Cyp1b1 affects external control of mouse hepatocytes, fatty acid homeostasis and signaling involving HNF4? and PPAR?.
ABSTRACT: Cytochrome P450 1b1 (Cyp1b1) is expressed in endothelia, stellate cells and pre-adipocytes, but not hepatocytes. Deletion alters liver fatty acid metabolism and prevents obesity and hepatic steatosis. This suggests a novel extra-hepatocyte regulation directed from cells that express Cyp1b1. To characterize these mechanisms, microarray gene expression was analyzed in livers of normal and congenic Cyp1b1-ko C57BL/6 J mice fed either low or high fat diets. Cyp1b1-ko gene responses indicate suppression of endogenous PPAR? activity, a switch from triglyceride storage to mitochondrial fatty acid oxidation and decreased oxidative stress. Many gene responses in Cyp1b1-ko are sexually dimorphic and correspond to increased activity of growth hormone mediated by HNF4?. Male responses stimulated by GH pulses are enhanced, whereas responses that decline exhibit further suppression, including Cyp regulation by PPAR?, CAR and PXR. These effects of Cyp1b1 deletion overlap with effects caused by deletion of the small heterodimeric partner, a suppressor of these nuclear factors. Redirection of gene expression associated with liver fat homeostasis in Cyp1b1-ko mice that directs hypothalamic control of GH and leptin. Cyp1b1-ko suppresses neonatal Scd1 and delays adult maturation of dimorphic GH/HNF4? signaling. Alternatively, deletion may diminish hypothalamic metabolism of estradiol, which establishes adult GH regulation.
Project description:Cytochrome P450 1b1 (Cyp1b1) expression is absent in mouse hepatocytes, but present in liver endothelia and activated stellate cells. Increased expression during adipogenesis suggests a role of Cyp1b1 metabolism in fatty acid homeostasis. Wild-type C57BL/6j (WT) and Cyp1b1-null (Cyp1b1-ko) mice were provided low or high fat diets (LFD and HFD, respectively). Cyp1b1-deletion suppressed HFD-induced obesity, improved glucose tolerance and prevented liver steatosis. Suppression of lipid droplets in sinusoidal hepatocytes, concomitant with enhanced glycogen granules, was a consistent feature of Cyp1b1-ko mice. Cyp1b1 deletion altered the in vivo expression of 560 liver genes, including suppression of PPAR?, stearoyl CoA desaturase 1 (Scd1) and many genes stimulated by PPAR?, each consistent with this switch in energy storage mechanism. Ligand activation of PPAR? in Cyp1b1-ko mice by WY-14643 was, nevertheless, effective. Seventeen gene changes in Cyp1b1-ko mice correspond to mouse transgenic expression that attenuated diet-induced diabetes. The absence of Cyp1b1 in mouse hepatocytes indicates participation in energy homeostasis through extra-hepatocyte signaling. Extensive sexual dimorphism in hepatic gene expression suggests a developmental impact of estrogen metabolism by Cyp1b1. Suppression of Scd1 and increased leptin turnover support enhanced leptin participation from the hypothalamus. Cyp1b1-mediated effects on vascular cells may underlie these changes.
Project description:Distinct male and female patterns of pituitary GH secretion produce sexually differentiated hepatic gene expression profiles, thereby influencing steroid and xenobiotic metabolism. We used a fully automated system to obtain serial nocturnal blood samples every 15 minutes from cannulated wild-type (WT) and somatostatin knockout (Sst-KO) mice to determine the role of SST, the principal inhibitor of GH release, in the generation of sexually dimorphic GH pulsatility. WT males had lower mean and median GH values, less random GH secretory bursts, and longer trough periods between GH pulses than WT females. Each of these parameters was feminized in male Sst-KO mice, whereas female Sst-KO mice had higher GH levels than all other groups, but GH pulsatility was unaffected. We next performed hepatic mRNA profiling with high-density microarrays. Male Sst-KO mice exhibited a globally feminized pattern of GH-dependent mRNA levels, but female Sst-KO mice were largely unaffected. Among the differentially expressed female-predominant genes was Serpina6, which encodes corticosteroid-binding globulin (CBG). Increased CBG was associated with elevated diurnal peak plasma corticosterone in unstressed WT females and both sexes of Sst-KO mice compared with WT males. Sst-KO mice also had exaggerated ACTH and corticosterone responses to acute restraint stress. However, consistent with their lack of phenotypic signs of excess glucocorticoids, cerebrospinal fluid concentrations of free corticosterone in Sst-KO mice were not elevated. In summary, SST is necessary for the prolonged interpulse troughs that define masculinized pituitary GH secretion. SST also contributes to sexual dimorphism of the hypothalamic-pituitary-adrenal axis via GH-dependent regulation of hepatic CBG production.
