Adenosine 2A receptor antagonist prevented and reversed liver fibrosis in a mouse model of ethanol-exacerbated liver fibrosis.

The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl4, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl4) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl4, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl4. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis.Moderate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl4. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol

Progression of fibrosis with moderate ethanol intake and superimposed CCl₄ liver injury.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections for 2 weeks or 5 weeks. (<b>A</b>) Collagen 1 staining was performed on frozen liver sections at 2 weeks and 5 weeks. (<b>B</b>) Paraffin-embedded liver sections were de-paraffinized followed by Sirius red staining to assess ECM deposition at 2 weeks and 5 weeks. Representative images are shown. (Solid arrow: immunostaining pattern consistent with bridging fibrosis. Open arrow: immunostaining pattern less than bridging fibrosis.) Morphometric analysis with Image-Pro-Plus software was used for semi-quantification. Value represents mean ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p

Effect of moderate ethanol intake on CYP2E1 and CCl₄ hepatotoxicity.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) (11% calories) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections over 72 hours, 2 weeks or 5 weeks. (<b>A</b>) Plasma ALT was measured in the acute time course to assess hepatic injury. (<b>B</b>) Plasma AST and ALT were measured at different time points. (<b>C</b>) CYP2E1 protein level in liver was measured by Western blot using whole liver extracts. HSC 70 was used as loading control. Insets show representative image of CYP2E1 Western blots. (<b>D</b>) Activity of CYP2E1 in liver was measured by the hydroxylation of <i>p</i>-nitrophenol in whole liver extracts. Values represent means ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p

Effect of A2AR antagonist on hepatic angiogenic response and sinusoidal capillarization.

<p>(<b>A</b>) C57BL/6J wild-type (WT) mice were allowed free access to diets with increasing concentrations of 2% (vol/vol) ethanol or pair-fed controls for 4 days. All animals were injected intraperitoneally with pimonidazole (120 mg/Kg, PMD) 1 hour before euthanasia. Paraffin-embedded liver sections were de-paraffinized and stained for PMD-adducts. Representative images are shown. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. (Solid black arrow: increased pimonidazole adducts accumulation.) (<b>B</b>) A short time course study using single injection of CCl₄ with pretreatment of KW-6002 or saline in C57BL/6J wild-type (WT) mice. ANGP1, TNF-α and MIP2 mRNA in liver was measured after KW-6002 administration as an intermediate marker of angiogenesis and inflammation. (* indicates statistical significance between KW-6002 and saline control, p<0.05) (<b>C</b>) C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections with KW-6002 for 2 weeks in the prevention model or with KW-6002 in the last week of the 5 weeks treatment model. Frozen liver sections were used for CD31 immunofluorescent staining to study sinusoidal capillarization and angiogenic response. (Solid arrow: increased CD31 expression in sinusoidal space.) Representative images are shown. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet.</p

Effect of A2AR antagonist in prevention and treatment of CCl₄-induced HSC activation and liver fibrosis.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and exposed to intraperitoneal CCl₄ injections with KW-6002 for 2 weeks in the prevention model or with KW-6002 in the last week of the 5 weeks treatment model. (<b>A</b>) α-SMA was used as a marker for activated HSC in the 2 week and 5 week models. Representative images are shown. (<b>B</b>) ECM deposition was measured by Sirius red staining in the 2 week and 5 week models. Representative images are shown. (<b>C, D</b>) Morphometric analysis with Image-Pro-Plus software was used for semi-quantification. Values represent means ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05. (<b>E</b>) Hepatic hydroxyproline concentration was measured using a colorimetric assay in liver acid–hydrolysates in the 5 week model. Values represent means ± SEM. n = 3–8 in each group. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p

Effect of A2AR antagonist on liver injury.

<p>Plasma AST and ALT levels in pair-fed and ethanol-fed C57BL/6J mice exposed to CCl₄ with KW-6002 or saline control at the 2 week and 5 week time points. Values represent mean ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet.</p

Effect of moderate ethanol intake on HSC activation in response to CCl₄.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections over 72 hours, 2 weeks or 5 weeks. (<b>A</b>) Hepatic accumulation of Col1A1, Col1A2 and α-SMA mRNA was used as intermediate biomarkers of HSC activation and fibrosis at 72 hours. (<b>B, C</b>) Paraffin-embedded liver sections were de-paraffinized followed by α-SMA staining at 2 weeks (<b>B</b>) and 5 weeks (<b>C</b>). Representative images are shown. Morphometric analysis with Image-Pro-Plus software was used for semi-quantification. Values represent means ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p

Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice[S]

The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARγ binding site in the promoter of Pck1 (PPARE−/−). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE−/− mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE−/− mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention