HCV core-induced nonobese hepatic steatosis is associated with hypoadiponectinemia and is ameliorated by adiponectin administration

Obesity-related hepatic steatosis is commonly associated with central fat accumulation and alterations in adipocytokine secretion; however, the connection between nonobese hepatic steatosis and adipocytokines remains unclear. We aim to investigate this connection using an animal model of conditional hepatitis C virus (HCV) core-transgenic mice. Double transgenic mice (DTM) with doxycycline (dox)-regulated hepatic overexpression of the HCV core protein were fed standard rodent chow ad libitum following 1 month of a dox-rich diet. The mice exhibited nonobese hepatic steatosis at 2 months of age. The levels of leptin and adiponectin were assessed in 2-month-old DTM (i.e., HCV core-tetracycline transactivator (tTA)) and single transgenic mice (STM; i.e., tTA). The total fat mass and the body fat distribution of the mice were evaluated using dual-energy X-ray absorptiometry (DEXA) and magnetic resonance imaging (MRI). Microarray analyses and quantitative real-time PCR were conducted using RNA obtained from the visceral fat of paired DTM and STM. Adiponectin was administered intraperitoneally to the 2-month-old DTM. No significant differences of the various fat components were noted between the DTM and STM. Leptin mRNA was downregulated in the visceral fat of DTM (P = 0.011), and serum adiponectin protein levels were reduced in the DTM compared with those in the STM (P = 0.035). Adiponectin treatment also significantly ameliorated hepatic steatosis in the DTM compared to the controls (P = 0.024). In conclusion, HCV core-induced nonobese hepatic steatosis is associated with downregulation of the leptin gene in visceral fat and concurrent hypoadiponectinemia; however, these effects may be ameliorated by adiponectin treatment.     

Alpha-lipoic acid attenuates methionine choline deficient diet-induced steatohepatitis in C57BL/6 mice

AimsNon-alcoholic steatohepatitis (NASH) is a liver disease that causes fat accumulation, inflammation and fibrosis. Increased oxidative stress contributes to hepatic inflammation and fibrosis by upregulation of Cytochrome P450 2E1 (CYP2E1), endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) activity. This study examined whether alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, prevents steatohepatitis through the inhibition of several pathways involved in hepatic inflammation and fibrosis.Main MethodsC57BL/6 mice were fed an MCD diet with or without ALA for 4 weeks. Liver sections from mice on control or MCD diets with or without ALA were stained with hematoxylin-eosin, oil red O, and anti-4-HNE antibody. The effects of ALA on methionine-choline deficient MCD-diet induced plasma AST and ALT as well as tissue TBARS were measured. The effects of ALA on CYP2E1 expression, ER stress, MAPK levels, and NF-κB activity in MCD diet-fed mice liver were measured by northern and western blot analysis.Key findingsDietary supplementation with ALA reduced MCD diet-induced hepatic lipid accumulation, hepatic inflammation, TBARS, 4-HNE, and plasma ALT and AST levels. These effects were associated with a reduced expression of CYP2E1 and reduced ER stress and MAPK and NF-κB activity.SignificanceTaken together, the results of the present study indicate that ALA attenuates steatohepatitis through inhibition of several pathways, and provide the possibility that ALA can be used to prevent the development and progression of non-alcoholic fatty liver disease in patients who have strong risk factors for NASH.     

Vitamin E Attenuates the Progression of Non-Alcoholic Fatty Liver Disease Caused by Partial Hepatectomy in Mice

Background and Aim

The progression of non-alcoholic fatty liver disease (NAFLD) likely involves a ‘multiple hit’ mechanism. We hypothesized that partial hepatectomy, a procedure performed frequently in patients with NAFLD, would accelerate the progression of disease.

Methods

C57BL/6JolaHsd mice were fed a choline-deficient L-amino acid-defined diet (CD-AA) or a choline-sufficient L-amino acid-defined control diet (CS-AA). Part of the mice in the CD-AA group received a diet enriched in vitamin E (~20 mg /day). Two weeks after the start of the diet, mice underwent a partial hepatectomy or a sham operation.

Results

In the CD-AA group, NAFLD activity scores were significantly higher at 7 days after partial hepatectomy compared to the sham operated mice (3.7 ± 1.3 vs. 1.8 ± 0.7; P<0.05). In addition, TBARS, a measure for oxidative stress, in liver tissue of the CD-AA group were significantly higher at day 1, 3 and 7 after partial hepatectomy compared to the sham operated mice (P<0.05). Vitamin E therapy significantly reduced TBARS level at day 7 after partial hepatectomy compared to the CD-AA diet group (P< 0.05). Vitamin E suppletion reduced NAFLD activity score at day 7 after partial hepatectomy compared to the CD-AA group (2.3 ± 0.8 vs. 3.8 ± 1.0; P<0.05).

Conclusion

Partial hepatectomy accelerates the progression of NAFLD. Disease progression induced by partial hepatectomy is substantially attenuated by vitamin E.     

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, P_TF, was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P_TF to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P_NSE) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed.     

Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy

Although the translocation of metallothionein (MT) from cytoplasm to nucleus has been demonstrated in liver during times of high requirement for zinc (fetal development and the neonatal period), the role of MT in cellular growth is not well understood. In this study, a potential role of MT in liver regeneration was investigated in wild type (WT) and MT-I and MT-II gene knockout (MT-null) mice after 35% partial hepatectomy (PH) or sham laparotomy. Hepatic MT levels and proliferation index were measured at 0, 5, 15, 24, 36, 48, and 60 hrs after PH and 48 hrs after sham laparotomy (control). MT levels were increased in WT mice (peak at 24 hrs after PH) and declined to normal levels by 60 hrs after PH. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24 hrs after PH, whereas MT was present mainly in the cytoplasm at 36-60 hrs after PH and 48 hrs after sham laparotomy. Hepatic proliferation index in both WT and MT-null mice, as determined by argyrophilic nucleolar organizing region staining and proliferating cell nuclear antigen immunohistochemical staining, reached a peak at 48 hrs and declined by 60 hrs after PH. Cell proliferation was significantly less in MT-null mice as compared to WT mice during liver regeneration after PH. These results suggest that MT may play a positive role in hepatic regeneration after PH.     

Cytokine characteristics of jaundice in mouse liver

OBJECTIVE: The aim of this study is to clarify the perioperative cytokine changes and their mechanism in jaundiced liver. MATERIALS AND METHODS: Obstructive jaundice was induced using a common bile duct ligation (CBDL) and a two-thirds hepatectomy (HEP) was performed in six- to seven-week-old male C3H/HeN mice. When hepatectomy was added to CBDL, it was carried out 2 to 5 days after CBDL. The serum interleukin 6 (IL-6) levels and heat shock protein (HSP)-70 expression were evaluated. One mg per mouse of methylprednisolone (MPL) was intraperitonealy administered in some mice of CBDL+HEP group. RESULTS: The post-hepatectomy IL-6 values at 2 and 3 days after CBDL were significantly lower than those in the HEP group, while those at 5 days after CBDL were significantly higher than those in HEP group. The serum IL-6 value of the steroid group was significantly lower than that of non-steroid group in HEP group. However, no steroid effects were recognized on post-hepatectomy IL-6 values at 3 days after CBDL, steroid inhibited post-hepatectomy IL-6 production at 5 days after CBDL. No expression of HSP70 protein was observed in the control group, but HSP70 protein was expressed in both the hepatocytes and Kupffer cells 3 days after CBDL, then at 5 days after CBDL, no HSP70 protein was expressed in the Kupffer cells. CONCLUSIONS: In the early period of obstructive jaundice, the IL-6 level after hepatectomy did not increase in comparison to HEP group, and steroid had no effect on IL-6 level. According to the progression of obstructive jaundice, the IL-6 level after hepatectomy increased to a higher level than before, and the effect of MPL was restored. HSP70 is thus suggested to have an important role in cytokine production.     

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.     

