Peroxisome proliferator-activated receptor (PPAR) α is a transcription factor that regulates genes involved in fatty acid catabolism. Here we provide evidence that PPARα is constitutively expressed in nuclei of hippocampal neurons and surprisingly controls calcium influx and the expression of various plasticity-related genes via direct transcriptional regulation of CREB. Accordingly, Ppara-null, but not Pparb-null, mice are deficient in CREB and memory-associated genes, and have decreased spatial learning and memory. While shRNA knockdown of PPARα in the hippocampus suppressed CREB and NR2A rendering wild type animals markedly poor in consolidating spatial memory, introduction of PPARα to the hippocampus of Ppara-null mice increased hippocampal CREB and NR2A and improved spatial learning and memory. These results together with detailed analyses of CREB, NR2A and spatial learning and memory in bone marrow chimeric animals lacking PPARα in the CNS describe a novel mechanism for transcriptional control of Creb and associated plasticity genes by PPARα.
Background & Aims
Ethanol-inducible cytochrome P450 2E1 (CYP2E1) activity contributes to oxidative stress. However, CYP2E1 may have an important role in the pathogenesis of high-fat mediated nonalcoholic steatohepatitis (NASH). Thus, the role of CYP2E1 in high-fat mediated NASH development was evaluated.
Male wild-type (WT) and Cyp2e1-null mice were fed a low-fat diet (LFD, 10% energy-derived) or high-fat diet (HFD, 60% energy-derived) for 10 weeks. Liver histology and tissue homogenates were examined for various parameters of oxidative stress and inflammation.
Liver histology showed that only WT mice fed a HFD developed NASH despite increased steatosis in both WT and Cyp2e1-null mice fed HFD. Markers of oxidative stress such as elevated CYP2E1 activity and protein amounts, lipid peroxidation, protein carbonylation, nitration, and glycation with increased phospho-JNK were all markedly elevated only in the livers of HFD-fed WT mice. Furthermore, while the levels of inflammation markers osteopontin and F4/80 were higher in HFD-fed WT mice, TNFα and MCP-1 contents were lower compared to the corresponding LFD-fed WT. Finally, only HFD-fed WT mice exhibited increased insulin resistance and impaired glucose tolerance.
These data suggest that CYP2E1 is critically important in NASH development by promoting oxidative/nitrosative stress, protein modifications, inflammation and insulin resistance.
Liver; CYP2E1; null mice; high-fat diet; NAFLD; NASH; oxidative stress; protein modifications; inflammation; insulin resistance
Rifaximin, a nonsystemic antibiotic that exhibits low gastrointestinal absorption, is a potent agonist of human pregnane X receptor (PXR), which contributes to its therapeutic efficacy in inflammatory bowel disease. To investigate the effects of long-term administration of rifaximin on the liver, PXR-humanized mice were administered rifaximin for 6 months; wild-type and Pxr-null mice were treated in parallel as controls. Histological analysis revealed time-dependent intense hepatocellular fatty degeneration and increased hepatic triglycerides in PXR-humanized mice and not in wild-type and Pxr-null mice. After long-term treatment, PXR target genes were induced in small intestine and liver, with significant up-regulation in the expression of hepatic genes related to triglyceride synthesis and lipid accumulation. However, no significant hepatic accumulation of rifaximin was found, even after 6 months of treatment, in PXR-humanized mice. Genes in the small intestine that are involved in the uptake of fatty acids and triglycerides were induced along with increased triglyceride accumulation in intestinal epithelial cells of PXR-humanized mice; this was not observed in wild-type and Pxr-null mice. These findings suggest that long-term administration of rifaximin could lead to PXR-dependent hepatocellular fatty degeneration as a result of activation of genes involved in lipid uptake, thus indicating a potential adverse effect of rifaximin on liver function after long-term exposure.
rifaximin; hepatotoxicity; human pregnane X receptor; steatosis
Mass spectrometry-based metabolomics has previously demonstrated utility for identifying biomarkers of ionizing radiation exposure in cellular, mouse and rat in vivo radiation models. To provide a valuable link from small laboratory rodents to humans, γ-radiation-induced urinary biomarkers were investigated using a nonhuman primate total-body-irradiation model. Mass spectrometry-based metabolomics approaches were applied to determine whether biomarkers could be identified, as well as the previously discovered rodent biomarkers of γ radiation. Ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry analysis was carried out on a time course of clean-catch urine samples collected from nonhuman primates (n = 6 per cohort) exposed to sham, 1.0, 3.5, 6.5 or 8.5 Gy doses of 60Co γ ray (~0.55 Gy/min) ionizing radiation. By multivariate data analysis, 13 biomarkers of radiation were discovered: N-acetyltaurine, isethionic acid, taurine, xanthine, hypoxanthine, uric acid, creatine, creatinine, tyrosol sulfate, 3-hydroxytyrosol sulfate, tyramine sulfate, N-acetylserotonin sulfate, and adipic acid. N-Acetyltaurine, isethionic acid, and taurine had previously been identified in rats, and taurine and xanthine in mice after ionizing radiation exposure. Mass spectrometry-based metabolomics has thus successfully revealed and verified urinary biomarkers of ionizing radiation exposure in the nonhuman primate for the first time, which indicates possible mechanisms for ionizing radiation injury.
Rifampicin and isoniazid co–therapy frequently causes liver injury in humans. A pregnane X receptor–humanized mouse model revealed that rifampicin and isoniazid co–treatment causes accumulation of protoporphyrin IX, an endogenous hepatotoxin, in the liver via a pregnane X receptor–mediated alteration of heme biosynthesis pathway. These results provide novel insight into the mechanism of rifampicin and isoniazid–induced liver injury that may be applied to clinical management of the hepatotoxicity associated with tuberculosis chemotherapy.
Single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that harbors the coagulation factor XI (F11), prekallikrein (KLKB1), and a cytochrome P450 family member (CYP4V2) genes are associated with deep venous thrombosis (DVT). These SNPs exert their effect on DVT by modifying the circulating levels of FXI. However, SNPs associated with DVT were not necessarily all in F11, but also in KLKB1 and CYP4V2. Here, we searched for evidence for common regulatory elements within the 4q35.2 locus, outside the F11 gene, that might control FXI plasma levels and/or DVT risk. To this end, we investigated the regulation of the orthologous mouse gene cluster under several metabolic conditions that impact mouse hepatic F11 transcription. In livers of mice in which HNF4α, a key transcription factor controlling F11, was ablated, or reduced by siRNA, a strong decrease in hepatic F11 transcript levels was observed that correlated with Cyp4v3 (mouse orthologue of CYP4V2), but not by Klkb1 levels. Estrogens induced hepatic F11 and Cyp4v3, but not Klkb1 transcript levels, whereas thyroid hormone strongly induced hepatic F11 transcript levels, and reduced Cyp4v3, leaving Klkb1 levels unaffected. Mice fed a high-fat diet also had elevated F11 transcription, markedly paralleled by an induction of Klkb1 and Cyp4v3 expression. We conclude that within the mouse F11, Klkb1, Cyp4v3 gene cluster, F11 and Cyp4v3 frequently display striking parallel transcriptional responses suggesting the presence of shared regulatory elements.
Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. The nuclear farnesoid X receptor (FXR) and the membrane G protein-coupled receptor, TGR5, regulate bile acid (BA) homeostasis and inflammation. Therefore, we hypothesized that activation of FXR and/or TGR5 could ameliorate liver injury in Mdr2−/− (Abcb4−/−) mice, a model of chronic cholangiopathy. Hepatic inflammation, fibrosis, as well as bile secretion and key genes of BA homeostasis were addressed in Mdr2−/− mice fed either a chow diet or a diet supplemented with the FXR agonist, INT-747, the TGR5 agonist, INT-777, or the dual FXR/TGR5 agonist, INT-767 (0.03% w/w). Only the dual FXR/TGR5 agonist, INT-767, significantly improved serum liver enzymes, hepatic inflammation, and biliary fibrosis in Mdr2−/− mice, whereas INT-747 and INT-777 had no hepatoprotective effects. In line with this, INT-767 significantly induced bile flow and biliary HCO3− output, as well as gene expression of carbonic anhy-drase 14, an important enzyme able to enhance HCO3− transport, in an Fxr-dependent manner. In addition, INT-767 dramatically reduced bile acid synthesis via the induction of ileal Fgf15 and hepatic Shp gene expression, thus resulting in significantly reduced biliary bile acid output in Mdr2−/− mice.
This study shows that FXR activation improves liver injury in a mouse model of chronic cholangiopathy by reduction of biliary BA output and promotion of HCO3−-rich bile secretion.
Stress is a risk factor for several cardiovascular pathologies. PPARα holds a fundamental role in control of lipid homeostasis by directly regulating genes involved in fatty acid transport and oxidation. Importantly, PPARα 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 PPARα and HNF4α 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 Pparα and its target genes in the liver, including Acox, Acot1, Acot4, Cyp4a10, Cyp4a14 and Lipin2, an effect that was highly correlated with Hnf4α. 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 PPARα and HNF4α regulation. Notably, restraint stress, independent of PPARα, 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 PPARα and HNF4α in stress-induced lipid metabolism.
Periplakin (PPL) is a rod-shaped cytolinker protein thought to connect cellular adhesion junctional complexes to cytoskeletal filaments. PPL serves as a structural component of the cornified envelope in the skin and interacts with various types of proteins in cultured cells; its level decreases dramatically during tumorigenic progression in human epithelial tissues. Despite these intriguing observations, the physiological roles of PPL, especially in non-cutaneous tissues, are still largely unknown. Because we observed a marked fluctuation of PPL expression in mouse liver in association with the bile acid receptor farnesoid X receptor (FXR) and cholestasis, we sought to characterize the role of PPL in the liver and determine its contributions to the etiology and pathogenesis of cholestasis.
Time- and context-dependent expression of PPL in various mouse models of hepatic and renal disorders were examined by immunohistochemistry, western blotting, and quantitative real-time polymerase chain reactions.
The hepatic expression of PPL was significantly decreased in Fxr−/− mice. In contrast, the expression was dramatically increased during cholestasis, with massive PPL accumulation observed at the boundaries of hepatocytes in wild-type mice. Interestingly, the hepatic accumulation of PPL resulting from cholestasis was reversible. In addition, similar accumulation of PPL at cellular boundaries was found in epithelial cells around renal tubules upon ureteral obstruction.
PPL may be involved in the temporal accommodation to fluid stasis in different tissues. Further examination of the roles for PPL may lead to the discovery of a novel mechanism for cellular protection by cytolinkers that is applicable to many tissues and in many contexts.
Periplakin; Cholestasis; Bile acids; Farnesoid X receptor; Urinary stasis
Acetaminophen (APAP) overdose causes acute liver failure in humans and rodents due in part to the destruction of mitochondria as a result of increased oxidative stress followed by hepatocellular necrosis. Activation of the peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor superfamily that controls the expression of genes encoding peroxisomal and mitochondrial fatty acid β-oxidation enzymes, with the experimental ligand Wy-14,643 or the clinically-used fibrate drug fenofibrate, fully protects mice from APAP-induced hepatotoxicity. PPARα-humanized mice were also protected while Ppara-null mice were not, thus indicating that the protection extends to human PPARα and is PPARα-dependent. This protection is due in part to induction of the PPARα target gene encoding mitochondrial uncoupling protein 2 (UCP2). Forced overexpression of UCP2 protected wild-type mice against APAP-induced hepatotoxicity in the absence of PPARα activation. Ucp2-null mice, however, were sensitive to APAP induced hepatotoxicity despite activation of PPARα with Wy-14,643. Protection against hepatotoxicity by UCP2-induction through activation of PPARα, is associated with decreased APAP-induced c-jun and c-fos expression, decreased phosphorylation of JNK and c-jun, lower mitochondrial H2O2 levels, increased mitochondrial glutathione in liver, and decreased levels of circulating fatty acyl-carnitines. These studies indicate that the PPARα target gene UCP2 protects against elevated reactive oxygen species generated during drug-induced hepatotoxicity and suggest that induction of UCP2 may also be a general mechanism for protection of mitochondria during fatty acid β-oxidation.
