for 72 h. The concentration of TAM was determined by MTT assay.

Cell treatment

OA was dissolved in DMSO then diluted with DMEM containing 10% BSA and filter sterilized (0.22 mm Millipore filter, Millipore, United States). Cells were seeded to appropriate density on 6-well plates, and TAM (10 μM) were applied along with OA (50 μM) to HepG2 cells for 72 h.

Biochemical detection

Rat blood samples were centrifuged at 3,500 rpm/min for 10 min at 4 °C and the supernatants were collected for the serum lipid analysis. The serum, liver, and cells TGs, total cholesterol (TC), serum nonesterified fatty acid (NEFA), and very low density lipoprotein (VLDL) were examined by corresponding assay kits (Jiancheng, Nanjing, China) and were carried out as per the manufacturer’s instructions. Serum aspartate aminotransferase (AST) and alanine transaminase (ALT) were detected with assay kits (Whitman Biotech, Nanjing, China) according to the manufacturer’s instructions to evaluate liver injury.

Oil red O staining

Liver slices from rats were made into oil red O staining frozen sections. All these manufactured sections were supplied by Jiangsu hospital of integrated Traditional Chinese and Western Medicine (Nanjing, China).

Western blotting

The total protein was extracted from 70 mg of rat liver sample using RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor (Beyotime Biotechnology, Jiangsu, China). The protein concentration was determined with the BCA protein assay reagent kit. The 40 μg of total protein was separated by 6–10% SDS-PAGE and was transferred to PVDF membrane (Millipore Corp, Bedford, MA). After blocking with Tris-buffered saline containing 5% bovine serum albumin for 1 h, the membranes were incubated overnight with targeted primary antibodies at 4 °C. After washing with TBST for 3 times, the PVDF membranes were incubated with HRP-conjugated secondary antibodies for 1.5 h and were developed using an enhanced chemiluminescence system (ECL; Thermo Scientific, United States).

Statistical analysis

All data are presented as the mean ± SEM. The statistical significant differences among groups were determined by 1-way ANOVA or Student’s t-tests using GraphPad Prism Software (La Jolla, CA, United States). The differences were considered to be statistically significant at P < 0.05.

Results

TAM aggravated hepatic steatosis in HFD rats

After 15 weeks of HFD, obese rats were treated with TAM for 14 days. As reported, TAM is an appetite suppressor,⁴ and we observed that the weight of rats treated with TAM decreased significantly compared to the HFD-control rats (Fig. 1A). However, TAM significantly increased the liver index and decreased the fat index in periuterine and ventral dorsal (Fig. 1B). The results of biochemical tests showed that TAM could notably increase the content of NEFA and VLD and decrease TC, HDL-C, and LDL-c in serum but had no significant effect on TG (Fig. 1C). Additionally, hepatic TC and TG significantly increased after TAM administration (Fig. 1D). The lipid droplets in rat liver were increased distinctly after TAM treatment (Fig. 1E). At the same time, we detected the level of serum transaminase to indicate the cell damage of liver. The results displayed that the levels of AST, ALT, and ALP in serum had no significant change after administration (Fig. 1F). These results evidenced that TAM had already caused fatty liver and blood lipid changes before it caused obvious liver damage in obese rats.

Fig. 1.

TAM aggravated hepatic steatosis in HFD rats. A) Body weight change of animals. B) Effects of TAM on liver, periuterine, and ventral dorsal fat coefficients of HFD rats. C) Effect of TAM on serum TC, TG, HDL-c, LDL-c, VLDL, and NEFA of rats among different groups. D) Effect of TAM on hepatic TC and TG levels. E) Histopathological view of rat livers via oil red O staining: (a) CD-control, (b) HFD-control, (c) HFD-TAM-L (4 mg/kg), (d) HFD-TAM-H (8 mg/kg); oil red O sections were photographed at original 200 × magnification. F) Serum AST, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured among groups. The rats were treated with TAM (4, 8 mg/kg) intragastrically or not after CD or HFD for 15 weeks. The data are the mean ± SEM (n = 6). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to the CD-control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the HFD-control group.

