Begoña Ochoa

Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C

Background Fatty acid translocase CD36 (FAT/CD36) mediates uptake and intracellular transport of long-chain fatty acids in diverse cell types. While the pathogenic role of FAT/CD36 in hepatic steatosis in rodents is well-defined, little is known about its significance in human liver diseases. Objective To examine the expression of FAT/CD36 and its cellular and subcellular distribution within the liver of patients with non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C virus (HCV) infection. Patients 34 patients with non-alcoholic steatosis (NAS), 30 with non-alcoholic steatohepatitis (NASH), 66 with HCV genotype 1 (HCV G1) and 32 with non-diseased liver (NL). Methods Real-time PCR and western blot analysis were used to assess hepatic FAT/CD36 expression. Computational image analysis of immunostained liver biopsy sections was performed to determine subcellular distribution and FAT/CD36 expression index. Results Compared with NL, hepatic mRNA and protein levels of FAT/CD36 were significantly higher in patients with NAS (median fold increase 0.84 (range 0.15–1.61) and 0.66 (range 0.33–1.06), respectively); NASH (0.91 (0.22–1.81) and 0.81 (0.38–0.92), respectively); HCV G1 without steatosis (0.30 (0.17–1.59) and 0.33 (0.29–0.52), respectively); and HCV G1 with steatosis (0.85 (0.15–1.98) and 0.87 (0.52–1.26), respectively). In contrast to NL, FAT/CD36 was predominantly located at the plasma membrane of hepatocytes in patients with NAFLD and HCV G1 with steatosis. A significant correlation was observed between hepatic FAT/CD36 expression index and plasma insulin levels, insulin resistance (HOMA-IR) and histological grade of steatosis in patients with NASH (r=0.663, r=0.735 and r=0.711, respectively) and those with HCV G1 with steatosis (r=0.723, r=0.769 and r=0.648, respectively). Conclusions Hepatic FAT/CD36 upregulation is significantly associated with insulin resistance, hyperinsulinaemia and increased steatosis in patients with NASH and HCV G1 with fatty liver. Translocation of this fatty acid transporter to the plasma membrane of hepatocytes may contribute to liver fat accumulation in patients with NAFLD and HCV.

HEDGEHOG SIGNALING IS CRITICAL FOR NORMAL LIVER REGENERATION AFTER PARTIAL HEPATECTOMY IN MICE

Distinct mechanisms are believed to regulate growth of the liver during fetal development and after injury in adults, because the former relies on progenitors and the latter generally involves replication of mature hepatocytes. However, chronic liver injury in adults increases production of Hedgehog (Hh) ligands, developmental morphogens that control progenitor cell fate and orchestrate various aspects of tissue construction during embryogenesis. This raises the possibility that similar Hh‐dependent mechanisms also might regulate adult liver regeneration. The current analysis of murine liver regeneration after 70% partial hepatectomy (PH), an established model of adult liver regeneration, demonstrated that PH induced production of Hh ligands and activated Hh signaling in liver cells. Treatment with a specific Hh signaling inhibitor interfered with several key components of normal liver regeneration, significantly inhibiting progenitor responses, matrix remodeling, proliferation of hepatocytes and ductular cells, and restoration of liver mass. These global inhibitory effects on liver regeneration dramatically reduced survival after PH. Conclusion: Mechanisms that mediate liver organogenesis, such as Hh pathway activation, are retained and promote reconstruction of adult livers after injury. Hepatology 2010

Profiling and Imaging of Lipids on Brain and Liver Tissue by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Using 2-Mercaptobenzothiazole as a Matrix

2-Mercaptobenzothiazole (MBT) is employed for the first time as a matrix for the analysis of lipids from tissue extracts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We demonstrate that the performance of MBT is superior to that of the matrixes commonly employed for lipids, due to its low vapor pressure, its low acidity, and the formation of small crystals, although because of the strong background at low m/z, it precludes detection of species below approximately 500 Da. This inconvenience can be partly overcome with the formation of Cs adducts. Using a polymer-based dual calibration, a mass accuracy of approximately 10 ppm in lipid extracts and of approximately 80 ppm in tissues is achieved. We present spectra from liver and brain lipid extracts where a large amount of lipid species is identified, in both positive and negative ion modes, with high reproducibility. In addition, the above-mentioned special properties of MBT allow its employment for imaging mass spectrometry. In the present work, images of brain and liver tissues showing different lipid species are presented, demonstrating the advantages of the employment of MBT.