Plasma Lipid Peroxides in Murine Sepsis —Sex Differences and Effect of Antioxidative/Anti-Inflammatory Therapy

In order to investigate the influence of antioxidative anti-inflammatory combination therapy (AACT) with dimethyl sulfoxide (DMSO). chlorpromaittic (CPZ) and vitamin E upon the activity of the inflammation. plasma lipid peroxide was measured as thiobarbituric acid reactive substance (TBARS) 12hrs postoperatively in the moclitied cecal ligation sepsis model in the mouse.

Significantly higher TBARS levels were found in the male control group (13.7 ± 0.7nmol MDA/ml) than in the female control group (11.6 ± 0.6nmol MDA/ml).

The operated male group had significantly higher TBARS levels (16.2 ± 0.6 nmol MDA/ml) than the unoperdted male control group (13.7 ± 0.7nmol MDA/ml). No increase of TBARS levels was observed in the operated female group.

Both male and female operated group. when postoperatively treated with AACT had the same TBARS level as the not operated male or female control group.

Survival curves of operated male and female group did not demonstrate any significant difference. The survival was better in an operated male and an operated female group. when postoperatively treated with AACT.

It was concluded that the applied TBARS test IS too insensitive to follow the activity of the inflammation and has no predictive value for the outcome of sepsis in this model.     

Expression of an HCV Core Antigen Coding Gene in Tobacco (N. tabacum L.)

Abstract

Hepatitis C virus (HCV) is the major agent causing chronic liver disease. The core gene is the most conserved sequence in the HCV genome and proved immunoreactive when expressed in bacteria and antigenic in humans. In order to test the ability of plants to express the core gene for the production of core antigen, transgenic tobacco plants carrying the core gene were generated. The core protein was stably synthesized in T₀ and T₁ generations and was found to be immunoreactive, not only with anti-core polyclonal and monoclonal antibodies, but also was able to recognize the HCV virus in infected human serum. The prospects of producing a plant based vaccine and/or a food vaccine for this important virus are discussed.     

Serum hormone levels following partial hepatectomy in the rat.

Serum levels of insulin, glucagon, growth hormone (somatotrophin) and thyroxine (TT₄) were measured by radioimmunoassay following both sham operation and 70% partial hepatectomy in the rat to evaluate changes in hormone levels during liver regeneration. An eleven fold increase in glucagon was observed (from 112 ± 10 pg/ml to 1500 ± 200 pg/ml) 6 hours following partial hepatectomy but not sham operation. In contrast, insulin levels remained unchanged compared to sham controls for up to 72 hr while growth hormone fell to low levels, 6 to 48 hr after partial hepatectomy. Both total thyroxine and free thyroxine levels also fell 24–72 hours after hepatectomy. These studies suggest that growth hormone, thyroxine and insulin are not primary stimulants of hepatic regeneration although the data suggests that glucagon may modify this growth process.     

Organisation and expression of a cluster of yolk protein genes in the Australian sheep blowfly, Lucilia cuprina

The Australian sheep blowfly Lucilia cuprina is a major pest for the Australian and New Zealand sheep industries. With the long-term aim of making a strain of L. cuprina suitable for a genetic control program, we previously developed a tetracycline-repressible female lethal genetic system in Drosophila. A key part of this system is a female-specific promoter from a yolk protein (yp) gene controlling expression of the tetracycline-dependent transactivator (tTA). Here we report the sequence of a 14.2 kb genomic clone from L. cuprina that contains a cluster of three complete yp genes and one partial yp gene. The Lcyp genes are specifically expressed in females that have received a protein meal. A bioinformatic analysis of the promoter of one of the yp genes (LcypA) identified several putative binding sites for DSX, a known regulator of yp gene expression in other Diptera. A transgenic strain of L. cuprina was made that contained the LcypA promoter driving the expression of the Escherichia coli lacZ reporter gene. Transgenic females express high levels of β-galactosidase after a protein meal. Thus the LcypA promoter could be used to obtain female-specific expression of tTA in transgenic L. cuprina.     

Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.     

Increased oxidative stress alters nucleosides metabolite levels in sickle cell anemia

Objectives: This study was conducted to assess the markers of oxidative stress, myeloperoxidase (MPO), acetylcholinesterase (AChE) and xanthine oxidase (XO) activities as well as the levels of nucleotide metabolites in sickle cell anemia (SCA) patients.