TAM increased lipid synthesis and liver lipid uptake, decreased TG export in HFD rat liver

To ascertain the underlying molecular mechanism of TAM-induced liver steatosis, we first measured the expression level of major lipoproteins. Western blotting results showed that the radio of m-SREBP1c/p-SREBP1c was rised obviously and the expression hydroxytrimethyl coenzyme a reductase (HMGCR) were significantly increased in TAM-treated rats (Fig. 2A), which are, respectively, the major transcriptional regulator of lipogenesis gene and the key enzyme for cholesterol synthesis. Meanwhile, the protein expression of ELOVL6, which catalyzes the carbon chain extension of long-chain fatty acids, and SCD1, which catalyzes the synthesis of monounsaturated fatty acids, increased markedly (Fig. 2B). However, the protein levels of ACC and fatty acid synthase (FASN) decreased dramatically (Fig. 2C). There was no significant difference between the CD-control and HFD-control group. These results indicated that TAM increased cholesterol synthesis, prolonged carbon chain, and desaturation of fatty acids in liver.

Fig. 2.

TAM increased lipid synthesis and liver lipid uptake, decreased TG export in HFD rat liver. A) the protein expression of p-SREBP1c, m-SREBP1c, and HMGCR were investigated by western blot. B) The protein expression of SCD1 and ELOVL6 were investigated by western blot. C) The protein expressions of ACC, p-ACC, and FAS were investigated by western blot. D) The protein expressions of CD36, MTTP, and LDLR were investigated by western blot. Band intensity was quantified by densitometry analysis. The rats were treated with TAM (4, 8 mg/kg) intragastrically or not after CD or HFD for 15 weeks. The results are the mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to the CD-control group. ^#P < 0.05, ^##P < .01, ^###P < 0.001 compared to the HFD-control group.

To determine the transport of lipid between blood and liver, we further detected the change of lipid transporters. Western blotting results showed that the protein expression of MTTP, which is responsible for liver lipid export, decreased significantly by TAM (Fig. 2D). TAM significantly upregulated the levels of protein CD36 and LDLR involved in liver lipid uptake (Fig. 2D). It was indicated that TAM increased liver lipid uptake and decreased TG export.

TAM downregulated the expression of SIRT1 in vivo and in vitro

SIRT1 is an important regulator in the process of lipid metabolism.^18,19 In this study, we analyzed the expression of SIRT1 protein levels by western blotting. As shown in Fig. 3A, SIRT1 was markedly downregulated in TAM-treated HFD rat liver.

Fig. 3.

TAM downregulated the expression of SIRT1 in vivo and in vitro. A) The protein expression of SIRT1 was investigated by western blot. B) Cells viability in different TAM concentrations. ^****P < 0.0001 compared to the control group. C) Intracellular TG levels of HepG2 cells. ^**P < 0.01 compared to the OA group. D) The protein expression of SIRT1 in HepG2 cells. ^*P < 0.05 compared to the OA group. Band intensity was quantified by densitometry analysis. The HepG2 cells were treated with corresponding concentration of TAM for 72 h. The rats were treated with TAM (4, 8 mg/kg) intragastrically or not after CD or HFD for 15 weeks. The results are the mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to the CD-control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the HFD-control group.

HepG2 cells was used to determine whether TAM could inhibit SIRT1 in hepatocytes. MTT assay was used to test the cell viability of HepG2 cells exposed to different concentrations of TAM for 72 h. As shown in Fig. 3B, the cell viability decreased by about 50% at 15 μM TAM. Consequently, we set up a TAM (5 and 10 μM) monotherapy group and a combination group with OA (50 μM) to establish a high-fat model. Intracellular TG levels were measured after 72 h dosing and showed no obvious increase of TG in the TAM alone group, while 10 μM of TAM significantly increased intracellular TG levels under the condition of OA (Fig. 3C). The expression of SIRT1 was decreased obviously after the treatment of TAM (Fig. 3D). These results indicated that the increase of lipid synthetase might be caused by the downregulation of SIRT1.

