HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

Impact of various high fat diets on gene expression and the microbiome across the mouse intestines.

High fat diets (HFDs) have been linked to several diseases including obesity, diabetes, fatty liver, inflammatory bowel disease (IBD) and colon cancer. In this study, we examined the impact on intestinal gene expression of three isocaloric HFDs that differed only in their fatty acid composition-coconut oil (saturated fats), conventional soybean oil (polyunsaturated fats) and a genetically modified soybean oil (monounsaturated fats). Four functionally distinct segments of the mouse intestinal tract were analyzed using RNA-seq-duodenum, jejunum, terminal ileum and proximal colon. We found considerable dysregulation of genes in multiple tissues with the different diets, including those encoding nuclear receptors and genes involved in xenobiotic and drug metabolism, epithelial barrier function, IBD and colon cancer as well as genes associated with the microbiome and COVID-19. Network analysis shows that genes involved in metabolism tend to be upregulated by the HFDs while genes related to the immune system are downregulated; neurotransmitter signaling was also dysregulated by the HFDs. Genomic sequencing also revealed a microbiome altered by the HFDs. This study highlights the potential impact of different HFDs on gut health with implications for the organism as a whole and will serve as a reference for gene expression along the length of the intestines.

Impact of Various High Fat Diets on Gene Expression and the Microbiome Across the Mouse Intestines.

High fat diets (HFDs) have been linked to several diseases including obesity, diabetes, fatty liver, inflammatory bowel disease (IBD) and colon cancer. In this study, we examined the impact on intestinal gene expression of three isocaloric HFDs that differed only in their fatty acid composition - coconut oil (saturated fats), conventional soybean oil (polyunsaturated fats) and a genetically modified soybean oil (monounsaturated fats). Four functionally distinct segments of the mouse intestinal tract were analyzed using RNA-seq - duodenum, jejunum, terminal ileum and proximal colon. We found considerable dysregulation of genes in multiple tissues with the different diets, including those encoding nuclear receptors and genes involved in xenobiotic and drug metabolism, epithelial barrier function, IBD and colon cancer as well as genes associated with the microbiome and COVID-19. Network analysis shows that genes involved in metabolism tend to be upregulated by the HFDs while genes related to the immune system are downregulated; neurotransmitter signaling was also dysregulated by the HFDs. Genomic sequencing also revealed a microbiome altered by the HFDs. This study highlights the potential impact of different HFDs on gut health with implications for the organism as a whole and will serve as a reference for gene expression along the length of the intestines.

HNF4α isoforms: the fraternal twin master regulators of liver function.

In the more than 30 years since the purification and cloning of Hepatocyte Nuclear Factor 4 (HNF4α), considerable insight into its role in liver function has been gleaned from its target genes and mouse experiments. HNF4α plays a key role in lipid and glucose metabolism and intersects with not just diabetes and circadian rhythms but also with liver cancer, although much remains to be elucidated about those interactions. Similarly, while we are beginning to elucidate the role of the isoforms expressed from its two promoters, we know little about the alternatively spliced variants in other portions of the protein and their impact on the 1000-plus HNF4α target genes. This review will address how HNF4α came to be called the master regulator of liver-specific gene expression with a focus on its role in basic metabolism, the contributions of the various isoforms and the intriguing intersection with the circadian clock.

Multiple roles and regulatory mechanisms of the transcription factor HNF4 in the intestine.

Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors.

Diet high in linoleic acid dysregulates the intestinal endocannabinoid system and increases susceptibility to colitis in Mice.

Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro. These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

Hepatic circadian and differentiation factors control liver susceptibility for fatty liver disease and tumorigenesis.

