CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis.

BACKGROUND: TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 (cluster of differentiation) and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis. METHODS: Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC Cd47 in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms. RESULTS: Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF (vascular endothelial growth factor)-C-induced AKT and eNOS activation in LEC and attenuated NO (nitric oxide) production. Further, CD47 silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV (adeno-associated virus) 8-PCSK9-injected LEC-specific Cd47 knockout mice (Cd47ΔLEC) had reduced atherosclerosis in both aorta and aortic root compared with control mice (Cd47ΔWT). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in Cd47ΔLEC mice in comparison to controls. CONCLUSIONS: These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC Cd47 attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.

Pregnane X receptor promotes ethanol-induced hepatosteatosis in mice.

The pregnane X receptor (PXR, NR1I2) is a xenobiotic-sensing nuclear receptor that modulates the metabolic response to drugs and toxic agents. Both PXR activation and deficiency promote hepatic triglyceride accumulation, a hallmark feature of alcoholic liver disease. However, the molecular mechanism of PXR-mediated activation of ethanol (EtOH)-induced steatosis is unclear. Here, using male wildtype (WT) and Pxr-null mice, we examined PXR-mediated regulation of chronic EtOH-induced hepatic lipid accumulation and hepatotoxicity. EtOH ingestion for 8 weeks significantly (1.8-fold) up-regulated Pxr mRNA levels in WT mice. The EtOH exposure also increased mRNAs encoding hepatic constitutive androstane receptor (3-fold) and its target, Cyp2b10 (220-fold), in a PXR-dependent manner. Furthermore, WT mice had higher serum EtOH levels and developed hepatic steatosis characterized by micro- and macrovesicular lipid accumulation. Consistent with the development of steatosis, lipogenic gene induction was significantly increased in WT mice, including sterol regulatory element-binding protein 1c target gene fatty-acid synthase (3.0-fold), early growth response-1 (3.2-fold), and TNFα (3.0-fold), whereas the expression of peroxisome proliferator-activated receptor α target genes was suppressed. Of note, PXR deficiency suppressed these changes and steatosis. Protein levels, but not mRNAs levels, of EtOH-metabolizing enzymes, including alcohol dehydrogenase 1, aldehyde dehydrogenase 1A1, and catalase, as well as the microsomal triglyceride transfer protein, involved in regulating lipid output were higher in Pxr-null than in WT mice. These findings establish that PXR signaling contributes to ALD development and suggest that PXR antagonists may provide a new approach for ALD therapy.

Role of the pregnane X receptor in binge ethanol-induced steatosis and hepatotoxicity.

The pregnane X receptor (PXR, NR1I2) is a xenobiotic-sensing nuclear receptor that defends against toxic agents. We have shown that PXR promotes chronic ethanol (EtOH)-induced steatosis. Therefore, we examined the role of PXR in binge EtOH-induced hepatotoxicity. Male wild type (WT) and Pxr-null mice were orally administered three binge doses of EtOH (4.5 g/kg, every 12 hours) and euthanized four hours after the final dose. Pxr-null mice displayed higher basal mRNA levels of hepatic lipogenic transcription factor sterol regulatory element binding protein 1c (Srebp-1c) and its target stearoyl-CoA desaturase 1 (Scd1) and the lipid peroxide detoxifying aldo-keto reductase 1b7 (Akr1b7) and higher protein levels of EtOH-metabolizing alcohol dehydrogenase 1 (ADH1). In both genotypes, binge EtOH-induced triglyceride accumulation was associated with inhibition of fatty acid ß-oxidation and upregulation of Srebp-1c- regulated lipogenic genes and hepatic CYP2E1 protein. Unexpectedly, gene expression of Cyp2b10, a constitutive androstane receptor target gene, implicated in EtOH hepatotoxicity, was PXR-dependent upregulated by binge EtOH. Also, PXR-dependent was the binge EtOH-induced inhibition of hepatic Akr1b8 mRNA, and protein levels of aldehyde dehydrogenase (ALDH) 1A1 and anti-apoptotic Bcl-2, but increased pro-apoptotic Bax protein expression, leading to increases in residual EtOH concentration and the cellular oxidative stress marker, malondialdehyde. In contrast, Pxr-null mice displayed increased Akr1b7 gene and ADH1 protein expression and hypertriglyceridemia following binge EtOH exposure. Taken together, this study demonstrates that PXR ablation prevents EtOH induced upregulation of Cyp2b10 and that PXR potentiates binge EtOH-induced oxidative stress and inhibition of EtOH catabolism, but protects against alcoholic hyperlipidemia.

