The metabolomic window into hepatobiliary disease

The emergent discipline of metabolomics has attracted considerable research effort in hepatology. Here we review the metabolomic data for non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), alcoholic liver disease (ALD), hepatitis B and C, cholecystitis, cholestasis, liver transplantation, and acute hepatotoxicity in animal models. A metabolomic window has permitted a view into the changing biochemistry occurring in the transitional phases between a healthy liver and hepatocellular carcinoma or cholangiocarcinoma. Whether provoked by obesity and diabetes, alcohol use or oncogenic viruses, the liver develops a core metabolomic phenotype (CMP) that involves dysregulation of bile acid and phospholipid homeostasis. The CMP commences at the transition between the healthy liver (Phase 0) and NAFLD/NASH, ALD or viral hepatitis (Phase 1). This CMP is maintained in the presence or absence of cirrhosis (Phase 2) and whether or not either HCC or CCA (Phase 3) develops. Inflammatory signalling in the liver triggers the appearance of the CMP. Many other metabolomic markers distinguish between Phases 0, 1, 2 and 3. A metabolic remodelling in HCC has been described but metabolomic data from all four Phases demonstrate that the Warburg shift from mitochondrial respiration to cytosolic glycolysis foreshadows HCC and may occur as early as Phase 1. The metabolic remodelling also involves an upregulation of fatty acid β-oxidation, also beginning in Phase 1. The storage of triglycerides in fatty liver provides high energy-yielding substrates for Phases 2 and 3 of liver pathology. The metabolomic window into hepatobiliary disease sheds new light on the systems pathology of the liver.

The story of HCC in NAFLD: from epidemiology, across pathogenesis, to prevention and treatment.

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. An increasing number of reports describe HCC in the setting of obesity and diabetes, two major risk factors for non-alcoholic fatty liver disease (NAFLD). The increasing incidence of these conditions and the emerging evidence of HCC in non-cirrhotic NAFLD prioritize a better understanding of NAFLD-related HCC epidemiology and pathogenesis in order to target screening policies and develop preventive-therapeutic strategies. In this review, we focus on the epidemiological impact of this condition, suggesting a possible link between HCC in cryptogenic cirrhosis and NAFLD. Furthermore, we analyse the suggested pathogenic mechanisms and the possible preventive-therapeutic strategies.

Hepatocellular carcinoma and lifestyles.

The majority of hepatocellular carcinoma occurs over pre-existing chronic liver diseases that share cirrhosis as an endpoint. In the last decade, a strong association between lifestyle and hepatocellular carcinoma has become evident. Abundance of energy-rich food and sedentary lifestyles have caused metabolic conditions such as obesity and diabetes mellitus to become global epidemics. Obesity and diabetes mellitus are both tightly linked to non-alcoholic fatty liver disease and also increase hepatocellular carcinoma risk independent of cirrhosis. Emerging data suggest that physical activity not only counteracts obesity, diabetes mellitus and non-alcoholic fatty liver disease, but also reduces cancer risk. Physical activity exerts significant anticancer effects in the absence of metabolic disorders. Here, we present a systematic review on lifestyles and hepatocellular carcinoma.

Nutrição na fisiopatologia e tratamento do fígado gordo não-alcoólico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Fígado gordo não-alcoólico : uma entidade emergente com potencial oncogénico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Ferroportin Disease (Haemochromatosis-type IV): a case report

Introdution: Haemochromatosis-type IV, the ferroportin disease, is characterized by an autosomal-dominant transmission and early iron accumulation in macrophages. It is caused by mutations in the transmembrane iron exporter protein ferroportin1 (SLC40A1 gene). In form A (classic), ferroportin loss of function mutants are unable to export iron from cells leading to cellular iron accumulation with decreased availability of iron for serum transferrin (TS). We present a Portuguese rare clinical case of HH-IV. Materials and Methods: A 41-year-old woman with hyperferritinemia and normal TS. Causes of hyperferritinemia (inflammation, chronic alcohol consumption, metabolic syndrome, cell necrosis, non-alcoholic fatty liver disease and aceruloplasminemia) were assessed. Liver iron, evaluated by magnetic resonance imaging (MRI) was carried out. Screening for mutation in HFE and SCL40A1 genes were performed by Sanger sequencing. Baseline: Ferritin:708ng/ml; TS: 27%; MRI:85µmol/g; Hb:13,6g/dl. Therapy: weekly 450ml Therapeutic Phlebotomies (TP) until ferritin≤50ng/ml. Results: Hyperferritinemia comorbidities and common genetic mutations for haemochromatosis were negative. However, sequencing of the patient SLC40A1 gene has revealed the presence in heterozygosity of the variant c.238G>A; p.Gly80Ser. Due to low tolerance to TP, we adopted smaller phlebotomies every three weeks. Conclusion: This patient has a rare autosomal-dominant Ferroportin disease due to a mutated ferroportin which is predicted to be defective in iron cellular export. In agreement, she presents hyperferritinemia, with normal TS and liver iron overload. The genotype/phenotype association allowed to diagnosis this rare FD case. Although a mild form A, we decided to start TP. Her father also has been treated for iron overload.

