Dysregulated Choline, Methionine, and Aromatic Amino Acid Metabolism in Patients with Wilson Disease: Exploratory Metabolomic Profiling and Implications for Hepatic and Neurologic Phenotypes.

Wilson disease (WD) is a genetic copper overload condition characterized by hepatic and neuropsychiatric symptoms with a not well-understood pathogenesis. Dysregulated methionine cycle is reported in animal models of WD, though not verified in humans. Choline is essential for lipid and methionine metabolism. Defects in neurotransmitters as acetylcholine, and biogenic amines are reported in WD; however, less is known about their circulating precursors. We aimed to study choline, methionine, aromatic amino acids, and phospholipids in serum of WD subjects. Hydrophilic interaction chromatography-quadrupole time-of-flight mass spectrometry was employed to profile serum of WD subjects categorized as hepatic, neurologic, and pre-clinical. Hepatic transcript levels of genes related to choline and methionine metabolism were verified in the Jackson Laboratory toxic milk mouse model of WD (tx-j). Compared to healthy subjects, choline, methionine, ornithine, proline, phenylalanine, tyrosine, and histidine were significantly elevated in WD, with marked alterations in phosphatidylcholines and reductions in sphingosine-1-phosphate, sphingomyelins, and acylcarnitines. In tx-j mice, choline, methionine, and phosphatidylcholine were similarly dysregulated. Elevated choline is a hallmark dysregulation in WD interconnected with alterations in methionine and phospholipid metabolism, which are relevant to hepatic steatosis. The elevated phenylalanine, tyrosine, and histidine carry implications for neurologic manifestations and are worth further investigation

The role of hepatic macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is becoming common chronic liver disease because of the increasing global prevalence of obesity and consequently Nonalcoholic fatty liver disease (NAFLD). However, the mechanism for progression of NAFLD to NASH and then cirrhosis is not completely understood, yet. The triggering of these hepatic diseases is thought from hepatocyte injury caused by over-accumulated lipid toxicity. Injured hepatocytes release damage-associated molecular patterns (DAMPs), which can stimulate the Kupffer cells (KCs), liver-resident macrophages, to release pro-inflammatory cytokines and chemokines, and recruit monocyte-derived macrophages (MDMs). The increased activation of KCs and recruitment of MDMs accelerate the progression of NAFLD to NASH and cirrhosis. Therefore, characterization for activation of hepatic macrophages, both KCs and MDMs, is a baseline to figure out the progression of hepatic diseases. The purpose of this review is to discuss the current understanding of mechanisms of NAFLD and NASH, mainly focusing on characterization and function of hepatic macrophages and suggests the regulators of hepatic macrophages as the therapeutic target in hepatic diseases.ope

Beneficial Effects of Viable and Heat-Inactivated Lactobacillus rhamnosus GG Administration on Oxidative Stress and Inflammation in Diet-Induced NAFLD in Rats

Oxidative stress and inflammation are well-known triggers of NAFLD onset and progression. The aim of this study is to compare the potential benefits of a viable probiotic (Lactobacillus rhamnosus GG) and its parabiotic (heat-inactivated) on oxidative stress, inflammation, DNA damage and cell death pathways in the liver of rats featuring diet-induced NAFLD. The consumption of the steatotic diet led to increased final body and liver weights, higher hepatic triacylglycerol content, altered serum transaminase levels and enhanced oxidative and inflammatory status. Administration of the probiotic and the parabiotic partially prevented the body weight increase induced by the steatotic diet, whereas the probiotic caused more effective decreasing hepatic triglyceride content. Sharp but nonstatistically significant decreases in serum transaminase levels were also observed for both treatments. The reduction in antioxidant enzyme activities found in the nontreated animals fed the steatotic diet was partially prevented by both treatments (GPx activity). Similarly, the reductions in nonenzymatic antioxidant protection (GSH content) and total antioxidant capacity (ORAC) found in the nontreated rats were restored by the administration of both treatments. These results show that both viable and heat-inactivated Lactobacillus rhamnosus GG administration partially prevent steatotic diet-induced liver oxidative stress and inflammation induced in rats.This study was supported by Instituto de Salud Carlos III (CIBERobn) under Grant CB12/03/30007, The Basque Government under Grant IT1482-22 and Synergic R&D Projects in New and Emerging Scientific Areas on the Frontier of Science and Interdisciplinary Nature of The Community of Madrid (METAINFLAMATIONY2020/BIO-6600). Laura Isabel Arellano-García is a recipient of a doctoral fellowship from the Gobierno Vasco

