We have previously demonstrated a role for the aryl hydrocarbon receptor (AHR) in the attenuation of the cholesterol biosynthesis pathway. This regulation did not require that the AHR binds to its cognate response element. Based on these observations and other reports depicting a role for AHR in lipid metabolism, we chose to investigate the involvement of the receptor in the regulation of the fatty acid synthesis pathway in mice and humans. For this purpose, C57BL/6J, liver-specific transgenic DRE-binding mutant AhR (A78D-AhrTtrCreAlbAhrfx/fx) and CreAlbAhrfx/fx mice were treated with an AHR ligand, and hepatic mRNA expression levels of key fatty acid genes (e.g., Acaca, Fasn, Scd1) were measured. The basal levels of those genes were also compared between C57BL6/J and hepatic AHR-deficient mice, as well as between Ahb and Ahd congenic mice. To extend these results to humans, fatty acid gene expression in human cells were compared with AHR-silenced cells. In addition, primary human hepatocytes were treated with an AHR ligand to assess alterations in gene expression and fatty acid synthesis. These studies indicated that the AHR constitutively attenuates the expression of key fatty acid synthesis genes in the absence of binding to its cognate response element. In addition, activation of AHR led to further repression of the expression of these genes and a decrease in overall fatty acid synthesis and secretion in human hepatocytes. Based on our results, we can conclude that increased AHR activity represses fatty acid synthesis, suggesting it may be a future therapeutic target.
The aryl hydrocarbon receptor (AHR) is a ligand-activated basic helix-loop-helix Per-Arnt-Sim transcription factor. AHR is most known for binding 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), and it has since been found to have numerous other AHR ligands which mediate xenobiotic metabolism. Following agonist binding, the AHR translocates to the nucleus where it dimerizes with its partner Ah receptor nuclear translocator (ARNT) to bind dioxin response elements (DREs/XREs) of dioxin responsive genes such as CYP1A1, CYP1B1, and CYP1A2. On the other hand, the phenotype exhibited by mice specifically lacking the AHR links the receptor to many endogenous biological functions (Beischlag et al., 2008). This endobiotic role is further supported by the identification of several endogenous agonists for the receptor (Chiaro et al., 2008; Schroeder et al., 2010). In addition, activated AHR has been observed to interact with other transcription factors, namely the nuclear factor kappa-light-chain-enhancer (NF-κB), the retinoblastoma protein, and the estrogen receptor, thereby modulating their transcriptional regulation of target genes (Beischlag et al., 2008).
Microarray studies assessing modulation of gene expression following TCDD treatment in rodents revealed AHR involvement in the regulation of the circadian rhythm, fatty acid synthesis, cholesterol biosynthesis, and glucose metabolism genes in the liver. Among the genes altered in mice, results demonstrated a reduction in the mRNA expression of two critical lipogenic genes, Acaca and Fasn (Sato et al., 2008), although resulting protein levels were not examined. In humans, industrial exposure to dioxin has been associated with lipid metabolism disruption and high circulating cholesterol and triglyceride levels (Pelclova et al., 2002). AHR involvement in hepatotoxicity, hepatic steatosis, and the wasting syndrome has already been documented in rodents, further linking the receptor to hepatic lipid metabolism (Angrish et al., 2011; Beischlag et al., 2008).
Based on our recent findings showing the role of AHR in the regulation of the cholesterol biosynthesis pathway, as well as studies pointing to lipid metabolism disruption associated with AHR activation, we set out to examine the relationship between the AHR and the transcriptional regulation of enzymes involved in the fatty acid synthesis pathway. In particular, we focused on the enzymes which are key regulators of fatty acid synthesis, such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). ACC is the rate-limiting enzyme for the synthesis of long-chain fatty acids through the carboxylation of acetyl CoA into malonyl CoA, which is believed to be a player in the pathogenesis of insulin resistance in muscle tissue of obese patients (Ruderman et al., 1999). FAS is a critical enzyme responsible for the terminal catalytic step involving the synthesis of saturated fatty acids (SFAs). FAS inhibitors have been proven to be effective anti-obesity agents in rodents (Loftus et al., 2000). The major product of FAS is palmitate, a 16-carbon long-chain fatty acid (C16:0) which can undergo elongation by FAS III-forming stearate (C18:0); this product is a key substrate for the rate-limiting enzyme SCD1. SCD1 catalyzes the conversion of SFAs into the mono unsaturated fatty acids such as oleate (C18:1). This makes the SCD1 a critical mediator of fatty acid synthesis and a key regulatory factor of body adiposity. Mice with targeted disruption of SCD1 exhibit a decrease in the content of their hepatic triglycerides and cholesterol esters (Miyazaki et al., 2001) coupled with very low levels of triglycerides in the very low-density lipoprotein and low-density lipoprotein fraction compared with their wild-type (WT) counterparts. In addition, they show enhanced insulin sensitivity in liver, adipose tissue, and skeletal muscle (Gutierrez-Juarez et al., 2006; Rahman et al., 2005). Although leptin-deficient ob/ob mice are obese and accumulate high amounts of lipid in the liver, SCD1 deficiency completely corrects the hypometabolic phenotype and hepatic steatosis in these mice (Cohen et al., 2002). Conversely, elevated SCD1 levels in humans positively correlate with higher plasma triglycerides, increased BMI, and high insulin levels (Attie et al., 2002).
We have previously demonstrated the constitutive endogenous regulation of the hepatic cholesterol biosynthesis pathway by AHR in mice and humans (Tanos et al., 2012). The fact that both cholesterol and lipid synthesis genes are regulated by a common transcription factor family (i.e., SREBP1/2) led us to test whether fatty acid synthesis genes are regulated by AHR in a similar manner. For this purpose, we used an AHR agonist, β-naphthoflavone (BNF), to activate the receptor in C57BL/6J and a transgenic mouse model expressing a hepatic DRE-binding mutant AHR (A78D-AHR). As seen with the cholesterol synthesis genes, activation of AHR was able to attenuate the expression of lipogenic genes Acaca, Fasn, and Scd1, through a DRE-independent mechanism. A similar repression was observed in primary human hepatocytes treated with BNF, accompanied by lower fatty acid production and secretion. Finally, we also demonstrated that AHR plays a constitutive role in the repression of fatty acid synthesis in mice and humans.
MATERIALS AND METHODS
Cell culture. Hep3B cells, a human hepatoma-derived cell line, were maintained in α-minimal essential medium (Sigma, St Louis, MO), supplemented with 10% fetal bovine serum (HyClone Labs, Logan, UT), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma) in a humidified incubator at 37°C, with an atmospheric composition of 95% air/5% CO2.
Primary human hepatocytes. Primary human hepatocytes were obtained from the University of Pittsburgh, through the Liver Tissue Cell Distribution System, NIH Contract #N01-DK-7-0004/HHSN267200700004C. Culture details have been reported previously (Tanos et al., 2012). About 48h following Matrigel (BD Biosciences, San Jose, CA) addition, cells were exposed to BNF (10µM) or carrier solvent for 48h. BNF treatment was replenished every 12h.
RNA isolation and reverse transcription. RNA samples were isolated from cell cultures and mouse livers using TRI Reagent according to the manufacturer’s specifications (Sigma Aldrich). cDNA was generated using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA).
Quantitative PCR. PerfeCTa SYBR Green SuperMix for iQ (Quanta Biosciences, Gaithersburg, MD) was used and analysis was conducted using MyIQ software (Bio-Rad Laboratories, Hercules, CA). Sequences of primers designed to detect specific mRNA levels are given in Supplementary table 1.
Gene silencing. AHR and ARNT levels were decreased in Hep3B cells using siRNA as previously described (Tanos et al., 2012). RNA and protein samples were isolated 72h postelectroporation.
