Role of EpCAM+ CD133+ extracellular vesicles in steatosis to steatohepatitis transition in NAFLD.

BACKGROUND AND AIMS: Extracellular vesicles (EVs) have emerged as a potential source of circulating biomarkers in liver disease. We evaluated circulating AV+ EpCAM+ CD133+ EVs as a potential biomarker of the transition from simple steatosis to steatohepatitis. METHODS: EpCAM and CD133 liver proteins and EpCAM+ CD133+ EVs levels were analysed in 31 C57BL/6J mice fed with a chow or high fat, high cholesterol and carbohydrates diet (HFHCC) for 52 weeks. The hepatic origin of MVs was addressed using AlbCrexmT/mG mice fed a Western (WD) or Dual diet for 23 weeks. Besides, we assessed plasma MVs in 130 biopsy-proven NAFLD patients. RESULTS: Hepatic expression of EpCAM and CD133 and EpCAM+ CD133+ EVs increased during disease progression in HFHCC mice. GFP+ MVs were higher in AlbCrexmT/mG mice fed a WD (5.2% vs 12.1%) or a Dual diet (0.5% vs 7.3%). Most GFP+ MVs were also positive for EpCAM and CD133 (98.3% and 92.9% respectively), suggesting their hepatic origin. In 71 biopsy-proven NAFLD patients, EpCAM+ CD133+ EVs were significantly higher in those with steatohepatitis compare to those with simple steatosis (286.4 ± 61.9 vs 758.4 ± 82.3; p < 0.001). Patients with ballooning 367 ± 40.6 vs 532.0 ± 45.1; p = 0.01 and lobular inflammation (321.1 ± 74.1 vs 721.4 ± 80.1; p = 0.001), showed higher levels of these EVs. These findings were replicated in an independent cohort. CONCLUSIONS: Circulating levels of EpCAM+ CD133+ MVs in clinical and experimental NAFLD were increased in the presence of steatohepatitis, showing high potential as a non-invasive biomarker for the evaluation and management of these patients.

Pancreas fat content, insulin homeostasis and circulating endothelial microparticles in male essential hypertensive patients.

The pancreas fat content has been poorly investigated in essential hypertension. The authors aim to relate pancreas and liver fat content with parameters measuring insulin resistance, beta-cell function and also with markers of endothelial dysfunction and platelet or endothelial cell destruction. The authors studied a group of 40 male hypertensive patients with well-controlled blood pressure, maintaining a stable weight, and having not changed their medication during the last year. Pancreas fat content was correlated with HOMA-IR (r = .616, p < .001), HOMA-S (r = -.439, p < .005), beta cell function parameter (r = .457, p < .005), and QUICKI (r = .412, p < .01), whereas liver fat was not patients in the highest quartile of pancreas fat content had more circulating endothelial microparticles than patients in the other quartiles (median 129 [94.3-200] vs. 60.9 [49.4-88.8], p = .002). However, patients in the highest quartile of the pancreas fat content distribution did not differ from the lowest in hyperemic response after ischemia nor circulating platelet microparticles count. Liver fat content was not related to any of the parameters studied. In a multivariate stepwise binary logistic regression analysis (Wald Method) circulating endothelial microparticles remain significantly associated with pancreas fat content after adjusting for confounding factors, such as tobacco, diabetes mellitus, hypercholesterolemia, or metabolic syndrome. Our results reflect that in essential hypertension, pancreas fat content is superior to liver fat to study beta-cell functionality and insulin resistance. Moreover, the authors described for the first time that pancreas fat content is related to endothelial cell destruction. Further studies are needed to confirm this point.

Extracellular Vesicles as Biomarkers in Liver Disease.

