Pathways In Liver Tissue Research Articles

Hepatic expression patterns in psychosocially high-stressed pigs suggest mechanisms following allostatic principles

Psychosocial challenges are known to introduce cellular and humoral adaptations in various tissues and organs, including parts of the sympatho-adrenal-medullary system and hypothalamic–pituitary–adrenal axis as well as other peripheral tissue being responsive to cortisol and catecholamines. The liver is of particular interest given its vital roles in maintaining homeostasis and health as well as regulating nutrient utilization and overall metabolism. We aimed to evaluate whether and how response to psychosocial stress is reflected by physiological molecular pathways in liver tissue. A pig mixing experiment was conducted to induce psychosocial stress culminating in skin lesions which reflect the involvement in aggressive behavior and fighting. At 27weeks of age, animals prone to psychosocially low- and high-stress were assigned to mixing groups. Skin lesions were counted before mixing and after slaughter on the carcass. Individual liver samples (n=12) were taken. The isolated RNA was hybridized on Affymetrix GeneChip porcine Genome Arrays. Relative changes of mRNA abundances were estimated via variance analyses. Molecular routes related to tRNA charging, urea cycle, acute phase response, galactose utilization, and steroid receptor signaling were found to be increased in psychosocially high-stressed animals, whereas catecholamine degradation and cholesterol biosynthesis were found to be decreased. In particular, psychosocially high-stressed animals show decreased expression of catechol-O-methyltransferase (COMT) which has been linked to molecular mechanisms regulating aggressiveness and stress response. The expression patterns of high-stressed animals revealed metabolic alterations of key genes related to energy-mobilizing processes at the expense of energy consuming processes. Thus, the coping following psychosocial challenges involves transcriptional alterations in liver tissue which may be summarized with reference to the concept of allostasis, a strategy which is critical for survival.

High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway

Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)α, LXRβ, and retinoid X receptor (RXR)β. Exogenous expression and activation of LXRα/β increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis.

A fast method for the quantitation of key metabolites of the methionine pathway in liver tissue by high-resolution mass spectrometry and hydrophilic interaction ultra-performance liquid chromatography

We developed an assay for the extraction and simultaneous quantitation of five key metabolites of the methionine metabolic pathway in liver tissue. The metabolites included were 5'-methylthioadenosine, methionine, homocysteine, S-adenosyl-L-homocysteine, and S-adenosyl-L-methionine. The metabolites were extracted using a bead-based homogenization method, and quantitation was carried out using hydrophilic interaction chromatography and time-of-flight mass spectrometry. The extraction procedure was optimized by testing the effect of various solvent combinations. The chromatographic method was optimized for peak shape, signal intensity, and carry-over. With a total chromatographic run time of 5 min, this assay is suitable for the analysis of large sample sets. Time-of-flight mass spectrometry provided high mass accuracy which, combined with isotope pattern matching and use of chemical standards, guarantees high specificity. Moreover, by operating the mass spectrometer in enhanced duty cycle mode the signal strength for the analytes increased three- to tenfold in comparison with the generic full-scan mode. For quantitation, a matrix-spiked calibration method was used. The lowest analyte levels detected and quantified using our method were within the range of concentrations found in the liver. The inter-day coefficients of variance for the analytes were between 5 and 15% in pooled tissue samples. Interestingly, the CVs between individual liver tissue aliquots were about twice as high. Additional experiments suggested that this higher variability was caused by uneven distribution of the analytes within the liver. In conclusion, an optimized and robust assay is now available for the extraction and quantification of key metabolites in the methionine metabolic pathway.

Oxidative stress pathways in noncancerous human liver tissue to predict hepatocellular carcinoma recurrence: A prospective, multicenter study

The prediction of cancer recurrence holds the key to improvement of the postoperative prognosis of patients. In this study, the recurrence of early-stage hepatocellular carcinoma (HCC) after curative hepatectomy was analyzed by the genome-wide gene-expression profiling on cancer tissue and the noncancerous liver tissue. Using the training set of 78 cases, the cytochrome P450 1A2 (CYP1A2) gene in noncancerous liver tissue was identified as the predictive candidate for postoperative recurrence (hazard ratio [HR], 0.447; 95% confidence interval [CI], 0.249-0.808; P = 0.010). Multivariate analysis revealed the statistically significant advantage of CYP1A2 down-regulation to predict recurrence (odds ratio, 0.534; 95% CI, 0.276-0.916; P = 0.036), and the expression of CYP1A2 protein was confirmed immunohistochemically. An independently multi-institutional cohort of 211 patients, using tissue microarrays, validated that loss of expression of CYP1A2 in noncancerous liver tissue as the only predictive factor of recurrence after curative hepatectomy for early-stage HCC (HR, 0.480; 95% CI, 0.256-0.902; P = 0.038). Gene set-enrichment analysis revealed close association of CYP1A2 down-regulation with oxidative stress pathways in liver tissue (P < 0.001, false discovery rate [FDR] = 0.042; P = 0.006, FDR = 0.035). Our results indicate these pathways as the molecular targets to prevent recurrence, as well as the potential prediction of the super high-risk population of HCC using liver tissue.

