Expression of CYP3A in chronic ethanol-fed mice is mediated by endogenous pregnane X receptor ligands formed by enhanced cholesterol metabolism.

Pregnane X receptor (PXR) is a nuclear receptor that plays a key regulatory role in xenobiotic metabolism in a ligand-dependent manner. Recently, ethanol was reported to be either an inducer or inhibitor of Cytochrome P450 (CYP) 3A expression. According to our recent microarray data, chronic ethanol upregulates the expression of the genes associated with oxidative phase I drug metabolism, phase II conjugation reaction and phase III xenobiotic transport, most of which are known to be regulated by PXR. In this study, we investigated the effects of chronic ethanol on the expression and activity of CYP3A11 in mice and the role of PXR. Ethanol was administrated to male ICR mice by feeding a standard Lieber-DeCarli diet containing 36 % ethanol for 4 weeks. Ethanol significantly increased hepatic mRNA expression of Pxr and Cyp3a11. Treatment of mice with ethanol increased nuclear translocation of PXR. Consistent with the increase in nuclear PXR, ethanol significantly increased the binding of PXR to the Cyp3a11 promoter. Hepatic cholesterol level and bile acid synthesis are increased by ethanol treatment. The level of some cholesterol metabolites, such as 5ß-cholestane-3α,7α,12α-triol, 7α-hydroxy-4-cholestene-3-one and lithocholic acid, that have been identified as potent PXR agonists are increased in the livers of ethanol-treated mice. In summary, chronic ethanol upregulates the expression of Pxr and Cyp3a11 mRNAs and proteins in mice by PXR activation mediated by enhanced cholesterol metabolism and bile acid synthesis. Our data provide some critical information needed to understand the molecular mechanisms of ethanol-induced CYP3A expression.

Analysis of hepatic gene expression during fatty liver change due to chronic ethanol administration in mice.

Chronic consumption of ethanol can cause cumulative liver damage that can ultimately lead to cirrhosis. To explore the mechanisms of alcoholic steatosis, we investigated the global intrahepatic gene expression profiles of livers from mice administered alcohol. Ethanol was administered by feeding the standard Lieber-DeCarli diet, of which 36% (high dose) and 3.6% (low dose) of the total calories were supplied from ethanol for 1, 2, or 4 weeks. Histopathological evaluation of the liver samples revealed fatty changes and punctate necrosis in the high-dose group and ballooning degeneration in the low-dose group. In total, 292 genes were identified as ethanol responsive, and several of these differed significantly in expression compared to those of control mice (two-way ANOVA; p<0.05). Specifically, the expression levels of genes involved in hepatic lipid transport and metabolism were examined. An overall net increase in gene expression was observed for genes involved in (i) glucose transport and glycolysis, (ii) fatty acid influx and de novo synthesis, (iii) fatty acid esterification to triglycerides, and (iv) cholesterol transport, de novo cholesterol synthesis, and bile acid synthesis. Collectively, these data provide useful information concerning the global gene expression changes that occur due to alcohol intake and provide important insights into the comprehensive mechanisms of chronic alcoholic steatosis.

Identification of novel synthetic peptide showing angiogenic activity in human endothelial cells.

A novel synthetic hexapeptide (SFKLRY-NH(2)) that displays angiogenic activity has been identified by positional scanning of a synthetic peptide combinatorial library (PS-SPCL). The peptide induced proliferation, migration, and capillary-like tube formation in primary cultured HUVECs, and augmented vessel sprouting ex vivo while attenuated by the treatment with pertussis toxin (PTX) or U73122 (PLC-inhibitor) suggesting the influence of PTX-sensitive G-proteins and PLC. In addition, SFKLRY-NH(2) up-regulated the expression of VEGF-A in HUVECs and the neutralizing antibody against VEGF suppressed SFKLRY-NH(2)-induced tube formation activity. Taken together, these results suggest that SFKLRY-NH(2) may induce blood vessel formation by PTX-sensitive G protein-coupled receptor-PLC-Ca(2+) signaling cascade leading into VEGF-A expression in HUVECs.

Chitosan dosage regimen to trap fecal oil excretion after peroral lipase inhibitor administration in mice.

This study was designed to investigate the oil entrapment and systemic oil absorption-reducing activities of chitosan. High-molecular-weight chitosan formed gel aggregates with oil and bile salts in vitro. The oil/chitosan ratio and the molecular weight of chitosan were optimized for the in vivo study, and a molecular weight >100,000 was effective in reducing the oil contamination of mouse fur. The oil/chitosan weight ratio required for effective oil entrapment was less than 13 and 5 in the in vitro and in vivo experiments, respectively. Chitosan administration was most effective during meals, and high-molecular-weight chitosan could trap and facilitate the reduction of systemic absorption of oil droplets separated by orlistat. The activity of the lipase inhibitor was not altered by chitosan as evidenced by thin layer chromatography, and orlistat was not absorbed systemically by the co-administration of chitosan.