Serotonin induced hepatic steatosis is associated with modulation of autophagy and notch signaling pathway.

BACKGROUND: Besides its neurotransmitter and vasoconstriction functions, serotonin is an important mediator of numerous biological processes in peripheral tissues including cell proliferation, steatosis, and fibrogenesis. Recent reports indicate that serotonin may promote tumor growth in liver cancer, however, the molecular mechanisms remain elusive. n this study, we investigated the role and molecular signaling mechanisms mediated by serotonin in liver cancer cell survival, drug resistance, and steatosis. METHODS: Effect of serotonin on modulation of cell survival/proliferation was determined by MTT/WST1 assay. Effect of serotonin on the regulation of autophagy biomarkers and lipid/fatty acid proteins expression, AKT/mTOR and Notch signaling was evaluated by immunoblotting. The role of serotonin in normal human hepatocytes and liver cancer cell steatosis was analyzed by Oil Red O staining. The mRNA expression levels of lipid/fatty acid proteins and serotonin receptors were validated by qRT-PCR. The important roles of autophagy, Notch signaling, serotonin receptors and serotonin re-uptake proteins on serotonin-mediated cell steatosis were investigated by using selective inhibitors or antagonists. The association of peripheral serotonin, autophagy, and hepatic steatosis was also investigated using chronic EtOH fed mouse model. RESULTS: Exposure of liver cancer cells to serotonin induced Notch signaling and autophagy, independent of AKT/mTOR pathway. Also, serotonin enhanced cancer cell proliferation/survival and drug resistance. Furthermore, serotonin treatment up-regulated the expression of lipogenic proteins and increased steatosis in liver cancer cells. Inhibition of autophagy or Notch signaling reduced serotonin-mediated cell steatosis. Treatment with serotonin receptor antagonists 5-HTr1B and 5-HTr2B reduced serotonin-mediated cell steatosis; in contrast, treatment with selective serotonin reuptake inhibitors (SSRIs) increased steatosis. In addition, mice fed with chronic EtOH resulted in increased serum serotonin levels which were associated with the induction of hepatic steatosis and autophagy. CONCLUSIONS: Serotonin regulates liver cancer cell steatosis, cells survival, and may promote liver carcinogenesis by activation of Notch signaling and autophagy.

Role of pregnane X receptor in obesity and glucose homeostasis in male mice.

Clinical obesity is a complex metabolic disorder affecting one in three adults. Recent reports suggest that pregnane X receptor (PXR), a xenobiotic nuclear receptor important for defense against toxic agents and for eliminating drugs and other xenobiotics, may be involved in obesity. Noting differences in ligand specificities between human and mouse PXRs, the role of PXR in high fat diet (HFD)-induced obesity was examined using male PXR-humanized (hPXR) transgenic and PXR-knock-out (PXR-KO) mice in comparison to wild-type (WT) mice. After 16 weeks on either a control diet or HFD, WT mice showed greater weight gain, whereas PXR-KO mice gained less weight due to their resistance to HFD-induced decreases in adipose tissue peroxisome proliferator-activated receptor α and induction of hepatic carnitine palmitoyltransferase 1, suggesting increased energy metabolism. Interestingly, control-fed PXR-KO mice exhibited hepatomegaly, hyperinsulinemia, and hyperleptinemia but hypoadiponectinemia and lower adiponectin receptor R2 mRNA levels relative to WT mice. Evaluation of these biologic indicators in hPXR mice fed a control diet or HFD revealed further differences between the mouse and human receptors. Importantly, although HFD-fed hPXR mice were resistant to HFD-induced obesity, both PXR-KO and hPXR mice exhibited impaired induction of glucokinase involved in glucose utilization and displayed elevated fasting glucose levels and severely impaired glucose tolerance. Moreover, the basal hepatic levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 were increased in hPXR mice compared with WT mice. Altogether, although the mouse PXR promotes HFD-induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and thus provides a model for exploring the role of human PXR in the metabolic syndrome.