Methods: Fifteen SCA treated patients and 30 health subjects (control group) were selected. The markers of oxidative stress (levels of reactive oxygen species (ROS), plasma proteins, carbonyl content, lipid peroxidation (TBARS), total thiols (T-SH), glutathione and catalase activity), MPO, AChE and XO activities as well as the levels of nucleotide metabolites were measured in SCA patients.

Results: ROS, thiobarbituric acid-reactive substances (TBARS) and T-SH levels as well as the activities of catalase and MPO were significantly increased while glutathione level was reduced in SCA patients. Furthermore, a significant (P?P?P?P?Discussion: The altered parameters in SCA patients suggest that the generation and impairment of oxidative stress in this disease as well as antioxidant markers are contributory factors towards cellular redox homeostasis and alteration of purine metabolites.     

NR2B‐deficient mice are more sensitive to the locomotor stimulant and depressant effects of ethanol

The NR2B subunit of N‐methyl d ‐aspartate glutamate receptors influences pharmacological properties and confers greater sensitivity to the modulatory effects of ethanol. This study examined behavioral responses to acute ethanol in a conditional knockout mouse model that allowed for a delayed genetic deletion of the NR2B subunit to avoid mouse lethality. Mice lacking the NR2B gene (knockout) were produced by mating NR2B[f/f] mice with CAMKIIa‐driven tTA transgenic mice and the tetO‐CRE transgenic mice. Adult male and female offspring representing each of the resultant genotypes (knockout, CAM, CRE and wildtype mice) were tested for open‐field locomotor activity following acute low‐ and high‐dose ethanol challenge as well as loss of righting reflex. Findings indicate that male and female mice lacking the NR2B subunit exhibited greater overall activity in comparison to other genotypes during the baseline locomotor activity test. NR2B knockout mice exhibited an exaggerated stimulant response to 1.5 g/kg (i.p.) and an exaggerated depressant response to 3.0 g/kg (i.p.) ethanol challenge. In addition, NR2B knockout mice slept longer following a high dose of ethanol (4.0 g/kg, i.p.). To evaluate pharmacokinetics, clearance rates of ethanol (1.5, 4.0 g/kg, i.p.) were measured and showed that female NR2B knockouts had a faster rate of metabolism only at the higher ethanol dose. Western blot analyses confirmed significant reduction in NR2B expression in the forebrain of knockout mice. Collectively, these data indicate that the NR2B subunit of the N‐methyl d ‐aspartate glutamate receptor is involved in regulating low‐dose stimulant effects of ethanol and the depressant/hypnotic effects of ethanol.     

Fas ligand is responsible for CXCR3 chemokine induction in CD4+ T cell-dependent liver damage

Immune-mediated hepatic damage has been demonstrated in the pathogenesis of hepatitis C virus (HCV) and other hepatotrophic infections. Fas/Fas ligand (FasL) interaction plays a critical role in immune-mediated hepatic damage. To understand the molecular mechanism(s) of FasL-mediated liver inflammation, we examined the effect of CD4(+) T cells expressing high levels of FasL on the initiation of hepatic damage through analysis of chemokine and chemokine receptor expression in HCV core x TCR (DO11.10) double-transgenic mice. In vivo antigenic stimulation triggers a marked influx of core-expressing Ag-specific CD4(+) T cells into the liver of the immunized core(+) TCR mice but not their core(-) TCR littermates. Strikingly, the inflammatory process in the liver of core(+) TCR mice was accompanied by a dramatic increase in IFN-inducible protein 10 and monokine induced by IFN-gamma production. The intrahepatic lymphocytes were primarily CXCR3-positive and anti-CXCR3 Ab treatment abrogates migration of CXCR3(+) lymphocytes into the liver and hepatic damage. Importantly, the blockade of Fas/FasL interaction reduces the expression of IFN-inducible protein 10 and monokine induced by IFN-gamma and cellular infiltration into the liver. These findings suggest that activated CD4(+) T cells with elevated FasL expression are involved in promoting liver inflammation and hepatic damage through the induction of chemokines.