Extracellular vesicles (EVs) are membrane-derived vesicles released by a variety of cell types, including hepatocytes, hepatic stellate cells, and immune cells in normal and pathological conditions. Depending on their biogenesis, there is a complex repertoire of EVs that differ in size and origin. EVs can carry lipids, proteins, coding and non-coding RNAs, and mitochondrial DNA causing alterations to the recipient cells, functioning as intercellular mediators of cell-cell communication (auto-, para-, juxta-, or even endocrine). Nevertheless, many questions remain unanswered in relation to the function of EVs under physiological and pathological conditions. The development and optimization of methods for EV isolation are crucial for characterizing their biological functions, as well as their potential as a treatment option in the clinic. In this manuscript, we will comprehensively review the results from different studies that investigated the role of hepatic EVs during liver diseases, including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic liver disease, fibrosis, and hepatocellular carcinoma. In general, the identification of patients with early-stage liver disease leads to better therapeutic interventions and optimal management. Although more light needs to be shed on the mechanisms of EVs, their use for early diagnosis, follow-up, and prognosis has come into the focus of research as a high-potential source of 'liquid biopsies', since they can be found in almost all biological fluids. The use of EVs as new targets or nanovectors in drug delivery systems for liver disease therapy is also summarized.

Emerging pharmacological treatment options for MAFLD.

Metabolic dysfunction-associated fatty liver disease (MAFLD) prevalence and incidence is rising globally. It is associated with metabolic comorbidities, obesity, overweight, type 2 diabetes mellitus, and at least two metabolic risk factors, such as hypertension, hypertriglyceridemia, hypercholesterolemia, insulin resistance, and cardiovascular risk, increasing the risk of mortality. The excessive accumulation of fat comprises apoptosis, necrosis, inflammation and ballooning degeneration progressing to fibrosis, cirrhosis, and liver decompensations including hepatocellular carcinoma development. The limitation of approved drugs to prevent MAFLD progression is a paradigm. This review focuses on recent pathways and targets with evidence results in phase II/III clinical trials.

Impact of a Loss-of-Function Variant in HSD17B13 on Hepatic Decompensation and Mortality in Cirrhotic Patients.

A common splice variant in HSD17B13 (rs72613567:TA) was recently found to be associated with a reduced risk of developing chronic liver disease in NAFLD patients and a reduced risk of progression to advanced fibrosis and cirrhosis. In this study, we aimed to evaluate the prognosis of cirrhotic patients harboring this variant. We performed a retrospective analysis on 483 prospectively recruited patients from four different hospitals in Spain, followed-up for at least 5 years. We collected clinical, demographic, and biochemical data, and we performed a genotyping analysis for common variants previously associated with liver disease risk (HSD17B13 rs72613567:TA and PNPLA3 rs738409). Patients homozygous for the TA allele showed a higher MELD score (p = 0.047), Child−Turcotte−Pugh score (p = 0.014), and INR levels (p = 0.046), as well as decreased albumin (p = 0.004) at baseline. After multivariate analysis, patients with the "protective" variant indeed had an increased risk of hepatic decompensation [aHR 2.37 (1.09−5.06); p = 0.029] and liver-related mortality [aHR 2.32 (1.20−4.46); p = 0.012]. Specifically, these patients had an increased risk of developing ascites (Log-R 11.6; p < 0.001), hepatic encephalopathy (Log-R 10.2; p < 0.01), and higher mortality (Log-R 14.1; p < 0.001) at 5 years of follow-up. Interactions with the etiology of the cirrhosis and with the variant rs738409 in PNPLA3 are also described. These findings suggest that the variant rs72613567:TA in HSD17B13 has no protective effect, but indeed increases the risk of decompensation and death in patients with advanced chronic liver disease.

Long non-coding RNA H19 as a biomarker for hepatocellular carcinoma.

