Mechanisms, biomarkers and targets for therapy in alcohol-associated liver injury: From Genetics to nutrition: Summary of the ISBRA 2018 symposium.

The symposium "Mechanisms, Biomarkers and Targets for Therapy in Alcohol-associated Liver Injury: From Genetics to Nutrition" was held at the 19th Congress of International Society for Biomedical Research on Alcoholism on September 13th, 2018 in Kyoto, Japan. The goal of the symposium was to discuss the importance of genetics and nutrition in alcoholic liver disease (ALD) development from mechanistic and therapeutic perspectives. The following is a summary of this session addressing the gene polymorphisms in ALD, the role of zinc in gut-liver axis perturbations associated with ALD, highlighting the importance of dietary fat in ALD pathogenesis, the hepatic n6 and n3 PUFA oxylipin pattern associated with ethanol-induced liver injury, and finally deliberating on new biomarkers for alcoholic hepatitis and their implications for diagnosis and therapy. This summary of the symposium will benefit junior and senior faculty currently investigating alcohol-induced organ pathology as well as undergraduate, graduate, and post-graduate students and fellows.

Microbiome dysbiosis and alcoholic liver disease☆.

Microbiome dysbiosis is strongly associated with alcoholic liver disease (ALD). Recent studies on comprehensive analyses of microbiome compositional and functional changes have begun to uncover the mechanistic relation between microbiome and the pathogenesis of ALD. Importantly, targeting the microbiome has become a potential strategy for the prevention and treatment of ALD. In this review, we summarize the clinical evidence of microbiome dysbiosis in ALD patients, and experimental advances in microbiome and metabolomic functional changes in animals with different species and genetic backgrounds in ALD. We also summarize the studies in humanized intestinal microbiome and fecal microbiota transplantation in mice. We introduce new developments in the studies on the role of the circulating bacterial microbiome, oral bacterial microbiome and fungal microbiome in the development of ALD. We highlight the potential mechanisms by which microbiome dysbiosis contributes to ALD, including short chain fatty acid changes, bile acid metabolism, intestinal barrier function, release of bacterial and fungal products, and inflammation. In addition, we summarize the recent developments targeting the microbiome in prevention and treatment of ALD, including dietary nutrient interference, herbal medicine, antibiotics, anti-fungal agents, probiotics, engineered bacterial therapy, fecal transplantation and oral hygiene. Although recent preclinical studies have advanced our understanding of the microbiome and ALD, clinical studies, especially prospective studies with large samples, are needed to better understand the cause-effect of microbiome dysbiosis in ALD. Identifying new precision-based strategies targeting the microbiome are expected to be developed as more effective therapies in ALD.

Fibroblast growth factor 21 deficiency exacerbates chronic alcohol-induced hepatic steatosis and injury.

Fibroblast growth factor 21 (FGF21) is a hepatokine that regulates glucose and lipid metabolism in the liver. We sought to determine the role of FGF21 in hepatic steatosis in mice exposed to chronic alcohol treatment and to discern underlying mechanisms. Male FGF21 knockout (FGF21 KO) and control (WT) mice were divided into groups that were fed either the Lieber DeCarli diet containing 5% alcohol or an isocaloric (control) diet for 4 weeks. One group of WT mice exposed to alcohol received recombinant human FGF21 (rhFGF21) in the last 5 days. Liver steatosis and inflammation were assessed. Primary mouse hepatocytes and AML-12 cells were incubated with metformin or rhFGF21. Hepatic genes and the products involved in in situ lipogenesis and fatty acid ß-oxidation were analyzed. Alcohol exposure increased circulating levels and hepatic expression of FGF21. FGF21 depletion exacerbated alcohol-induced hepatic steatosis and liver injury, which was associated with increased activation of genes involved in lipogenesis mediated by SREBP1c and decreased expression of genes involved in fatty acid ß-oxidation mediated by PGC1α. rhFGF21 administration reduced alcohol-induced hepatic steatosis and inflammation in WT mice. These results reveal that alcohol-induced FGF21 expression is a hepatic adaptive response to lipid dysregulation. Targeting FGF21 signaling could be a novel treatment approach for alcoholic steatohepatitis.