Abstract 3870: Distinct and cooperative functions of the RB and p53 tumor suppressors in genotoxic response and tumorigenesis

Abstract The compound inactivation of RB and p53 is a frequent occurrence in human cancers. At a molecular level, the RB and p53 pathways intersect at multiple points that have critical implications for cell cycle control and tumorigenesis. To examine the cooperation of these proteins in the response to genotoxic damage, we used a combination of genetic deletion and dominant negative strategies in vitro, and liver-specific genetic deletion in vivo. In vitro, RB proficiency was sufficient to maintain cell cycle checkpoint in response to genotoxic stress, independent of p53 status. In contrast, RB-deficient cells displayed deregulated cell growth, resulting in aggressive recovery from DNA damage. Strikingly, while disruption of the RB and p53 pathways alone does not result in transformation, the RB-deficient, recurred cell populations were tumorigenic. These in vitro findings indicate that RB is a critical suppressor of tumorigenesis, beyond that of p53, and a significant factor in the evolution of cells during genotoxic challenge. To further examine the cooperation of RB and p53 in vivo, a model of liver tumorigenesis was employed. Interestingly, these in vivo studies confirmed that while compound inactivation of the RB and p53 pathways in liver tissue promoted deregulated cell cycle control, this was not sufficient for transformation. However, loss of RB and p53 cooperated in promoting aberrant cell proliferation and mitosis in the presence of genotoxic challenge by DEN (diethyl-nitrosamine), ultimately resulting in enhanced tumorigenesis. Strikingly, loss of RB alone promoted S-phase entry as a result of genotoxic challenge, but there was an uncoupling of this event from productive mitosis. In contrast, the combined loss of RB/p53 resulted in further deregulation of DNA replication that was accompanied by a productive mitotic entry. These data indicate that, while both RB and p53 can contribute to appropriate replicative control, p53 plays a dominant role in mediating G2/M regulation. Furthermore, RB/p53-deficient livers displayed rapid progression to highly aggressive HCC within the span of six months, which was not observed in either RB-deficient or p53-deficient livers. Combined, these studies indicate that RB and p53 play distinct and cooperating roles in modulating response to genotoxic stress and tumorigenesis. Discovering the mechanism behind this cooperation could uncover a critical facet of tumor etiology and provide a means by which to treat this complex disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3870.

526 HYPERAMMONEMIA AND INFLAMMATION IN THE INDUCTION OF MINIMAL HEPATIC ENCEPHALOPHATY

plays an important role in the TGF-b1/ Smad pathway. Boers W et al identified genes involved in the transformation of HSC into myofibroblasts and discovered IGFBP-rP1 showed the highest expression in the midphase of the transdifferentiation process. To investigate the effect of IGFBPrP1 and its relationship with TGF-b1/Smad3 pathway in liver tissue from patients with hepatic cirrhosis, we designed this project. Methods: 31 patients included 16 cases of liver cirrhosis and 15 cases of hemangiomas of liver surrounding healthy controls. The cirrhotic group has 10 males and 6 female, between the age of 43–61 (48.63±9.08); the control group contains 6 males and 9 females, aged from 41 to 91 (53.13±13.73). These paraffin samples of liver tissue were carried out HE and Masson straining. The expressions of IGFBPrP1, TGF-b1 and Smad3 were detected by immunohistochemistry staining. Results: HE and Masson staining showed the content of collagen was much higher in the cirrhotic group than that of control group. Immunohistochemical staining showed very few hepatocyte and sinus hepaticus had positive expression of TGF-b1 in the normal liver; sinus hepaticus, portal area and fibrous interval had stronger positive staining in the cirrhotic group. Smad3 had a wide and low level of expression in hepatocytes of normal liver tissue; in the cirrhotic group, positive expression increased in hepatocytes, along with stellate cell plasma in sinus hepaticus, portal area and fibrous intervals. IGFBPrP1 expressed in a few hepatocytes in the normal liver; in the cirrhotic group, plasma of HSC, Kupffer cell, fibroblasts in sinus hepaticus, portal area and fibrous intervals and some hepatocytes had stronger positive expression. The expressions of TGF-b1, Smad3 and IGFBPrP1 were largely increased in cirrhotic group, compared with normal control. IGFBPrP1’s expression has significant positive correlation to that of TGF-b1, Smad3(r = 0.886, 0.900; P < 0.01). Serum HA levels of the 16 patients in the cirrhotic group are much higher than normal. Conclusion: IGFBPrP1 may play an important role in the occurrence and development of liver cirrhosis, which is probably related to TGF-b1/Smad3 pathway.