BACKGROUND AND AIMS: Liver cancer stem cells (CSCs) could be involved in the carcinogenesis, recurrence, metastasis and chemoresistance of hepatocellular carcinoma (HCC). The aim of this study was to explore the role of lncRNA-H19 as a biomarker for liver cancer. METHODS: LncRNA-H19 expression levels and the functional assays were conducted in EpCAM+ CD133+ CSCs and C57BL/6J mice fed with a high-fat high-cholesterol carbohydrate (HFHCC) or standard diet for 52 weeks. Liver tissue and plasma samples from patients with cirrhosis, with or without HCC, were used for the analyses of gene expression and circulating lncRNA-H19 levels in an estimation and validation cohort. RESULTS: EpCAM+ CD133+ cells showed a stem cell-like phenotype, self-renewal capacity, upregulation of pluripotent gene expression and overexpressed lncRNA-H19 (p < .001). Suppression of lncRNA-H19 by antisense oligonucleotide treatment significantly reduced the self-renewal capacity (p < .001). EpCAM, CD133 and lncRNA-h19 expression increased accordingly with disease progression in HFHCC-fed mice (p < .05) and also in liver tissue from HCC patients (p = .0082). Circulating lncRNA-H19 levels were significantly increased in HCC patients in both cohorts (p = .013; p < .0001). In addition, lncRNA-H19 levels increased accordingly with BCLC staging (p < .0001) and decreased after a partial and complete therapeutic response (p < .05). In addition, patients with cirrhosis who developed HCC during follow-up showed higher lncRNA-H19 levels (p = .0025). CONCLUSION: LncRNA-H19 expression was increased in CSCs, in liver tissue and plasma of patients with HCC and decreased after partial/complete therapeutic response. Those patients who developed HCC during the follow-up showed higher levels of lncRNA-H19. LncRNA-H19 could constitute a new biomarker of HCC.

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation.

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr -/-). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m2), diabetes mellitus and ALT were found to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears.

Impact of COVID-19 on liver disease: From the experimental to the clinic perspective.

Coronavirus disease 2019 (COVID-19) has caused a global pandemic unprecedented in over a century. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a predominantly respiratory infection, various degrees of liver function abnormalities have been reported. Pre-existing liver disease in patients with SARS-CoV-2 infection has not been comprehensively evaluated in most studies, but it can critically compromise survival and trigger hepatic decompensation. The collapse of the healthcare services has negatively impacted the diagnosis, monitoring, and treatment of liver diseases in non-COVID-19 patients. In this review, we aim to discuss the impact of COVID-19 on liver disease from the experimental to the clinic perspective.

Wilson's disease: Revisiting an old friend.

Wilson's disease (WD) is a rare condition caused by copper accumulation primarily in the liver and secondly in other organs, such as the central nervous system. It is a hereditary autosomal recessive disease caused by a deficiency in the ATP7B transporter. This protein facilitates the incorporation of copper into ceruloplasmin. More than 800 mutations associated with WD have been described. The onset of the disease frequently includes manifestations related to the liver (as chronic liver disease or acute liver failure) and neurological symptoms, although it can sometimes be asymptomatic. Despite it being more frequent in young people, WD has been described in all life stages. Due to its fatal prognosis, WD should be suspected in all patients with unexplained biochemical liver abnormalities or neurological or psychiatric symptoms. The diagnosis is established with a combination of clinical signs and tests, including the measurement of ceruloplasmin, urinary copper excretion, copper quantification in liver biopsy, or genetic assessment. The pharmacological therapies include chelating drugs, such as D-penicillamine or trientine, and zinc salts, which are able to change the natural history of the disease, increasing the survival of these patients. In some cases of end-stage liver disease or acute liver failure, liver transplantation must be an option to increase survival. In this narrative review, we offer an overview of WD, focusing on the importance of clinical suspicion, the correct diagnosis, and treatment.

Development and Validation of a Clinical-Genetic Risk Score to Predict Hepatic Encephalopathy in Patients With Liver Cirrhosis.