Ethanol and dietary unsaturated fat (corn oil/linoleic acid enriched) cause intestinal inflammation and impaired intestinal barrier defense in mice chronically fed alcohol.

Alcohol and dietary fat both play an important role in alcohol-mediated multi-organ pathology, including gut and liver. In the present study we hypothesized that the combination of alcohol and dietary unsaturated fat (USF) would result in intestinal inflammatory stress and mucus layer alterations, thus contributing to disruption of intestinal barrier integrity. C57BL/6N mice were fed Lieber-DeCarli liquid diets containing EtOH and enriched in USF (corn oil/linoleic acid) or SF (medium chain triglycerides: beef tallow) for 8 weeks. Intestinal histology, morphometry, markers of inflammation, as well as levels of mucus protective factors were evaluated. Alcohol and dietary USF triggered an intestinal pro-inflammatory response, characterized by increase in Tnf-α, MCP1, and MPO activity. Further, alcohol and dietary USF, but not SF, resulted in alterations of the intestinal mucus layer, characterized by decreased expression of Muc2 in the ileum. A strong correlation was observed between down-regulation of the antimicrobial factor Cramp and increased Tnf-α mRNA. Therefore, dietary unsaturated fat (corn oil/LA enriched) is a significant contributing factor to EtOH-mediated intestinal inflammatory response and mucus layer alterations in rodents.

Protective effect of FGF21 on type 1 diabetes-induced testicular apoptotic cell death probably via both mitochondrial- and endoplasmic reticulum stress-dependent pathways in the mouse model.

Fibroblast growth factor 21 (FGF21) is a novel member identified and was reported to express predominantly in pancreas, liver and adipose tissue, and relatively less in other organs, such as the testis. However, the role of FGF21 in the testis has never been addressed. The present study examined FGF21 expression at mRNA level by real-time RT-PCR assay in the testis of fasting and non-fasting mice or mice with type 1 diabetes that was induced with streptozotocin. We also examined the effect of Fgf21 gene deletion or supplementation of the exogenous FGF21 on the testicular apoptotic cell death spontaneously or induced by type 1 diabetes in FGF21 knockout (FGF21-KO) mice. Deletion of Fgf21 gene does not affect testicular cell proliferation, but significantly increases the spontaneous incidence of testicular TUNEL positive cells with increases in the Bax/Bcl2 expression ratio and apoptosis-inducing factor (AIF) expression. Diabetes induced significant increases in testicular TUNEL positive cells, Bax/Bcl2 expression ratio, AIF expression, CHOP and cleaved caspase-12 expression, and oxidative damage, but did not change the expression of cleaved caspase-3 and caspase-8. Deletion of Fgf21 gene also significantly enhances diabetes-induced TUNEL positive cells along with the increased expression of Bax/Bcl2 ratio, AIF, CHOP, cleaved caspase-12, and oxidative damage, which was significantly prevented by the supplementation of exogenous FGF21. These results suggest that Fgf21 gene may involve in maintaining normal spermatogenesis and also protect the germ cells from diabetes-induced apoptotic cell death probably via the prevention of diabetes-induced oxidative damage.

Cardiac protection by basic fibroblast growth factor from ischemia/reperfusion-induced injury in diabetic rats.

