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Background & aimsWe recently showed that alcoholic hepatitis (AH) is characterized by dedifferentiation of hepatocytes and loss of mature functions. Glucose metabolism is tightly regulated in healthy hepatocytes. We hypothesize that AH may lead to metabolic reprogramming of the liver, including dysregulation of glucose metabolism.MethodsWe performed integrated metabolomic and transcriptomic analyses of liver tissue from patients with AH or alcoholic cirrhosis or normal liver tissue from hepatic resection. Focused analyses of chromatin immunoprecipitation coupled to DNA sequencing was performed. Functional in vitro studies were performed in primary rat and human hepatocytes and HepG2 cells.ResultsPatients with AH exhibited specific changes in the levels of intermediates of glycolysis/gluconeogenesis, the tricarboxylic acid cycle, and monosaccharide and disaccharide metabolism. Integrated analysis of the transcriptome and metabolome showed the used of alternate energetic pathways, metabolite sinks and bottlenecks, and dysregulated glucose storage in patients with AH. Among genes involved in glucose metabolism, hexokinase domain containing 1 (HKDC1) was identified as the most up-regulated kinase in patients with AH. Histone active promoter and enhancer markers were increased in the HKDC1 genomic region. High HKDC1 levels were associated with the development of acute kidney injury and decreased survival. Increased HKDC1 activity contributed to the accumulation of glucose-6-P and glycogen in primary rat hepatocytes.ConclusionsAltered metabolite levels and messenger RNA expression of metabolic enzymes suggest the existence of extensive reprogramming of glucose metabolism in AH. Increased HKDC1 expression may contribute to dysregulated glucose metabolism and represents a novel biomarker and therapeutic target for AH.

Background & Aims: Recent studies have suggested that the kallikrein-kinin system regulates tissue fibrogenesis. We hypothesize that bradykinin (BK), the main effector peptide of this system, regulates hepatic fibrogenesis., Methods: Kallikrein-kinin system components were studied by quantitative reverse-transcription polymerase chain reaction analysis, immunohistochemistry, and Western blotting. The effect of bradykinin on liver injury was studied by infusing saline or bradykinin (1 and 100 ng/kg/min) through a subcutaneous pump into carbon tetrachloride-treated rats and mice treated with Fas-stimulating antibody. Bradykinin effects were studied in cultured hepatic stellate cells (HSCs) and hepatocytes., Results: Bradykinin receptors and kallikrein-1 were detected in both normal and fibrotic human livers and HSCs. BK receptors were up-regulated in fibrotic livers and activated HSCs. Bradykinin infusion reduced liver damage, as indicated by decreased aminotransferase serum levels and reduced histologic necroinflammatory score without inducing changes in arterial pressure. Moreover, bradykinin attenuated hepatic fibrosis, as indicated by reduced collagen accumulation, smooth muscle alpha-actin content, as well as decreased pro-collagen-alpha1(I) and transforming growth factor-beta1 gene expression. Bradykinin infusion reduced hepatocellular apoptosis induced by anti-Fas-receptor antibody. HSCs responded to bradykinin with intracellular calcium mobilization. Bradykinin reduced procollagen-alpha1(I) and transforming growth factor-beta1 gene expression and induced matrix metalloproteinase-2 activation. Finally, BK induced prosurvival and proliferative intracellular signaling in primary hepatocytes., Conclusions: Bradykinin attenuates liver damage and fibrosis development in a rat model of chronic liver injury. Therefore, activation of the kallikrein-kinin system may be a new therapeutic approach to the management of chronic liver disease.

Background & Aims: Alcoholic hepatitis (AH) is a form of acute-on-chronic liver failure for which current therapy is not fully effective. We investigated the hepatic expression of candidate genes in patients with AH to identify new targets for therapy., Methods: Hepatic expression of candidate genes (n = 46) was assessed by quantitative polymerase chain reaction in patients with AH (n = 23) and in normal livers (n = 6). Disease severity was assessed by the Maddrey's discriminant function and the occurrence of clinical complications. Histologic analysis included the assessment of myofibroblasts (smooth muscle alpha-actin), collagen deposition (Sirius red), and inflammatory infiltrate (CD43). Portal hypertension was assessed by hepatic venous pressure gradient. Predictive association between gene expression and disease severity was assessed by k-nearest neighbor analysis., Results: Patients with AH showed profound hepatocellular dysfunction advanced fibrosis, and severe portal hypertension. Livers with AH showed up-regulation of genes encoding extracellular matrix proteins (procollagen I), fibrogenesis mediators, inflammatory cytokines, and apoptosis regulators. Key components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were markedly up-regulated, whereas cytochrome p450 2E1 and angiotensinogen were down-regulated. The expression of tissue inhibitor of metalloproteinases-1, growth-related oncogene alpha, and several components of NADPH oxidase (dual oxidases 1 and 2) correlated with histologic findings and parameters indicative of disease severity., Conclusions: Genes involved in hepatic fibrogenesis, inflammatory response, and oxidative stress are overexpressed in AH. Some candidate genes correlate with histologic findings and disease severity, suggesting that they may be potential targets for therapy.

Background & Aims: The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II., Methods: Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry., Results: Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1., Conclusions: After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.