INTRODUCTION: We aimed to define the impact of the genetic background on overt hepatic encephalopathy (HE) in patients with liver cirrhosis by developing a combined clinical-genetic risk score. METHODS: Patients suffering from liver cirrhosis from the outpatient clinics of 4 hospitals (n = 600) were included and followed up for at least 5 years until HE bouts, liver transplant, or death. Patients were genotyped for 60 candidate single nucleotide polymorphisms together with the microsatellite in the promoter region of the gene GLS. RESULTS: Single nucleotide polymorphisms rs601338 (FUT2), rs5743836 (TRL9), rs2562582 (SLC1A3), rs313853 (SLC1A5), and GLS microsatellite did predict independently the incidence and severity of overt HE and were included as genetic score. Competing risk analysis revealed that bilirubin (subhazard ratio [sHR] 1.30 [1.15-1.48], P < 0.001), albumin (sHR 0.90 [0.86-0.93], P < 0.001), genetic score (sHR 1.90 [1.57-2.30], P < 0.001), and previous episodes of overt HE (sHR 2.60 [1.57-4.29], P < 0.001) were independently associated to HE bouts during the follow-up with an internal (C-index 0.83) and external validation (C-index 0.74). Patients in the low-risk group had 5% and 12% risk of HE at 1 (log-rank 92.1; P < 0.001) and 5 (log-rank 124.1; P < 0.001) years, respectively, whereas 36% and 48% in the high-risk group. DISCUSSION: The genetic background influenced overt HE risk and severity. The clinical-genetic HE Risk score, which combined genetic background together with albumin, bilirubin, and previous episodes of overt HE, could be a useful tool to predict overt HE in patients with cirrhosis.

Combination of squamous cell carcinoma antigen immunocomplex and alpha-fetoprotein in mid- and long-term prediction of hepatocellular carcinoma among cirrhotic patients.

BACKGROUND: The combination of alpha-fetoprotein (AFP) and squamous cell carcinoma antigen immunocomplex (SCCA-IgM) have been proposed for its use in the screening of hepatocellular carcinoma (HCC). Current screening programs for all cirrhotic patients are controversial and a personalized screening is an unmet need in the precision medicine era. AIM: To determine the role of the combination of SCCA-IgM and AFP in predicting mid- and long-term appearance of HCC. METHODS: Two-hundred and three cirrhotic patients (Child A 74.9%, B 21.2%, C 3.9%) were followed-up prospectively every six months to screen HCC by ultrasound and AFP according to European Association for the Study of the Liver guidelines. The estimation cohort was recruited in Italy (30.5%; 62/203) and validation cohort from Spain (69.5%; 141/203). Patients underwent to evaluate SCCA-IgM by enzyme-linked immunosorbent assay (Hepa-IC, Xeptagen, Italy) and AFP levels at baseline. Patients were followed-up for 60 mo, being censored at the time of the appearance of HCC. RESULTS: There were 10.8% and 23.1% of HCC development at two- and five-years follow-up. Patients with HCC showed higher levels of SCCA-IgM than those without it (425.72 ± 568.33 AU/mL vs 195.93 ± 188.40 AU/mL, P = 0.009) during the five-year follow-up. In multivariate analysis, after adjusting by age, sex, aspartate transaminase and Child-Pugh, the following factors were independently associated with HCC: SCCA-IgM [Hazard ratio (HR) = 1.001, 95%CI: 1.000-1.002; P = 0.003], AFP (HR = 1.028, 95%CI: 1.009-1.046; P = 0.003) and creatinine (HR = 1.564 95%CI: 1.151-2.124; P = 0.004). The log-rank test of the combination resulted in 7.488 (P = 0.024) in estimation cohort and 11.061 (P = 0.004) in the validation cohort, and a 100% of correctly classified rate identifying a low-risk group in both cohorts in the two-year follow-up. CONCLUSION: We have constructed a predictive model based on the combination of SCCA-IgM and AFP that provides a new HCC screening method, which could be followed by tailored HCC surveillance for individual patients, especially for those cirrhotic patients belonging to the subgroup identified as low-risk of HCC development.