Project description:Stress is a risk factor for several cardiovascular pathologies. PPAR? holds a fundamental role in control of lipid homeostasis by directly regulating genes involved in fatty acid transport and oxidation. Importantly, PPAR? agonists are effective in raising HDL-cholesterol and lowering triglycerides, properties that reduce the risk for cardiovascular diseases. This study investigated the role of stress and adrenergic receptor (AR)-related pathways in PPAR? and HNF4? regulation and signaling in mice following repeated restraint stress or treatment with AR-antagonists administered prior to stress to block AR-linked pathways. Repeated restraint stress up-regulated Ppar? and its target genes in the liver, including Acox, Acot1, Acot4, Cyp4a10, Cyp4a14 and Lipin2, an effect that was highly correlated with Hnf4?. In vitro studies using primary hepatocyte cultures treated with epinephrine or AR-agonists confirmed that hepatic AR/cAMP/PKA/CREB- and JNK-linked pathways are involved in PPAR? and HNF4? regulation. Notably, restraint stress, independent of PPAR?, suppressed plasma triglyceride levels. This stress-induced effect could be attributed in part to hormone sensitive lipase activation in the white adipose tissue, which was not prevented by the increased levels of perilipin. Overall, this study identifies a mechanistic basis for the modification of lipid homeostasis following stress and potentially indicates novel roles for PPAR? and HNF4? in stress-induced lipid metabolism.
Project description:PPAR? is one of the three isoforms of the Peroxisome Proliferator-Activated Receptors (PPARs). PPAR? is activated by thiazolidinediones such as pioglitazone and is targeted to treat insulin resistance. PPAR? is densely expressed in brain areas involved in regulation of motivational and emotional processes. Here, we investigated the role of PPAR? in the brain and explored its role in anxiety and stress responses in mice. The results show that stimulation of PPAR? by pioglitazone did not affect basal anxiety, but fully prevented the anxiogenic effect of acute stress. Using mice with genetic ablation of neuronal PPAR? (PPAR?NestinCre), we demonstrated that a lack of receptors, specifically in neurons, exacerbated basal anxiety and enhanced stress sensitivity. The administration of GW9662, a selective PPAR? antagonist, elicited a marked anxiogenic response in PPAR? wild-type (WT), but not in PPAR?NestinCre knock-out (KO) mice. Using c-Fos immunohistochemistry, we observed that acute stress exposure resulted in a different pattern of neuronal activation in the amygdala (AMY) and the hippocampus (HIPP) of PPAR?NestinCre KO mice compared with WT mice. No differences were found between WT and KO mice in hypothalamic regions responsible for hormonal response to stress or in blood corticosterone levels. Microinjection of pioglitazone into the AMY, but not into the HIPP, abolished the anxiogenic response elicited by acute stress. Results also showed that, in both regions, PPAR? colocalizes with GABAergic cells. These findings demonstrate that neuronal PPAR? is involved the regulation of the stress response and that the AMY is a key substrate for the anxiolytic effect of PPAR?.Peroxisome Proliferator-Activated Receptor Gamma (PPAR?) is a classical target for antidiabetic therapies with thiazolidinedione compounds. PPAR? agonists such as rosiglitazone and pioglitazone are in clinical use for the treatment of insulin resistance. PPAR? has recently attracted attention for its involvement in the regulation of CNS immune response and functions. Here, we demonstrate that neuronal PPAR? activation prevented the negative emotional effects of stress and exerted anxiolytic actions without influencing hypothalamic-pituitary-adrenal axis function. Conversely, pharmacological blockade or genetic deletion of PPAR? enhanced anxiogenic responses and increased vulnerability to stress. These effects appear to be controlled by PPAR? neuronal-mediated mechanisms in the amygdala.