Research progress of the impact of nonalcoholic fatty liver disease on chronic hepatitis B infection

Chronic hepatitis B (CHB) is an infectious disease caused by persistent infection with the hepatitis B virus (HBV) and is highly prevalent worldwide. Non-alcoholic fatty liver disease (NAFLD) is a group of liver diseases related to metabolic abnormalities, excluding those caused by alcohol consumption or other liver injury factors. In recent years, with improvement of living standards and changes in lifestyle, the incidence of NAFLD has been increasing substantially, becoming the most common type of liver diseases in China and Western countries, and the second leading cause of liver transplantation in the West. The rising prevalence of NAFLD has also led to an increase in the incidence of NAFLD in patients with chronic HBV infection. However, there is considerable controversy both domestically and internationally regarding the relationship between these two diseases, including the disease progression, pathogenesis, impact on antiviral treatment efficacy, and prognosis of these concomitant CHB and NAFLD patients. Currently, both domestic and international guidelines lack detailed descriptions of diagnostic and treatment strategies for these conditions. This article summarizes the recent research progress in concomitant CHB and NAFLD, including epidemiology, diagnostic criteria, the impact of NAFLD on the virology of HBV infection, potential mechanisms of NAFLD-induced negative regulation of HBV, the effect of NAFLD on antiviral therapy efficacy, and prognosis. This article aims to gain a deeper understanding of the diseases themselves and provide new insights for basic and clinical research as well as diagnostic and treatment approaches

Role of microRNAs in the main molecular pathways of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignant neoplasia. HCC is characterized by a poor prognosis. The need to find new molecular markers for its diagnosis and prognosis has led to a progressive increase in the number of scientific studies on this topic. MicroRNAs (miRNAs) are small noncoding RNA that play a role in almost all main cellular pathways. miRNAs are involved in the regulation of expression of the major tumor-related genes in carcinogenesis, acting as oncogenes or tumor suppressor genes. The aim of this review was to identify papers published in 2017 investigating the role of miRNAs in HCC tumorigenesis. miRNAs were classified according to their role in the main molecular pathways involved in HCC tumorigenesis: (1) mTOR; (2) Wnt; (3) JAK/STAT; (4) apoptosis; and (5) MAPK. The role of miRNAs in prognosis/response prediction was taken into consideration. Bearing in mind that the analysis of miRNAs in serum and other body fluids would be crucial for clinical management, the role of circulating miRNAs in HCC patients was also investigated. The most represented miRNA-regulated pathway in HCC is mTOR, but apoptosis, Wnt, JAK/STAT or MAPK pathways are also influenced by miRNA expression levels. These miRNAs could thus be used in clinical practice as diagnostic, prognostic or therapeutic targets for HCC treatment

Identification and functional characterization of proteins involved in hepatic triglyceride metabolism