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, and the most common primary liver malignancy to present in the clinic. With the exception of liver transplant, treatment options for advanced HCC are limited, but improved tumor stratification could open the door to new treatment options. Previously, we demonstrated that the circadian regulator Aryl Hydrocarbon-Like Receptor Like 1 (ARNTL, or Bmal1) and the liver-enriched nuclear factor 4 alpha (HNF4α) are robustly co-expressed in healthy liver but incompatible in the context of HCC. Faulty circadian expression of HNF4α- either by isoform switching, or loss of expression- results in an increased risk for HCC, while BMAL1 gain-of-function in HNF4α-positive HCC results in apoptosis and tumor regression. We hypothesize that the transcriptional programs of HNF4α and BMAL1 are antagonistic in liver disease and HCC. Here, we study this antagonism by generating a mouse model with inducible loss of hepatic HNF4α and BMAL1 expression. The results reveal that simultaneous loss of HNF4α and BMAL1 is protective against fatty liver and HCC in carcinogen-induced liver injury and in the "STAM" model of liver disease. Furthermore, our results suggest that targeting Bmal1 expression in the absence of HNF4α inhibits HCC growth and progression. Specifically, pharmacological suppression of Bmal1 in HNF4α-deficient, BMAL1-positive HCC with REV-ERB agonist SR9009 impairs tumor cell proliferation and migration in a REV-ERB-dependent manner, while having no effect on healthy hepatocytes. Collectively, our results suggest that stratification of HCC based on HNF4α and BMAL1 expression may provide a new perspective on HCC properties and potential targeted therapeutics.

Palmitic acid negatively regulates tumor suppressor PTEN through T366 phosphorylation and protein degradation.

Chronic elevated free fatty (FFA) levels are linked to metabolic disorders and tumorigenesis. However, the molecular mechanism by which FFAs induce cancer remains poorly understood. Here, we show that the tumor suppressor PTEN protein levels were decreased in high fat diet (HFD) fed mice. As palmitic acid (PA, C16:0) showed a significant increase in the HFD fed mice, we further investigated its role in PTEN down regulation. Our studies revealed that exposure of cells to high doses of PA induced mTOR/S6K-mediated phosphorylation of PTEN at T366. The phosphorylation subsequently enhanced the interaction of PTEN with the E3 ubiquitin ligase WW domain-containing protein 2 (WWP2), which promoted polyubiquitination of PTEN and protein degradation. Consistent with PTEN degradation, exposure of cells to increased concentrations of PA also promoted PTEN-mediated AKT activation and cell proliferation. Significantly, a higher level of S6K activation, PTEN T366 phosphorylation, and AKT activation were also observed in the livers of the HFD fed mice. These results provide a molecular mechanism by which a HFD and elevated PA regulate cell proliferation through inactivation of tumor suppressor PTEN.

Dysregulation of Hypothalamic Gene Expression and the Oxytocinergic System by Soybean Oil Diets in Male Mice.

Soybean oil consumption has increased greatly in the past half-century and is linked to obesity and diabetes. To test the hypothesis that soybean oil diet alters hypothalamic gene expression in conjunction with metabolic phenotype, we performed RNA sequencing analysis using male mice fed isocaloric, high-fat diets based on conventional soybean oil (high in linoleic acid, LA), a genetically modified, low-LA soybean oil (Plenish), and coconut oil (high in saturated fat, containing no LA). The 2 soybean oil diets had similar but nonidentical effects on the hypothalamic transcriptome, whereas the coconut oil diet had a negligible effect compared to a low-fat control diet. Dysregulated genes were associated with inflammation, neuroendocrine, neurochemical, and insulin signaling. Oxt was the only gene with metabolic, inflammation, and neurological relevance upregulated by both soybean oil diets compared to both control diets. Oxytocin immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus was reduced, whereas plasma oxytocin and hypothalamic Oxt were increased. These central and peripheral effects of soybean oil diets were correlated with glucose intolerance but not body weight. Alterations in hypothalamic Oxt and plasma oxytocin were not observed in the coconut oil diet enriched in stigmasterol, a phytosterol found in soybean oil. We postulate that neither stigmasterol nor LA is responsible for effects of soybean oil diets on oxytocin and that Oxt messenger RNA levels could be associated with the diabetic state. Given the ubiquitous presence of soybean oil in the American diet, its observed effects on hypothalamic gene expression could have important public health ramifications.

HNF4α-Deficient Fatty Liver Provides a Permissive Environment for Sex-Independent Hepatocellular Carcinoma.

