The overall objective of this research was to investigate the status of tumor necrosis factor-?? (TNF-??), and molecules associated with its signaling, in the pathological state of hepatic steatosis. The effect of NSAID piroxicam, a cancer preventive agent also known to affect TNF-?? signaling on hepatic steatosis, was also investigated. The biological state of the tissue was assessed by examining the expression of TNF-?? signaling molecule in whole tissue, as well as in hepatic lipid raft. Lipid rafts are dynamic assemblies of cholesterol and sphingolipids, microdomains that form in the exoplasmic leaflet of the biological membranes shown to play a role in compartmentalization, modulation and integration of the cell signaling. In the present research, Zucker obese rats were used as a model of human obesity and insulin resistant state. These rats exhibit hepatic steatosis in adulthood similar to those noted in obese individuals. Female Zucker obese and lean rats (5 weeks old) were fed a semisynthetic diet with or without piroxicam (150 ppm). Zucker lean counterparts served as control. After 8 weeks of feeding, rats were euthanized and liver from each animal was collected. Liver tissue from each animal was processed for histology and biochemical analysis which included lipids and proteins (COX-1 and 2, TNF-??, TNF-RI and RII, IKK-??, I??B-?? and NF-??B). Liver histology and the level of total lipids confirmed that Zucker obese rats had hepatic steatosis, which was further augmented by piroxicam treatment. Whole tissue protein expression, using western blot, showed that the steatotic liver differed from non-steatotic livers by having lower levels of TNF-RII. TNF-RII showed a trend which was inversely proportional to the pathological state of the tissue. The obese-piroxicam liver had the lowest level of TNF-RII and lean livers had the highest (p<0.05). The total NF-??B level was higher in the obese and obese-piroxicam groups compared to the lean or lean-piroxicam groups (p<0.05). Piroxicam treatment lowered the level of NF-??B in obese and lean livers. I??B-?? was higher in obese livers than in lean livers. The nuclear level of NF-??B by western blot analysis showed the same pattern as noted in the whole tissue homogenate. However, the difference in the level between obese and lean was marked. The obese nuclei contained two to three fold higher levels of NF-??B protein than the lean liver nuclei. I??B-?? level was significantly higher in the obese liver tissues and nuclei than their lean counterparts. While transcriptionally active NF-??B was higher (p<0.05) in the obese livers than in the lean livers, the difference between obese and lean groups was not as significant as that noted for the level of NF-??B assessed by western blot. This suggests that the proportion of active NF-??B present in the nuclear fraction is much higher in the lean than in the obese nuclei. Lipid raft was extracted and identified successfully from obese and lean livers. The total caveolin and flotillin levels were significantly higher in the liver lipid rafts of the obese-piroxicam than that of the other groups. This is the group that also exhibited higher steatosis. Piroxicam treatment significantly decreased the level of caveolin in the lean liver and significantly increased the level of flotillin in the obese liver. While COX-1 was not detectable, however, the level of COX-2 and TNF-RII in lipid raft was opposite to the level noted in the whole tissue homogenate. TNFRII was highest in the obese-piroxicam lipid raft and lowest in the lean-piroxicam lipid raft. TNF-RII, COX-2, I??B-?? and NF-??B proteins were the molecules profoundly affected by the pathological state of the tissue and piroxicam treatment. This research is the first to report the presence of I??B-?? in the nuclear compartment with a higher level in the nuclei and whole tissue in the obese liver than in the lean liver. This research demonstrates that TNF-?? to NF-??B axis is altered in steatotic liver, and analysis of lipid rafts in steatotic and non-steatotic liver demonstrates that lipid rafts play a distinct role in modifying the biological availability of key proteins in the pathological state of liver steatosis.