SRT1720 attenuated TAM-induced hepatic steatosis

In order to verify whether SIRT1 is a target of TAM-induced hepatic steatosis, SRT1720 (Fig. 4A), a selective agonist of SIRT1, was used for the further study. Compared with the control group, TAM significantly decreased the expression of SIRT1 in rat liver, while SRT1720 dramatically increased its expression (Fig. 4B). Oil red O staining of liver slices was to confirm whether SRT1720 improved liver lipid accumulation induced by TAM. TAM strikingly increased the quantity of lipid droplets in liver. Compared with TAM treatment group, the lipid droplets in SRT1720 low-dose combination group decreased significantly, while in high-dose combination group, it basically disappeared (Fig. 4C). Meanwhile, compared with TAM-alone treatment group, hepatic TG was reduced in a dose-dependent manner after combining with SRT1720, but TC only had a reduced trend without significance (Fig. 4D). Moreover, we also detected the levels of AST and ALT in rat liver to indicate liver injury. The results showed that TAM alone or in combination with SRT1720 did not cause liver dysfunction (Fig. 4E). These results suggested that SRT1720 could alleviate TAM-induced liver lipid accumulation without aggravating liver injury.

Fig. 4.

SRT1720 attenuated TAM-induced hepatic steatosis. A) The SRT1720 chemical structure. B) The protein expression of SIRT1 was investigated by western blot. C) Histopathological view of rat livers via oil red O staining: (a) control, (b) TAM, (c) TAM + SRT1720 (15 mg/kg), (d) TAM + SRT1720 (30 mg/kg); oil red O sections were photographed at original 200 × magnification. D) Hepatic levels of TC and TG in rats. E) Serum ALT, AST were measured among groups. The rats were treated with TAM (8 mg/kg) or TAM combined with SRT1720 (15 mg/kg or 30 mg/kg) after 15 weeks of HFD. The data are the mean ± SEM (n = 6). ^*P < 0.05, ^**P < 0.01 compared to the control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the TAM treatment group.

SRT1720 alleviated TAM-induced changes of lipid synthetases instead of lipid transporters

As the previous results showed, TAM affected lipid synthesis and transport in rat liver, we wonder whether SIRT1720 alleviates TAM-induced liver lipid accumulation by improving these 2 ways. First, we detected the expression of proteins related to lipid synthesis, and the results showed that SRT1720 reversed the upregulation of SREBP 1c and CHREBP protein levels by TAM (Fig. 5A). Next, we examined whether SRT1720 impacted the effect of TAM on liver lipid transporters. However, as shown in the Fig. 5B, SRT1720 could not reverse the remarkably decrease of serum TC, TG and HDL-c levels caused by TAM. The results also showed that SRT1720 could not regulate CD36 and MTTP protein expression (Fig. 5C). The above results manifested that SRT1720 could improve the effect of TAM on liver lipid synthesis instead of lipid transport, indicating SIRT1 may not be involved in lipid transport pathway.

Fig. 5.

SRT1720 alleviated TAM-induced changes of lipid synthetases instead of lipid transporters. A) The protein expressions of SREBP1c and CHREBP were investigated by western blot. B) Serum TC, TG, HDL-c of rats among different groups. C) The protein expressions of CD36 and MTTP were investigated by western blot. Band intensity was quantified by densitometry analysis. The rats were treated with TAM (8 mg/kg) or TAM combined with SRT1720 (15 or 30 mg/kg) after 15 weeks of HFD. The data are the mean ± SEM (n = 6). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to the control group. ^###P < 0.001 compared to the TAM treatment group.