Triglycerides are the main form of energy in the tissues and liver, along with the adipose tissue, is the main organ of triglyceride metabolism and storage in the lipid-droplet organelles. A number of proteins are involved in the regulation of the triglyceride metabolism in human liver, however their specific role is still not thoroughly known. The aim of this thesis is to evaluate the functional role of three proteins in triglyceride regulation in an experimental model of human liver. In Paper I we identified the gene Transmembrane 6 superfamily member 2 (TM6SF2) as the putative cause for the association between the 19p12 locus with plasma triglyceride levels and non-alcoholic fatty liver disease, by employing expression studies and expression quantitative trait locus analysis in 206 human liver samples. TM6SF2 encodes a protein of 351 amino acids localized in the Endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment, as investigated in human hepatoma cells. Functional studies showed that TM6SF2 siRNA inhibition led to reduced secretion of triglyceride-rich lipoproteins (TRLs) and increased cellular triglyceride concentration and number of lipid-droplets, however the putative pathophysiological mechanism of these observations is still unclear. In Paper II we investigated the physiological functions of Patatin-like phospholipase domain containing proteins 2, 3 and 4 (PNPLA2, PNPLA3 and PNPLA4), as potential triglyceride hydrolases in Huh7 and HepG2 human hepatomas. We found that siRNA inhibition of PNPLA3 or PNPLA4 is not associated with changes in triglyceride hydrolysis, TRL secretion or cellular triglyceride accumulation. However, PNPLA2 siRNA inhibition reduced intracellular triglyceride hydrolysis and decreased TRL secretion, both in the absence or presence of oleate-containing medium or of the PNPLA2 inhibitor Atglistatin. In contrast, we found no effects of PNPLA2 inhibition on lipid-droplet homeostasis. Visualization analysis with confocal microscopy found significant co-localization of PNPLA2 with the ER, but no clear evidence for PNPLA2 localization around the lipid-droplets. This data indicates that PNPLA2 hydrolyses a triglyceride compartment comprising of very small lipid-droplets that are involved in the regulation of TRL secretion, but are not detectable by confocal microscopy. In Paper III we studied the likely role of Abhydrolase domain-containing 5 (ABHD5) as the co-activator of PNPLA2 in the regulation of hepatic triglyceride metabolism. We employed siRNA inhibition techniques in Huh7 hepatoma cells and showed that ABHD5 siRNA inhibition reduced triglyceride hydrolysis and decreased TRL secretion while there was no effect on cellular triglyceride content. These results are similar to the effects of PNPLA2 siRNA inhibition on triglyceride metabolism as examined in Paper II. We also found no additive effects of combined ABHD5-PNPLA2 siRNA inhibition in hepatic triglyceride metabolism. We employed confocal microscopy analysis and observed localization of ABHD5 in the ER, but not in Golgi or around the lipid-droplets, while a significant colocalization of ABHD5 and PNPLA2 was observed. These observations suggest that ABHD5 is a co-activator of PNPLA2 with no separate triglyceride hydrolysis activity in human hepatocytes. Overall, this Thesis identifies TM6SF2 as a membrane protein regulating the TRL secretion in Huh7 and HepG2 hepatoma cells. It also demonstrates the role of triglyceride hydrolysis in the regulation of TRL secretion where PNPLA2 is the main triglyceride hydrolase activated by ABHD5. Finally, it suggests the existence of very small lipid-droplets containing the substrate compartment of the PNPLA2- and ABHD5-mediated triglyceride hydrolysis

Identification of a novel mechanism driving NAFLD progression and therapeutic strategies

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries with an increasing prevalence of approximately 25 %. NAFLD comprises several stages, starting as benign steatosis and progressing to non-alcoholic steatohepatitis (NASH), and in some cases to liver cirrhosis and hepatocellular carcinoma (HCC). Although several emerging therapies are currently in clinical trials, so far there are no approved drugs for treatment of NASH. The overarching goal of this thesis was to investigate the mechanisms of NAFLD stage transitions, and to establish preventive measures for the progression from benign steatosis to NASH. First, a mouse model of NAFLD progression was established by long-term feeding of male C57Bl/6N mice with western-style diet (WD) up to 54 weeks. The disease progression was evaluated time-dependently by biochemical, histopathological, and immunohistochemical analyses as well as by intravital two-photon-based imaging. This analyses revealed six stages in NAFLD progression: (1) benign steatosis, (2) macrophage crown-like structure formation, (3) macropinocytosis of bile, (4) ductular reaction, (5) dedifferentiation and functional shutdown, and (6) tumor nodule formation. The novel finding of this thesis was the identification of stage 3, where a retrograde vesicular uptake of bile from bile canaliculi to hepatocytes led to toxic accumulation of bile acids in the liver tissue, providing a link between NAFLD and cholestasis. The phenomenon was further identified as macropinocytosis by treating the mice with a macropinocytosis-specific inhibitor imipramine. As a result, a single application of imipramine efficiently blocked macropinocytosis in WD-fed mice. Interestingly, a long-term application of imipramine for 8 weeks decreased the bile acid concentrations in the liver tissue and led to significant NAFLD amelioration. Moreover, bile macropinocytosis was also found relevant in human NAFLD patients as detected by the presence of fragments of bile canaliculi within steatotic hepatocytes. In conclusion, an NAFLD mouse model recapitulating the different stages of human NAFLD progression to NASH and eventually to HCC was successfully established. Moreover, a novel mechanism possibly driving NALFD progression was identified as macropinocytosis of bile from bile canaliculi back to hepatocytes