Akt is translocated to the mitochondria during etoposide-induced apoptosis of HeLa cells.

Akt, or protein kinase B, is a key serine-threonine kinase, which exerts anti-apoptotic effects and promotes cell proliferation in response to various stimuli. Recently, however, it was demonstrated that Akt exhibits a proapoptotic role in certain contexts. During etoposide­induced apoptosis of HeLa cells, Akt enhances the interaction of second mitochondria­derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) and X­linked inhibitor of apoptosis protein by phosphorylating Smac at serine 67, and thus promotes apoptosis. However, the detailed mechanisms underlying Akt regulation in etoposide­mediated apoptosis remain to be determined. The present study investigated whether etoposide triggers the translocation of Akt into the mitochondria. It was found that Akt activity was increased and sustained during apoptosis triggered by etoposide in HeLa cells. During apoptosis, Akt was translocated from the cytoplasm into the mitochondria in a phosphoinositide 3­kinase-dependent manner at the early and late stages of apoptosis. Concomitantly, the depletion of Akt in the nuclear fraction was observed after etoposide treatment from analysis of confocal microscopy. The results suggest that etoposide­stimulated Akt is translocated into the mitochondria, thereby possibly enhancing its interaction with Smac and promoting apoptosis in HeLa cells. These results indicate that Akt may be a promising candidate for a pro-apoptotic approach in cancer treatment.

LXR-α antagonist meso-dihydroguaiaretic acid attenuates high-fat diet-induced nonalcoholic fatty liver.

Collaborative regulation of liver X receptor (LXR) and sterol regulatory element binding protein (SREBP)-1 are main determinants in hepatic steatosis, as shown in both animal models and human patients. Recent studies indicate that selective intervention of overly functional LXRα in the liver shows promise in treatment of fatty liver disease. In the present study, we evaluated the effects of meso-dihydroguaiaretic acid (MDGA) on LXRα activation and its ability to attenuate fatty liver in mice. MDGA inhibited activation of the LXRα ligand-binding domain by competitively binding to the pocket for agonist T0901317 and decreased the luciferase activity in LXRE-tk-Luc-transfected cells. MDGA significantly attenuated hepatic neutral lipid accumulation in T0901317- and high fat diet (HFD)-induced fatty liver. The effect of MDGA was so potent that treatment with 1mg/kg for 2 weeks completely reversed the lipid accumulation induced by HFD feeding. MDGA reduced the expression of LXRα co-activator protein RIP140 and LXRα target gene products associated with lipogenesis in HFD-fed mice. These results demonstrate that MDGA has the potential to attenuate nonalcoholic steatosis mediated by selective inhibition of LXRα in the liver in mice.

Magnolia officinalis reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element-binding protein-1c.

The generally accepted hypothesis for the pathogenesis of alcoholic liver disease (ALD) is the two-hit model, which proposes that fat accumulation in the liver increases the sensitivity of the liver to a second hit that leads to inflammatory liver cell damage. In this study we evaluated the effects of Magnolia officinalis (MO), which contains honokiol and magnolol as the primary pharmacological components, to eradicate fatty liver in rats fed an ethanol diet. In vitro studies showed that MO was able to protect RAW 264.7 cells from ethanol-induced production of tumor necrosis factor-alpha, reactive oxygen species, and superoxide anion radicals; the activation of NADPH oxidase; and subsequent cell death. We also investigated the therapeutic effects of MO on alcoholic fatty liver in Lieber-DeCarli ethanol diet-fed rats. MO treatment of the rats for the last 2 weeks of ethanol feeding completely reversed all the serum, hepatic parameters, and fatty liver changes. The increased maturation of sterol regulatory element-binding protein-1c in the liver by ethanol treatment was completely inhibited by treatment with MO. Therefore, MO may be a promising candidate for development as a therapeutic agent for ALD.

Phosphorylation of Smac by JNK3 attenuates its interaction with XIAP.

Here we demonstrate that JNK3 can phosphorylate Smac. Smac phosphorylation attenuates its ability to activate apoptosome activity in HeLa S-100 cell lysates. Addition of the X-linked inhibitor of apoptosis protein (XIAP) to the S-100 markedly suppresses apoptosome activity, and this suppressive effect of XIAP is neutralized by adding unphosphorylated Smac, but not phosphorylated Smac. Furtherover, JNK3-mediated phosphorylation of Smac markedly attenuates the interaction between Smac and XIAP, as measured by BIACORE assays and non-denaturing gel shift assays. When JNK3 activity is down-regulated in etoposide-induced HeLa cells by transiently overexpressing a dominant negative version of JNK3 (DN-JNK3), the caspase-3 activity as well as PARP cleavages are markedly enhanced. And the interaction of Smac with XIAP also increases by down-regulating JNK3 activity under the same conditions. These results suggest that JNK3 activity can attenuate the progression of apoptosis through a novel mechanism of action, the down-regulation of interaction between Smac and XIAP.