Proliferative Suppression by CDK4/6 Inhibition: Complex Function of the Retinoblastoma Pathway in Liver Tissue and Hepatoma Cells

Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide; current chemotherapeutic interventions for this disease are largely ineffective. The retinoblastoma tumor suppressor (RB) is functionally inactivated at relatively high frequency in hepatocellular carcinoma and hepatoma cell lines. Here, we analyzed the ability of CDK4/6 inhibition to inhibit hepatocyte proliferation and the effect of RB status on this process. Hepatoma cell lines and xenograft models harboring RB knockdown and mice harboring liver-specific Rb deletion were used to define the role of RB function in response to CDK4/6 inhibition. Our study shows that CDK4/6-dependent cell cycle progression in hepatoma cells was readily arrested by inhibition of CDK4/6 by PD-0332991 or p16ink4a irrespective of RB status. Interestingly, upon CDK4/6 inhibition, p107 protein stability was dramatically increased as a function of RB loss. This engagement of compensatory mechanisms was critical for cell cycle inhibition in the absence of RB, because both the E1A oncoprotein and overexpression of E2F proteins were capable of overcoming the effect of CDK4/6 inhibition. These findings were recapitulated in xenograft models. Furthermore, to determine how these findings relate to hepatocyte proliferation in vivo, mice were exposed to carbon tetrachloride to induce liver regeneration followed by treatment with PD-0332991. This treatment significantly inhibited hepatocyte proliferation. Strikingly, this facet of PD-0332991 function was retained even in RB-deficient livers. These data show that CDK4/6 inhibition is a potent mediator of cytostasis and that RB loss can be readily compensated for in the context of both hepatoma cell lines and liver tissue.

Hepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis

Background & Aims: The leukotrienes C4/D4/E4 (cysteinyl-LTs) are 5-lipoxygenase (5-LO)-derived eicosanoids with potent vasoconstrictor, proliferative, and profibrogenic properties that may participate in key pathophysiologic events in liver cirrhosis. We examined the cysteinyl-LT biosynthetic pathway in liver tissue and purified liver cells isolated from rats with carbon tetrachloride–induced cirrhosis, and assessed the vasoactive properties of LTD4 in hepatic stellate cells (HSCs) and anesthetized rats. Methods & Results: Liver homogenates from cirrhotic rats had increased 5-LO mRNA and cysteinyl-LT content, as determined by Northern blot and enzyme immunoassay, respectively. In isolated rat liver cells, 5-LO mRNA expression was found to be restricted to Kupffer cells. However, among the liver cells (i.e., hepatocytes, Kupffer cells, HSCs, and sinusoidal endothelial cells), hepatocytes exhibited the highest ability to generate cysteinyl-LTs from the unstable intermediate LTA4. Hepatocytes from cirrhotic rats showed an enhanced baseline generation of cysteinyl-LTs, but their ability to synthesize cysteinyl-LTs from exogenous LTA4 was found to be similar to that of hepatocytes from normal animals. Both LTD4 and hepatocyte-conditioned medium increased intracellular Ca2+ concentration and induced contraction in HSCs, suggesting that hepatocyte-derived cysteinyl-LTs could act in a paracrine fashion on nearby nonparenchymal liver cells. The relevance of these in vitro findings was further established in vivo by the observation that LTD4 significantly increased portal pressure in anesthetized rats. Conclusions: These data suggest a role for hepatocyte-derived cysteinyl-LTs in mediating hepatic vascular tone abnormalities in cirrhosis.GASTROENTEROLOGY 2000;119:794-805

Hormonal regulation of amino acid accumulation in human fetal liver explants Effects of dibutyryl cyclic AMP, glucagon and insulin

α-Aminoisobutyrate accumulation by human fetal liver explants in organ culture is stimulated by dibutyryl cyclic AMP ( N 6, 2′O- dibutyryl adenosine 3′–5′: cyclic monophosphate), glucagon or insulin. Theophylline increased the effect of submaximal concentrations of dibutyryl cyclic AMP or glucagon. Maximal concentrations of glucagon and dibutyryl cyclic AMP yielded the same results as either agent alone. A period of about 4–6 h was required to observe the stimulatory effect of dibutyryl cyclic AMP or insulin, which could be completely prevented by simultaneous incubation with cycloheximide. Maximal effects of either dibutyryl cyclic AMP or glucagon plus insulin produced additive results. These data support the hypothesis that insulin acts via a mechanism independent of the glucagon—cyclic AMP pathway in liver tissue. In addition, the pharmacologic receptor for glucagon was detected in liver explants from a 30-mm (crown - rump) specimen (6 weeks gestation). The liver had the competence to respond to dibutyryl cyclic AMP by the 36-mm stage. Tissue from a 36-mm specimen did not respond to insulin, but a clear response was elicited from a specimen at the 48-mm stage. These data demonstrate the ability of human fetal liver to respond to hormones at a very early stage in gestation.