Pregnane X Receptor-Humanized Mice Recapitulate Gender Differences in Ethanol Metabolism but Not Hepatotoxicity.

Both human and rodent females are more susceptible to developing alcoholic liver disease following chronic ethanol (EtOH) ingestion. However, little is known about the relative effects of acute EtOH exposure on hepatotoxicity in female versus male mice. The nuclear receptor pregnane X receptor (PXR; NR1I2) is a broad-specificity sensor with species-specific responses to toxic agents. To examine the effects of the human PXR on acute EtOH toxicity, the responses of male and female PXR-humanized (hPXR) transgenic mice administered oral binge EtOH (4.5 g/kg) were analyzed. Basal differences were observed between hPXR males and females in which females expressed higher levels of two principal enzymes responsible for EtOH metabolism, alcohol dehydrogenase 1 and aldehyde dehydrogenase 2, and two key mediators of hepatocyte replication and repair, cyclin D1 and proliferating cell nuclear antigen. EtOH ingestion upregulated hepatic estrogen receptor α, cyclin D1, and CYP2E1 in both genders, but differentially altered lipid and EtOH metabolism. Consistent with higher basal levels of EtOH-metabolizing enzymes, blood EtOH was more rapidly cleared in hPXR females. These factors combined to provide greater protection against EtOH-induced liver injury in female hPXR mice, as revealed by markers for liver damage, lipid peroxidation, and endoplasmic reticulum stress. These results indicate that female hPXR mice are less susceptible to acute binge EtOH-induced hepatotoxicity than their male counterparts, due at least in part to the relative suppression of cellular stress and enhanced expression of enzymes involved in both EtOH metabolism and hepatocyte proliferation and repair in hPXR females.

Pregnane X receptor knockout mitigates weight gain and hepatic metabolic dysregulation in female C57BL/6 J mice on a long-term high-fat diet.

Obesity is a significant risk factor for several chronic diseases. However, pre-menopausal females are protected against high-fat diet (HFD)-induced obesity and its adverse effects. The pregnane X receptor (PXR, NR1I2), a xenobiotic-sensing nuclear receptor, promotes short-term obesity-associated liver disease only in male mice but not in females. Therefore, the current study investigated the metabolic and pathophysiological effects of a long-term 52-week HFD in female wild-type (WT) and PXR-KO mice and characterized the PXR-dependent molecular pathways involved. After 52 weeks of HFD ingestion, the body and liver weights and several markers of hepatotoxicity were significantly higher in WT mice than in their PXR-KO counterparts. The HFD-induced liver injury in WT female mice was also associated with upregulation of the hepatic mRNA levels of peroxisome proliferator-activated receptor gamma (Pparg), its target genes, fat-specific protein 27 (Fsp27), and the liver-specific Fsp27b involved in lipid accumulation, apoptosis, and inflammation. Notably, PXR-KO mice displayed elevated hepatic Cyp2a5 (anti-obesity gene), aldo-keto reductase 1b7 (Akr1b7), glutathione-S-transferase M3 (Gstm3) (antioxidant gene), and AMP-activated protein kinase (AMPK) levels, contributing to protection against long-term HFD-induced obesity and inflammation. RNA sequencing analysis revealed a general blunting of the transcriptomic response to HFD in PXR-KO compared to WT mice. Pathway enrichment analysis demonstrated enrichment by HFD for several pathways, including oxidative stress and redox pathway, cholesterol biosynthesis, and glycolysis/gluconeogenesis in WT but not PXR-KO mice. In conclusion, this study provides new insights into the molecular mechanisms by which PXR deficiency protects against long-term HFD-induced severe obesity and its adverse effects in female mice.

High-fat diet induced obesity promotes inflammation, oxidative stress, and hepatotoxicity in female FVB/N mice.

Although obesity and subsequent liver injury are increasingly prevalent in women, female mouse models have generally shown resistance to high-fat diet (HFD)-induced obesity. We evaluated control and HFD-fed male and female FVB/N mice, a strain well-suited to transgenic analyses, for phenotypic, histological, and molecular markers related to control of glucose, lipids, and inflammation in serum, liver, and perigonadal white adipose tissues. Unlike many mouse models, HFD-fed FVB/N females gained more perigonadal and mesenteric fat mass and overall body weight than their male counterparts, with increased hepatic expression of lipogenic PPARγ target genes (Cd36, Fsp27, and Fsp27ß), oxidative stress genes and protein (Nqo1 and CYP2E1), inflammatory gene (Mip-2), and the pro-fibrotic gene Pai-1, along with increases in malondialdehyde and serum ALT levels. Further, inherent to females (independently of HFD), hepatic antioxidant heme oxygenase-1 (HMOX1, HO-1) protein levels were reduced compared to their male counterparts. In contrast, males may have been relatively protected from HFD-induced oxidative stress and liver injury by elevated mRNA and protein levels of hepatic antioxidants BHMT and Gpx2, increased fatty acid oxidation genes in liver and adipocytes (Pparδ), despite disorganized and inflamed adipocytes. Thus, female FVB/N mice offer a valuable preclinical, genetically malleable model that recapitulates many of the features of diet-induced obesity and liver damage observed in human females.

Molecular targets of PXR-dependent ethanol-induced hepatotoxicity in female mice.

The pregnane X receptor (PXR, NR1I2), a xenobiotic-sensing nuclear receptor signaling potentiates ethanol (EtOH)-induced hepatotoxicity in male mice, however, how PXR signaling modulates EtOH-induced hepatotoxicity in female mice is unknown. Wild type (WT) and Pxr-null mice received 5 % EtOH-containing diets or paired-fed control diets for 8 weeks followed by assessment of liver injury, EtOH elimination rates, histology, and changes in gene and protein expression; microarray and bioinformatic analyses were also employed to identify PXR targets in chronic EtOH-induced hepatotoxicity. In WT females, EtOH ingestion significantly increased serum ethanol and alanine aminotransferase (ALT) levels, hepatic Pxr mRNA, constitutive androstane receptor activation, Cyp2b10 mRNA and protein, oxidative stress, endoplasmic stress (phospho-elF2α) and pro-apoptotic (Bax) protein expression. Unexpectedly, EtOH-fed female Pxr-null mice displayed increased EtOH elimination and elevated levels of hepatic acetaldehyde detoxifying aldehyde dehydrogenase 1a1 (Aldh1a1) mRNA and protein, EtOH-metabolizing alcohol dehydrogenase 1 (ADH1), and lipid suppressing microsomal triglyceride transport protein (MTP) protein, aldo-keto reductase 1b7 (Akr1b7) and Cyp2a5 mRNA, but suppressed CYP2B10 protein levels, with evidence of protection against chronic EtOH-induced oxidative stress and hepatotoxicity. While liver injury was not different between the two WT sexes, female sex may suppress EtOH-induced macrovesicular steatosis in the liver. Several genes and pathways important in retinol and steroid hormone biosynthesis, chemical carcinogenesis, and arachidonic acid metabolism were upregulated by EtOH in a PXR-dependent manner in both sexes. Together, these data establish that female Pxr-null mice are resistant to chronic EtOH-induced hepatotoxicity and unravel the PXR-dependent and -independent mechanisms that contribute to EtOH-induced hepatotoxicity.

MicroRNA-483-5p Inhibits Hepatocellular Carcinoma Cell Proliferation, Cell Steatosis, and Fibrosis by Targeting PPARα and TIMP2.