A decrease in dkk1, a Wnt inhibitor, contributes to placental and fetal lipid accumulation in an obesity-prone rat model

Placenta, as the sole transport mechanism between mother and fetus, links the maternal physical state and the immediate and life-long outcomes of the offspring. The present study examined the mechanisms behind the effect of maternal obesity on placental lipid accumulation and metabolism. Pregnant Obese Prone (OP) and Obese Resistant (OR) rat strains were fed a control diet throughout gestation. Placentas were collected on gestational d21 for analysis and frozen placental sections were analyzed for fat accumulation as well as β-Catenin and Dkk1 localization. Additionally, DKK1 was overexpressed in JEG3 trophoblast cells, followed by treatment with NEFA and Oil Red O stain quantification and mRNA analysis to determine the relationship between placental DKK1 and lipid accumulation. Maternal plasma and placental NEFA and TG were elevated in OP dams, and offspring of OP dams were smaller than OR. Placental Dkk1 mRNA content was 4-fold lower in OP placentas, and there was a significant increase in β-Catenin accumulation as well as mRNA content of fat transport and TG synthesis enzymes, including Ppar-delta, Fatp1, Fat/Cd36, Lipin1, and Lipin3. There was significant lipid accumulation within the decidual zones in OP but not OR placentas, and the thickness of the decidual and junctional zones was significantly smaller in OP than OR placentas. Overexpression of DKK1 in JEG3 cells decreased lipid accumulation and the mRNA content of PPAR-Delta, FATP1, FAT/CD36, LIPIN1, and LIPIN3. Our results indicate that Dkk1 may be regulating placental lipid metabolism through Wnt-mediated mechanisms. Additionally, recent studies have suggested that maternal obesity may also program early development of non-alcoholic fatty liver disease (NAFLD), rates of which have correlated with the increase in the obesity epidemic. In the current study, livers of OP offspring had significantly increased TG content (P<0.05) and lipid accumulation when compared to offspring of OR dams. Additionally, hepatic Dkk1 mRNA content was significantly decreased in OP livers when compared to OR (P<0.05), and treating H4IIECR rat hepatocyte cells with NEFA showed that Dkk1 mRNA was also decreased in NEFA-treated cells (P<0.05) that also had lipid accumulation. Chromatin Immunoprecipitation (ChIP) analysis of the Dkk1 promoter in fetal livers showed a pattern of histone modifications associated with decreased gene transcription in OP offspring, which agrees with our gene expression data. These results demonstrate that the hepatic Dkk1 gene is epigenetically regulated via histone modification in neonatal offspring in the current model of gestational obesity, and future studies will be needed to determine whether these changes contribute to excessive hepatic lipid accumulation in offspring of obese dams.