Microcystin Exposure and Liver Injury Outcomes in NAFLD

Non-alcoholic fatty liver disease (NAFLD) is an emerging worldwide pandemic which is highly prevalent among obese individuals including children and adults. In a NAFLD condition, exposure to environmental contaminants or toxins can act as a second/ multiple hit, which leads to the progression of simple steatosis to NASH and ultimately may result in liver cirrhosis. With climate change today, elevated levels of microcystins are an emerging problem in fresh water bodies, which are a source of drinking water. Individuals are therefore at risk of exposure to microcystin through consuming contaminated water. In the progression of NAFLD from one clinical stage to another, microcystin can play an important role. Research has proved that the main mechanism of the Microcystin-LR is to work as an exogenous Protein phosphatase 2A (PP2A) inhibitor. In this thesis we test the hypothesis that microcystin activates Kupffer cells as well as the hepatic stellate cells, which are the crucial mediators in hepatic inflammation and fibrogenesis via NOX2 dependent pathway in NAFLD condition. Results showed that microcystin exposure via intraperitoneal routes in mice that have mild steatosis will lead to advanced histopathological characteristics of NAFLD. Further, we looked at the effect of early microcystin exposure in young mice, which were then fed a high fat diet to induce obesity/NAFLD. Early exposure to microcystin resulted in exacerbation of NAFLD clinical pathology with increased expression of proinflammatory cytokines, compared to vehicle control treated mice. This process has been shown to be reduced in mice that lacked miR21, p47 phox , or NLRP3 genes. In addition, inhibition of AKT pathway reduced microcystin -induced NOX2 activation as well as upregulation of miR21. All things considered, it was observed that microcystin exposure exacerbated NAFLD pathology via activating NOX2-dpendent peroxynitare generation by increasing miR21 levels. Moreover, early childhood exposure to microcystin will exacerbate adult hepatic injury following HFD via NLRP3 inflammasomes

The pathophysiological role of physical inactivity and fat-diet to development of metabolic syndrome in an animal model

