Abstract
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is widely considered as the most toxic and common carcinogen in the world. Exposure to TCDD causes liver lipid metabolism disorder and steatosis. However, the molecular mechanism of TCDD-induced liver lipid accumulation is not completely clear. Here, we found that a 5 μg/kg TCDD exposure for 3 weeks induced hepatocyte lipid deposition, increased CD36 expression, and promoted AMP-activated protein kinase (AMPK) ɑ phosphorylation in the liver of C57BL/6J mice. Furthermore, sulfo-N-succinimidyl oleate, a CD36 inhibiter, blunted TCDD-induced lipid deposition in Huh7 cells, confirming the critical role of CD36 in TCDD-induced hepatic steatosis. In terms of molecular mechanisms, we found that TCDD exposure increased reactive oxygen species (ROS) levels in Huh7 cells, which activated AMPK. Moreover, the activated AMPK upregulated CD36 expression. Therefore, we can see that the increase in CD36 expression induced by TCDD was regulated by ROS/AMPK/CD36 signaling pathway. Our results help to clarify the molecular mechanism of TCDD-induced hepatic steatosis.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most notorious environmental pollutants, accumulates readily in human population and wildlife animals due to its high lipophilicity and resistance to metabolism (Van den Berg et al., 1994). It has been shown that exposure to TCDD leads to various problems, including immunotoxicity, cancer, abnormal development, cardiovascular diseases, endocrine toxicity, and neurological disruption (Birnbaum, 1994; White and Birnbaum, 2009). TCDD toxicity is largely mediated by aryl hydrocarbon receptor (AhR), a cytosolic ligand-activated transcription factor (Mandal, 2005). Upon binding with TCDD, AhR translocates into the nucleus and dimerizes with the AhR nuclear translocator to regulate the transcriptional activation of target genes (Rijo et al., 2021).
Nonalcoholic fatty liver disease (NAFLD), occurring when there is excess lipid accumulation in the liver, affects approximately 25% of people, resulting in a considerable global disease burden (Powell et al., 2021). Epidemiological studies have shown that exposure to persistent organic pollutants can contribute to lipid metabolism disorders and increase the risk of NAFLD (Cave et al., 2010; Guo et al., 2018; Li et al., 2020). Moreover, TCDD increases the uptake and packaging of fatty acid, inhabits β-oxidation, and represses very-low-density lipoproteins (VLDL) assembly to impair lipid efflux to promote hepatic steatosis in mice (Nault et al., 2017). In addition, the incidences of liver cirrhosis in the TCDD-exposed population of Korean Vietnam War veterans and the Seveso population were much higher compared to unexposed people (Mocarelli et al., 1988; Yi et al., 2014).
However, the molecular mechanism underlying TCDD-induced excess lipid accumulation in the liver remains to be determined. CD36, a plasma membrane protein, mediates lipid uptake (Wang and Li, 2019) and plays a critical role in the pathogenesis of hepatic steatosis (Nassir et al., 2013). The CD36 expression in the liver is usually low under normal physiological conditions but significantly increased in patients and mice with hepatic steatosis (Inoue et al., 2005; Miquilena-Colina et al., 2011). Furthermore, hepatic steatosis was attenuated in CD36 knock-out mice (Wilson et al., 2016). Previous research also showed that TCDD-induced hepatic steatosis was associated with the upregulation of CD36, which transcription is promoted by AhR (Lee et al., 2010). Therefore, upregulation of CD36 expression may be involved in the pathogenesis of TCDD-induced liver lipid accumulation. Of interest, CD36 protein stability is regulated by ubiquitination modification. CD36 polyubiquitination mediated by Parkin promotes its protein degradation. AMP-activated protein kinase (AMPK), composed of a catalytic subunit (ɑ, AMPKɑ) and 2 regulatory subunits (β and γ) (Zaha and Young, 2012), upregulates the CD36 protein level by inhibiting Parkin mRNA stability in intestinal epithelial cells (Wu et al., 2020). However, little is known about the effects of AMPK in TCDD-induced hepatic steatosis.
This study examined the effects of the CD36 signaling pathway in TCDD-induced hepatic lipid metabolism disorders. It was found that CD36 was a key regulator of TCDD-induced liver lipid accumulation. Furthermore, the CD36 protein level was elevated by activated AMPK, which was activated by the increased reactive oxygen species (ROS) level induced by TCDD. This study provides a theoretical basis for further toxicological evaluation of TCDD and for coming up with scientific prevention and treatment measures for TCDD-induced hepatic steatosis.