Radiation metabolomics can be defined as the global profiling of biological fluids to uncover latent, endogenous small molecules whose concentrations change in a dose-response manner following exposure to ionizing radiation. In response to the potential threat of nuclear or radiological terrorism, the Center for High-Throughput Minimally Invasive Radiation Biodosimetry (CMCR) was established to develop field-deployable biodosimeters based, in principle, on rapid analysis by mass spectrometry of readily and easily obtainable biofluids. In this review, we briefly summarize radiation biology and key events related to actual and potential nuclear disasters, discuss the important contributions the field of mass spectrometry has made to the field of radiation metabolomics, and summarize current discovery efforts to use mass spectrometry-based metabolomics to identify dose-responsive urinary constituents, and ultimately to build and deploy a noninvasive high-throughput biodosimeter.
Biodosimetry; ionizing radiation; metabolomics
The current study tests the hypothesis that peroxisome proliferator-activated receptor β (PPARβ) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. The role of PPARβ in liver regeneration was studied using two-third partial hepatectomy (PH) in Wild-type (WT) and PPARβ-null (KO) mice. In KO mice, liver regeneration was delayed and the number of Ki-67 positive cells reached the peak at 60 hr rather than at 36–48 hr after PH shown in WT mice. RNA-sequencing uncovered 1344 transcriptomes that were differentially expressed in regenerating WT and KO livers. About 70% of those differentially expressed genes involved in glycolysis and fatty acid synthesis pathways failed to induce during liver regeneration due to PPARβ deficiency. The delayed liver regeneration in KO mice was accompanied by lack of activation of phosphoinositide-dependent kinase 1 (PDK1)/Akt. In addition, cell proliferation-associated increase of genes encoding E2f transcription factor (E2f) 1–2 and E2f7–8 as well as their downstream target genes were not noted in KO livers 36–48 hr after PH. E2fs have dual roles in regulating metabolism and proliferation. Moreover, transient steatosis was only found in WT, but not in KO mice 36 hr after PH. These data suggested that PPARβ-regulated PDK1/Akt and E2f signaling that controls metabolism and proliferation is involved in the normal progression of liver regeneration.
The mechanisms by which deregulated nuclear factor erythroid-2–related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and 13C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.
Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic β-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and β-cell ATP-dependent K+ (KATP) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by the FXR agonist GW4064 and suppressed by the FXR antagonist guggulsterone. TCDC and GW4064 stimulated the electrical activity of β-cells and enhanced cytosolic Ca2+ concentration ([Ca2+]c). These effects were blunted by guggulsterone. Sodium ursodeoxycholate, which has a much lower affinity to FXR than TCDC, had no effect on [Ca2+]c and insulin secretion. FXR activation by TCDC is suggested to inhibit KATP current. The decline in KATP channel activity by TCDC was only observed in β-cells with intact metabolism and was reversed by guggulsterone. TCDC did not alter insulin secretion in islets of SUR1-KO or FXR-KO mice. TCDC did not change islet cell apoptosis. This is the first study showing an acute action of BA on β-cell function. The effect is mediated by FXR by nongenomic elements, suggesting a novel link between FXR activation and KATP channel inhibition.
Pregnane X receptor (PXR; NR1I2), a member of the nuclear receptor superfamily, has a major role in the induction of genes involved in drug transport and metabolism. Recent studies in mice have provided insight into a novel function for PXR in inflammatory bowel disease (IBD). The mechanisms of the protective effect of PXR activation on IBD is not fully established, but is due in part to the attenuation of nuclear factor kappa B (NF-κB) signaling that results in lower expression of proinflammatory cytokines. Recent clinical trials with the antibiotic rifaximin, a PXR agonist in the gastrointestinal system, have revealed its potential therapeutic value in the treatment of intestinal inflammation in humans. Thus, PXR may be a novel target for IBD therapy.
Procainamide, a type I antiarrhythmic agent, is used to treat a variety of atrial and ventricular dysrhythmias. It was reported that long-term therapy with procainamide may cause lupus erythematosus in 25–30% of patients. Interestingly, procainamide does not induce lupus erythematosus in mouse models. To explore the differences in this side-effect of procainamide between humans and mouse models, metabolomic analysis using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) was conducted on urine samples from procainamide-treated humans, CYP2D6-humanized mice, and wild-type mice. Thirteen urinary procainamide metabolites, including nine novel metabolites, derived from P450-dependent, FMO-dependent oxidations and acylation reactions, were identified and structurally elucidated. In vivo metabolism of procainamide in CYP2D6-humanized mice as well as in vitro incubations with microsomes and recombinant P450s suggested that human CYP2D6 plays a major role in procainamide metabolism. Significant differences in N-acylation and N-oxidation of the drug between humans and mice largely account for the interspecies differences in procainamide metabolism. Significant levels of the novel N-oxide metabolites produced by FMO1 and FMO3 in humans might be associated with the development of procainamide-induced systemic lupus erythematosus. Observations based on this metabolomic study offer clues to understanding procainamide-induced lupus in humans and the effect of P450s and FMOs on procainamide N-oxidation.
Procainamide; Systemic lupus erythematosus; Metabolomics; N-Oxidation; Ultra-performance liquid chromatography; Time-of-flight mass spectrometry
Peroxisome proliferator-activated receptor γ (PPARγ) is widely expressed in vessel walls, and its activation by agonists showed beneficial effects in cardiovascular diseases. However, the role of endothelial cell (EC) PPARγ in atherogenesis is not fully understood.
Methods and Results
To assess the contribution of endothelial-specific PPARγ in atherosclerosis, EC-specific PPARγ disruption and low-density lipoprotein receptor (LDLR) double-knockout (PparγΔEC/Ldlr−/−) mice were developed. When challenged with a high-cholesterol diet for 4 weeks, PparγΔEC/Ldlr−/− mice exhibited severe atherosclerotic lesions compared to either their littermate controls or macrophage-specific PPARγ disruption and low-density lipoprotein receptor (LDLR) double knockout (PparγΔMϕ/Ldlr−/−) mice. Metabolic analysis showed severe dyslipidemia and significant increase in systolic blood pressure in the PparγΔMϕ/Ldlr−/− mice. Histological analysis and real-time quantitative PCR suggested an exacerbated inflammation in PparγΔEC/Ldlr−/− mice, as revealed by the increases of proinflammatory gene expression and macrophage infiltration in vivo and in vitro. Furthermore, in vivo endothelial permeability was also increased by endothelial PPARγ disruption. Bone-marrow transplantation studies, which reconstituted hematopoietic PPARγ, demonstrated that the accelerated atherogenesis was due to endothelial PPARγ deficiency.
