For dioxin-induced birth defects, mouse or human CYP1A2 in maternal liver protects whereas mouse CYP1A1 and CYP1B1 are inconsequential

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cleft palate and hydronephrosis in mice, when exposed in utero; these effects are mediated by the aryl hydrocarbon receptor. The Cyp1a1, Cyp1a2, and Cyp1b1 genes are up-regulated by the aryl hydrocarbon receptor. To elucidate their roles in dioxin-induced teratogenesis, we compared Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) knock-out mice with Cyp1(+/+) wild-type mice. Dioxin was administered (25 microg/kg, gavage) on gestational day 10, and embryos were examined on gestational day 18. The incidence of cleft palate and hydronephrosis was not significantly different in fetuses from Cyp1a1(-/-), Cyp1b1(-/-), and Cyp1(+/+) wild-type mice. To fetuses carried by Cyp1a2(-/-) dams, however, this dose of dioxin was lethal; this effect was absolutely dependent on the maternal Cyp1a2 genotype and independent of the embryonic Cyp1a2 genotype. Dioxin levels were highest in adipose tissue, mammary gland, and circulating blood of Cyp1a2(-/-) mothers, compared with that in the Cyp1(+/+) mothers, who showed highest dioxin levels in liver. More dioxin reached the embryos from Cyp1a2(-/-) dams, compared with that from Cyp1(+/+) dams. Fetuses from Cyp1a2(-/-) dams exhibited a approximately 6-fold increased sensitivity to cleft palate, hydronephrosis, and lethality. Using the humanized hCYP1A1_1A2 transgenic mouse (expressing the human CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a2 gene), the teratogenic effects of dioxin reverted to the wild-type phenotype. These data indicate that maternal mouse hepatic CYP1A2, by sequestering dioxin and thus altering the pharmacokinetics, protects the embryos from toxicity and birth defects; substitution of the human CYP1A2 trans-gene provides the same protection. In contrast, neither CYP1A1 nor CYP1B1 appears to play a role in dioxin-mediated teratogenesis.     

CYP2E1-catalyzed oxidation contributes to the sperm toxicity of 1-bromopropane in mice

1-bromopropane (1-BrP) induces dose- and time-dependent reproductive organ toxicity and reduced sperm motility in rodents. The contribution of cytochrome P4502E1 (CYP2E1) to both 1-BrP metabolism and the induction of male reproductive toxicity was investigated using wild-type (WT) and Cyp2e1-/- mice. In gas uptake inhalation studies, the elimination half-life of [1,2,3-(13)C]-1-BrP was longer in Cyp2e1-/- mice relative to WT (3.2 vs. 1.3 h). Urinary metabolites were identified by 13C nuclear magnetic resonance. The mercapturic acid of 1-bromo-2-hydroxypropane (2OHBrP) was the major urinary metabolite in WT mice, and products of conjugation of 1-BrP with glutathione (GSH) were insignificant. The ratio of GSH conjugation to 2-hydroxylation increased 5-fold in Cyp2e1-/- mice relative to WT. After 1-BrP exposure, hepatic GSH was decreased by 76% in WT mice vs. 47% in Cyp2e1-/- mice. Despite a 170% increase in 1-BrP exposure in Cyp2e1-/- vs. WT mice, sperm motility in exposed Cyp2e1-/- mice did not change relative to unexposed matched controls. This suggests that metabolites produced through CYP2E1-mediated oxidation may be responsible for 1-BrP-induced sperm toxicity. Both 1-BrP and 2OHBrP inhibited the motility of sperm obtained from WT mice in vitro. However, only 2OHBrP reduced the motility of sperm obtained from Cyp2e1-/- mice in vitro, suggesting that conversion of parent compound to 2OHBrP within the spermatozoa may contribute, at least in part, to reduced motility. Overall, these data suggest that metabolism of 1-BrP is mediated in part by CYP2E1, and activation of 1BrP via this enzyme may contribute to the male reproductive toxicity of this chemical.     

Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1α in vivo

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver, and liver injury. This study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild-type (WT), CYP2E1-knock-in (KI), and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair-fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolyl hydroxylase 2, which promotes HIF-1α degradation, were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol-fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were colocalized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells, which express CYP2E1, with ethanol plus arachidonic acid (AA) or ethanol plus buthionine sulfoximine (BSO), which depletes glutathione, caused loss of cell viability to a greater extent than in HepG2 C34 cells, which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than in C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced by ethanol/AA or ethanol/BSO in the E47 cells. These results suggest that CYP2E1 plays a role in ethanol-induced hypoxia, oxidative stress, and activation of HIF-1α and that HIF-1α contributes to CYP2E1-dependent ethanol-induced toxicity. Blocking HIF-1α activation and actions may have therapeutic implications for protection against ethanol/CYP2E1-induced oxidative stress, steatosis, and liver injury.     

Alterations of the classic pathway of complement in adipose tissue of obesity and insulin resistance

Adipose tissue inflammation has recently been linked to the pathogenesis of obesity and insulin resistance. C1 complex comprising three distinct proteins, C1q, C1r, and C1s, involves the key initial activation of the classic pathway of complement and plays an important role in the initiation of inflammatory process. In this study, we investigated adipose expression and regulation of C1 complement subcomponents and C1 activation regulator decorin in obesity and insulin resistance. Expression of C1q in epididymal adipose tissue was increased consistently in ob/ob mice, Zucker obese rats, and high fat-diet-induced obese (HF-DIO) mice. Decorin was found to increase in expression in Zucker obese rats and HF-DIO mice but decrease in ob/ob mice. After TZD administration, C1q and decorin expression was reversed in Zucker obese rats and HF-DIO mice. Increased expression of C1 complement and decorin was observed in both primary adipose and stromal vascular cells isolated from Zucker obese rats. Upregulation of C1r and C1s expression was also perceived in adipose cells from insulin-resistant humans. Furthermore, expression of C1 complement and decorin is dysregulated in TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes and cultured rat adipose cells as they become insulin resistant after 24-h culture. These data suggests that both adipose and immune cells are the sources for abnormal adipose tissue production of C1 complement and decorin in obesity. Our findings also demonstrate that excessive activation of the classic pathway of complement commonly occurs in obesity, suggesting its possible role in adipose tissue inflammation and insulin resistance.     

Alcohol steatosis and cytotoxicity: The role of cytochrome P4502E1 and autophagy

The goal of the current study was to evaluate whether CYP2E1 plays a role in binge-ethanol induced steatosis and if autophagy impacts CYP2E1-mediated hepatotoxicity, oxidative stress and fatty liver formation produced by ethanol. Wild type (WT), CYP2E1 knockin (KI) and CYP2E1 knockout (KO) mice were gavaged with 3g/kg body wt ethanol twice a day for four days. This treatment caused fatty liver, elevation of CYP2E1 and oxidative stress in WT and KI mice but not KO mice. Autophagy was impaired in ethanol-treated KI mice compared to KO mice as reflected by a decline in the LC3-II/LC3-I ratio and lower total LC-3 and Beclin-1 levels coupled to increases in P62, pAKT/AKT and mTOR. Inhibition of macroautophagy by administration of 3-methyladenine enhanced the binge ethanol hepatotoxicity, steatosis and oxidant stress in CYP2E1 KI, but not CYP2E1 KO mice. Stimulation of autophagy by rapamycin blunted the elevated steatosis produced by binge ethanol. Treatment of HepG2 E47 cells which express CYP2E1 with 100mM ethanol for 8 days increased fat accumulation and oxidant stress but decreased autophagy. Ethanol had no effect on these reactions in HepG2 C34 cells which do not express CYP2E1. Inhibition of autophagy elevated ethanol toxicity, lipid accumulation and oxidant stress in the E47, but not C34 cells. The antioxidant N-acetylcysteine, and CYP2E1 inhibitor chlormethiazole blunted these effects of ethanol. These results indicate that CYP2E1 plays an important role in binge ethanol-induced fatty liver. We propose that CYP2E1-derived reactive oxygen species inhibit autophagy, which subsequently causes accumulation of lipid droplets. Inhibition of autophagy promotes binge ethanol induced hepatotoxicity, steatosis and oxidant stress via CYP2E1.     

