Laura W. Schrum

Rodent models of alcoholic liver disease: Of mice and men

Alcoholic liver disease (ALD) is a major cause of acute and chronic liver disease worldwide. The progressive nature of ALD is well described; however, the complex interactions under which these pathologies evolve remain to be fully elucidated. Clinically there are no clear biomarkers or universally accepted, effective treatment strategies for ALD. Experimental models of ALD are an important component in identifying underlying mechanisms of alcohol-induced injury to develop better diagnostic markers, predictors of disease progression, and therapeutic targets to manage, halt, or reverse disease progression. Rodents remain the most accessible model for studying ALD pathology. Effective rodent models must mimic the natural history of ALD while allowing examination of complex interactions between multiple hepatic, and non-hepatic, cell types in the setting of altered metabolic or oxidative/nitrosative stress, inflammatory responses, and sensitivity to cytotoxic stress. Additionally, mode and duration of alcohol delivery influence hepatic response and present unique challenges in understanding disease pathology. This review provides an overview of rodent models of ALD, their strengths and weaknesses relative to human disease states, and provides insight of the potential to develop novel rodent models to simulate the course of human ALD.

Novel role of nuclear receptor rev‐erbα in hepatic stellate cell activation: Potential therapeutic target for liver injury

Hepatic stellate cell (HSC) transdifferentiation from a quiescent, adipocyte‐like cell to a highly secretory and contractile myofibroblast‐like phenotype contributes to negative pathological consequences, including fibrosis/cirrhosis with portal hypertension (PH). Antiadipogenic mechanisms have been shown to underlie activation of HSCs. We examined the role of heme‐sensing nuclear receptor Rev‐erbα, a transcriptional repressor involved in metabolic and circadian regulation known to promote adipogenesis in preadipocytes, in HSC transdifferentiation. We discovered that Rev‐erbα protein was up‐regulated in activated HSCs and injured livers; however, transcriptional repressor activity was not affected by fibrogenic treatments. Surprisingly, increased protein expression was accompanied with increased cytoplasmic accumulation of Rev‐erbα, which demonstrated distributions similar to myosin, the major cellular motor protein. Cells overexpressing a cytoplasm‐localized Rev‐erbα exhibited enhanced contractility. Ectopically expressed Rev‐erbα responded to both adipogenic ligand and fibrogenic transforming growth factor beta treatment. Rev‐erb ligand SR6452 down‐regulated cytoplasmic expression of Rev‐erbα, decreased expression of fibrogenic markers and the activated phenotype in HSCs, and ameliorated fibrosis and PH in rodent models. Conclusions: Up‐regulation of Rev‐erbα is an intrinsic fibrogenic response characterized by cytoplasmic accumulation of the protein in activated HSCs. Cytoplasmic expression of Rev‐erbα promotes a contractile phenotype. Rev‐erbα acts as a bifunctional regulator promoting either anti‐ or profibrogenic response, depending on milieu. Rev‐erb ligand SR6452 functions by a previously undescribed mechanism, targeting both nuclear activity and cytoplasmic expression of Rev‐erbα. Our studies identify Rev‐erbα as a novel regulator of HSC transdifferentiation and offers exciting new insights on the therapeutic potential of Rev‐erb ligands. (Hepatology 2014;59:2383–2396)

Silibinin (Milk Thistle) potentiates ethanol-dependent hepatocellular carcinoma progression in male mice.

Hepatocellular carcinoma (HCC) is a global health burden with limited treatment options and poor prognosis. Silibinin, an antioxidant derived from the Milk Thistle plant (Silybum marianum), is reported to exert hepatoprotective and antitumorigenic effects in vitro and in vivo by suppressing oxidative stress and proliferation. Using a DEN-initiated mouse model of HCC, this study examined the effects of dietary silibinin supplementation alone, or in combination with chronic ethanol consumption on HCC progression. Our data demonstrate silibinin exerted marginal hepatoprotective effects in early stages of hepatocarcinogenesis but, when co-administered with ethanol, exacerbated the promotional effects of ethanol in HCC bearing mice, but only in males.