Endothelial PPARγ plays an important protective role in atherogenesis.
atherosclerosis; endothelium; PPARγ; inflammation
Hypoxia-inducible factor (HIF), a key modulator of the transcriptional response to hypoxia, is increased in colon cancer. However, the role of HIF in colon carcinogenesis in vivo remains unclear. In this study, we found that intestinal epithelium-specific disruption of the von Hippel-Lindau tumor suppressor protein (VHL) resulted in constitutive HIF signaling, and increased HIF expression augmented colon tumorigenesis in the Apcmin/+ intestinal tumor model. Intestine-specific disruption of Vhl increased colon tumor multiplicity and progression from adenomas to carcinomas. These effects were ameliorated in mice with double disruption of Vhl and HIF-2α. Activation of HIF signaling resulted in increased cell survival in normal colon tissue, however tumor apoptosis was not affected. Interestingly, a robust activation of cyclin D1 was observed in tumors of Apcmin/+ mice in which HIF-2α was activated in the intestine. Consistent with this result, BrdU incorporation indicated that cellular proliferation was increased in colon tumors following HIF activation. Further analysis demonstrated that dysregulation of the intestinal iron absorption transporter divalent metal transporter-1 (DMT-1) was a critical event in HIF-2α-mediated colon carcinogenesis. These data provide a mechanistic basis for the widely reported link between iron accumulation and colon cancer risk. Together, our findings demonstrate that a chronic increase in HIF-2α in the colon initiates pro-tumorigenic signaling which may have important implications in developing preventive and therapeutic strategies for colon cancer.
hypoxia-inducible factor-2α; colon cancer; Apcmin/+; iron homeostasis; divalent metal transporter-1
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are involved in regulating glucose and lipid homeostasis, inflammation, proliferation and differentiation. Although all of these functions might contribute to the influence of PPARs in carcinogenesis, there is a distinct need for a balanced review of the literature and additional experimentation to determine the potential for targeting PPARs for cancer therapy and cancer chemoprevention. As PPAR agonists include drugs used for the treatment of metabolic diseases, a more complete understanding of the roles of PPARs in cancer will aid in determining any increased cancer risk for patients undergoing therapy with PPAR agonists.
Possible prevention and therapeutic intervention strategies to counteract acetaminophen (APAP) hepatotoxicity would be of great value. Wuzhi tablet (WZ, extract of Schisandrae sphenanthera) possesses hepatoprotective effects against hepatitis and the hepatic dysfunction induced by various chemical hepatotoxins. In this study, the protective effect of WZ on APAP-induced hepatic injury was evaluated and targeted metabolomics by LC-MS-based metabolomics was used to examine whether WZ influences hepatic metabolism. The results demonstrated significant hepatoprotection of WZ against APAP-induced liver injury; pretreatment with WZ prior to APAP administration blocks the increase in serum palmitoylcarnitine and oleoylcarnitine and thus restores the APAP-impaired fatty acid β-oxidation to normal levels. These studies further revealed a significant and prolonged upregulation of the PPARα target genes Cpt1 and Acot1 by WZ mainly contributing to the maintenance of normal fatty acid metabolism and thus potentially contributing to the hepatic protection of WZ against APAP-induced hepatic toxicity. Taken together, the current study provides new insights into understanding the hepatoprotective effect of WZ against APAP-induced liver toxicity.
Cytochrome P450 1B1 contributes to the development of angiotensin II-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the kidney, as well as in salt and water homeostasis, and blood pressure regulation, we determined the contribution of cytochrome P450 1B1 to renal dysfunction and injury associated with angiotensin II-induced hypertension in male Cyp1b1+/+ and Cyp1b1−/− mice. Angiotensin II infusion (700 ng/kg/min) given by miniosmotic pumps for 13 and 28 days increased systolic blood pressure in Cyp1b1+/+ mice; this increase was significantly reduced in Cyp1b1−/− mice. Angiotensin II increased renal Cyp1b1 activity, vascular resistance and reactivity to vasoconstrictor agents, and caused endothelial dysfunction in Cyp1b1+/+ but not Cyp1b1−/− mice. Angiotensin II increased water consumption and urine output, decreased urine osmolality, increased urinary Na+ and K+ excretion, and caused proteinuria and albuminuria in Cyp1b1+/+ mice that was diminished in Cyp1b1−/− mice. Infusion of angiotensin II for 28, but not 13 days, caused renal fibrosis, tubular damage and inflammation in Cyp1b1+/+ mice, which was minimized in Cyp1b1−/− mice. Angiotensin II increased levels of 12- and 20-hydroxyeicosatetraenoic acids; reactive oxygen species; and activity of NADPH oxidase, ERK1/2, p38 MAPK, and c-Src in the kidneys of Cyp1b1+/+ but not Cyp1b1−/− mice. These data suggest that increased thirst, renal dysfunction, and injury and inflammation associated with angiotensin II-induced hypertension in mice depend on cytochrome P450 1B1 activity thus indicating that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension.