MicroRNAs (miRNAs) are small non-coding RNA molecules that bind with the 3' untranslated regions (UTRs) of genes to regulate expression. Downregulation of miR-483-5p (miR-483) is associated with the progression of hepatocellular carcinoma (HCC). However, the significant roles of miR-483 in nonalcoholic fatty liver disease (NAFLD), alcoholic fatty liver diseases (AFLD), and HCC remain elusive. In the current study, we investigated the biological significance of miR-483 in NAFLD, AFLD, and HCC in vitro and in vivo. The downregulation of miR-483 expression in HCC patients' tumor samples was associated with Notch 3 upregulation. Overexpression of miR-483 in a human bipotent progenitor liver cell line HepaRG and HCC cells dysregulated Notch signaling, inhibited cell proliferation/migration, induced apoptosis, and increased sensitivity towards antineoplastic agents sorafenib/regorafenib. Interestingly, the inactivation of miR-483 upregulated cell steatosis and fibrosis signaling by modulation of lipogenic and fibrosis gene expression. Mechanistically, miR-483 targets PPARα and TIMP2 gene expression, which leads to the suppression of cell steatosis and fibrosis. The downregulation of miR-483 was observed in mice liver fed with a high-fat diet (HFD) or a standard Lieber-Decarli liquid diet containing 5% alcohol, leading to increased hepatic steatosis/fibrosis. Our data suggest that miR-483 inhibits cell steatosis and fibrogenic signaling and functions as a tumor suppressor in HCC. Therefore, miR-483 may be a novel therapeutic target for NAFLD/AFLD/HCC management in patients with fatty liver diseases and HCC.

Pregnane X receptor exacerbates nonalcoholic fatty liver disease accompanied by obesity- and inflammation-prone gut microbiome signature.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease due to the current epidemics of obesity and diabetes. The pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor known for trans-activating liver genes involved in drug metabolism and transport, and more recently implicated in energy metabolism. The gut microbiota can modulate the host xenobiotic biotransformation and contribute to the development of obesity. While the male sex confers a higher risk for NAFLD than women before menopause, the mechanism remains unknown. We hypothesized that the presence of PXR promotes obesity by modifying the gut-liver axis in a sex-specific manner. Male and female C57BL/6 (wild-type/WT) and PXR-knockout (PXR-KO) mice were fed control or high-fat diet (HFD) for 16-weeks. Serum parameters, liver histopathology, transcriptomic profiling, 16S-rDNA sequencing, and bile acid (BA) metabolomics were performed. PXR enhanced HFD-induced weight gain, hepatic steatosis and inflammation especially in males, accompanied by PXR-dependent up-regulation in hepatic genes involved in microbial response, inflammation, oxidative stress, and cancer; PXR-dependent increase in intestinal Firmicutes/Bacteroides ratio (hallmark of obesity) and the pro-inflammatory Lactobacillus, as well as a decrease in the anti-obese Allobaculum and the anti-inflammatory Bifidobacterum, with a PXR-dependent reduction of beneficial BAs in liver. The resistance to NAFLD in females may be explained by PXR-dependent decrease in pro-inflammatory bacteria (Ruminococcus gnavus and Peptococcaceae). In conclusion, PXR exacerbates hepatic steatosis and inflammation accompanied by obesity- and inflammation-prone gut microbiome signature, suggesting that gut microbiome may contribute to PXR-mediated exacerbation of NAFLD.

TNFAIP8 regulates autophagy, cell steatosis, and promotes hepatocellular carcinoma cell proliferation.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) expression has been linked to tumor progression in various cancer types, but the detailed mechanisms of TNFAIP8 are not fully elucidated. Here we define the role of TNFAIP8 in early events associated with development of hepatocellular carcinoma (HCC). Increased TNFAIP8 levels in HCC cells enhanced cell survival by blocking apoptosis, rendering HCC cells more resistant to the anticancer drugs, sorafenib and regorafenib. TNFAIP8 also induced autophagy and steatosis in liver cancer cells. Consistent with these observations, TNFAIP8 blocked AKT/mTOR signaling and showed direct interaction with ATG3-ATG7 proteins. TNFAIP8 also exhibited binding with fatty acids and modulated expression of lipid/fatty-acid metabolizing enzymes. Chronic feeding of mice with alcohol increased hepatic levels of TNFAIP8, autophagy, and steatosis but not in high-fat-fed obese mice. Similarly, higher TNFAIP8 expression was associated with steatotic livers of human patients with a history of alcohol use but not in steatotic patients with no history of alcohol use. Our data indicate a novel role of TNFAIP8 in modulation of drug resistance, autophagy, and hepatic steatosis, all key early events in HCC progression.

Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXRalpha-null mice.

Retinoid X receptor-alpha (RXRalpha) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXRalpha deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXRalpha-null (H-RXRalpha-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid beta-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXRalpha-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXRalpha-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXRalpha-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXRalpha-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXRalpha-null mice. In conclusion, these data suggest a critical role for RXRalpha in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.

NF-E2-related factor 2 inhibits lipid accumulation and oxidative stress in mice fed a high-fat diet.

NF-E2-related factor 2 (Nrf2) is a transcription factor that is activated by oxidative stress and electrophiles that regulates the expression of numerous detoxifying and antioxidant genes. Previous studies have shown that Nrf2 protects the liver from xenobiotic toxicity; however, whether Nrf2 plays a role in lipid homeostasis in liver is not known. Accordingly, wild-type and Nrf2-null mice were fed a high-fat diet (HFD) for up to 4 weeks. Hepatic gene expression and lipid profiles were analyzed for changes in fatty acid, triglyceride, and cholesterol status. It is interesting to note that HFD reduced the mRNA expression of Nrf2 and its target genes in wild-type mice. The mRNA expression of lipogenic and cholesterologenic transcriptional factors and their target genes, such as sterol regulatory element-binding proteins 1c and 2, fatty acid synthase, acetyl-CoA carboxylase 1, fatty acid elongase, 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase, and low-density lipoprotein receptor mRNA expression were higher in Nrf2-null mice compared with wild-type mice after feeding a HFD, suggesting that Nrf2 may suppress these pathways. Hepatic triglycerides and cholesterol levels were not different between genotypes, whereas concentrations of hepatic free fatty acid and malondialdehyde equivalents were higher in Nrf2-null mice compared with wild-type mice 4 weeks after HFD feeding. Overall, these results suggest that Nrf2 inhibits lipid accumulation and oxidative stress in mouse liver after feeding a HFD, probably by interfering with lipogenic and cholesterologenic pathways.

Hepatocyte retinoid X receptor alpha-dependent regulation of lipid homeostasis and inflammatory cytokine expression contributes to alcohol-induced liver injury.

Hepatocyte retinoid X receptor alpha (RXRalpha)-deficient mice are more sensitive to ethanol toxicity than wild-type mice. Because RXRalpha-mediated pathways are implicated in lipid homeostasis and the inflammatory response, we hypothesized that a compromise in lipid metabolism and associated production of proinflammatory mediators are responsible for the hepatotoxicity observed in ethanol-treated hepatocyte RXRalpha-deficient mice. Wild-type and hepatocyte RXRalpha-deficient mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. After ethanol treatment, serum ALT levels increased significantly (4-fold) in hepatocyte RXRalpha-deficient mice, but not in the wild-type mice. Hepatic liver fatty acid binding protein (L-FABP) mRNA and protein levels were reduced due to RXRalpha deficiency. Ethanol induced L-FABP mRNA and protein in wild-type mice and provided protection against nonesterified fatty acid toxicity; however, this effect was absent in the mutant mice. Accordingly, hepatic nonesterified fatty acid level was increased in ethanol-fed mutant mice. Ethanol increased nuclear factor (NF)-kappaB binding activity in hepatocyte RXRalpha-deficient mice, but not in wild-type mice. In agreement, hepatic mRNA levels of proinflammatory cytokines and chemokines were increased to a greater extent in the mutant than in wild-type mice. Furthermore, signal transducer and activator of transcription factor (STAT) 3 and associated Bcl-xL induction was observed in ethanol-fed wild-type mice but not in ethanol-fed hepatocyte RXRalpha-deficient mice. Taken together, after ethanol treatment, hepatocyte RXRalpha deficiency results in lack of L-FABP induction, increased hepatic free fatty acids, NF-kappaB activation, and proinflammatory cytokines production and a lack of STAT3 activation, which in part may contribute to alcohol-induced liver damage.