The incidence of hepatocellular carcinoma (HCC) is on the rise worldwide. Although the incidence of HCC in males is considerably higher than in females, the projected rates of HCC incidence are increasing for both sexes. A recently appreciated risk factor for HCC is the growing problem of nonalcoholic fatty liver disease, which is usually associated with obesity and the metabolic syndrome. In this study, we showed that under conditions of fatty liver, female mice were more likely to develop HCC than expected from previous models. Using an inducible knockout model of the tumor-suppressive isoform of hepatocyte nuclear factor 4 alpha ("P1-HNF4α") in the liver in combination with prolonged high fat (HF) diet, we found that HCC developed equally in male and female mice as early as 38 weeks of age. Similar sex-independent HCC occurred in the "STAM" model of mice, in which severe hyperglycemia and HF feeding results in rapid hepatic lipid deposition, fibrosis, and ultimately HCC. In both sexes, reduced P1-HNF4α activity, which also occurs under chronic HF diet feeding, increased hepatic lipid deposition and produced a greatly augmented circadian rhythm in IL6, a factor previously linked with higher HCC incidence in males. Loss of HNF4α combined with HF feeding induced epithelial-mesenchymal transition in an IL6-dependent manner. Collectively, these data provide a mechanism-based working hypothesis that could explain the rising incidence of aggressive HCC. SIGNIFICANCE: This study provides a mechanism for the growing incidence of hepatocellular carcinoma in both men and women, which is linked to nonalcoholic fatty liver disease.

Dimethyl Sulfoxide Decreases Levels of Oxylipin Diols in Mouse Liver.

Dimethylsulfoxide (DMSO) is widely used as a solvent and cryopreservative in laboratories and considered to have many beneficial health effects in humans. Oxylipins are a class of biologically active metabolites of polyunsaturated fatty acids (PUFAs) that have been linked to a number of diseases. In this study, we investigated the effect of DMSO on oxylipin levels in mouse liver. Liver tissue from male mice (C57Bl6/N) that were either untreated or injected with 1% DMSO at 18 weeks of age was analyzed for oxylipin levels using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). A decrease in oxylipin diols from linoleic acid (LA, C18:2n6), alpha-linolenic acid (ALA, C18:3n3) and docosahexeanoic acid (DHA, C22:6n3) was observed 2 h after injection with DMSO. In contrast, DMSO had no effect on the epoxide precursors or other oxylipins including those derived from arachidonic acid (C20:4n6) or eicosapentaenoic acid (EPA, C20:5n3). It also did not significantly affect the diol:epoxide ratio, suggesting a pathway distinct from, and potentially complementary to, soluble epoxide hydrolase inhibitors (sEHI). Since oxylipins have been associated with a wide array of pathological conditions, from arthritis pain to obesity, our results suggest one potential mechanism underlying the apparent beneficial health effects of DMSO. They also indicate that caution should be used in the interpretation of results using DMSO as a vehicle in animal experiments.

Incompatibility of the circadian protein BMAL1 and HNF4α in hepatocellular carcinoma.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of liver-specific gene expression with potent tumor suppressor activity, yet many liver tumors express HNF4α. This study reveals that P1-HNF4α, the predominant isoform expressed in the adult liver, inhibits expression of tumor promoting genes in a circadian manner. In contrast, an additional isoform of HNF4α, driven by an alternative promoter (P2-HNF4α), is induced in HNF4α-positive human hepatocellular carcinoma (HCC). P2-HNF4α represses the circadian clock gene ARNTL (BMAL1), which is robustly expressed in healthy hepatocytes, and causes nuclear to cytoplasmic re-localization of P1-HNF4α. We reveal mechanisms underlying the incompatibility of BMAL1 and P2-HNF4α in HCC, and demonstrate that forced expression of BMAL1 in HNF4α-positive HCC prevents the growth of tumors in vivo. These data suggest that manipulation of the circadian clock in HNF4α-positive HCC could be a tractable strategy to inhibit tumor growth and progression in the liver.

Conservation of DNA and ligand binding properties of retinoid X receptor from the placozoan Trichoplax adhaerens to human.