Materials and methods
Reagents, antibodies, and plasmids
TCDD (F-402S, Cerilliant Co, Darmstadt, Germany) was dissolved in dimethylsulfoxide (DMSO; D2650, Sigma, Darmstadt, Germany); β-Actin antibody (AB0035, Abways Technology, Shanghai, China, Rabbit, 1:10 000); AMPKɑ antibody (#5831, Cell Signaling Technology, MA, Rabbit, 1:1000); phosphor-AMPKɑ (Thr172) (p-AMPKɑ) antibody (#2535, Cell Signaling Technology, MA, Rabbit, 1:1000); CD36 antibody (18836-1-AP, Proteintech, Hubei, China, Rabbit, 1:1000; CY5796, Abways Technology, Rabbit, 1:1000); Flag antibody (M20008, Abmart, Shanghai, China, Mouse, 1:1000); Goat antirabbit IgG H&L (ab6721, Abcam, Cambridge, UK, Goat, 1:10 000); Goat antimouse IgG H&L (A00160, GenScript, Nanjing, China, Goat, 1:3000); sulfo-N-succinimidyl oleate (SSO; HY-112847, MedChemExpress, Shanghai, China); Compound C (HY-13418, MedChemExpress); N-acetyl-L-cysteine (NAC; ST1546, Beyotime, Shanghai, China); Nile red (A606340-0001; BBI Life Sciences Corporation, Shanghai, China); Z-Leu-Leu-Leu-al (MG132, HY-13259, MedChemExpress, New Jersey).
CD36 was amplified from Huh7 cDNA using primers: forward: 5′-AACGGGCCCTCTAGACTCGAGATGGGCTGTGACCGGAAC-3′, reverse: 5′-TAGTCCAGTGTGGTGGAATTCTTTTATTGTTTTCGATCTGCATG-3′, and cloned into the Xho and EcoR1 restriction sites of pCDNA3.1-Flag vector using One Step Cloning Kit (C112, Vazyme, Jiangsu, China).
Animal treatments
All animal experiments were conducted according to the ethical guidelines of the Ethics Committee of Laboratory Animal Care and Welfare, Nantong University. Six- to 8-week-old C57BL/6 mice were purchased from the Laboratory Animal Centre of Nantong University. Ten male mice were housed under a 12 h light/dark cycle and accessed to standard rodent chow and water ad libitum. After 1 week of adaptive feeding, all mice were randomly divided into 2 groups and intraperitoneally injected with either vehicle (corn oil) or TCDD (5 µg/kg·bw/week) (Duval et al., 2017). After 3 weeks of TCDD exposure, the mice were sacrificed, and the livers were isolated and processed for further analysis. A small piece of liver tissue was fixed in 4% paraformaldehyde for histological analysis, and the remaining tissue was stored at −80°C. All operations were performed with full consideration for animal welfare.
Cell culture and stimulation
Huh7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco's modified eagle medium (DMEM, C11965500BT, Gibco, Beijing, China) with 10% fetal bovine serum (04-001-1A, Biological Industries, Beijing, China) at 37°C in 5% CO2. Huh7 cells were treated with different concentrations of TCDD (0, 0.1, 1, and 10 nM), which dissolved in DMSO, or different times of 10 nM TCDD or vehicle (DMSO) when the cell density reached 50%. Samples were collected 48 h after treatment.
Huh7 cells were pretreated with CD36-specific inhibitor SSO (50 μM) or vehicle (DMSO) for 1 h, then treated with 10 nM TCDD for 48 h to analyze the effect of CD36 in TCDD-induced fat deposition. Huh7 cells were pretreated with 5 μM AMPK inhibitor compound C or vehicle (DMSO) for 1 h and then added 10 nM TCDD for 48 h to determine the role of AMPK in TCDD-induced CD36 expression. Huh7 cells were pretreated with ROS inhibitor NAC (10 μM) or vehicle (water) for 1 h before 10 nM TCDD treatment for 48 h to explore the effect of ROS in TCDD-activated AMPK/CD36 signaling. Huh7 cells were cotransfected with CD36-Flag and 4Ub-HA. After 24 h, the cells were exposed to 10 nM TCDD for 48 h and treated with 10 μM MG132 for 6 h before sample collection to analyze the effect of TCDD on CD36 ubiquitination.
Western blot analysis
The proteins were collected from the corresponding cell or mice liver sample and extracted using RIPA Lysis Buffer (P0013B, Beyotime) added phenylmethanesulfonyl fluoride (ST505, Beyotime). The protein concentration was measured using the BCA kit (P0012, Beyotime). Protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to vinylidene fluoride (PVDF) membranes. The PVDF membrane was sealed with 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, and 0.05% Tween-20) at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C. The secondary antibodies were then incubated with the membrane for 1 h at room temperature. Finally, the protein bands were visualized using chemiluminescence HRP substrate (WBKLS0500, Millipore, Massachusetts) and detected using an enhanced chemiluminescence system (Tanon 5200 Multi, Tianneng Technology Co, Shanghai, China).