Background: The increased incidence of obesity and associated metabolic diseases has driven research focused on genetically or pharmacologically alleviating metabolic dysfunction. These studies employ a range of fasting-refeeding models including 4-24 h fasts, "overnight" fasts, or meal feeding. Still, we lack literature that describes the physiologically relevant adaptations that accompany changes in the duration of fasting and re-feeding. Since the liver is central to whole body metabolic homeostasis, we investigated the timing of the fast-induced shift toward glycogenolysis, gluconeogenesis, and ketogenesis and the meal-induced switch toward glycogenesis and away from ketogenesis. Methods: Twelve to fourteen week old male C57BL/6J mice were fasted for 0, 4, 8, 12, or 16 h and sacrificed 4 h after lights on. In a second study, designed to understand the response to a meal, we gave fasted mice access to feed for 1 or 2 h before sacrifice. We analyzed the data using mixed model analysis of variance. Results: Fasting initiated robust metabolic shifts, evidenced by changes in serum glucose, non-esterified fatty acids (NEFAs), triacylglycerol, and beta-OH butyrate, as well as, liver triacylglycerol, non-esterified fatty acid, and glycogen content. Glycogenolysis is the primary source to maintain serum glucose during the first 8 h of fasting, while de novo gluconeogenesis is the primary source thereafter. The increase in serum a-OH butyrate results from increased enzymatic capacity for fatty acid flux through beta-oxidation and shunting of acetyl-CoA toward ketone body synthesis (increased CPT1 (Carnitine Palmitoyltransferase 1) and HMGCS2 (3-Hydroxy-3-Methylglutaryl-CoA Synthase 2) expression, respectively). In opposition to the relatively slow metabolic adaptation to fasting, feeding of a meal results in rapid metabolic changes including full depression of serum a-OH butyrate and NEFAs within an hour. Conclusions: Herein, we provide a detailed description of timing of the metabolic adaptations in response to fasting and re-feeding to inform study design in experiments of metabolic homeostasis. Since fasting and obesity are both characterized by elevated adipose tissue lipolysis, hepatic lipid accumulation, ketogenesis, and gluconeogenesis, understanding the drivers behind the metabolic shift from the fasted to the fed state may provide targets to limit aberrant gluconeogenesis and ketogenesis in obesity.

ntroduction: Fasciola hepatica is a parasite of the class Trematoda. It commonly has been found in developing countries. When it infects humans is characterized by a triad of fever, pain in right upper quadrant and peripheral eosinophilia. We present a 67-year-old female from a rural town of the north of Lima, Peru, it was found abdominal pain, eosinophilia and focal hepatic lesions. For this reason, a hepatic mass was the initial suspicion. The hepatic biopsy was performed and one of the findings was eosinophilia. Fasciola hepatica infection should be considered as part of differential diagnosis in hepatic tumors with eosinophilia when the origin of the patient is from endemic areas of F. hepatica. / Fasciola hepática es un parásito de la clase Trematoda común en países en desarrollo. La infección en el ser humano se caracteriza por la triada de fiebre, dolor abdominal en el cuadrante superior derecho y eosinofilia. Se presenta el caso de una mujer de 67 años procedente de una zona rural al norte de Lima, con historia de dolor abdominal de seis meses de evolución, con una imagen hipodensa hepática en el TAC abdominal y eosinofilia. La biopsia hepática mostró un infiltrado inflamatorio con eosinofilia. En el diagnóstico diferencial en pacientes con un tumor hepático y eosinofilia, se deben incluir infecciones parasitarias como F. hepatica; sobre todo en pacientes que proceden de áreas endémicas.