Relative roles of CYP2E1 and CYP1A2 in mouse uroporphyria caused by acetone

Porphyria cutanea tarda is a liver disease characterized by excess production of uroporphyrin. We previously reported that acetone, an inducer of CYP2E1, enhances hepatic uroporphyrin accumulation in mice treated with iron dextran (Fe) and 5-aminolevulinic acid (ALA). Cyp2e1(-/-) mice treated with Fe and ALA were used to investigate whether CYP2E1 is required for the acetone effect. Hepatic uroporphyrin accumulation was stimulated by acetone in Cyp2e1(-/-) mice to the same extent as in wild-type mice. In the absence of acetone, uroporphyrin accumulated in Cyp2e1(-/-) mice treated with Fe and ALA, but less than in wildtype mice. However, in Cypla2(-/-) mice, uroporphyrin accumulation caused by Fe and ALA, with or without acetone, was completely prevented. Acetone was not an inducer of hepatic CYP1A2 in the wild-type mice. Although acetone is an inducer of CYP2E1, CYP1A2 appears to have the essential role in acetone-enhancement of uroporphyria.     

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa^−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.     

Cyp1a2 protects against reactive oxygen production in mouse liver microsomes

H(2)O(2) production was evaluated in liver microsomes prepared from Cyp1a1/1a2(+/+) wild-type and Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice pretreated with 5 microg dioxin (TCDD)/kg body wt or vehicle alone. NADPH-dependent H(2)O(2) production in TCDD-induced microsomes from wild-type mice was about one-third of that in noninduced microsomes. In Cyp1a2(-/-) mice, H(2)O(2) production was the same for induced and noninduced microsomes, with levels significantly higher than those in wild-type mice. Cyp1a1(-/-) microsomes displayed markedly lower levels of H(2)O(2) production in both induced and noninduced microsomes, compared with those in wild-type and Cyp1a2(-/-) microsomes. The CYP1A2 inhibitor furafylline in vitro exacerbated microsomal H(2)O(2) production proportional to the degree of CYP1A2 inhibition, and the CYP2E1 inhibitor diethyldithiocarbamate decreased H(2)O(2) production proportional to the degree of CYP2E1 inhibition. Microsomal H(2)O(2) production was strongly correlated to NADPH-stimulated production of thiobarbituric acid-reactive substances, as well as to decreases in microsomal membrane polarization anisotropy, indicative of peroxidation of unsaturated membrane lipids. Our results suggest that possibly acting as an "electron sink," CYP1A2 might decrease CYP2E1-and CYP1A1-mediated H(2)O(2) production and oxidative stress. In this regard, CYP1A2 may be considered an antioxidant enzyme.     

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.     

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.     

The effect of ethanol-induced cytochrome p4502E1 on the inhibition of proteasome activity by alcohol

The present investigation was undertaken to determine the effect of CYP2E1 induction by ethanol on the inhibition of proteasomal activity in wild-type and CYP2E1 knockout C57 black mice. The proteasomal chymotrypsin-like activity decreased significantly in ethanol-fed wild-type mice liver, but was not reduced in ethanol-fed knockout mice liver. The 26S proteasomal activity was decreased more by ethanol feeding than was the 20S proteasomal fraction. Individual hepatocytes lost immunostaining of the proteasomes in the centrilobular zone in the livers of ethanol-fed wild-type mice and the knockout mouse liver. There was increased product of protein oxidation in the liver in the wild type but not in the knockout mice given ethanol. Taken together, these results suggest that CYP2E1 induction was responsible for the decrease in proteasome activity seen in the wild-type mice which head to the accumulation of oxidized proteins which were increased as the result of free radicals generated by CYP2E1 metabolism of ethanol.     

Basal and inducible CYP1 mRNA quantitation and protein localization throughout the mouse gastrointestinal tract

The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal.     