Hematoxylin and eosin staining
Liver tissue was fixed in a 4% paraformaldehyde solution and embedded in paraffin. Following, the tissue was cut into 3 μm sections. After deparaffination and rehydration, the slices were stained with hematoxylin for 5 min, washed with water for 5 min, transferred to 0.1% hydrochloric acid dissolved in ethanol for 30 s, washed again for 5 min with PBS, and immersed for 5 s in 95% ethanol. Then, place the slices in an eosin solution for 2 min before being wiped clean with water. After dehydration, the slices were sealed with neutral resin and observed under a light microscope (DM4000 B LED, Leica, Wetzlar, Germany).
Immunoprecipitation
Huh7 cells were lysed using 0.5% NP-40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, and pH 7.5) containing protease inhibitors for 30 min at 4°C. After centrifugation at 12 000 g for 5 min, the supernatant solution was incubated with anti-Flag M2 Affinity Gel (A2200, Sigma, Darmstadt, Germany) for 4 h at 4°C. After centrifugation at 3000 g for 3 min, removing the supernatant, the beads were washed 3 times with 0.5% NP-40 lysis buffer. Then, the beads were boiled in an SDS loading buffer for 10 min to isolate the target protein.
Oil red O staining
The liver tissue was cut into 6 μm sections in a cryostat according to standard protocol. Liver sections were stained with Oil red O solution (G1015, Servicebio, Hubei, China) for 8–10 min, washed with 60% isopropyl alcohol and pure water for 3–5 and 10 s, redyed with hematoxylin staining solution (G1004, Servicebio) and observed under a light microscope (DM4000 B LED, Leica). Quantitative analysis was performed using ImageJ software.
Nile red staining
Hu7h cells were cultured on glass coverslips in 12-well plates. After TCDD treatment, the cells were fixed with 4% paraformaldehyde for 30 min then rinsed with PBS before staining with 10 μM Nile red dissolved in DMSO for 30 min. After washing with PBS, the slides were blocked with 50% glycerol (diluted with PBS). Finally, the cells were observed by fluorescence microscopy (TI2-S-HU, Nikon, Tokyo, Japan) with a 530-nm excitation wavelength.
Real-time quantitative polymerase chain reaction
Total RNA was isolated from the liver using the Trizol reagent (9109, TakaRa, Dalian, China). Reverse transcription was performed using the cDNA synthesis kit (DRR047A, Threebio technology, Shanghai, China). Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Green PrimeScriptTM RT-PCR kit (QR0100, Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer's instructions. The β-actin (Actb) was chosen as the reference gene. The primers used in this experiment are shown in Table 1.
Primer information for qPCR analysis of the expression of target genes
| Gene names | Primer name sequence (5′–3′) |
|---|---|
| Actb | F: TTTCCAGCCTTCCTTCTTG |
| Actb | R: TGGCATAGAGGTCTTTACGG |
| Cd36 | F: TTTGGAGGCATTCTCATG |
| Cd36 | R: GATTTTGCTGCTGTTCTTT |
| Dgat1 | F: CCGATTCTTCCAAGGGAACTAT |
| Dgat1 | R: ATCGTAGTTGAGCACGTAGTAG |
| Mgl | F: GAATCCACGAAGCCTACC |
| Mgl | R: GAAGAATCGGACCTCTGC |
| Atgl | F: CAGAATGGCAAGAATGAAGA |
| Atgl | R: AAGGTAAAGAGCGGGTGA |
| Dgat2 | F: GCGAAAACAAGAATAAAGG |
| Dgat2 | R: ACCAGGAAGGATAGGACC |
| Fas | F: AGCCGTTGGGAGTGAAAG |
| Fas | R: GTCCCACAGAAGTCAGTAGGTC |
| Acc1 | F: AGCGATGTTCTGTTGGAC |
| Acc1 | R: AGACTCTGGGAATGTGGG |
| Hsl | F: GCAAAGATCAATCGGACC |
| Hsl | R: CACCTTCAGCGAGTAGCG |
| Mttp | F: TCTGGAACTACCAACGAGG |
| Mttp | R: TGGCTTTTGAACTGACACC |
| Fatp2 | F: TGTTCCGAGACGAGACGC |
| Fatp2 | R: CCATGAAGAGGGCTACGC |
| Cpt1 | F: CTCAGTGGGAGCGGATGTTT |
| Cpt1 | R: TGCTGTCTCTCATGTGCTGG |
| Acox1 | F: TGTGGAACCTGTTGGCCTCAAT |
| Acox1 | R: CTCGAAGATGAGTTCCGTGGC |
| Fatp5 | F: AGCTATACCAGCATGTCCGC |
| Fatp5 | R: ACCAGCCGTGACTTTACCAG |
| Gene names | Primer name sequence (5′–3′) |
|---|---|
| Actb | F: TTTCCAGCCTTCCTTCTTG |
| Actb | R: TGGCATAGAGGTCTTTACGG |
| Cd36 | F: TTTGGAGGCATTCTCATG |
| Cd36 | R: GATTTTGCTGCTGTTCTTT |
| Dgat1 | F: CCGATTCTTCCAAGGGAACTAT |
| Dgat1 | R: ATCGTAGTTGAGCACGTAGTAG |
| Mgl | F: GAATCCACGAAGCCTACC |
| Mgl | R: GAAGAATCGGACCTCTGC |
| Atgl | F: CAGAATGGCAAGAATGAAGA |
| Atgl | R: AAGGTAAAGAGCGGGTGA |
| Dgat2 | F: GCGAAAACAAGAATAAAGG |
| Dgat2 | R: ACCAGGAAGGATAGGACC |
| Fas | F: AGCCGTTGGGAGTGAAAG |
| Fas | R: GTCCCACAGAAGTCAGTAGGTC |
| Acc1 | F: AGCGATGTTCTGTTGGAC |
| Acc1 | R: AGACTCTGGGAATGTGGG |
| Hsl | F: GCAAAGATCAATCGGACC |
| Hsl | R: CACCTTCAGCGAGTAGCG |
| Mttp | F: TCTGGAACTACCAACGAGG |
| Mttp | R: TGGCTTTTGAACTGACACC |
| Fatp2 | F: TGTTCCGAGACGAGACGC |
| Fatp2 | R: CCATGAAGAGGGCTACGC |
| Cpt1 | F: CTCAGTGGGAGCGGATGTTT |
| Cpt1 | R: TGCTGTCTCTCATGTGCTGG |
| Acox1 | F: TGTGGAACCTGTTGGCCTCAAT |
| Acox1 | R: CTCGAAGATGAGTTCCGTGGC |
| Fatp5 | F: AGCTATACCAGCATGTCCGC |
| Fatp5 | R: ACCAGCCGTGACTTTACCAG |
Primer information for qPCR analysis of the expression of target genes
| Gene names | Primer name sequence (5′–3′) |
|---|---|
| Actb | F: TTTCCAGCCTTCCTTCTTG |
| Actb | R: TGGCATAGAGGTCTTTACGG |
| Cd36 | F: TTTGGAGGCATTCTCATG |
| Cd36 | R: GATTTTGCTGCTGTTCTTT |
| Dgat1 | F: CCGATTCTTCCAAGGGAACTAT |
| Dgat1 | R: ATCGTAGTTGAGCACGTAGTAG |
| Mgl | F: GAATCCACGAAGCCTACC |
| Mgl | R: GAAGAATCGGACCTCTGC |
| Atgl | F: CAGAATGGCAAGAATGAAGA |
| Atgl | R: AAGGTAAAGAGCGGGTGA |
| Dgat2 | F: GCGAAAACAAGAATAAAGG |
| Dgat2 | R: ACCAGGAAGGATAGGACC |
| Fas | F: AGCCGTTGGGAGTGAAAG |
| Fas | R: GTCCCACAGAAGTCAGTAGGTC |
| Acc1 | F: AGCGATGTTCTGTTGGAC |
| Acc1 | R: AGACTCTGGGAATGTGGG |
| Hsl | F: GCAAAGATCAATCGGACC |
| Hsl | R: CACCTTCAGCGAGTAGCG |
| Mttp | F: TCTGGAACTACCAACGAGG |
| Mttp | R: TGGCTTTTGAACTGACACC |
| Fatp2 | F: TGTTCCGAGACGAGACGC |
| Fatp2 | R: CCATGAAGAGGGCTACGC |
| Cpt1 | F: CTCAGTGGGAGCGGATGTTT |
| Cpt1 | R: TGCTGTCTCTCATGTGCTGG |
| Acox1 | F: TGTGGAACCTGTTGGCCTCAAT |
| Acox1 | R: CTCGAAGATGAGTTCCGTGGC |
| Fatp5 | F: AGCTATACCAGCATGTCCGC |
| Fatp5 | R: ACCAGCCGTGACTTTACCAG |
| Gene names | Primer name sequence (5′–3′) |
|---|---|
| Actb | F: TTTCCAGCCTTCCTTCTTG |
| Actb | R: TGGCATAGAGGTCTTTACGG |
| Cd36 | F: TTTGGAGGCATTCTCATG |
| Cd36 | R: GATTTTGCTGCTGTTCTTT |
| Dgat1 | F: CCGATTCTTCCAAGGGAACTAT |
| Dgat1 | R: ATCGTAGTTGAGCACGTAGTAG |
| Mgl | F: GAATCCACGAAGCCTACC |
| Mgl | R: GAAGAATCGGACCTCTGC |
| Atgl | F: CAGAATGGCAAGAATGAAGA |
| Atgl | R: AAGGTAAAGAGCGGGTGA |
| Dgat2 | F: GCGAAAACAAGAATAAAGG |
| Dgat2 | R: ACCAGGAAGGATAGGACC |
| Fas | F: AGCCGTTGGGAGTGAAAG |
| Fas | R: GTCCCACAGAAGTCAGTAGGTC |
| Acc1 | F: AGCGATGTTCTGTTGGAC |
| Acc1 | R: AGACTCTGGGAATGTGGG |
| Hsl | F: GCAAAGATCAATCGGACC |
| Hsl | R: CACCTTCAGCGAGTAGCG |
| Mttp | F: TCTGGAACTACCAACGAGG |
| Mttp | R: TGGCTTTTGAACTGACACC |
| Fatp2 | F: TGTTCCGAGACGAGACGC |
| Fatp2 | R: CCATGAAGAGGGCTACGC |
| Cpt1 | F: CTCAGTGGGAGCGGATGTTT |
| Cpt1 | R: TGCTGTCTCTCATGTGCTGG |
| Acox1 | F: TGTGGAACCTGTTGGCCTCAAT |
| Acox1 | R: CTCGAAGATGAGTTCCGTGGC |
| Fatp5 | F: AGCTATACCAGCATGTCCGC |
| Fatp5 | R: ACCAGCCGTGACTTTACCAG |
ROS detection