Matrix-assisted laser desorption ionization imaging mass spectrometry in lipidomics

The relevant structural, energetics, and regulatory roles of lipids are universally acknowledged. However, the high variability of lipid species and the large differences in concentrations make unraveling the role played by the different species in metabolism a titanic task. A recently developed technique, known as imaging mass spectrometry, may shed some light on the field, as it enables precise information to be obtained on the location of lipids in tissues. A review of the state of the art of the technique is presented in this manuscript, including detailed analysis of sample-preparation steps, data handling, and the identification of the species mapped so far.

A subset of dysregulated metabolic and survival genes is associated with severity of hepatic steatosis in obese Zucker rats

We aimed to characterize the primary abnormalities associated with fat accumulation and vulnerability to hepatocellular injury of obesity-related fatty liver. We performed functional analyses and comparative transcriptomics of isolated primary hepatocytes from livers of obese insulin-resistant Zucker rats (comprising mild to severe hepatic steatosis) and age-matched lean littermates, searching for novel genes linked to chronic hepatic steatosis. Of the tested genome, 1.6% was identified as steatosis linked. Overexpressed genes were mainly dedicated to primary metabolism (100%), signaling, and defense/acute phase (∼70%); detoxification, steroid, and sulfur metabolism (∼65%) as well as cell growth/proliferation and protein synthesis/transformation (∼70%) genes were downregulated. The overexpression of key genes involved in de novo lipogenesis, fatty acid and glycerolipid import and synthesis, as well as acetyl-CoA and cofactor provision was paralleled by enhanced hepatic lipogenesis and production of large triacylglycerol-rich VLDL. Greatest changes in gene expression were seen in those encoding the lipogenic malic enzyme (up to 7-fold increased) and cell-to-cell interacting cadherin 17 (up to 8-fold decreased). Among validated genes, fatty acid synthase, stearoyl-CoA desaturase 1, fatty acid translocase/Cd36, malic enzyme, cholesterol-7α hydroxylase, cadherin 17, and peroxisome proliferator-activated receptor α significantly correlated with severity of hepatic steatosis. In conclusion, dysregulated expression of metabolic and survival genes accompany hepatic steatosis in obese insulin-resistant rats and may render steatotic hepatocytes more vulnerable to cell injury in progressive nonalcoholic fatty liver disease.

Short- and long-term effects of atorvastatin, lovastatin and simvastatin on the cellular metabolism of cholesteryl esters and VLDL secretion in rat hepatocytes.

The short- and long-term in vitro effects of the hydroxymethylglutaryl-CoA reductase inhibitor atorvastatin, compared with lovastatin and simvastatin on VLDL secretion, and on the formation and the neutral and acid lysosomal hydrolysis of cholesteryl esters was investigated in rat liver hepatocytes maintained in suspension (2 or 4 h) or cultured in monolayers (24 h). All statins time-dependently reduced [14C]oleate incorporation into cholesteryl esters, but when exogenous cholesterol was added only atorvastatin caused an immediate transient decrease in hepatocyte ACAT activity. Activity of the lysosomal, microsomal and cytosolic CEH isoforms was unaffected by the hepatocyte treatments. Statins reduced free and esterified cholesterol mass in hepatocyte microsomes after 2 h, and this was followed by a modest decline in VLDL cholesteryl esters, whilst secretion of VLDL apoB and triglycerides was unaltered. However, after 24 h of treatment, statins caused generalized 20-40% decreases in the secretion of VLDL apoB, cholesterol and triglycerides, with the reduction in apoB48 secretion being significantly superior to that caused in apoB100. The mean diameter of secreted VLDL was not modified by either duration or drug treatment. Additional studies with subcellular fractions demonstrated that statins have a direct selective effect on the enzymes governing the cholesterol-cholesteryl ester cycle, with the exception of the microsomal CEH. Atorvastatin, lovastatin and simvastatin inhibited ACAT activity in microsomes by 50% at doses of 250, 100 and 50 microM, respectively. The cytosolic CEH elicited a biphasic profile of activity with activations up to 100 microM statin and inhibitions above 250 microM, and the lysosomal CEH was only inhibited by atorvastatin at a dose of 100 microM or more. We conclude that a prolonged, but not a short, limited availability of hepatocyte cholesterol derived from the endogenous synthesis reduces VLDL secretion, and that reactivity of statins at the cellular level are more similar than reactivity at the subcellular level as regards the cholesterol-cholesteryl ester cycle.

Lipid and fatty acid composition of canine lipoproteins.