Altered expression and function of regulator of G-protein signaling-17 (RGS17) in hepatocellular carcinoma

Guanine nucleotide regulatory proteins (G-proteins) are central to normal hepatocyte function and are implicated in hepatic disease initiation and progression. Regulators of G-protein signaling (RGS) are critical to defining G-protein-dependent signal fidelity, yet the role of RGS proteins in the liver is poorly defined. The aims of this study were to determine RGS17 expression in normal and transformed hepatic tissue and cells, and address the function of RGS17 in hepatic tumorgenicity. RGS17 expression was determined in human and rat HCC tissue and cell lines. Molecular approaches were used to alter RGS17 expression in HCC cells, effects on cell function measured, and RGS17 association with specific Gα-subunits determined. Using these approaches RGS17 mRNA, but not protein, was detectable in human and rat HCC tissue and cells. Conversely, RGS17 mRNA was not detected in normal tissue, isolated hepatocytes, or non-tumorigenic hepatic cells. Subsequent studies using transfected cells demonstrated that RGS17 proteins were not post-translationally modified in HCC cells, and RGS17 expression is governed by protein degradation and not via miRNAs. Notwithstanding inherently low RGS17 protein levels, altering RGS17 expression profoundly affected HCC cell mitogenesis and migration. Analysis of RGS17-G-protein interaction demonstrated RGS17 associates with both Giα- and Gqα-subunits in HCC cells of human and rat origin. In conclusion, these data demonstrate that, despite difficulties in measuring endogenous RGS protein expression, RGS17 is differentially expressed in HCC and plays a central role in regulating transformed hepatocyte tumorgenicity.

Mitigation of myocardial fibrosis by molecular cardiac surgery–mediated gene overexpression

OBJECTIVE Heart failure is accompanied by up-regulation of transforming growth factor beta signaling, accumulation of collagen and dysregulation of sarcoplasmic reticulum calcium adenosine triphosphatase cardiac isoform 2a (SERCA2a). We examined the fibrotic response in small and large myocardial infarct, and the effect of overexpression of the SERCA2a gene. METHODS Ischemic cardiomyopathy was induced via creation of large or small infarct in 26 sheep. Animals were divided into 4 groups: small infarct; large infarct with heart failure; gene-treated (large infarct with heart failure followed by adeno-associated viral vector, serotype 1.SERCA2a gene construct transfer by molecular cardiac surgery with recirculating delivery); and control. RESULTS Heart failure was significantly less pronounced in the gene-treated and small-infarct groups than in the large-infarct group. Expression of transforming growth factor beta signaling components was significantly higher in the large-infarct group, compared with the small-infarct and gene-treated groups. Both the angiotensin II type 1 receptor and angiotensin II were significantly elevated in the small- and large-infarct groups, whereas gene treatment diminished this effect. Active fibrosis with de novo collagen synthesis was evident in the large-infarct group; the small-infarct and gene-treated groups showed less fibrosis, with a lower ratio of de novo to mature collagen. CONCLUSIONS The data presented provide evidence that progression of fibrosis is mediated through increased transforming growth factor beta and angiotensin II signaling, which is mitigated by increased SERCA2a gene expression.

Rev-erb agonist and TGF-β similarly affect autophagy but differentially regulate hepatic stellate cell fibrogenic phenotype.