Hepatitis C Virus Clearance by Direct-Acting Antivirals Agents Improves Endothelial Dysfunction and Subclinical Atherosclerosis: HEPCAR Study.

INTRODUCTION: Hepatitis C virus (HCV) infection has been related to increased cardiovascular (CV) risk. The aim of this study was to analyze the impact of sustained virological response (SVR) on endothelial dysfunction and subclinical atherosclerosis in patients with hepatitis C virus treated with direct-acting antiviral agents. METHODS: A total of 114 patients were prospectively recruited and underwent CV risk assessment including (i) endothelial dysfunction determined through laser Doppler flowmetry and (ii) subclinical atherosclerosis, elucidated by the ankle-brachial index (ABI). Atherogenic lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides); markers of oxidative stress (oxidized low-density lipoprotein antibodies [OLAbs]), soluble markers of adhesion (vascular cell adhesion molecule [VCAM], e-selectin, and soluble markers of angiogenesis; and vascular endothelial growth factor, endothelial [EMPs] and platelet [PMPs] apoptotic microparticles, and cell-free DNA [cfDNA]) were measured. All determinations were performed at baseline, 12 weeks (SVR time), and 1 year after treatment. RESULTS: In patients with endothelial dysfunction, area of hyperemia improved after virus clearance (P = 0.013) and was related to significant decrease in VCAM, e-selectin (P < 0.001), and cfDNA (P = 0.017) and to increased OLAb levels (P = 0.001). In patients with subclinical atherosclerosis at baseline, a significantly improved ABI was seen after HCV clearance (P < 0.001). Levels of both EMPs and PMPs also decreased after SVR and at follow-up (P = 0.006 and P = 0.002, respectively). DISCUSSION: HCV clearance improved not only liver function but also endothelial dysfunction and subclinical atherosclerosis promoted by decrease in levels of VCAM, e-selectin, cfDNA, and PMPs and EMPs.

Relationship between the endothelial dysfunction and the expression of the ß1-subunit of BK channels in a non-hypertensive sleep apnea group.

STUDY OBJECTIVES: Vascular damage must be diagnosed early in patients with hypertension. In this regard, endothelial dysfunction (ED) is an early sign of vascular disease and a predictor of cardiovascular diseases. In obstructive sleep apnea (OSA), intermittent hypoxia triggers ED, but mechanisms are not clear. In this context, it has been described that BK channels regulates arterial tone and that chronic and intermittent hypoxia downregulates the expression of the BK channel ß1-subunit facilitating vasoconstriction. Thus, we investigated the relationship among hypoxemia, ED, and mRNA expression of the ß1-subunit in patients with severe OSA. We aimed to assess (1) ED in non-hypertensive patients with OSA using laser-Doppler flowmetry, (2) BK ß1-subunit mRNA expression, and (3) the impact of continuous positive airway pressure (CPAP) treatment on ED and ß1-subunit regulation. METHODS: OSA patients underwent 24-hour blood pressure monitoring to exclude hypertension. Laser-Doppler flowmetry was performed to assess ED, and ß1-subunit mRNA expression was evaluated using a blood test of peripheral blood leukocytes at baseline and after 3 months of CPAP treatment. RESULTS: In normotensive patients with OSA, endothelial function correlated with the severity of OSA. CPAP improved endothelial function in normotensive OSA patients and the speed of the arterial response was significantly correlated with ß1-subunit mRNA expression. ß1-subunit mRNA expression at baseline correlated inversely with its change after CPAP. CONCLUSIONS: Sleep apnea is related to ED in normotensive patients with OSA. CPAP therapy improves endothelial function and regulates ß1-subunit mRNA expression.

Metabolic characterization of two different non-alcoholic fatty liver disease pre-clinical mouse models

Introduction: non-alcoholic fatty liver disease is one of the most prevalent liver disorders in the developed world. Currently, there is no approved pharmacological therapy except for lifestyle intervention. Therefore, there is a need to increase the knowledge of preclinical models in order to boost novel discoveries that could lead to a better therapeutic management. Material and methods: this study characterized the effects of two different diets, a long-term high-fat high-fructose diet (HF-HFD) and a choline-deficient, methionine supplemented high-fat diet (CDA-HFD) in C57BL/6J mice for 52 weeks or 16 weeks, respectively. Body weight, lipid and hepatic profile were analyzed and liver histology was subsequently evaluated. Results: HF-HFD animals had an increased body weight and total cholesterol levels, whereas the opposite occurred in CDA-HFD. Both HF-HFD and CDA-HFD animals had higher ALT and AST levels. With regard to histology findings, HF-HFD and CDA-HFD diets induced an increased collagen deposit and intrahepatic steatosis accumulation. Conclusion: in conclusion, the comparison of these models aided in the selection of a long-term, more physiological model for physiopathology studies or a more rapid NASH model for novel molecule testing

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Metabolic characterization of two different non-alcoholic fatty liver disease pre-clinical mouse models.

INTRODUCTION: non-alcoholic fatty liver disease is one of the most prevalent liver disorders in the developed world. Currently, there is no approved pharmacological therapy except for lifestyle intervention. Therefore, there is a need to increase the knowledge of preclinical models in order to boost novel discoveries that could lead to a better therapeutic management. MATERIAL AND METHODS: this study characterized the effects of two different diets, a long-term high-fat high-fructose diet (HF-HFD) and a choline-deficient, methionine supplemented high-fat diet (CDA-HFD) in C57BL/6J mice for 52 weeks or 16 weeks, respectively. Body weight, lipid and hepatic profile were analyzed and liver histology was subsequently evaluated. RESULTS: HF-HFD animals had an increased body weight and total cholesterol levels, whereas the opposite occurred in CDA-HFD. Both HF-HFD and CDA-HFD animals had higher ALT and AST levels. With regard to histology findings, HF-HFD and CDA-HFD diets induced an increased collagen deposit and intrahepatic steatosis accumulation. CONCLUSION: in conclusion, the comparison of these models aided in the selection of a long-term, more physiological model for physiopathology studies or a more rapid NASH model for novel molecule testing.

miRNAs in patients with non-alcoholic fatty liver disease: A systematic review and meta-analysis.

BACKGROUND & AIMS: microRNAs (miRNAs) are deregulated in non-alcoholic fatty liver disease (NAFLD) and have been proposed as useful markers for the diagnosis and stratification of disease severity. We conducted a meta-analysis to identify the potential usefulness of miRNA biomarkers in the diagnosis and stratification of NAFLD severity. METHODS: After a systematic review, circulating miRNA expression consistency and mean fold-changes were analysed using a vote-counting strategy. The sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio and area under the curve (AUC) for the diagnosis of NAFLD or non-alcoholic steatohepatitis (NASH) were pooled using a bivariate meta-analysis. Deeks' funnel plot was used to assess the publication bias. RESULTS: Thirty-seven studies of miRNA expression profiles and six studies of diagnostic accuracy were ultimately included in the quantitative analysis. miRNA-122 and miRNA-192 showed consistent upregulation. miRNA-122 was upregulated in every scenario used to distinguish NAFLD severity. The miRNA expression correlation between the serum and liver tissue was inconsistent across studies. miRNA-122 distinguished NAFLD from healthy controls with an AUC of 0.82 (95% CI 0.75-0.89), and miRNA-34a distinguished non-alcoholic steatohepatitis (NASH) from non-alcoholic fatty liver (NAFL) with an AUC of 0.78 (95% CI 0.67-0.88). CONCLUSION: miRNA-34a, miRNA-122 and miRNA-192 were identified as potential diagnostic markers to segregate NAFL from NASH. Both miRNA-122, in distinguishing NAFLD from healthy controls, and miRNA-34a, in distinguishing NASH from NAFL, showed moderate diagnostic accuracy. miRNA-122 was upregulated in every scenario of NAFL, NASH and fibrosis. LAY SUMMARY: microRNAs are deregulated in non-alcoholic fatty liver disease. The microRNAs, miRNA-34a, miRNA-122 and miRNA-192, were identified as potential biomarkers of non-alcoholic fatty liver and non-alcoholic steatohepatitis, at different stages of disease severity. The correlation between miRNA expression in the serum and in liver tissue was inconsistent, or even inverse.