Lipid classes and their fatty acids were studied in the major lipoprotein fractions from canine, in comparison with human, plasma. In dogs, high-density-lipoprotein (HDL), the main carrier of plasma phospholipid (PL), cholesterol ester (CE) and free cholesterol, was the most abundant lipoprotein, followed by low and very-low density lipoproteins (LDL and VLDL). Notably, LDL and VLDL contributed similarly to the total dog plasma triacylglycerol (TG). The PL composition was similar in all three lipoproteins, dominated by phosphatidylcholine (PC). Even though the content and composition of lipids within and among lipoproteins differed markedly between dog and man, the total amount of circulating lipid was similar. All canine lipoproteins were relatively richer than those from humans in long-chain (C20-C22) n-6 and n-3 polyunsaturated fatty acids (PUFA) but had comparable proportions of total saturated and monoenoic fatty acids, with 18:2n-6 being the main PUFA in both mammals. The fatty acid profile of canine and human lipoproteins differed because they had distinct proportions of their major lipids. There were more n-3 and n-6 long-chain PUFA in canine than in human plasma, because dogs had more HDL, their HDL had more PC and CE, and both these lipids were richer in such PUFA.

Methionine adenosyltransferase 1A gene deletion disrupts hepatic very low-density lipoprotein assembly in mice†‡

Very low‐density lipoprotein (VLDL) secretion provides a mechanism to export triglycerides (TG) from the liver to peripheral tissues, maintaining lipid homeostasis. In nonalcoholic fatty liver disease (NAFLD), VLDL secretion disturbances are unclear. Methionine adenosyltransferase (MAT) is responsible for S‐adenosylmethionine (SAMe) synthesis and MAT I and III are the products of the MAT1A gene. Deficient MAT I and III activities and SAMe content in the liver have been associated with NAFLD, but whether MAT1A is required for normal VLDL assembly remains unknown. We investigated the role of MAT1A on VLDL assembly in two metabolic contexts: in 3‐month‐old MAT1A‐knockout mice (3‐KO), with no signs of liver injury, and in 8‐month‐old MAT1A‐knockout mice (8‐KO), harboring nonalcoholic steatohepatitis. In 3‐KO mouse liver, there is a potent effect of MAT1A deletion on lipid handling, decreasing mobilization of TG stores, TG secretion in VLDL and phosphatidylcholine synthesis via phosphatidylethanolamine N‐methyltransferase. MAT1A deletion also increased VLDL– apolipoprotein B secretion, leading to small, lipid‐poor VLDL particles. Administration of SAMe to 3‐KO mice for 7 days recovered crucial altered processes in VLDL assembly and features of the secreted lipoproteins. The unfolded protein response was activated in 8‐KO mouse liver, in which TG accumulated and the phosphatidylcholine‐to‐phosphatidylethanolamine ratio was reduced in the endoplasmic reticulum, whereas secretion of TG and apolipoprotein B in VLDL was increased and the VLDL physical characteristics resembled that in 3‐KO mice. MAT1A deletion also altered plasma lipid homeostasis, with an increase in lipid transport in low‐density lipoprotein subclasses and decrease in high‐density lipoprotein subclasses. Conclusion: MAT1A is required for normal VLDL assembly and plasma lipid homeostasis in mice. Impaired VLDL synthesis, mainly due to SAMe deficiency, contributes to NAFLD development in MAT1A‐KO mice. (HEPATOLOGY 2011

High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway.

BackgroundHyperhomocysteinemia (HHcy) causes increased oxidative stress and is an independent risk factor for cardiovascular disease. Oxidative stress is now believed to be a major contributory factor in the development of non alcoholic fatty liver disease, the most common liver disorder worldwide. In this study, the changes which occur in homocysteine (Hcy) metabolism in high fat-diet induced non alcoholic fatty liver disease (NAFLD) in rats were investigated.Methods and resultsAfter feeding rats a standard low fat diet (control) or a high fat diet (57% metabolisable energy as fat) for 18 weeks, the concentration of homocysteine in the plasma was significantly raised while that of cysteine was lowered in the high fat as compared to the control diet fed animals. The hepatic activities of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGS), the enzymes responsible for the breakdown of homocysteine to cysteine via the transsulphuration pathway in the liver, were also significantly reduced in the high fat-fed group.ConclusionsThese results indicate that high fat diet-induced NAFLD in rats is associated with increased plasma Hcy levels caused by down-regulation of hepatic CBS and CGL activity. Thus, HHcy occurs at an early stage in high fat diet-induced NAFLD and is likely to contribute to the increased risk of cardiovascular disease associated with the condition.

TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

Background & Aims Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. Methods To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. Results In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12–8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. Conclusions TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.