Hu7h cells were cultured in 96-well plates. After TCDD (10 nM) or vehicle (DMSO) treatment for 48 h, the ROS levels in Huh7 were detected using the ROS Assay kit (S0033S, Beyotime). Briefly, the cells were treated with 10 μM DCFH-DA dissolved in DMEM for 20 min at 37°C. Then, the cells were washed with DMEM 3 times. Finally, they were observed by fluorescence microscope (TI2-S-HU, Nikon) with a 488 nm excitation wavelength.
Statistical analysis
Data were presented as the mean ± standard error of the mean (SEM) of at least 3 biologically independent experiments. All data were analyzed with GraphPad Prism 8.0 (GraphPad, San Diego, California). The comparisons of 2 groups were analyzed using the 2-tailed unpaired Student’s t test. The comparisons of 3 or more groups were analyzed using the ordinary 1-way ANOVA, followed by Tukey's multiple comparisons post hoc test. p values < .05 were considered statistically significant.
Results
TCDD promotes hepatic steatosis in mice
To understand the effect of TCDD on fat accumulation in the liver, C57BL/6J mice were exposed to TCDD of 5 µg/kg·bw for 3 weeks. The liver histopathological morphology and fatty droplets were detected by hematoxylin and eosin (HE) and Oil red O staining, respectively. As indicated by HE staining, no significant changes in liver histopathology were observed in the control group. However, a greater extent of fat vacuolar degeneration was observed in the TCDD group (Fig. 1A). In line with this, liver lipid droplets were significantly higher in TCDD-exposed mice (Figures 1B and 1C) compared to the control group. These results show that TCDD exposure induced lipid accumulation in mouse livers.
TCDD induces liver lipid metabolism dysfunction in vivo. A, HE staining of mouse liver sections. B, Oil red O staining of mouse liver sections. C, Quantification of Oil red O staining positive area. D, qPCR analysis of lipid metabolism-related genes in mouse liver. E, Western blot analysis of CD36 protein expression and quantitative analysis of CD36/ACTB in mouse liver tissue. The results were presented as mean ± SEM, n = 3. Significance was calculated by 2-tailed unpaired Student’s t test (C, D, E). *p < .05 versus the vehicle group.
To explore the molecular mechanism of TCDD-induced hepatic steatosis, the expression of genes related to fatty acid synthesis, fatty acid β oxidation, fatty acid uptake, triglyceride synthesis, triglyceride decomposition, and triglyceride exportation were detected using qPCR. As shown in Figure 1D, TCDD had no significant effects on the expression of Acc1, Fas, Mgl, Acox1, Cpt1, Dgat1, Dgat2, Mttp, Fatp2, and Fatp5 (p > .05). However, while Atgl expression level was significantly enhanced, Hsl expression was inhibited. In addition, TCDD significantly enhanced the expression of fatty acid transport-related gene Cd36, with an about 2.7-fold increase (p < .05) (Figure 1D). Consistently, the Western blot results showed that TCDD significantly enhanced the expression of CD36 protein in mouse liver tissue (Figure 1E), indicating that TCDD may promote hepatic steatosis by upregulating CD36.
CD36 is involved in the accumulation of hepatic lipids caused by TCDD
To determine the role of CD36 in TCDD-induced mouse livers lipid metabolism disorder, Huh7 cells were treated with 50 μM CD36 specific inhibitor SSO for 1 h before 10 nM TCDD exposure. After 48 h, the lipid droplets were detected using Nile red staining. The results showed that TCDD promoted lipid accumulation, and SSO blunted the TCDD-induced lipid droplets in Huh7 cells (Figures 2A and 2B). Therefore, we speculate that CD36 is critical in TCDD-induced excess liver lipid accumulation in vitro.