We demonstrated that ligand-activated nuclear receptor Rev-erbα mitigates CCl4-induced liver fibrosis. Rev-erbα is also a novel regulator of autophagy, a crucial eukaryotic catabolic system in which lysosomes degrade substrates for energy generation. In hepatic stellate cells (HSC) autophagy is reportedly required for this purpose to activate HSCs during fibrogenesis. Here, we examined whether pharmacological activation of Rev-erb with its synthetic ligand SR9009 or treatment with the pro-fibrotic cytokine, TGF-β, each differentially modulate autophagy to regulate the HSC phenotype. We measured the effects of SR9009 on autophagy markers in a CCl4-induced liver fibrosis model. Using primary and immortalized HSCs in vitro, we quantified SR9009 and TGF-β effects on autophagy flux. Compared with vehicle-treated controls, livers from CCl4-treated mice exhibited lower AMPK, higher P70S6K phosphorylation, elevated P62 and lower levels of ATG proteins, indicating a disruption of autophagosome (AV) formation. SR9009 treatment prevented CCl4-induced P70S6K phosphorylation but did not affect CCl4-induced changes in AMPK, ATG proteins or P62. Analysis of autophagy markers and autophagy flux in primary HSCs or an immortalized human HSC line (LX2), revealed that SR9009 exposure down-regulated AV biogenesis. These events were associated with lower levels of fibrogenic gene expression, P70S6K phosphorylation and HSC proliferation. However, HSC exposure to TGF-β enhanced fibrogenic gene expression, P70S6K phosphorylation and HSC proliferation, while it simultaneously decelerated AV synthesis. The autophagy activator rapamycin and the autophagy inhibitor wortmannin each decreased HSC activation, P70S6K phosphorylation and HSC proliferation. Furthermore, knock-down of P70S6K using siRNA blocked basal and TGF-β-induced cell proliferation in human activated LX2. We conclude that SR9009 and TGF-β both similarly affected autophagy but, differentially regulated HSC fibrogenic phenotype through modulation of P70S6K, which is crucial for cell proliferation and fibrogenesis.

Dietary fructose augments ethanol-induced liver pathology

Certain dietary components when combined with alcohol exacerbate alcohol-induced liver injury (ALI). Here, we tested whether fructose, a major ingredient of the western diet, enhances the severity of ALI. We fed mice ethanol for 8 weeks in the following Lieber-DeCarli diets: (a) Regular (contains olive oil); (b) corn oil (contains corn oil); (c) fructose (contains fructose and olive oil) and (d) corn+fructose (contains fructose and corn oil). We compared indices of metabolic function and liver pathology among the different groups. Mice fed fructose-free and fructose-containing ethanol diets exhibited similar levels of blood alcohol, blood glucose and signs of disrupted hepatic insulin signaling. However, only mice given fructose-ethanol diets showed lower insulin levels than their respective controls. Compared with their respective pair-fed controls, all ethanol-fed mice exhibited elevated levels of serum ALT; the inflammatory cytokines TNF-α, MCP-1 and MIP-2; hepatic lipid peroxides and triglycerides. All the latter parameters were significantly higher in mice given fructose-ethanol diets than those fed fructose-free ethanol diets. Mice given fructose-free or fructose-containing ethanol diets each had higher levels of hepatic lipogenic enzymes than controls. However, the level of the lipogenic enzyme fatty acid synthase (FAS) was significantly higher in livers of mice given fructose control and fructose-ethanol diets than in all other groups. Our findings indicate that dietary fructose exacerbates ethanol-induced steatosis, oxidant stress, inflammation and liver injury, irrespective of the dietary fat source, to suggest that inclusion of fructose in or along with alcoholic beverages increases the risk of more severe ALI in heavy drinkers.

Data on the effect of pro-fibrotic cytokine TGF-β on hepatic stellate cell autophagy

Our data describe autophagic flux in primary rat hepatic stellate cells (rHSCs) treated with pro-fibrotic growth factor, transforming growth factor beta (TGF-β). An autophagy flux experiment determines the rate of synthesis and degradation of the autophagosome marker, LC3-II in the presence and absence of the lysosomal inhibitor bafilomcyin, which blocks LC3-II degradation in lysosomes. The effects of a test agent on LC3-II flux through the autophagic pathway is determined immunochemically by its relative amounts detected in lysates of cells treated with and without bafilomycin. This measurement helps to validate whether exposure to an agent affects the biogenesis or the degradation of autophagosomes during autophagy, a major macromolecular degrading mechanism in eukaryotic cells. (“Rev-erb Agonist and TGF-β Similarly Affect Autophagy but Differentially Regulate Hepatic Stellate Cell Fibrogenic Phenotype” (Thomes et al., in press) [1].

Advances in Alcoholic Liver Disease

This special issue reflects on multiple factors/mechanisms involved in the pathogenesis of ALD. Alcoholic liver injury is known to cause a broad range of liver abnormalities. Alcohol is primarily metabolized in the hepatocyte leading to increased secretion of inflammatory mediators, which, in turn, activate and/or influence the response of the nonparenchymal cells (NPCs) (hepatic stellate cells, Kupffer cells, and sinusoidal endothelial cells) and subsequently control the degree of liver injury.