Mice. Transgenic A78D-AhrTtr CreAlbAhrfx/fx mice were generated as described previously (Tanos et al., 2012). Transgenic CreAlbAhrfx/fxwas a kind gift from Christopher Bradfield, University of Wisconsin. Congenic Ahd and WT mice (C57BL/6J) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed on corncob bedding in a temperature- and light-controlled facility and given access to food and water ad libitum. Mice were maintained in a pathogen-free facility and treated with humane care with approval from the Animal Care and Use Committee of the Pennsylvania State University. Adult (10–12 weeks) female mice of different genotypes were injected intraperitoneally with BNF at 50mg/kg dissolved in corn oil or with corn oil alone. After 5h mice were sacrificed and livers harvested.
Protein and RNA preparation. Mouse liver samples were collected and frozen immediately in liquid nitrogen before storage at −80°C; RNA was isolated using TRI Reagent (Sigma). Livers and Hep3B extracts were prepared as reported previously (Tanos et al., 2012).
Immunoblot analysis. Mouse liver and cell extracts were resolved on 8% SDS-tricine polyacrylamide gels. Proteins were transferred to polyvinylidene floride membrane and detected using the AHR antibody RPT1 (Thermo Scientific), ACC, SCD1 antibodies (Santa Cruz Biotechnology), or FAS antibody (BD Biosciences). All antibodies were visualized using the appropriate biotinylated secondary antibody and [I125]streptavidin followed by autoradiography. Relative protein levels were determined using image J software obtained from the National Institutes of Health.
Triglyceride and GC-MS analysis. The level of triglycerides in serum from mice was determined using a LabAssay triglyceride analysis kit (Wako Pure Chemical Industries, Ltd.). Primary human hepatocytes were treated for 48h with BNF (10 µM), which was replenished at 12h intervals. Lipids were extracted from the media and analyzed by GC-MS as described (Tanos et al., 2012). Heptadecanoic acid (Sigma) was used as an internal standard. Values represent an average of three wells run in duplicate.
Statistical analysis. Data were analyzed using t-test in GraphPad Prism (v.5.01) software to determine statistical significance between treatments. p values <0.05 were considered statistically significant (*p < 0.05; **p < 0.01;***p < 0.001).
AHR Regulates Fatty Acid Synthesis Gene Expression in C57BL/6J Mice
As a first approach to examine the regulation of lipogenic genes by AHR, we treated C57BL/6J mice with the AHR agonist BNF for 5h. The agonist BNF was chosen because of our prior experience with this ligand and its apparent nontoxicity (Hollingshead et al., 2006). The dose of 50mg/kg was utilized because it yields a near maximal induction of CYP1A1 in the liver. The time point was chosen because longer treatment of mice with a strong AHR agonist leads to downregulation of AHR levels in the liver. In our preliminary 24-h treatment experiments, a lower level of a repression of fatty acid synthesis gene expression was observed. Hepatic mRNA analysis revealed a significant and collective attenuation of the expression levels of the three rate-limiting enzymes of fatty acid synthesis Acc, Fas, and Scd1. Cyp1a1 mRNA levels were determined as a positive control to illustrate the level of receptor activation (Fig. 1A). In contrast, BNF failed to alter mRNA levels of the transcription regulator SREBP1c, the master regulator of fatty acid synthesis. Given the short time of treatment, these observations might suggest that AHR is directly influencing the regulation of the three genes examined at the promoter level.
Regulation of Key Genes Involved in Fatty Acid Synthesis by the AHR Is DRE-independent
Our previous work, as well as that of other laboratories, has demonstrated the ability of AHR to function through multiple mechanisms other than DRE-mediated activity (Beischlag et al., 2008; Patel et al., 2009; Tanos et al., 2012); for this reason, we opted to test whether this regulation is DRE specific. We have established a suitable transgenic mouse model to test such a hypothesis. Using a hepatocyte-specific transtherytin promoter, a transgenic mouse (A78D-AhrTtrCreAlbAhrfx/fx) was designed to express the DRE-binding mutant form of the receptor (A78D-AHR) in the liver, which also has the endogenous AHR gene deleted. In response to BNF treatment, these transgenic mice showed a similar marked attenuation in the expression levels of the genes of interest (Fig. 1B), establishing the ability of the receptor to repress Scd1 and Fas expression independent of DRE-mediated activity in hepatocytes. As shown in Figure 1C, these repressive events were not observed in CreAlbAhrfx/fx mice which lack AHR expression in hepatocytes, demonstrating that the observed effect exerted by BNF in C57BL/6 and A78D-AhrTtrCreAlbAhrfx/fx mice is mediated through the AHR.
AHR Constitutively Represses the Expression of Fatty Acid Synthesis Genes and Triglyceride Levels in Mice
Mice with a targeted global deletion of AHR exhibit a set of phenotypes, including decreased liver weight and compromised immune and reproductive systems, which points to an endogenous role for the receptor independent of its xenobiotic function. In order to probe for a constitutive role of the receptor in fatty acid synthesis, we looked for differences in the basal expression level of hepatic fatty acid genes between C57BL/6J and CreAlbAhrfx/fx mice. Remarkably, mRNA expression levels of the Acaca, Fasn, and Scd1 genes were significantly lower when AHR was present, suggesting a constitutive activity of the receptor in the repression of those genes (Fig. 2A). Serum triglyceride levels were also assessed in C57BL/6J and CreAlbAhrfx/fx mice; the results revealed a significant elevation in triglyceride levels in CreAlbAhrfx/fx mice (Fig. 2B).
Several AHR endogenous ligands (Chiaro et al., 2008; Schroeder et al., 2010) have been identified in our laboratory and shown to modulate the receptor’s activity. To investigate the presence of an endogenous ligand exhibiting agonist activity, we compared the mRNA levels of fatty acid genes in mice expressing the Ahd allele (low ligand affinity) in a congenic C57BL6/J background and C57BL6/J mice, which express the Ahb allele (high ligand affinity). Relatively higher mRNA levels were noted in Ahd congenic mice especially for SCD1 (Fig. 2C), suggesting a role for an endogenous ligand in the constitutive regulation of those genes by AHR.
AHR Constitutively Represses the Expression of Fatty Acid Synthesis Genes in a Human Hepatoma Cell Line
To examine whether AHR-mediated regulation of the fatty acid synthesis genes is relevant in humans, we largely ablated AHR levels in Hep3B cells, a human hepatoma cell line, using siRNA, and measured levels of fatty acid synthesis gene expression using quantitative real-time PCR and Western blot. We observed higher levels of the three fatty acid synthesis gene transcripts (Fig. 3A) and their respective proteins (Figs. 3B and 3C) upon repression of AHR levels. These results are consistent with the data obtained in mice, indicating the AHR constitutively represses the expression of fatty acid synthesis genes in humans as well.
AHR Represses Fatty Acid Synthesis Gene Expression Independent of ARNT Expression
DRE-mediated AHR activity is known to take place through heterodimerization with its partner ARNT, which renders the receptor capable of binding its cognate response element in the promoter region of target genes. Figure 3D shows that unlike AHR, ablation of ARNT expression in Hep 3B cells has no effect on the expression of the fatty acid synthesis genes. Constitutive CYP1A1 mRNA levels were almost totally repressed by ARNT siRNA treatment, demonstrating that ARNT expression was ablated. This further supports the idea that AHR is capable of mediating regulation of the fatty acid synthesis genes in the absence of heterodimer formation and thus independent of DRE-binding in humans.
AHR Is Capable of Suppressing Fatty Acid Synthesis Gene Expression in Primary Human Hepatocytes
Because AHR has been shown to behave differently in humans than in mice, we sought to examine whether activation of the receptor by an exogenous ligand could also modulate the expression of fatty acid synthesis genes in humans and if this in turn translates to attenuation of fatty acid synthesis. Similar to our results in rodents, ACACA, FASN and SCD1 mRNA expression levels were markedly reduced following AHR activation in primary human hepatocytes accompanied by unchanged SREBP1c mRNA levels (Fig. 4A). This experiment was performed after 48h exposure to obtain decreases in both mRNA and protein. These enzymes require a considerable amount of time to turnover, which then would lead to changes in fatty acid secretion.