CD36 regulates TCDD-induced lipid deposition in vitro. A, Huh7 cells were treated with SSO (50 μM) or vehicle (DMSO) before TCDD exposure, and lipid droplets were detected using Nile red staining. B, Quantitative analysis of mean fluorescence intensity for (A). The results were presented as mean ± SEM, n = 5. Significance was calculated by 1-way ANOVA with Tukey’s multiple comparisons test (B). *p < .05 versus the vehicle group. #p < .05 versus the TCDD-treated group. +p < .05 versus the SSO-treated group.
TCDD increases CD36 expression via downregulating its ubiquitin modification
In order to elucidate the molecular mechanism of the CD36 upregulation caused by TCDD, the CD36 protein expression was analyzed in Huh7 cells. The results showed that TCDD enhanced CD36 protein expression in a concentration-dependent manner and induced the highest expression level in 10 nM (Figure 3A). Hence, 10 nM TCDD was chosen to treat Huh7 cells for different durations. As shown in Figure 3B, CD36 protein expression was significantly increased when treated with 10 nM TCDD for 48 h. Consistent with previous reports, the CD36 mRNA expression in hepatocytes was also remarkably upregulated by treated with 10 nM TCDD for 48 h (Figure 3C). Previous studies have shown that CD36 protein level is regulated by ubiquitination. CD36 ubiquitination was measured via immunoprecipitation after TCDD exposure. The results confirmed that TCDD inhibited CD36 ubiquitination (Figure 3D). Consistent with this, compared with the MG132 treated group, the protein expression of CD36 was not significantly increased in the MG132 and TCDD added group, indicating TCDD-induced upregulation of CD36 was inhibited when the proteasome was inhibited (Figures 3E and 3F). These results demonstrate that TCDD can upregulate CD36 expression via changing its ubiquitin modification.
TCDD upregulates CD36 protein level in vitro. A, Huh7 cells were treated with TCDD at different concentrations (0, 0.1, 1, and 10 nM) for 48 h. The CD36 protein expression levels were analyzed by Western blot and quantitative analysis of CD36/ACTB. B, Huh7 cells were exposed to 10 nM TCDD for different times (0, 6, 12, 24, and 48 h), and the CD36 protein expression levels were measured by western blot and quantitative analysis of CD36/ACTB. C, qPCR analysis of Cd36 mRNA expression in Huh7 cells treated with 10 nM TCDD for 48 h. D, CD36-Flag and 4Ub-HA plasmids were cotransfected into Huh7 cells. Twenty-four hours after transfection, the cells were treated with TCDD for 48 h. The ubiquitination levels of CD36 were detected by immunoblot. E, Huh7 cells were exposed to 10 nM TCDD for 48 h, and 10 μM MG132 or vehicle (DMSO) were added for 6 h before sample collection. The CD36 protein expression levels were then measured by Western blot. F, Quantitative results of CD36/ACTB for (E). The results are presented as mean ± SEM, n = 3. Significance was calculated by 1-way ANOVA with Tukey’s multiple comparisons test (A, B, F) or 2-tailed unpaired Student’s t test (C). *p < .05 versus the vehicle group. ns, no significant difference, versus the MG132-treated group.
TCDD increases CD36 protein level via activating AMPK
AMPK plays a crucial role in lipid metabolism and regulates fatty acid uptake, lipid oxidation, and lipid absorption. It was further assessed whether AMPK activation was involved in TCDD-induced liver lipid accumulation. It was found that after TCDD exposure, the levels of phospho-AMPKɑ (Thr172) (p-AMPKɑ), an indicator of AMPK activation state (Zaha and Young, 2012), were significantly increased in mouse liver tissue (Figure 4A). Moreover, the levels of p-AMPKɑ were significantly upregulated after 12, 24, and 48 h of 10 nM TCDD treatment in Huh7 cells (Figure 4B). AMPK has been reported to increase the level of CD36 protein through inhibiting Parkin-mediated CD36 polyubiquitination (Wu et al., 2020). To examine whether the upregulation of CD36 induced by TCDD is related to increased AMPK activation, Huh7 cells were treated with AMPK inhibitor, compound C (1–5 μM), for 48 h; 5 μM compound C decreased p-AMPKɑ and CD36 protein expression (Figure 4C). However, inhibition of AMPK activation had an indistinctive effect on CD36 mRNA expression (Figure 4D), suggesting that AMPK may affect CD36 protein level through post-transcription regulation. Furthermore, the upregulation of CD36 induced by TCDD was blunted by inhibiting AMPK activation (Figs. 4E–G). These results indicate that the TCDD-induced increase of CD36 is regulated by AMPK activation.