RESUMO Esta tese é suportada por uma revisão narrativa da literatura, perspetivando fornecer uma visão geral dos principais aspetos etiológicos, fisiopatológicos e clínicos da síndrome metabólica, assim como por três artigos originais, utilizando um modelo animal, que objetivaram identificar a contribuição isolada e conjugada da inatividade física e de uma dieta hiperlipídica no desenvolvimento da síndrome metabólica. Para tal, ratos Wistar machos foram alimentados com dieta hiperlipídica e/ou tiveram a atividade física restrita ao espaço da gaiola por 21 semanas. Os resultados revelaram que a dieta hiperlipídica e a inatividade física induziram um característico fenótipo adulto, com ratos maiores e mais leves, possuindo órgãos mais leves e uma expressiva deposição de gordura retroperitoneal sem, contudo, ganho de peso. Os trabalhos empíricos também revelaram a predominância da dieta gorda sobre a inatividade física na indução de desarranjos metabólicos, uma vez que apenas os animais alimentados com dieta gorda desenvolveram síndrome metabólica, independentemente dos níveis de atividade física, com maladaptações hepáticas e do pâncreas endócrino evidenciando uma intensa resposta inflamatória, aumento do conteúdo local de colágeno e da apoptose celular em ambos os órgãos. Estes resultados permitem concluir que a dieta rica em gordura é um fator patogénico predominante na indução da síndrome metabólica e das maladaptações hepáticas e pancreáticas em um modelo animal, comparativamente à inatividade física. Ainda assim, comparativamente à dieta gorda, mais determinante na indução da intolerância à glicose, a inatividade física parece ser mais decisiva no comprometimento da sensibilidade à insulina. Níveis mais elevados de atividade física não parecem prevenir o desenvolvimento da síndrome metabólica e das alterações orgânicas associadas após 21 semanas de protocolo experimental, mas atenuam os transtornos metabólicos e estruturais induzidos pela dieta gorda. Palavras Chave: Physical inactivity, fat diet, metabolic syndrome, liver disorder, and endocrine pancreas disarrangement.This thesis is supported by a literature narrative review aiming to provide an overview of the metabolic syndrome main etiological, pathophysiological and clinical aspects, as well as three original articles using an animal model that aimed to identify the physical inactivity and the hyperlipidic diet isolated and conjugated contribution in the metabolic syndrome development. For this, male Wistar rats were fed with a high-fat diet and/or had the physical activity restricted to the cage space for 21 weeks. The results revealed that the high-fat diet and physical inactivity induced a singular adult phenotype, with larger and lighter rats, having lighter organs and an expressive retroperitoneal fat deposition without, however, gaining weight. The empirical studies also revealed the high-fat diet predominance over physical inactivity in inducing metabolic disorders, since only high-fat diet fed-animals developed metabolic syndrome, regardless of physical activity levels, with the hepatic and endocrine pancreas maladaptations evidencing an intense inflammatory response, increased local collagen content and cellular apoptosis in both organs. These results allow us to conclude that the high-fat diet is a predominant pathogenic factor in inducing the metabolic syndrome and the hepatic and pancreatic maladaptations in an animal model, compared to physical inactivity. Nevertheless, in comparison to the high-fat diet, which is more important in the glucose intolerance induction, physical inactivity seems to be more decisive in the impairment of insulin sensitivity. Higher levels of physical activity do not seem to prevent the metabolic syndrome development and related organic changes after 21 weeks of experimental protocol but attenuate the metabolic and structural disorders induced by the high-fat diet

The small GTPase RAB18: Insights into cellular steatosis, lipophagy and the non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease is a rapidly growing concern for public health. Its most prevalent marker is steatosis, which is the accumulation of large lipid storage organelles called lipid droplets (LD) in the hepatocytes. RAB18, a member of the Rab family, localizes to the LD membrane. Rab family proteins are regulators of cellular membrane trafficking, therefore RAB18 is expected to play a role regulating LD biology. The presented work aims to elucidate this role as well as the mechanisms behind the localization of RAB18 to the LD membrane. The localization of RAB18 was investigated by overexpressing mutant RAB18 variants in HepG2 cells. RAB18 localization was observed to depend on the reversible cyclical palmitoylation of its C-terminus. Using FRAP experiments, it could be shown that targeting the palmitoylation machinery with small molecule inhibitors modulated RAB18 localization. This coincided with changes in LD size in cells treated with de-palmitoylation inhibitors. An overall increase in LD size was observed in HepG2 cells with RAB18 downregulation. The wild type LD size in these cells was restored by the inhibition of autophagy. This size reduction was due to newly created LDs. Inhibition of autophagy prior to LD accumulation was subsequently tested in vitro on primary human hepatocytes in sandwich culture. Inhibition of autophagy by chloroquine resulted in a dose dependent rise in LD number and a decrease in average LD size in these cells. These effects could be translated to the in vivo situation in mice. Daily chloroquine injection of mice on a steatogenic diet resulted in a significant decrease of LD size in vivo. Conversely, no changes were detected in the blood-values of treated mice compared with the control. This thesis demonstrates, that RAB18 localizes to the LD via a C-terminal acylation cycle. RAB18 reduces the size of LDs by modulating autophagy of newly formed LDs. This mechanism is important for LD number and size regulation in hepatocytes