AHR Activation Represses Fatty Acid Secretion From Primary Human Hepatocytes
Analysis of fatty acid composition by GC-MS of lipids extracted from the primary human hepatocyte media revealed that BNF-induced alterations in several fatty acid products such that the secretion of saturated (myristic acid C14:0, palmitic acid C16:0, and stearic acid C18:0) and monounsaturated (palmitoleic acid C16:1, oleic acid C18:1) fatty acids were significantly reduced (Fig. 4B). Thus, these results suggest that lower enzyme levels led to reduced fatty acid synthesis and subsequent reduced fatty acid secretion. These results establish a functional consequence to the observed repression of fatty acid synthesis gene expression observed in primary human cells after AHR activation. To ensure that the decrease in fatty acid secretion observed was not due to cellular toxicity, the level of lactate dehydrogenase was assessed in the cell medium and no difference was seen (Fig. 4C).
Previous studies examining the role of AHR in the control of the lipogenic pathway have focused on the effect of TCDD exposure in rodents (Fletcher et al., 2005; Sato et al., 2008). However, there have been no investigations into the constitutive role of the AHR in rodents or the effect of AHR activation on de novo lipogenesis in humans. Here, we have reported that the AHR-mediated repression of three rate-limiting enzymes of the fatty acid synthesis pathway, ACC, FAS and SCD1, both in mice and humans, which leads to a subsequent decrease in fatty acid production. Furthermore, we demonstrated that AHR involvement in this regulation occurs in a DRE-independent manner. Indeed, AHR involvement in fatty acid metabolism is consistent with both the wasting syndrome, characterized by body fat loss, and the hypoinsulinemia observed in TCDD-treated animals (Beischlag et al., 2008), as well as reports describing disruption of fatty acid and triglyceride levels in workers exposed to dioxin (Pelclova et al., 2002). Microarray and serum data from rats injected with TCDD have shown repression of Fasn expression and triglyceride levels (Fletcher et al., 2005). Paradoxically, a mouse study showed repression of Acaca on microarrays while Fasn transcript levels were increased in liver (Sato et al., 2008). In addition, TCDD exposure in mice for 24h has revealed a twofold increase in both Scd1 mRNA and protein levels and that Scd1 plays a role in TCDD-mediated steatosis (Angrish et al., 2011). One possible explanation for this result is a high dose of TCDD likely leads to a dramatic reduction in total AHR levels and in cytoplasmic AHR in hepatocytes. This in turn would limit the ability of the AHR which can enter into protein–protein interactions, leading to a result similar to what is seen when the AHR is knockdown with siRNA. Indeed, a reduction in receptor levels in Hep3B cells results in an increase in SCD1, as shown in Figure 3. It is also important to note here that previous studies exploring a role for AHR in fat metabolism and transport involved the use of TCDD. Although this compound is known to be a highly potent ligand for AHR, multiple reports argue for an independent role for this compound. In fact, dioxin alone has been shown to bind and disrupt the three-dimensional structure of lipoprotein molecules, rendering them incapable of binding to their cognate receptor (Arehart et al., 2004) and attenuating insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes, all in an AHR-independent manner (Hsu et al., 2010). Hence, the development of insulin resistance, hepatic steatosis, and elevated triglyceride levels following exposure to TCDD might be occurring at least in part through a mechanism independent of AHR. In addition, the highly toxic properties of TCDD complicate the interpretation of gene expression data.
There is a marked diurnal rhythm in the rate of fatty acid synthesis in the liver of lean mice (Hems et al., 1975). Likewise, AHR is known to exhibit rhythmic expression with its highest peak levels coinciding with the lowest fatty acid synthesis rates (Shimba and Watabe, 2009). Moreover, the rise in fatty acid synthesis at the beginning of the dark cycle occurs after the decrease in AHR levels. This pattern seems even more pronounced in genetically obese (ob/ob) mice (Hems et al., 1975). Considering that liver AHR levels ebb and flow according to a circadian rhythm which inversely correlates with fatty acid synthesis, our current results would support the idea of a constitutive role for AHR in the repression of fatty acid synthesis both in mice and humans.