Disruption of mouse cytochrome p450 4f14 (Cyp4f14 gene) causes severe perturbations in vitamin E metabolism

Vitamin E is a family of naturally occurring and structurally related lipophilic antioxidants, one of which, α-tocopherol (α-TOH), selectively accumulates in vertebrate tissues. The ω-hydroxylase cytochrome P450-4F2 (CYP4F2) is the only human enzyme shown to metabolize vitamin E. Using cDNA cloning, cell culture expression, and activity assays, we identified Cyp4f14 as a functional murine ortholog of CYP4F2. We then investigated the effect of Cyp4f14 deletion on vitamin E metabolism and status in vivo. Cyp4f14-null mice exhibited substrate-specific reductions in liver microsomal vitamin E-ω-hydroxylase activity ranging from 93% (γ-TOH) to 48% (γ-tocotrienol). In vivo data obtained from metabolic cage studies showed whole-body reductions in metabolism of γ-TOH of 90% and of 68% for δ- and α-TOH. This metabolic deficit in Cyp4f14(-/-) mice was partially offset by increased fecal excretion of nonmetabolized tocopherols and of novel ω-1- and ω-2-hydroxytocopherols. 12'-OH-γ-TOH represented 41% of whole-body production of γ-TOH metabolites in Cyp4f14(-/-) mice fed a soybean oil diet. Despite these counterbalancing mechanisms, Cyp4f14-null mice fed this diet for 6 weeks hyper-accumulated γ-TOH (2-fold increase over wild-type littermates) in all tissues and appeared normal. We conclude that CYP4F14 is the major but not the only vitamin E-ω-hydroxylase in mice. Its disruption significantly impairs whole-body vitamin E metabolism and alters the widely conserved phenotype of preferential tissue deposition of α-TOH. This model animal and its derivatives will be valuable in determining the biological actions of specific tocopherols and tocotrienols in vivo.     

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.     

Activation of peroxisome proliferator-activated receptor-γ by rosiglitazone improves lipid homeostasis at the adipose tissue-liver axis in ethanol-fed mice

The development of alcohol-induced fatty liver is associated with a reduction of white adipose tissue (WAT). Peroxisome proliferator-activated receptor (PPAR)-γ prominently distributes in the WAT and plays a crucial role in maintaining adiposity. The present study investigated the effects of PPAR-γ activation by rosiglitazone on lipid homeostasis at the adipose tissue-liver axis. Adult C57BL/6 male mice were pair fed liquid diet containing ethanol or isocaloric maltose dextrin for 8 wk with or without rosiglitazone supplementation to ethanol-fed mice for the last 3 wk. Ethanol exposure downregulated adipose PPAR-γ gene and reduced the WAT mass in association with induction of inflammation, which was attenuated by rosiglitazone. Ethanol exposure stimulated lipolysis but reduced fatty acid uptake capacity in association with dysregulation of lipid metabolism genes. Rosiglitazone normalized adipose gene expression and corrected ethanol-induced lipid dyshomeostasis. Ethanol exposure induced steatosis and upregulated inflammatory genes in the liver, which were attenuated by rosiglitazone. Hepatic peroxisomal fatty acid β-oxidation was suppressed by ethanol in associated with inhibition of acyl-coenzyme A oxidase 1. Rosiglitazone elevated plasma adiponectin level and normalized peroxisomal fatty acid β-oxidation rate. However, rosiglitazone did not affect ethanol-reduced very low-density lipoprotein secretion from the liver. These results demonstrated that activation of PPAR-γ by rosiglitazone reverses ethanol-induced adipose dysfunction and lipid dyshomeostasis at the WAT-liver axis, thereby abrogating alcoholic fatty liver.     