As a critical parameter in pharmacokinetics, prediction of clearance is an integral aspect of drug discovery programmes. Since the liver is the major site of xenobiotic biotransformation, accurate prediction of hepatic clearance (CLh) is vital. The use of cellular and subcellular in vitro systems for this purpose is common practice; however, prediction accuracy tends to be poor. The aim of this thesis was to explore potential contributing factors to the underprediction of in vivo clearance, specifically with relation to the in vitro methodology of hepatocyte clearance assays, which is largely unstandardised. Literature data analyses highlighted an overall clearance-dependent trend of underprediction in both human and rat hepatocytes, indicating a fundamental in vitro system bias which is independent of species. During initial investigation of incubation conditions, the format of hepatocyte assays (suspension in microcentrifuge tubes, 96-well plates, 24-well plates and short-term monolayer culture) was demonstrated to influence determined intrinsic clearance (CLint). Differences in midazolam CLint were observed not only between suspended and short-term cultured hepatocytes, but also between suspended hepatocytes in different vessels/plate formats. The applicability of 1 µM as a generic substrate incubation concentration for determination of CLint by substrate depletion was evaluated in rat hepatocytes using nine well-characterised drugs. For seven of the nine drugs, a statistically significantly (p < 0.05) higher CLint was observed in determinations of 0.1 µM substrate as opposed to 1 µM, highlighting the potential for false determinations using current practices. Cofactor depletion in isolated hepatocytes was investigated based on previous speculation as the cause of clearance-dependent underprediction of in vivo clearance. Although moderate increases in CLint were observed with the addition of NADPH to hepatocyte incubations, this was subsequently attributed to the replenishment of NADPH in membrane-damaged hepatocytes. Retained functionality of metabolic enzymes in cells which would generally be considered non-viable by trypan blue exclusion was indicated in comparisons of unpurified and Percoll-purified cryopreserved hepatocytes. This phenomenon was conclusively demonstrated in incubations of permeabilised hepatocytes supplemented with NADPH, revealing a need for re-evaluation of the use of plasma membrane integrity (trypan blue exclusion) as a measure of viability in metabolic studies. ATP content was considered as a potential alternative measure; however no significant correlations were found between ATP content, trypan blue exclusion and the CLint of nine drugs in associated preparations. The effect of shaking on CLint in rat hepatocytes was also examined. For 10 out of 12 drugs, CLint determined at 900 rpm was significantly (p < 0.05) higher than in static incubations. Three potential mechanisms were hypothesised: plasma membrane damage, increased substrate distribution throughout the bulk medium and reduction in the depth of the unstirred water layer (UWL) surrounding cells. Shaking of saponin-permeabilised hepatocytes (supplemented with NADPH to maintain metabolism) also increased the determined CLint of saquinavir, indicating a rate-limitation other than membrane permeation. However, shaking of ultra-sonicated hepatocytes in which the plasma membrane was entirely destroyed (also supplemented with NADPH) did not change the determined CLint of saquinavir, revealing the rate-limitation of UWL permeation in both intact and permeabilised cells. The depth of such an UWL in vitro is likely to be artificially greater than in vivo; therefore reduction of UWL depth through incubation shaking is proposed as a physiologically sound approach by which to increase in vitro CLint. In addition, a framework of experiments and related equations is presented by which intact and permeabilised hepatocytes in static and shaken conditions may be utilised to identify the rate-determining process and contribution of individual processes to the in vitro CLint of a drug. The effects of substrate concentration and shaking were also evaluated in human hepatocytes. Significant increases in determined CLint of drugs with use of 0.1 µM substrate (as opposed to 1 µM) and shaking at 900 rpm were demonstrated, confirming equivalent potential in vitro sources of underprediction between rat and human. These highly significant findings reveal the existing limitations of in vitro assays and potential flaws in current practice in in vitro determinations of CLint. Appropriate consideration of the properties of in vitro systems, including the presence of the UWL, should lead to improved predictions of in vivo clearance.

Class of 2007 Abstract / Objectives: To characterize the difference in hepatic gene expression between Cyp2e1 +/+ and Cyp2e1 -/- mice after exposure to hydrazine in order to elucidate the functional pathway(s) for hydrazine-induced steatosis. Methods: The project was designed by Dr. Charlene McQueen and consisted of the following aims: (1) to characterize the hepatic pathology induced by hydrazine in CYP2E1 +/+ and -/- mice, (2) to evaluate hepatic gene expression profiles following exposure to hydrazine, and (3) to determine the expression of CYP2E1 and CYP4A14. The animal exposure and data collection have been completed and aim #2 is awaiting data analysis. Aim #2 consisted of treating CYP2E1 +/+ and CYP2E1 -/- mice to saline and hydrazine at doses of 100 mg/kg. Livers were collected at six and 24 hours and the mRNA was isolated with an Absolutely RNA RT-PCR Miniprep Kit. The transcriptome was determined using the Affymetrix GeneChip Expression Analysis System using total mouse genome GeneChips. The GeneChips were scanned using an Agilent GeneArray Scanner and the image was quantitated and archived awaiting data analysis. The data was collected by the SWEHSC Microarray Facility on June 20, 2005 was analyzed. The data analysis was completed by both Kristjon Lindgren and Dana Seng with the help and training from Dr. George Watts. The six sets of data from aim #2 was analyzed using Agilent's GeneSpring 7.3.1 software to characterize the two-fold differences in mice (n = 2 per group) hepatic gene expression. Genes of interest were identified as containing the keywords cyp, fatty, glutathione, hepat, lipid, liver, oxid, perox, steroid, and phosphatidylinositol in the Gene Ontology Biological Process, Cellular Component, or Molecular Function descriptions. Lastly, pathway mining of/for genes of interest was performed using Bioresource for array of genes (BioRag) available at www.biorag.org and maintained by the AzCC/SWEHSC Bioinformatics Facility. Results: The amount of information extracted from this research project is too immense to be described or summarized on this form. For more information, please obtain a copy of this research project from the University of Arizona College of Pharmacy or from the project co-authors Kristjon Lindgren (kristjon.lindgren@gmail.com) or Dana Seng (dana.seng@gmail.com). Conclusions: The effects of Cyp2e1 on hepatic gene expression in 129/Sv-Cyp2e1tm1Gonz/J and 129S1/SvImJ mice exposed to hydrazine was analyzed. Data showing that Cyp2e1 was protective against HD-induced hepatotoxicity was consistent with the proposed hypothesis. Hepatic gene expression results show that Cyp2e1 -/- mice have decreased expression of microsomal ω-oxidation genes (Cyp4a10 and Cyp4a14) compared to Cyp2e1 +/+ at 6h (both increased at 24h) and peroxisomal β–oxidation genes (Ehhadh) at 6h like Cyp2e1 +/+ (but increased at 24h only in Cyp2e1 -/-). Conversely, an increased expression of mitochondrial β-oxidation genes (Cpt1a) in both genotypes at 6 and 24h and cholesterol synthesis genes (Fdft1, Hmgcr, Hmgcs1, Idi, Lss, Mvk, Nsdhl, Sc4mol, and Sqle) in Cyp2e1 -/- at 24h was observed. These results support mechanisms by which ω-oxidation or PPARγ is protective or peroxisomal β- oxidation is damaging. Additional studies are needed to further eludidate the mechanisms of HD-induced steatosis.