SRT1720 alleviated TAM-induced hepatic steatosis by inhibiting phosphorylated-FoxO1/LXRα-SREBP1c pathway

In order to explore which regulator get involved in the inhibitory effect of SRT1720 on TAM-induced liver lipid synthesis, we detected the protein expression of phosphorylation levels of AMPK and FoxO1, which are known downstream proteins of SIRT1 which participated in lipid metabolism. TAM treatment increased the expression of hepatic phosphorylated-FoxO1 (p-FoxO1), while combined treatment with SRT1720 restored the level of p-FoxO1 protein (Fig. 6). Whereas there was no significant difference in phosphorylated AMPK expression in TAM with and without SRT1720 groups (Fig. 6), indicating that the SIRT1-AMPK pathway may not be involved in TAM-induced hepatic lipid accumulation. Furthermore, we detected the expression of LXRα, an important regulator for SREBP1c and CHREBP. The result showed TAM treatment increased LXRα expression, while SRT1720 inhibited this phenomenon (Fig. 6). These results suggested that FoxO1/LXRα signaling played an important role in TAM-induced liver fat accumulation through SIRT1 inhibition.

Fig. 6.

SRT1720 alleviated TAM-induced hepatic steatosis by inhibiting p-FoxO1/LXRα-SREBP1c pathway. The protein expressions of p-FoxO1/FoxO1, p-AMPK/AMPK and LXRα were investigated by western blot. Band intensity was quantified by densitometry analysis. The rats were treated with TAM (8 mg/kg) or TAM combined with SRT1720 (15 or 30 mg/kg) after 15 weeks of HFD. The data are the mean ± SEM (n = 6). ^*P < 0.05, ^***P < 0.001 compared to the control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the TAM treatment group.

Discussion

In this study, we elucidated that TAM induced hepatic steatosis by altering hepatic lipid synthesis and transport pathways and downregulated the expression of SIRT1. Administration of SIRT1 agonist SRT1720 could inhibit the promotion of lipid synthesis by TAM, alleviating liver lipid accumulation, which was based on its function of increasing FoxO1 transcription activity and enhancing LXRα expression.

Obesity is not only a well-established risk factor for breast cancer incidence and mortality^21,22 but is also an independent risk factor for TAM-associated lipid accumulation in the liver.²³ Most of the models currently used in correlational studies are normal animals,⁴ while we attempted to explore the molecular mechanism of TAM causing this side effect in the high-fat model. Wistar female rats fed with high fat for 15 weeks could successfully build obese model, which can slightly increases liver lipid, while TAM could enhance liver lipid accumulation (Fig. 1D and E). The serum transaminase level was basically unchanged (Fig. 1F), which indicated that the model was merely fatty liver without causing liver dysfunction. However, it was still uncertain whether patients with TAM-induced fatty liver accompany with elevated serum transaminases in clinical.²⁴ And then, we established a high-fat model with OA in vitro. The results showed that TAM was more likely to increase the lipid content of HepG2 cells in the presence of OA (Fig. 3C), which seemed to be related to its upregulation of intracellular lipid synthesis by inhibition of SIRT1 (Fig. 3D).

In the present study, we demonstrated that TAM treatment increased hepatic lipid synthesis and lipid transport to the liver, inversely decreased hepatic lipid export (Fig. 2), which is consistent with some previous reports.^10,14 Interestingly, our results showed that ACC and FASN were dramatically downregulated after TAM administration (Fig. 2C). We speculated this was due to the increased intrahepatic transport of exogenous fatty acids, leading to feedback inhibition of endogenous synthesis via pathways other than SREBP1c.