Diabetes impairs the expression and function of endogenous growth factors, leading to increased cardiovascular events in diabetic patients. Supplementation of fibroblast growth factors (FGFs) protected the heart from ischemia/reperfusion (I/R)-induced injury in animal models. However, it has not yet been tested in diabetic heart. The present study was thus to clarify whether basic fibroblast growth factor (bFGF) could protect the heart from I/R-induced damage under diabetic conditions using a rat model. Male Sprague Dawley rats were used to induce diabetes by intraperitoneal injection of streptozotocin. Eight weeks later, I/R injury was generated in diabetic rats and age-matched non-diabetic rats. All I/R rats were administrated bFGF or saline through intramyocardial injection. Seven days after I/R, cardiac infarction, structural changes, cell death and blood vessel density, serum malondialdehyde (MDA) and cardiac enzyme lactate dehydrogenase (LDH) were examined. We found that I/R induced significant increases in the cardiac infarction, blood MDA contents and LDH activities, and the expression of caspase-3. Treatment of I/R rats with bFGF simultaneously with reperfusion significantly attenuated I/R-induced pathological changes, along with a significant increase in the cardiac blood vessel density in both diabetic and non-diabetic rates. The protective effects of bFGF on I/R-induced cardiac injury in diabetic group are less than those in non-diabetic group. The results indicated that bFGF provide a protection of the heart against I/R-induced oxidative damage, cell death and infarction under diabetic conditions.

Expression and Purification of glutathione transferase-small ubiquitin-related modifier-metallothionein fusion protein and its neuronal and hepatic protection against D-galactose-induced oxidative damage in mouse model.

The present study aimed to produce and pathophysiologically evaluate the metallothionein (MT) fusion protein. A recombinant plasmid containing DNA segment coding the pET-glutathione transferase (GST)-small ubiquitin-related modifier (SUMO)-MT fusion protein was inserted into Escherichia coli for expression. The expression level of the fusion protein was very high, reaching to 38.4% of the total supernatant proteins from the organism. Subsequent filtration through glutathione Sepharose 4B gel and Sephadex G-25 yielded an MT fusion protein with purity more than 95%. When exposed to metals, E. coli containing the GST-SUMO-MT fusion protein showed an increased accumulation of Cd(2+), Zn(2+), or Cu(2+) at approximately 4.2, 4.0, or 1.6 times higher, respectively, than those containing the control protein. Administration of GST-SUMO-MT to mice that were also treated with D-galactose to induce neuronal and hepatic damage showed a significant improvement of animal learning and memory capacity, which was depressed in mice treated by D-galactose alone. Administration of MT fusion protein also prevented D-galactose-increased malondialdehyde contents and histopathological changes in the brain and liver. Furthermore, supplement of the fusion protein significantly prevented D-galactose-increased nitric oxide contents and -decreased superoxide dismutase activity in the brain, liver, and serum. The fusion protein was also able to prevent ionizing radiation-induced DNA damage of the mouse thymus. The present study indicates that GST-SUMO-MT has a normal metal binding feature and also significantly protects the multiple tissues against oxidative damage in vivo caused by chronic exposure to D-galactose and by ionizing radiation. Therefore, GST-SUMO-MT may be a potential candidate to be developed for the clinical application.

Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice.

Sustained pressure overload causes cardiac hypertrophy and the transition to heart failure. We show here that dietary supplementation with physiologically relevant levels of copper (Cu) reverses preestablished hypertrophic cardiomyopathy caused by pressure overload induced by ascending aortic constriction in a mouse model. The reversal occurs in the continued presence of pressure overload. Sustained pressure overload leads to decreases in cardiac Cu and vascular endothelial growth factor (VEGF) levels along with suppression of myocardial angiogenesis. Cu supplementation replenishes cardiac Cu, increases VEGF, and promotes angiogenesis. Systemic administration of anti-VEGF antibody blunts Cu regression of hypertrophic cardiomyopathy. In cultured human cardiomyocytes, Cu chelation blocks insulin-like growth factor (IGF)-1- or Cu-stimulated VEGF expression, which is relieved by addition of excess Cu. Both IGF-1 and Cu activate hypoxia-inducible factor (HIF)-1alpha and HIF-1alpha gene silencing blocks IGF-1- or Cu-stimulated VEGF expression. HIF-1alpha coimmunoprecipitates with a Cu chaperone for superoxide dismutase-1 (CCS), and gene silencing of CCS, but not superoxide dismutase-1, prevents IGF-1- or Cu-induced HIF-1alpha activation and VEGF expression. Therefore, dietary Cu supplementation improves the condition of hypertrophic cardiomyopathy at least in part through CCS-mediated HIF-1alpha activation of VEGF expression and angiogenesis.