This study tested the ability of a novel adipose tissue derived cytokine, C1q TNF Related Protein 3 (CTRP3), to prevent alcohol-induced hepatic lipid accumulation, or alcoholic fatty liver disease (ALD). Previous work has demonstrated that CTRP3 is effective at preventing high fat diet-induced fatty liver, however, the potential of CTRP3 to inhibit ALD has not been explored. To test the potential protective effects of CTRP3, transgenic mice overexpressing CTRP3 (Tg) or wildtype littermates (WT) were subjected to one of two different models of ALD. In the first model, known as the NIAAA model, mice were fed control or alcohol-containing liquid diets (5% v/v) for 10 days followed by a single gavage of ethanol (5 g/kg). In the second model, the chronic model, mice were fed control or alcohol-containing diets for 6 weeks with no gavage. This study found that CTRP3 reduced triglyceride accumulation in the chronic model of alcohol consumption by ~50%, whereas no reduction was observed in the NIAAA model. Further analysis of isolated primary hepatocytes from WT and Tg mice demonstrated that CTRP3 increased oxygen consumption in the presence of fatty acids, indicating that CTRP3 increases hepatic fatty acid utilization. In conclusion, this study indicates that CTRP3 attenuates hepatic triglyceride accumulation in response to long-term chronic but not short-term alcohol consumption.

No description available.

Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-£]1 (TGF-£]1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-£]1 (TGF-£]1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-£]1 superfamily, has been known to regulate cell proliferation, differentiation, and bone morphogenesis. Our previous study demonstrated that BMP-2 was downregulated in fibrotic liver of mice, supporting its antagonistic role in hepatic fibrogenesis. Downregulation of BMP-2 in fibrotic livers may lose its ability to antagonize the pro-fibrogenic action of TGF-£]1. The purpose of this study was to determine whether mutual regulatory mechanisms exist between BMP-2 and TGF-£]1. Treatment of recombinant protein, TGF-£]1, significantly suppressed BMP-2 expression in hepatocytes clone-9 and HSC-T6 cells. Moreover, treatment of BMP-2 in both cell also attenuated TGF-£]1protein levels in a dose-dependent manner. This finding supports that TGF-£]1 and BMP-2 mutually modulated their expression not only in vivo, but also in vitro. Western blotting analysis showed that TGF-£]1 and BMP-2 exerted different kinase phosphorylation profiles of signaling activities. However, the signal pathways and detail mechanisms of the interactions of BMP-2 and TGF-£]1 in the fibrogenic action will need to further evaluate.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成7年12月20日 北川喜己氏の博士論文として提出された

Bone morphogenetic proteins-2/4/7 (BMP-2/4/7) are important cytokines in systemic tissue morphogenesis. It has been demonstrated BMPs may have positive effects on liver repair and regeneration after hepatic injury. However, their function in the liver still remains unclear. D-galactosamine (D-gal) is a hepatotoxin used to induce hepatic failure. We employed D-gal and rat hepatoma cell line (1548) to investigate BMP-2/4/7 expression in hepatic injury induced by D-gal and probe their relations with liver repair and regeneration in hepatic injury. LDH release, mRNA and protein expression were detected. Results indicated that BMP-2/4/7 expression was activated by injury of rat hepatoma cells. It is indicative that repair and regeneration of the liver after hepatic injury and morphogenesis in early embryos seem to proceed through the same process. BMPs may be not only associated with hepatic injury after repair and regeneration, but also involved in chronic liver.