Nuclear receptors are a superfamily of transcription factors restricted to animals. These transcription factors regulate a wide variety of genes with diverse roles in cellular homeostasis, development, and physiology. The origin and specificity of ligand binding within lineages of nuclear receptors (e.g., subfamilies) continues to be a focus of investigation geared toward understanding how the functions of these proteins were shaped over evolutionary history. Among early-diverging animal lineages, the retinoid X receptor (RXR) is first detected in the placozoan, Trichoplax adhaerens. To gain insight into RXR evolution, we characterized ligand- and DNA-binding activity of the RXR from T. adhaerens (TaRXR). Like bilaterian RXRs, TaRXR specifically bound 9-cis-retinoic acid, which is consistent with a recently published result and supports a conclusion that the ancestral RXR bound ligand. DNA binding site specificity of TaRXR was determined through protein binding microarrays (PBMs) and compared with human RXRɑ. The binding sites for these two RXR proteins were broadly conserved (∼85% shared high-affinity sequences within a targeted array), suggesting evolutionary constraint for the regulation of downstream genes. We searched for predicted binding motifs of the T. adhaerens genome within 1000 bases of annotated genes to identify potential regulatory targets. We identified 648 unique protein coding regions with predicted TaRXR binding sites that had diverse predicted functions, with enriched processes related to intracellular signal transduction and protein transport. Together, our data support hypotheses that the original RXR protein in animals bound a ligand with structural similarity to 9-cis-retinoic acid; the DNA motif recognized by RXR has changed little in more than 1 billion years of evolution; and the suite of processes regulated by this transcription factor diversified early in animal evolution.

Omega-6 and omega-3 oxylipins are implicated in soybean oil-induced obesity in mice.

Soybean oil consumption is increasing worldwide and parallels a rise in obesity. Rich in unsaturated fats, especially linoleic acid, soybean oil is assumed to be healthy, and yet it induces obesity, diabetes, insulin resistance, and fatty liver in mice. Here, we show that the genetically modified soybean oil Plenish, which came on the U.S. market in 2014 and is low in linoleic acid, induces less obesity than conventional soybean oil in C57BL/6 male mice. Proteomic analysis of the liver reveals global differences in hepatic proteins when comparing diets rich in the two soybean oils, coconut oil, and a low-fat diet. Metabolomic analysis of the liver and plasma shows a positive correlation between obesity and hepatic C18 oxylipin metabolites of omega-6 (ω6) and omega-3 (ω3) fatty acids (linoleic and α-linolenic acid, respectively) in the cytochrome P450/soluble epoxide hydrolase pathway. While Plenish induced less insulin resistance than conventional soybean oil, it resulted in hepatomegaly and liver dysfunction as did olive oil, which has a similar fatty acid composition. These results implicate a new class of compounds in diet-induced obesity-C18 epoxide and diol oxylipins.

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer.

HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like ß, RELMß, and are extremely sensitive to DSS) are crossed with Retnlb(-/-) mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMß promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer.

Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells.

The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/ß-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/ß-catenin/TCF4 and AP-1 pathways.

Soybean Oil Is More Obesogenic and Diabetogenic than Coconut Oil and Fructose in Mouse: Potential Role for the Liver.

The obesity epidemic in the U.S. has led to extensive research into potential contributing dietary factors, especially fat and fructose. Recently, increased consumption of soybean oil, which is rich in polyunsaturated fatty acids (PUFAs), has been proposed to play a causal role in the epidemic. Here, we designed a series of four isocaloric diets (HFD, SO-HFD, F-HFD, F-SO-HFD) to investigate the effects of saturated versus unsaturated fat, as well as fructose, on obesity and diabetes. C57/BL6 male mice fed a diet moderately high in fat from coconut oil and soybean oil (SO-HFD, 40% kcal total fat) showed statistically significant increases in weight gain, adiposity, diabetes, glucose intolerance and insulin resistance compared to mice on a diet consisting primarily of coconut oil (HFD). They also had fatty livers with hepatocyte ballooning and very large lipid droplets as well as shorter colonic crypt length. While the high fructose diet (F-HFD) did not cause as much obesity or diabetes as SO-HFD, it did cause rectal prolapse and a very fatty liver, but no balloon injury. The coconut oil diet (with or without fructose) increased spleen weight while fructose in the presence of soybean oil increased kidney weight. Metabolomics analysis of the liver showed an increased accumulation of PUFAs and their metabolites as well as γ-tocopherol, but a decrease in cholesterol in SO-HFD. Liver transcriptomics analysis revealed a global dysregulation of cytochrome P450 (Cyp) genes in SO-HFD versus HFD livers, most notably in the Cyp3a and Cyp2c families. Other genes involved in obesity (e.g., Cidec, Cd36), diabetes (Igfbp1), inflammation (Cd63), mitochondrial function (Pdk4) and cancer (H19) were also upregulated by the soybean oil diet. Taken together, our results indicate that in mice a diet high in soybean oil is more detrimental to metabolic health than a diet high in fructose or coconut oil.