Although many reports regarding the effects of TAM on hepatic lipid metabolism, there are no unified treatment guidelines or effective drugs currently, so it is of great clinical significance and necessity to seek effective preventive drugs. Therefore, our aim was to find the promising therapeutic target of TAM-induced liver fat accumulation. To identify the target of TAM, we focused on SIRT1, which has been found to be downregulated in NAFLD patients.²⁵ Therefore, we explored the role of SIRT1 in TAM-induced lipid accumulation in the liver and whether it is a promising therapeutic target. Our results showed that TAM could downregulate the expression of SIRT1 in the liver of HFD rats (Fig. 3A). Administration of SRT1720, a SIRT1 agonist, could activate SIRT1 (Fig. 4B) and inhibit the stimulation by TAM on SREBP1c and CHREBP (Fig. 5A), important transcription factors that mediate hepatic lipid synthesis, thereby improving TAM-induced hepatic lipid accumulation (Fig. 4C and D). Nevertheless, SRT1720 failed to allay the lipid-lowering effect of TAM and its impact on the expression of MTTP and CD36 proteins (Fig. 5C), which are responsible for mediating hepatic lipid secretion and NEFA transport to the liver, respectively.^26,27 Subsequently, we detected the expression of CPT-1 and PPARα related to lipid oxidation, and the results showed that slightly upregulated the gene and protein expression of CPT-1 and PPARα, while TAM further aggravated these changes (Supplementary Fig. 1). This might be a compensatory result from the raised hepatic lipid, which illustrated that TAM hardly affected the oxidation of fatty acids under the HFD condition. Collectively, it was indicated that SIRT1 might mediate TAM-induced hepatic lipid accumulation by increasing hepatic lipid production, but it does not participate in the lipid transport and oxidation pathway. The effect of TAM on lipid transport needs further study.

As FoxO1, LXRα, and AMPK are key lipid regulators regulated by SIRT1 deacetylase activity,^28,29 we paid attention to them. Phosphorylation causes FoxO1 transferred from the nucleus to the cytoplasm, downregulating its transcriptional activity, while acetylation weakens the ability of FoxO1 to bind to homologous DNA sequences, which promotes the phosphorylation of FoxO1.³⁰ SIRT1 deacetylates FoxO1 to reduce its phosphorylation, thereby stabilizing FoxO1 in the nucleus and promoting its transcriptional activity.³¹ It has been covered that FoxO1 could efficaciously inhibit the expression of SREBP1c gene and the activity of CHREBP,^32,33 which was in line with our results that TAM increased its phosphorylation level and SREBP1c expression while inhibiting SIRT1 expression, whereas the phosphorylation of FoxO1 and the expression of SREBP1c were suppressed after SIRT1 was activated (Fig. 6). SREBP1c and CHREBP are also direct targets of LXR,^34,35 and deacylation of SIRT1 leads to increased expression of LXRs.¹⁵ Our study showed TAM significantly increased the hepatic expression of LXRα and SRT1720 restrained this phenomenon (Fig. 6). In addition, evidence demonstrated that decreased SIRT1 activity mitigated the activation of AMPKα so as to promote SREBP1 activities.³⁶ Conversely, we revealed that the liver AMPK expression between groups had no marked difference (Fig. 6). These results indicated that TAM regulated liver lipogenesis by reducing FoxO1 transcription activity and increasing LXRα expression through SIRT1 but did not affect AMPK activity.

Conclusion

Taken together, the results from this study suggested that TAM-induced liver lipid accumulation was result from the increase of hepatic lipid synthesis via the upregulation of LXRα/FoxO1-SREBP1c pathway. The inhibition of SIRT1 was a main reason for the activation of lipid synthetase which could be alleviated by SRT1720. Our research provides a new idea for solving the clinical problems of TAM-induced fatty liver disease which indicated SIRT1 might be a potential therapeutic target.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81773827), Scholar of the 14th batch of “Six Talents Peak” High-Level Talent Selection (No. SWYY-094), “Double First-Class” University Project (No. CPU2018GY33), the Postgraduate Research Practice Innovation Program of Jiangsu Province (No. KYCX19_0763), and Natural Science Foundation of Jiangsu Province (No. BK20210430).

Conflict of interest statement

The authors report no declarations of interests.

Authors’ contributions

Q.Y., Z.J., and L.Z. conceived of the study. M.L., Y.C., and X.C. performed data collection, data analysis, and produced the figured and scripts, with overall guidance from Q.Y., Z.J.. and L.Z. M.L. wrote preliminary draft, and other authors participated in the revision of the manuscript. Q.Y., Z.J., and M.L. deposited the data.