Cytochrome P-450 CYP2E1 knockout mice are protected against high-fat diet-induced obesity and insulin resistance

Conventional (whole body) CYP2E1 knockout mice displayed protection against high-fat diet-induced weight gain, obesity, and hyperlipidemia with increased energy expenditure despite normal food intake and spontaneous locomotor activity. In addition, the CYP2E1 knockout mice displayed a marked improvement in glucose tolerance on both normal chow and high-fat diets. Euglycemic-hyperinsulinemic clamps demonstrated a marked protection against high-fat diet-induced insulin resistance in CYP2E1 knockout mice, with enhanced adipose tissue glucose uptake and insulin suppression of hepatic glucose output. In parallel, adipose tissue was protected against high-fat diet-induced proinflammatory cytokine production. Taken together, these data demonstrate that the CYP2E1 deletion protects mice against high-fat diet-induced insulin resistance with improved glucose homeostasis in vivo.     

Alternate pathways of bile acid synthesis in the cholesterol 7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding

Bile acids are synthesized via the classic pathway initiated by cholesterol 7alpha-hydroxylase (CYP7A1), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27). These studies used mice lacking cholesterol 7alpha-hydroxylase (Cyp7a1(-/-)) to establish whether the loss of the classic pathway affected cholesterol homeostasis differently in males and females, and to determine if the rate of bile acid synthesis via alternate pathways was responsive to changes in the enterohepatic flux of cholesterol and bile acids. In both the Cyp7a1(-/-) males and females, the basal rate of bile acid synthesis was only half of that in matching Cyp7a1(+/+) animals. Although bile acid pool size contracted markedly in all the Cyp7a1(-/-) mice, the female Cyp7a1(-/-) mice maintained a larger, more cholic acid-rich pool than their male counterparts. Intestinal cholesterol absorption in the Cyp7a1(-/-) males fell from 46% to 3%, and in the matching females from 58% to 17%. Bile acid synthesis in Cyp7a1(+/+) males and females was increased 2-fold by cholesterol feeding, and 4-fold by cholestyramine treatment, but was not changed in matching Cyp7a1(-/-) mice by either of these manipulations. In the Cyp7a1(-/-) mice fed cholesterol, hepatic cholesterol concentrations increased only marginally in the males, but rose almost 3-fold in the females. CYP7A1 activity and mRNA levels were greater in females than in males, and were increased by cholesterol feeding in both sexes. CYP27 activity and mRNA levels did not vary as a function of CYP7A1 genotype, gender, or dietary cholesterol intake. We conclude that in the mouse the rate of bile acid synthesis via alternative pathways is unresponsive to changes in the enterohepatic flux of cholesterol and bile acid, and that factors governing gender-related differences in bile acid synthesis, pool size, and pool composition play an important role in determining the impact of CYP7A1 deficiency on cholesterol homeostasis in this species.     

CYP2E1 enhances ethanol-induced lipid accumulation but impairs autophagy in HepG2 E47 cells

The regulation and function of autophagy and lipid metabolism have recently been reported to be reciprocally related. Macroautophagy mediates the breakdown of lipids stored in lipid droplets. An inhibition of autophagy leads to the development of a fatty liver. We evaluated the ability of CYP2E1 to modulate the effects of ethanol on lipid accumulation and autophagy in vitro. The E47 HepG2 cell which expresses CYP2E1 was treated with ethanol at 50, 100 and 150 mM for 4 or 5 days. Ethanol-induced lipid accumulation and an increase of triglycerides (TG) in E47 cells to a greater extent than in control C34 cells which do not express CYP2E1. In contrast, autophagy (LC3 II/LC3 I ratio) was significantly induced by ethanol in C34 cells to a greater extent than in E47 cells. P62 was significantly increased in E47 cells after ethanol treatment. Thus, there is a reciprocal relationship between the effects of ethanol on lipid accumulation and autophagy in the CYP2E1-expressing cells. Inhibition of autophagy by 3-methyladenine (3MA), increased lipid accumulation and TG levels in C34 cells which display elevated autophagy, but enhanced lipid accumulation and TG level to a lesser extent in E47 cells which displayed lower autophagy. Ethanol induced CYP2E1 activity and oxidative stress in E47 cells compared with C34 cells. These experiments suggest that the expression of CYP2E1 may impair autophagy formation which contributes to lipid accumulation in the liver. We hypothesize that CYP2E1-induced oxidative stress promotes the accumulation of lipid droplets by ethanol and this may be responsible for the suppression of autophagy in the liver.