TCDD increases CD36 protein level via activating AMPK. A, Western blot analysis of the AMPKɑ and p-AMPKɑ protein expression levels and quantitative results of p-AMPKɑ/AMPKɑ in mouse liver tissue. B, Huh7 cells were exposed to 10 nM TCDD for different durations (0, 6, 12, 24, and 48 h), and the AMPKɑ and p-AMPKɑ expression levels were measured by Western blot. C, Hu7h cells were treated with 1–5 µM compound C or vehicle (DMSO) for 48 h. The p-AMPKɑ, AMPKɑ, and CD36 protein expression levels were analyzed by Western blot. D, qPCR analysis of Cd36 mRNA expression in Huh7 cells treated with 5 µM compound C or vehicle. E, Hu7h cells were treated with 5 µM compound C or vehicle for 1 h before 10 nM TCDD exposure for 48 h, and the p-AMPKɑ, AMPKɑ, and CD36 protein expression levels were measured by Western blot. F, Quantitative results of CD36/ACTB for (E). G, Quantitative results of p-AMPKɑ/AMPKɑ for (E). The results are presented as mean ± SEM, n = 3. Significance was calculated by 2-tailed unpaired Student’s t test (A, D) or 1-way ANOVA with Tukey’s multiple comparisons test (B, F, G). *p < .05 versus the vehicle group. #p < .05 versus the TCDD-treated group.
TCDD activates AMPK/CD36 signal through upregulating ROS levels
ROS plays a central role in TCDD-induced toxicities (Lin et al., 2007; Palanisamy et al., 2015). Moreover, ROS has been reported to activate AMPK indirectly (Hinchy et al.,2018). In order to reveal the role of ROS in TCDD-induced AMPK activation, the p-AMPKɑ and CD36 protein expression were detected after adding NAC (10 μM), an active oxygen scavenger. The results showed that TCDD significantly increased the ROS content in Huh7 cells (Figures 5A and 5B). Moreover, TCDD-induced upregulations of p-AMPKɑ and CD36 protein expression were inhibited by adding NAC (Figs. 5C–E), implying that TCDD promotes AMPK/CD36 signaling by increasing ROS levels.
TCDD activates AMPK/CD36 signaling via upregulating ROS levels. A, Huh7 cells were treated with 10 nM TCDD or vehicle (DMSO) for 48 h, then added 10 μM DCFH-DA for 20 min. The ROS levels were observed by fluorescence microscope. B, Quantification of ROS mean fluorescence intensity for (A). C, Huh7 cells were treated with 10 µM NAC or vehicle (water) for 1 h before 10 nM TCDD exposure for 48 h. The p-AMPKɑ, AMPKɑ, and CD36 protein expression levels were measured by Western blot. D, Quantitative results of CD36/ACTB for (C). E, Quantitative results of p-AMPKɑ/AMPKɑ for (C). The results are presented as mean ± SEM, n = 3. Significance was calculated by 2-tailed unpaired Student’s t test (B) or 1-way ANOVA with Tukey’s multiple comparisons test (D, E). *p < .05 versus the vehicle group. #p < .05 versus the TCDD-treated group.
Discussion
TCDD exposure induces severe hepatotoxicity, including liver lipid accumulation, inflammation, and liver fibrosis (Fader et al., 2015). In this mouse model, liver lipid droplets were enhanced after treatment with 5 µg/kg·bw TCDD once a week for 3 weeks, and the CD36 protein and p-AMPKɑ levels were increased after TCDD exposure in vivo. Moreover, through examining the relationship between CD36 and AMPK in vitro, it was found that TCDD activated AMPK to increase CD36 protein levels. In addition, TCDD-induced AMPK activation was induced by ROS in Huh7 cells. These results suggest that the TCDD-induced hepatic steatosis was related to the activation of ROS/AMPK/CD36 signaling.
The mechanisms of TCDD-induced hepatic steatosis are complex. Although previous studies reveal TCDD suppresses β-oxidation, de novo fatty acid synthesis, and increases fatty acid packaging to triglyceride, the molecular mechanisms are still unclear. In this study, the mRNA expression of these biological processes related to key genes, Acc1, Fas, Acox, Cpt1, Dgat1, and Dgat2, were not statistically significant differences after TCDD treatment. Protein expression or other lipid metabolism-related genes may better reflect the effects of TCDD on hepatic steatosis. The mRNA expressions of Atal and Hsl, critical triglyceride hydrolysis enzymes, were significantly upregulated and downregulated. However, the mRNA expression of Mgl, another critical enzyme of triglyceride catabolism, was not significantly changed after TCDD exposure. In order to uncover the mechanism of TCDD on triglyceride breakdown, the protein expression or enzyme activity is required for further investigation.