The sterol element binding protein 1c (SREBP1c) is the central regulator of the transcriptional activity of genes involved in fatty acid biosynthesis. Our results indicate a downregulation of those genes in AHR ligand-treated mice and primary human hepatocytes and their increased expression in the absence of AHR, suggesting a possible interaction between AHR and SREBP1c. Indeed a recent report demonstrated a physical interaction between the two factors in murine T cells, with a possible inhibition of SREBP1c activity by the presence of the AHR (Cui et al., 2011). In addition, we have previously demonstrated that AHR constitutively regulates the transcriptional targets of SREBP2 (Tanos et al., 2012) in a similar fashion, making it highly possible that the AHR interacts and/or affects the activity of both SREBP family members. However, one should not dismiss a possible involvement of other factors implicated in the expression of fatty acid synthesis genes, such as PPARα and LXR (Jump et al., 2005). Clearly, further studies are needed to precisely define the mechanism of AHR-mediated repression of fatty acid synthesis.
Recent studies have extensively focused on the promise of fatty acid synthesis enzyme inhibitors in the treatment of metabolic syndrome, hepatic steatosis, and diabetes. Inhibitors of ACC were effective in reducing plasma triglyceride levels and body fat mass, inhibiting fatty acid biosynthesis, stimulating fatty acid oxidation, and, most importantly, improving insulin sensitivity in rodents (Harwood, 2004). On the other hand, experimental results from animals treated with an FAS inhibitor showed significant reduction in food intake and adipose tissue (Zhao et al., 2011). Finally, SCD1 inhibitors have been tested in mice and revealed protection against obesity, hepatic steatosis, and improved insulin sensitivity, along with preclinical antidiabetic and antidyslipidemic efficacy (Oballa et al., 2011). However, despite the fact that the effects of SCD1 inhibitors are quite striking, they do not always result in lower plasma triglyceride levels as seen with ACC inhibition (Flowers et al., 2006). Our present investigation clearly demonstrates that AHR has a major impact on the lipogenesis pathway by directly regulating the expression of three rate-limiting enzymes. Although to the best of our knowledge no study has attempted to inhibit more than one enzyme at a time, the above observations suggest that reducing the level of a number of FA regulatory enzymes might be a particularly effective treatment for fatty liver disease. Such an approach would also help prevent the excessive depletion of any key cell–signaling fatty acid molecule and/or the accumulation of any metabolite resulting from a single-enzyme targeting strategy. Furthermore, the use of chemical inhibitors of a given ubiquitous enzyme (e.g., SCD1) may have an effect in every tissue and cell type. In contrast, an AHR ligand will exert its activity dependent on the expression pattern of AHR in various tissues. The AHR is expressed at a relatively high level in liver compared with many tissues (e.g., brain). In addition, the AHR exhibits very low levels in mature adipose tissue, and there is almost no AHR expression in skeletal muscle. Thus, activation of the AHR will have a more targeted effect on liver fatty acid synthesis.
Our results are the first to establish the role of the AHR in the hepatic regulation of three rate-limiting hepatic fatty acid synthesis genes, as well as the role of the receptor and the effect of its activation on the production of fatty acid synthesis products in primary human hepatocytes. Moreover, we demonstrate the complexity of AHR activation by an agonist and its effect on the expression of a variety of genes through DRE and non-DRE mechanisms. In conclusion, evidence of DRE-independent AHR regulation of the lipid biosynthesis pathways shows promise for the therapeutic use of the AHR for the treatment of nonalcoholic fatty liver disease, obesity, and cancer.
National Institutes of Health (ES004869 and ES019964) and Bristol-Myers Squibb fellowship.
The authors thank Dr Stephan C. Strom and Dr Curtis J. Omiecinski for the primary human hepatocytes. We thank Marcia H. Perdew for excellent editorial assistance. We also thank Dr Christopher Bradfield for providing the Ahrfx/fxCreAlb mice.