Project description:The liver is the main organ involved in the metabolism of amino acids (AA), which are oxidized by amino acid catabolizing enzymes (AACE). Peroxisome proliferator-activated receptor-? (PPAR?) stimulates fatty acid ?-oxidation, and there is evidence that it can modulate hepatic AA oxidation during the transition of energy fuels. To understand the role and mechanism of PPAR?'s regulation of AA catabolism, the metabolic and molecular adaptations of Ppara-null mice were studied. The role of PPAR? on AA metabolism was examined by in vitro and in vivo studies. In wild-type and Ppara-null mice, fed increasing concentrations of the dietary protein/carbohydrate ratio, we measured metabolic parameters, and livers were analyzed by microarray analysis, histology and Western blot. Functional enrichment analysis, EMSA and gene reporter assays were performed. Ppara-null mice presented increased expression of AACE in liver affecting AA, lipid and carbohydrate metabolism. Ppara-null mice had increased glucagon/insulin ratio (7.2-fold), higher serum urea (73.1 %), lower body protein content (19.7 %) and decreased several serum AA in response to a high-protein/low-carbohydrate diet. A functional network of differentially expressed genes, suggested that changes in the expression of AACE were regulated by an interrelationship between PPAR? and HNF4?. Our data indicated that the expression of AACE is down-regulated through PPAR? by attenuating HNF4? transcriptional activity as observed in the serine dehydratase gene promoter. PPAR? via HNF4? maintains body protein metabolic homeostasis by down-regulating genes involved in amino acid catabolism for preserving body nitrogen.
Project description:Fibroblast growth factor (FGF) 21 is an endocrine factor that normalizes glucose homeostasis and reduces insulin resistance in diabetes. Although the pancreas is an FGF21 target organ, its role in pancreatic islets remains obscure. This study aimed to elucidate the physiological role of FGF21 in pancreatic islets using FGF21-knockout (FGF21-KO) mice. Twenty-four-week-old male global FGF21-KO mice were used in this study. Glucose and insulin tolerance were assessed. Expression of genes and proteins related to islet function and underlying mechanisms were also examined. Islet morphology and insulin-secreting capacity were further evaluated. FGF21-KO mice exhibited insulin resistance while being normoglycemic, associated with increases in beta-cell proliferation and insulin synthesis, acting as compensatory responses. This phenotype probably results from enhanced growth hormone (GH) sensitivity in FGF21-KO mouse islets. In addition, ex vivo FGF21 treatment in normal C57BL/6J mouse islets reduced GH signaling, probably via upregulation of peroxisome proliferator-activated receptor gamma (PPAR?) and cytokine-inducible SH-2 containing (CIS) protein, whereas KO mouse islets displayed reduced PPAR? and CIS expression. FGF21 treatment also reversed GH-induced insulin expression, beta-cell proliferation and GH-impaired glucose-stimulated insulin secretion (GSIS) in islets. Furthermore, distorted islet morphology and impaired GSIS were observed in KO mice, suggestive of islet dysfunction, whereas the enhanced insulin expression and impaired GSIS in FGF21-KO mouse islets could be reversed by blockade of GH signaling. Our data indicate that FGF21 is important in the regulation of beta-cell proliferation and insulin synthesis, probably via modulation of GH signaling. These findings provide evidence that FGF21 is an obligatory metabolic regulator in pancreatic islets and shed new light onto the role of endogenous FGF21 in the pathogenesis of insulin resistance and islet dysfunction.
Project description:Mutations of the HNF4A gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of pancreatic ?-cell function. HNF4? is a transcription factor belonging to the nuclear receptor superfamily (NR2A1), but its target genes in pancreatic ?-cells are largely unknown. Here, we report that ankyrin repeat and sterile ? motif domain containing 4b (Anks4b) is a target of HNF4? in pancreatic ?-cells. Expression of Anks4b was decreased in both ?HNF4? KO islets and HNF4? knockdown MIN6 ?-cells, and HNF4? activated Anks4b promoter activity. Anks4b bound to glucose-regulated protein 78 (GRP78), a major endoplasmic reticulum (ER) chaperone protein, and overexpression of Anks4b enhanced the ER stress response and ER stress-associated apoptosis of MIN6 cells. Conversely, suppression of Anks4b reduced ?-cell susceptibility to ER stress-induced apoptosis. These results indicate that Anks4b is a HNF4? target gene that regulates ER stress in ?-cells by interacting with GRP78, thus suggesting that HNF4? is involved in maintenance of the ER.