CD36, known as a fatty acid translocase, promotes long-chain fatty acids uptake (Habets et al., 2007) and binds oxidized low-density lipoprotein (LDL), acetylated LDL, and the native lipoproteins HDL, LDL, and VLDL (Calvo et al., 1998; Jay et al., 2015) to regulate lipid metabolism. It has also been shown that CD36 knock-out can inhibit TCDD-induced liver steatosis (Lee et al., 2010), indicating that CD36 plays a critical role in TCDD-induced liver lipid metabolism disorders. As such, studying the mechanism of TCDD-induced CD36 upregulation is important to explain the hepatotoxicity of TCDD. Consistent with previous results (Duval et al., 2017), we found that Cd36 mRNA expression was upregulated by TCDD in vivo and in vitro. Moreover, the CD36 protein level was also increased by TCDD. Previous research demonstrated that TCDD increases CD36 transcription by binding to the TCDD-responsive elements in its gene promoter (Lee et al., 2010). However, it was unknown whether other molecules participated in the TCDD-induced CD36 expression. In this study, we found that CD36 was regulated by ubiquitination and AMPK activation induced by TCDD. These studies illustrate the complexity of the mechanisms of TCDD-induced CD36 upregulation and deserve further research.
AMPK is a critical metabolic regulator in energy homeostasis regulation (Huet et al., 2020). It reinforces fatty acid uptake by regulating CD36 signaling in intestinal epithelial cells and hepatocytes (Choi et al., 2017; Wu et al., 2020). Past studies have also shown that AMPK promotes CD36 expression via downregulating its polyubiquitination (Choi et al., 2017; Wu et al., 2020). Moreover, CD36 membrane translocation is enhanced by activating AMPK signaling (Samovski et al., 2012). In this study, we demonstrated that TCDD activated AMPK in vivo and in vitro, and inhibition of AMPK activation blunted TCDD-induced CD36 upregulation in Huh7 cells. To our knowledge, this is the first study to prove the role of AMPK in TCDD-exposed hepatocytes. In the traditional view, activation of AMPK reduces fat deposition by reinforcing lipolysis (Kim et al., 2016). However, TCDD-induced hepatic steatosis in mice was associated with the upregulation of AMPK activity. This inconsistency may be induced by the various effects of TCDD on lipid metabolism, including repressing β-oxidation, increasing dietary fat uptake, reducing adipose tissue LPL activity, and inhibiting lipoprotein export (Angrish et al., 2012; Brewster and Matsumura, 1984; Cholico et al., 2021).
AhR plays a critical role in TCDD-induced toxicity. The AhR-TCDD complex modulates target genes transcription by forming a heterodimer with AhR nuclear translocator (Rijo et al., 2021). Moreover, the activated AhR can regulate TCDD toxicity by the nongenomic pathway, such as TCDD-induced rapid inflammatory cellular response depending on AhR-activated calcium signaling (Matsumura, 2009). In addition, AhR can activate protein phosphorylation. TCDD stimulated Src kinase phosphorylation dependents on AhR in colon cancer cells (Xie et al., 2012), and the Src upregulates AMPK activation in cancer cells (Mizrachy-Schwartz et al., 2011). The selective AhR modulators SGA315 and SGA360 increase AMPK activity and restrict lipogenesis in Sebocytes (Muku et al., 2019). In this study, our results suggested that TCDD increased AMPK activity. However, the role of AhR in TCDD-mediated AMPK activity has not been elucidated, which deserves further investigation.
Oxidative stress is a common pathogenic mechanism for many diseases and plays a vital role in TCDD toxicity (Lin et al., 2007). TCDD-induced ROS upregulation induces DNA oxidative damage in breast cancer cells and promotes inflammatory responses (Lin et al., 2007; Palanisamy et al., 2015). TCDD also increases ROS in fetal hematopoietic stem cells, resulting in impaired long-term self-renewal function of these cells (Laiosa et al., 2016). Additionally, previous reports demonstrated that ROS could directly or indirectly activate AMPK (Hinchy et al., 2018; Zmijewski et al., 2010). Consistent with these findings, in our study, TCDD activated AMPK by increasing ROS in Huh7 cells, and scavenging ROS reversed TCDD-induced upregulations of p-AMPKɑ and CD36.
Several limitations of the current research should be noted and addressed in future studies. Firstly, the molecular mechanism by which AMPK regulates CD36 has not been elucidated in this study. Secondly, the role of ROS in TCDD-induced hepatic steatosis in the animal has not been studied in this article. Despite the above limitations, our results demonstrated that TCDD-induced hepatic steatosis is associated with the upregulation of p-AMPKɑ and CD36 in vivo, and through upregulating ROS level, TCDD activates AMPK to upregulate CD36 expression in vitro.
Conclusion
Our results provided insight into the molecular mechanisms of TCDD-induced hepatic steatosis. It showed that the upregulations of CD36 and p-AMPKɑ were related to TCDD-induced hepatic steatosis in vivo, and the TCDD-induced increase in CD36 was regulated by activating the ROS/AMPK signaling pathway in vitro. This study provides a novel target for treating TCDD-induced liver toxicity and a new reference for future studies on drug-induced liver injury.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The National Natural Science Foundation of China (21377062), a graduate innovation project in Jiangsu province (KYCX19_2083).
References
Author notes
Yewen Cong and Yujing Hong contributed equally to this study.
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