Exploring the role of miR-34a in regulating adipose inflammation during obesity

Background: Obesity is not a new disease, with roots that can be traced back to 400 BC. However, with the staggering increase in individuals that are overweight and obese since the 1980s, now over a quarter of individuals in Europe and the Americas are classed as obese. This presents a global health problem that needs to be addressed with novel therapies. It is now well accepted that obesity is a chronic, low-grade inflammatory condition that could predispose individuals to a number of comorbidities. Obesity is associated with cardiovascular diseases (CVDs) and type 2 diabetes (T2D) as part of “the metabolic syndrome,” and as first identified by Dr Vauge, central distribution of white adipose tissue (WAT) is an important risk factor in the development of these diseases. Subsequently, visceral WAT (vWAT) was shown to be an important factor in this association with CVDs and T2D, and increasing inflammation. As the obese WAT expands, mainly through hypertrophy, there is an increase in inflammation that recruits numerous immune cells to the tissue that further exacerbate this inflammation, causing local and systemic inflammatory and metabolic effects. One of the main types of immune cell involved in this pathogenic process is pro-inflammatory M1 adipose tissue macrophages (ATMs). MicroRNAs (miRNAs) are a species of small RNAs that post-transcriptionally regulate gene expression by targeting gene mRNA, causing its degradation or translational repression. These miRNAs are promiscuous, regulating numerous genes and pathways involved in a disease, making them useful therapeutic targets, but also difficult to study. miR-34a has been shown to increase in the serum, liver, pancreas, and subcutaneous (sc)WAT of patients with obesity, non- alcoholic fatty liver disease (NAFLD) and T2D. Additionally, miR-34a has been shown to regulate a number of metabolic and inflammatory genes in numerous cell types, including those in macrophages. However, the role of miR-34a in regulating vWAT metabolism and inflammation is poorly understood. Hypothesis: miR-34a is dysregulated in the adipose tissue during obesity, causing dysregulation of metabolic and inflammatory pathways in adipocytes and ATMs that contribute to adipose inflammation and obesity’s comorbidities, particularly T2D. Method/Results: The role of miR-34a in adipose inflammation was investigated using a murine miR-34a-/- diet-induced obesity model, and primary in vitro models of adipocyte differentiation and inflammatory bone marrow-derived macrophages (BMDMs). miR-34a was shown to be ubiquitously expressed throughout the murine epididymal (e)WAT of obese high-fat diet (HFD)-fed WT mice and ob/ob mice, as well as omental WAT from patients with obesity. Additionally, miR-34a transcripts were increased in the liver and brown adipose tissue (BAT) of ob/ob and HFD-fed WT mice, compared to WT controls. When miR-34a-/- mice were fed HFD ad libitum for 24 weeks they were significantly heavier than their WT counterparts by the end of the study. Ex vivo examinations showed that miR-34a-/- eWAT had a smaller adipocyte area on chow, which significantly increased to WT levels during HFD-feeding. Additionally, miR-34a-/- eWAT showed basal increases in cholesterol and fatty acid metabolism genes Cd36, Hmgcr, Lxrα, Pgc1α, and Fasn. miR-34a-/- iBAT showed basal reductions in Cebpα and Cebpβ, with increased Pgc1α expression during HFD- feeding. The miR-34a-/- liver additionally showed increased basal transcript expression of Pgc1α, suggesting miR-34a may broadly regulate PGC1α. Accompanying the ex vivo changes in cholesterol and fatty acid metabolism genes, in vitro miR-34a-/- white adipocytes showed increased lipid content. An F4/80high macrophage population was identified in HFD-fed miR-34a-/- eWAT, with increased Il-10 transcripts and serum IL-5 protein. Following these ex vivo observations, BMDMs from WT mice upregulated miR-34a expression in response to TNFα stimulation. Additionally, miR-34a-/- BMDMs showed an ablated CXCL1 response to TNFα. Conclusion: These findings suggest miR-34a has a multi-factorial role in controlling a susceptibility to obesity, by regulating inflammatory and metabolic pathways, potentially through regulation of PGC1α.

Guía de práctica clínica para la enfermedad hepática grasa no alcohólica

Objetivo: Brindar una guía de práctica clínica basada con la evidencia más reciente para el diagnóstico y tratamiento de la Enfermedad Hepática Grasa No alcohólica teniendo en cuenta la efectividad y seguridad de las intervenciones dirigidas a pacientes, personal asistencial, administrativo y entes gubernamentales de cualquier servicio de atención en Colombia. Materiales y métodos: Esta guía fue desarrollada por un equipo multidisciplinario con apoyo de la Asociación Colombiana de Gastroenterología, el Grupo Cochrane ITS y el Instituto de Investigaciones Clínicas de la Universidad Nacional de Colombia. Se desarrollaron preguntas clínicas relevantes y se realizó la búsqueda de guías nacionales e internacionales en bases de datos especializadas. Las guías existentes fueron evaluadas en términos de calidad y aplicabilidad. Una guía cumplió los criterios de adaptación, por lo que se decidió adaptar 3 preguntas clínicas. El Grupo Cochrane realizó la búsqueda sistemática de la literatura. Las tablas de evidencia y recomendaciones fueron realizadas con base a la metodología GRADE. Las recomendaciones de la guía fueron socializadas en una reunión de expertos con entes gubernamentales y pacientes. Resultados: Se desarrolló una guía de práctica clínica basada en la evidencia para el diagnóstico y tratamiento de la Enfermedad Hepática Grasa No alcohólica en Colombia Conclusiones: El diagnóstico y manejo oportuno de la Enfermedad Hepática Grasa No alcohólica contribuirá a disminuir la carga de la enfermedad en Colombia y las enfermedades asociadas

Theobroma cacao (cocoa) supplementation in the treatment of Non-alcoholic Steatohepatitis

Non-alcoholic Steatohepatitis (NASH) is a chronic disease that results from accumulation of fat within the liver that subsequently stimulates free radicals to damage the cells of the liver. Cocoa is a rich source of antioxidants and it was found that its consumption could slow the development of NASH.