The effect of ethanol, ethanol metabolizing enzyme inhibitors, and Vitamin E on regulating glutathione, glutathione S-transferase, and S-adenosylmethionine in mouse primary hepatocyte.

We studied changes in the antioxidant systems involved in hepatoprotection after ethanol exposure in primary culture of mouse hepatocytes. Ethanol decreased glutathione (GSH) levels and the S-adenosylmethionine (SAMe) to S-adenosylhomocysteine (SAH) ratio by 53% and 22%, respectively. Cytosolic glutathione S-transferase (GST) activity was significantly lower in ethanol exposed hepatocytes, which was accompanied by an increase in GST activity in the culture medium. When specific substrates for mu- and pi-class GST were utilized, ethanol significantly decreased the mu- and pi-class GST activity by 53% and 13%, respectively. Lipid peroxidation (LPO), assessed by the thiobarbituric acid assay, increased to 221% of control by ethanol and was potentiated by cyanamide, an aldehyde dehydrogenase inhibitor. The changes in LPO, cytosolic GST activity, GSH levels and SAMe/SAH ratio in ethanol exposed hepatocytes were completely or partially reversed by either Vitamin E or 4-methylpyrazole, an alcohol dehydrogenase (ADH) inhibitor. Retinoid X receptor alpha-deficient (RXRalpha KO) mice, which are more susceptible to ethanol-induced liver toxicity, have decreased pi-class GST (56%), mu-class GST (28%), and glutathione peroxidase (35%) activities compared with wild type. Taken together, primary hepatocyte provides a valuable model to analyze ethanol-induced oxidative stress. The inhibition of mu-class GST activity by ethanol and the decreased pi-class GST activity in RXRalpha KO mice implicate the importance of these isozymes in ethanol detoxification process.

Antioxidant properties of Thonningianin A, isolated from the African medicinal herb, Thonningia sanguinea.

The antioxidant properties of Thonningianin A (Th A), an ellagitannin, isolated from the methanolic extract of the African medicinal herb, Thonningia sanguinea were studied using the NADPH and Fe2+/ascorbate-induced lipid peroxidation (LPO), electron spin resonance spectrometer and the deoxyribose assay. Th A at 10 microM inhibited both the NADPH and Fe2+/ascorbate-induced LPO in rat liver microsomes by 60% without inhibitory effects on cytochrome P450 activity. Th A was similar to the synthetic antioxidant, tannic acid, as an inhibitor of both the NADPH and Fe2+/ascorbate-induced LPO but potent than gallic acid, vitamin C and vitamin E. While Th A poorly scavenged the hydroxyl radical generated by the Fenton reaction it dose-dependently scavenged 1,1-diphenyl-2-picrylhydrazyl, superoxide anion and peroxyl radicals with IC50 of 7.5, 10 and 30 microM, respectively. Furthermore, Th A showed inhibitory effects on the activity of xanthine oxidase with an IC50 of 30 microM. In the deoxyribose assay both T. sanguinea and its methanolic component Th A showed only site-specific (Fe3+ + H2O2) but not non-site-specific (Fe3+ + EDTA + H2O2) hydroxyl radical scavenging suggesting chelating ability for iron ions. Spectroscopic studies showed that Th A enhanced absorbance in the visible region in the presence of Fe2+ ions. These results indicate that the antioxidant properties of Th A involve radical scavenging, anti-superoxide formation and metal chelation.

Selective suppression of cytochrome P450 gene expression by the medicinal herb, Thonningia sanguinea in rat liver.