angiotensin II; CYP1B1; Cyp1b1−/− mice; renal function; oxidative stress
Endothelium is a crucial blood–tissue interface controlling energy supply according to organ needs. We investigated whether peroxisome proliferator‐activated receptor‐γ (PPARγ) induces expression of fatty acid–binding protein 4 (FABP4) and fatty acid translocase (FAT)/CD36 in capillary endothelial cells (ECs) to promote FA transport into the heart.
Methods and Results
Expression of FABP4 and CD36 was induced by the PPARγ agonist pioglitazone in human cardiac microvessel ECs (HCMECs), but not in human umbilical vein ECs. Real‐time PCR and immunohistochemistry of the heart tissue of control (Ppargfl/null) mice showed an increase in expression of FABP4 and CD36 in capillary ECs by either pioglitazone treatment or 48 hours of fasting, and these effects were not found in mice deficient in endothelial PPARγ (Pparg∆EC/null). Luciferase reporter constructs of the Fabp4 and CD36 promoters were markedly activated by pioglitazone in HCMECs through canonical PPAR‐responsive elements. Activation of PPARγ facilitated FA uptake by HCMECs, which was partially inhibited by knockdown of either FABP4 or CD36. Uptake of an FA analogue, 125I‐BMIPP, was significantly reduced in heart, red skeletal muscle, and adipose tissue in Pparg∆EC/null mice as compared with Ppargfl/null mice after olive oil loading, whereas those values were comparable between Ppargfl/null and Pparg∆EC/null null mice on standard chow and a high‐fat diet. Furthermore, Pparg∆EC/null mice displayed slower triglyceride clearance after olive oil loading.
These findings identified a novel role for capillary endothelial PPARγ as a regulator of FA handing in FA‐metabolizing organs including the heart in the postprandial state after long‐term fasting.
capillaries; cardiac metabolism; endothelium; fatty acids; transcription factors
Hypoxia-inducible factor 1 (HIF-1) is a heterodimer composed of HIF-1α and HIF-1β subunits. HIF-1 is known to promote tissue vascularization by activating the transcription of genes encoding angiogenic factors, which bind to receptors on endothelial cells (ECs) and bone marrow-derived angiogenic cells (BMDACs). In this study, we analysed whether HIF-1 activity in the responding ECs and BMDACs is also required for cutaneous vascularization during burn wound healing.
Methods and results
We generated mice with floxed alleles at the Hif1a or Arnt locus encoding HIF-1α and HIF-1β, respectively. Expression of Cre recombinase was driven by the Tie2 gene promoter, which is expressed in ECs and bone marrow cells. Tie2Cre+ and Tie2Cre− mice were subjected to burn wounds of reproducible diameter and depth. Deficiency of HIF-1α or HIF-1β in Tie2-lineage cells resulted in delayed wound closure, reduced vascularization, decreased cutaneous blood flow, impaired BMDAC mobilization, and decreased BMDAC homing to burn wounds.
HIF-1 activity in Tie2-lineage cells is required for the mobilization and homing of BMDACs to cutaneous burn wounds and for the vascularization of burn wound tissue.
Hypoxia; Wound healing; Conditional knockout; Angiogenesis
Previously, we showed that the cytochrome P450 1B1 inhibitor, 2,3´,4,5´-tetramethoxystilbene, reversed DOCA-salt induced hypertension and minimized endothelial and renal dysfunction in the rat. This study was conducted to test the hypothesis that cytochrome P450 1B1 contributes to cardiac dysfunction, and renal damage and inflammation associated with DOCA-salt-induced hypertension, via increased production of reactive oxygen species, and modulation of neurohumoral factors and signaling molecules. DOCA-salt increased systolic blood pressure, cardiac and renal cytochrome P450 1B1 activity, and plasma levels of catecholamines, vasopressin, and endothelin-1 in wild type (Cyp1b1+/+) mice that were minimized in Cyp1b1−/− mice. Cardiac function, assessed by echocardiography, showed that DOCA-salt increased the thickness of the left ventricular posterior and anterior walls during diastole, the left ventricular internal diameter, and end-diastolic and end-systolic volume in Cyp1b1+/+ but not Cyp1b1−/− mice; stroke volume was not altered in either genotype. DOCA-salt increased renal vascular resistance and caused vascular hypertrophy, renal fibrosis, increased renal infiltration of macrophages and T-lymphocytes, caused proteinuria, increased cardiac and renal NADPH oxidase activity, production of reactive oxygen species, and activities of ERK1/2, p38 MAPK and c-Src; these were all reduced in DOCA-salt-treated Cyp1b1−/− mice. Renal and cardiac levels of eicosanoids were not altered in either genotype of mice. These data suggest that in DOCA-salt hypertension in mice, cytochrome P450 1B1 plays a pivotal role in cardiovascular dysfunction, renal damage and inflammation, and increased levels of catecholamines, vasopressin, and endothelin-1, consequent to generation of reactive oxygen species and activation of ERK1/2, p38 MAPK, and c-Src independent of eicosanoids.
DOCA-salt; cytochrome P450 1B1; hypertension; oxidative stress; cardiac dysfunction; renal fibrosis; inflammation
Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) function and receptor cross-talk with other nuclear receptors, including PPARγ and retinoic acid receptors (RARs), was examined using stable human HaCaT keratinocyte cell lines over-expressing PPARβ/δ or PPARγ. Enhanced ligand-induced expression of two known PPAR target genes, adipocyte differentiation-related protein (ADRP) and angiopoietin-like protein 4 (ANGPTL4), was found in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ. Over-expression of PPARβ/δ did not modulate the effect of a PPARγ agonist on up-regulation of ADRP or ANGPTL4 mRNA in HaCaT keratinocytes. All-trans retinoic acid (atRA) increased expression of a known RAR target gene, yet despite a high ratio of fatty acid binding protein 5 (FABP5) to cellular retinoic acid binding protein II, did not increase expression of ANGPTL4 or 3-phosphoinositide-dependent-protein kinase 1 (PDPK1), even in HaCaT keratinocytes expressing markedly higher levels of PPARβ/δ. While PPARβ/δ-dependent attenuation of staurosporine- or UVB-induced poly (ADP-ribose) polymerase (PARP) cleavage was not observed, PPARβ/δ- and PPARγ-dependent repression of UVB-induced expression and secretion of inflammatory cytokines was found in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ. These studies suggest that FABP5 does not transport atRA or GW0742 to PPARβ/δ and promote anti-apoptotic activity by increasing expression of PDPK1, or that PPARβ/δ interferes with PPARγ transcriptional activity. However, these studies demonstrate that stable over-expression of PPARβ/δ or PPARγ significantly increases the efficacy of ligand activation and represses UVB-induced expression of tumor necrosis factor α (TNFα), interleukin 6 (IL6), or IL8 in HaCaT keratinocytes, thereby establishing an excellent model to study the functional role of these receptors in human keratinocytes.
human keratinocytes; PPARβ/δ; PPARγ; retinoic acid; apoptosis; inflammation