Metformin modifies glutamine metabolism in an in vitro and in vivo model of hepatic encephalopathy

Aim: to analyze the effect of metformin on ammonia production derived from glutamine metabolism in vitro and in vivo. Methods: twenty male Wistar rats were studied for 28 days after a porto-caval anastomosis (n = 16) or a sham operation (n = 4). Porto-caval shunted animals were randomized into two groups (n = 8) and either received 30 mg/kg/day of metformin for two weeks or were control animals. Plasma ammonia concentration, Gls gene expression and K-type glutaminase activity were measured in the small intestine, muscle and kidney. Furthermore, Caco2 were grown in different culture media containing glucose/glutamine as the main carbon source and exposed to different concentrations of the drug. The expression of genes implicated in glutamine metabolism were analyzed. Results: metformin was associated with a significant inhibition of glutaminase activity levels in the small intestine of porto-caval shunted rats (0.277 ± 0.07 IU/mg vs 0.142 ± 0.04 IU/mg) and a significant decrease in plasma ammonia (204.3 ± 24.4 µg/dl vs 129.6 ± 16.1 µg/dl). Glucose withdrawal induced the expression of the glutamine transporter SLC1A5 (2.54 ± 0.33 fold change; p < 0.05). Metformin use reduced MYC levels in Caco2 and consequently, SLC1A5 and GLS expression, with a greater effect in cells dependent on glutaminolytic metabolism. Conclusion: metformin regulates ammonia homeostasis by modulating glutamine metabolism in the enterocyte, exerting an indirect control of both the uptake and degradation of glutamine. This entails a reduction in the production of metabolites and energy through this pathway and indirectly causes a decrease in ammonia production that could be related to a decreased risk of HE development

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Metformin modifies glutamine metabolism in an in vitro and in vivo model of hepatic encephalopathy.

AIM: to analyze the effect of metformin on ammonia production derived from glutamine metabolism in vitro and in vivo. METHODS: twenty male Wistar rats were studied for 28 days after a porto-caval anastomosis (n = 16) or a sham operation (n = 4). Porto-caval shunted animals were randomized into two groups (n = 8) and either received 30 mg/kg/day of metformin for two weeks or were control animals. Plasma ammonia concentration, Gls gene expression and K-type glutaminase activity were measured in the small intestine, muscle and kidney. Furthermore, Caco2 were grown in different culture media containing glucose/glutamine as the main carbon source and exposed to different concentrations of the drug. The expression of genes implicated in glutamine metabolism were analyzed. RESULTS: metformin was associated with a significant inhibition of glutaminase activity levels in the small intestine of porto-caval shunted rats (0.277 ± 0.07 IU/mg vs 0.142 ± 0.04 IU/mg) and a significant decrease in plasma ammonia (204.3 ± 24.4 µg/dl vs 129.6 ± 16.1 µg/dl). Glucose withdrawal induced the expression of the glutamine transporter SLC1A5 (2.54 ± 0.33 fold change; p < 0.05). Metformin use reduced MYC levels in Caco2 and consequently, SLC1A5 and GLS expression, with a greater effect in cells dependent on glutaminolytic metabolism. CONCLUSION: metformin regulates ammonia homeostasis by modulating glutamine metabolism in the enterocyte, exerting an indirect control of both the uptake and degradation of glutamine. This entails a reduction in the production of metabolites and energy through this pathway and indirectly causes a decrease in ammonia production that could be related to a decreased risk of HE development.