The effect of the administration of Thonningia sanguinea (T. S.) on the abundance of individual components of the cytochrome P450 monooxygenase enzyme was examined using Western blotting and competitive reverse-transcriptase-polymerase chain reaction (RT-PCR). We also investigated the time-course of inhibition of T. S. on drug metabolizing enzymes. A single intraperitoneal dose of T. S. extract (5 ml/kg) suppressed CYP, cytochrome b5 and NADPH-CYP reductase activity by 45%, 34% and 22% respectively 24 h after T. S. administration. While T. S. did not have any significant effect on microsomal glutathione S-transferase activity, it inhibited p-nitrophenol hydroxylase (PNPH, CYP2E1) and 7-methoxyresorufin O-demethylase (MROD, CYP 1A2) activities by 37% and 32% respectively at 12 h post-T. S. administration. PNPH, erythromycin N-demethylase (ERDM, CYP 3A1/2) and MROD activities were inhibited by 28-36% 24 h after T. S. injection. Consistent with these observations, the levels of CYP2E1, CYP1A2 and CYP3A2 proteins were also suppressed 24 h post-T. S. administration. While CYP2E1 mRNA was unaffected by T. S. administration, CYP1A2 and CYP3A2 mRNAs were decreased by T. S. Cytosolic glutathione S-transferase activity was increased by 30%, 6 h after T. S injection. These data demonstrate that administration of T. S. differentially affect CYP isoforms in the liver of rats and that T. S. selectively suppresses CYP3A2 and CYP1A2 gene expression.

Inhibition of glutathione S-transferases by thonningianin A, isolated from the African medicinal herb, Thonningia sanguinea, in vitro.

There is evidence that increased expression of glutathione S-transferase (EC: 2.5.1.18, GST) is involved in resistance of tumor cells against chemotherapeutic agents. In this study we investigated the inhibitory effects of thonningianin A (Th A), a novel antioxidant isolated from the medicinal herb, Thonningia sanguinea on uncharacterized rat liver GST and human GST P1-1. Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, rat liver cytosolic GST activity was inhibited by Th A in a concentration dependent manner with 50% inhibition concentration (IC50) of 1.1 microM. When Th A was compared with known potent GST inhibitors the order of inhibition was tannic acid>cibacron blue>hematin>Th A>ethacrynic acid with CDNB as substrate. Th A also exhibited non-competitive inhibition towards both CDNB and glutathione. Furthermore, using 1,2-dichloro-4-nitrobenzene, ethacrynic acid and 1,2-epoxy-3-(p-nitrophenoxy) propane as substrates Th A at 1.0 microM inhibited cytosolic GST by 2%, 12% and 36% respectively. Human GST P1-1 was also inhibited by Th A with an IC50 of 3.6 microM. While Th A showed competitive inhibition towards CDNB it exhibited non-competitive inhibition towards GSH of the human GST P1-1. These results suggest that Th A represents a new potent GST in vitro inhibitor.

Role of human pregnane X receptor in high fat diet-induced obesity in pre-menopausal female mice.

Obesity is a complex metabolic disorder that is more prevalent among women. Until now, the only relevant rodent models of diet-induced obesity were via the use of ovariectomized ("postmenopausal") females. However, recent reports suggest that the xenobiotic nuclear receptor pregnane X receptor (PXR) may contribute to obesity. Therefore, we compared the roles of mouse and human PXRs in diet-induced obesity between wild type (WT) and PXR-humanized (hPXR) transgenic female mice fed either control or high-fat diets (HFD) for 16 weeks. HFD-fed hPXR mice gained weight more rapidly than controls, exhibited hyperinsulinemia, and impaired glucose tolerance. Fundamental differences were observed between control-fed hPXR and WT females: hPXR mice possessed reduced estrogen receptor α (ERα) but enhanced uncoupling protein 1 (UCP1) protein expression in white adipose tissue (WAT); increased protein expression of the hepatic cytochrome P450 3A11 (CYP3A11) and key gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, and increased total cholesterol. Interestingly, HFD ingestion induced both UCP1 and glucokinase protein expression in WT mice, but inhibited these enzymes in hPXR females. Unlike WT mice, CYP3A11 protein, serum 17ß-estradiol levels, and WAT ERα expression were unaffected by HFD in hPXR females. Together, these studies indicate that the hPXR gene promotes obesity and metabolic syndrome by dysregulating lipid and glucose homeostasis while inhibiting UCP1 expression. Furthermore, our studies indicate that the human PXR suppresses the protective role of estrogen in metabolic disorders. Finally, these data identify PXR-humanized mice as a promising in vivo research model for studying obesity and diabetes in women.