Rapid development of non-alcoholic steatohepatitis in Psammomys obesus (Israeli Sand Rat)

Background and AimsA major impediment to establishing new treatments for non-alcoholic steatohepatitis is the lack of suitable animal models that accurately mimic the biochemical and metabolic characteristics of the disease. The aim of this study was to explore a unique polygenic animal model of metabolic disease as a model of non-alcoholic steatohepatitis by determining the effects of 2% dietary cholesterol supplementation on metabolic and liver endpoints in Psammomys obesus (Israeli sand rat).MethodsP. obesus were provided ad libitum access to either a standard rodent diet (20% kcal/fat) or a standard rodent diet supplemented with 2% cholesterol (w/w) for 4 weeks. Histological sections of liver from animals on both diets were examined for key features of non-alcoholic steatohepatitis. The expression levels of key genes involved in hepatic lipid metabolism were measured by real-time PCR.ResultsP. obesus fed a cholesterol-supplemented diet exhibited profound hepatomegaly and steatosis, and higher plasma transaminase levels. Histological analysis identified extensive steatosis, inflammation, hepatocyte injury and fibrosis. Hepatic gene expression profiling revealed decreased expression of genes involved in delivery and uptake of lipids, and fatty acid and triglyceride synthesis, and increased expression of genes involved in very low density lipoprotein cholesterol synthesis, triglyceride and cholesterol export.ConclusionsP. obesus rapidly develop non-alcoholic steatohepatitis when fed a cholesterol-supplemented diet that appears to be histologically and mechanistically similar to patients.

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. In the present study, the applicability of laser-induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Rats received a high-lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P < 0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non-invasive evaluation of the degree of steatosis.

Adenosine signaling contributes to ethanol-induced fatty liver in mice.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

{\it In vivo\/} magnetic resonance studies of experimental liver disease: Carbon tetrachloride hepatotoxicity and alcohol-induced fatty liver in rat

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) were used to non-invasively determine if cirrhosis induced by carbon tetrachloride (CCl$\sb4$) and phospholipase-D (PLD) could be distinguished from fatty infiltration in rat. MRS localization and water suppression methods were developed, implemented and evaluated in terms of their application to in vivo proton NMR studies of experimental liver disease. MRS studies were also performed to quantitate fatty infiltration resulting from carbon tetrachloride (CCl$\sb4$) or alcohol (ethanol) administration and the MRS results were confirmed using biochemical total lipid analysis and histology. $\rm T\sb1$ weighted MR images acquired weekly, 48 hours post administration, demonstrated only a slight increase in overall liver intensity with CCl$\sb4$ or alcohol administration, which is consistent with previously reported results. The MR images were able to detect nodules resulting from CCl$\sb4$+PLD induced cirrhosis as hypointense regions, also consistent with previous reports. Localized in vivo water and lipid proton $\rm T\sb1$ relaxation time measurements were performed and demonstrated no statistically significant trends for either agent. In vivo proton spectra were also acquired using stimulated echo techniques to quantitatively follow the changes in liver lipid content. The changes in liver lipid content observed using MRS were verified by total lipid analysis using the Folch technique and histology. The in vivo $\rm T\sb1$ and lipid quantification data str inconsistent with the previous hypothesis that the changes in $\rm T\sb1$ weighted images were the result of increased "free" water content and, therefore, increased water $\rm T\sb1$ relaxation times. These data indicate that the long term changes are more likely the result of changes in lipid content. The data are also shown to agree with the accepted hypothesis that the time course and mechanism of fatty infiltration are different for CCl$\sb4$ and alcohol. The hypothesis that the lipids resulting from either protocol are from the same lipid fraction(s), presumably triglycerides, is also supported. And lastly, on the basis of MR images and quantitative MRS lipid information, it was shown that cirrhosis could be distinguished from fatty infiltration. ^