Project description:Hepatocyte nuclear factor 4 alpha (HNF4?) is critical for hepatic differentiation. Recent studies have highlighted its role in inhibition of hepatocyte proliferation and tumor suppression. However, the role of HNF4? in liver regeneration (LR) is not known. We hypothesized that hepatocytes modulate HNF4? activity when navigating between differentiated and proliferative states during LR. Western blotting analysis revealed a rapid decline in nuclear and cytoplasmic HNF4? protein levels, accompanied with decreased target gene expression, within 1 hour after two-thirds partial hepatectomy (post-PH) in C57BL/6J mice. HNF4? protein expression did not recover to pre-PH levels until day 3. Hepatocyte-specific deletion of HNF4? (HNF4?-KO [knockout]) in mice resulted in 100% mortality post-PH, despite increased proliferative marker expression throughout regeneration. Sustained loss of HNF4? target gene expression throughout regeneration indicated that HNF4?-KO mice were unable to compensate for loss of HNF4? transcriptional activity. Deletion of HNF4? resulted in sustained proliferation accompanied by c-Myc and cyclin D1 overexpression and a complete deficiency of hepatocyte function after PH. Interestingly, overexpression of degradation-resistant HNF4? in hepatocytes delayed, but did not prevent, initiation of regeneration after PH. Finally, adeno-associated virus serotype 8 (AAV8)-mediated reexpression of HNF4? in hepatocytes of HNF4?-KO mice post-PH restored HNF4? protein levels, induced target gene expression, and improved survival of HNF4?-KO mice post-PH. Conclusion: In conclusion, these data indicate that HNF4? reexpression following initial decrease is critical for hepatocytes to exit from cell cycle and resume function during the termination phase of LR. These results indicate the role of HNF4? in LR and have implications for therapy of liver failure.
Project description:Central nervous system (CNS) lipid accumulation, inflammation and resistance to adipo-regulatory hormones, such as insulin and leptin, are implicated in the pathogenesis of diet-induced obesity (DIO). Peroxisome proliferator-activated receptors (PPAR ?, ?, ?) are nuclear transcription factors that act as environmental fatty acid sensors and regulate genes involved in lipid metabolism and inflammation in response to dietary and endogenous fatty acid ligands. All three PPAR isoforms are expressed in the CNS at different levels. Recent evidence suggests that activation of CNS PPAR? and/or PPAR? may contribute to weight gain and obesity. PPAR? is the most abundant isoform in the CNS and is enriched in the hypothalamus, a region of the brain involved in energy homeostasis regulation. Because in peripheral tissues, expression of PPAR? increases lipid oxidative genes and opposes inflammation, we hypothesized that CNS PPAR? protects against the development of DIO. Indeed, genetic neuronal deletion using Nes-Cre loxP technology led to elevated fat mass and decreased lean mass on low-fat diet (LFD), accompanied by leptin resistance and hypothalamic inflammation. Impaired regulation of neuropeptide expression, as well as uncoupling protein 2, and abnormal responses to a metabolic challenge, such as fasting, also occur in the absence of neuronal PPAR?. Consistent with our hypothesis, KO mice gain significantly more fat mass on a high-fat diet (HFD), yet are surprisingly resistant to diet-induced elevations in CNS inflammation and lipid accumulation. We detected evidence of upregulation of PPAR? and target genes of both PPAR? and PPAR?, as well as genes of fatty acid oxidation. Thus, our data reveal a previously underappreciated role for neuronal PPAR? in the regulation of body composition, feeding responses, and in the regulation of hypothalamic gene expression.
Project description:Human cytochrome P450 1B1 (CYP1B1)-mediated biotransformation of endobiotics and xenobiotics plays an important role in the progression of human breast cancer. In this study, we investigated the effects of WY-14643, a peroxisome proliferator-activated receptor ? (PPAR?) agonist, on CYP1B1 expression and the related mechanism in MCF7 breast cancer cells. We performed quantitative reverse transcription-polymerase chain reaction, transient transfection, and chromatin immunoprecipitation to evaluate the effects of PPAR? on peroxisome proliferator response element (PPRE)-mediated transcription. WY-14643 increased the protein and mRNA levels of CYP1B1, as well as promoter activity, in MCF-7 cells. Moreover, WY-14643 plus GW6471, a PPAR? antagonist, significantly inhibited the WY-14643-mediated increase in CYP1B1 expression. PPAR? knockdown by a small interfering RNA markedly suppressed the induction of CYP1B1 expression by WY-14643, suggesting that WY-14643 induces CYP1B1 expression via a PPAR?-dependent mechanism. Bioinformatics analysis identified putative PPREs (-833/-813) within the promoter region of the CYP1B1 gene. Inactivation of these putative PPREs by deletion mutagenesis suppressed the WY-14643-mediated induction of CYP1B1 promoter activation. Furthermore, WY-14643 induced PPAR? to assume a form capable of binding specifically to the PPRE-binding site in the CYP1B1 promoter. Our findings suggest that WY-14643 induces the expression of CYP1B1 through activation of PPAR?.