Liver Cholesterol Overload Aggravates Obstructive Cholestasis by Inducing Oxidative Stress and Premature Death in Mice

Nonalcoholic steatohepatitis is one of the leading causes of liver disease. Dietary factors determine the clinical presentation of steatohepatitis and can influence the progression of related diseases. Cholesterol has emerged as a critical player in the disease and hence consumption of cholesterol-enriched diets can lead to a progressive form of the disease. The aim was to investigate the impact of liver cholesterol overload on the progression of the obstructive cholestasis in mice subjected to bile duct ligation surgery. Mice were fed with a high cholesterol diet for two days and then were subjected to surgery procedure; histological, biochemical, and molecular analyses were conducted to address the effect of cholesterol in liver damage. Mice under the diet were more susceptible to damage. Results show that cholesterol fed mice exhibited increased apoptosis and oxidative stress as well as reduction in cell proliferation. Mortality following surgery was higher in HC fed mice. Liver cholesterol impairs the repair of liver during obstructive cholestasis and aggravates the disease with early fatal consequences; these effects were strongly associated with oxidative stress.This work was supported by grants of the CONACYT 252942, 236558, and 166042, CONACYT Fronteras de la Ciencia 1320, Asociación Mexicana de Hepatología, and SEP-PRODEP-913026-1461211, Universidad Autónoma Metropolitana Iztapalapa, by grants SAF-2011-23031 and SAF-2012-34831 from Plan Nacional de I+D, Spain, Fundació la Marató de TV3, La Mutua Madrileña, PI11/0325 (META) Grant from the Instituto Salud Carlos III, by the support of CIBEREHD, and by the Center Grant P50-AA-11999 from Research Center for Liver and Pancreatic Diseases funded by NIAAA/NIH.Peer Reviewe

Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system

The hepatocyte growth factor induces an anti-inflammatory and repairing response in the cholestasis-induced colon damage

Aim: Cholestasis remains a partially characterized disease. Evidence has been gained that it is a systemic disease that begins in the liver but significantly impacts other organs and systems such as the kidney, heart, and intestine, among others. One of the primary damage mechanisms is the generation of reactive oxygen species (ROS), which eventually leads to oxidative stress, impacting canalicular morphology and actin cytoskeleton changes that could worsen the problem. These characteristics are also observed in the kidney and intestine. The work focused on addressing the intestine effects of intrahepatic cholestasis induced by α-naphthyl isothiocyanate (ANIT) and the protective response of the hepatocyte growth factor (HGF). Methods: The 10- to 12-week-old CD1 male mice were treated with ANIT and then treated or not with HGF; intestine damage was addressed by histology, immunohistochemistry (IHC) of specific markers, oxidative stress, and apoptosis. Results: Results show changes in the intestine histology, particularly the colon and ileum, induced by the cholestasis. HGF treatment restored the histology presentation and reverted the oxidative damage, clearly indicating a healing response. This observation was supported by an increment in anti-inflammatory macrophages (CD163+) in the HGF treatment. Conclusions: The data prove that HGF induces a protective and repairing response in the intestine under cholestatic challenges

La sobrecarga hepática de colesterol agrava el daño inducido por la colestasis osbtructiva mediante la inducción de estrés oxidante

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Cholesterol overload enhances liver damage and impairs organ repair in mice subjected to bile duct ligation

Resumen del póster presentado en The International Liver Congress – 48th Annual meeting of the European Association for the Study of the Liver celebrado en Paises Bajos en abril de 2013.-- et al.Nonalcoholic fatty liver disease is the hepatic manifestation of the metabolic syndrome, defined by the deposition of fat in more than 5% of hepatocytes. It is well known that lipid overload, particularly cholesterol, sensitizes to hepatocellular damage. We were focused to figure out the effect of a high cholesterol diet (HC, 2% cholesterol and 0.5% sodium cholate) in the bile duct ligation (BDL)-induced cholestasis and the involvement of c-Met in the repair process. [Methods]: C57Bl/6 mice were fed with the HC diet for two days and after that were subject to BDL, parallel animals were fed with regular rodent chow diet (Chow) and BDL was performed. Hepatic enzymes in serum were measured. Western blotting of main survival pathways were analyzed and confocal immunofluorescence for c-Met was performed in primary hepatocytes isolated by the two-step collagenase perfusion. [Results]: Our data show that HC animals were more susceptible to both insults, all animals in the HC-BDL group (n = 6) die during the first 72 h after surgery, while all animals in Chow-BDL (n = 7) were alive. We proceeded to sacrifice animals at 24, 48 and 72 h after BDL of all groups. Liver macroscopic inspection of HC mice showed the characteristic pale color in steatosis and changes in gallbladder. Although AST, ALT and ALP were increased as a consequence of BDL, animals fed with the hypercholesterolemic diet increased significantly these values (ranging from 20 to 200- fold), these data were in agreement with an elevation on bilirubin, suggesting an exacerbation of cholestatic damage. Examination of the main signaling pathways involved in repair process, such as Akt, Stat3 and Erk1/2 in HC animals showed a downactivation in Erk1/2 and Akt, but some compensatory effect in Stat3 was observed. In vitro analysis of c-Met activation by Western blotting and immunofluorescence in hepatocytes from HC animals revealed a delayed activation of this receptor after HGF treatment. In conclusion our data suggest that cholesterol overload in hepatocytes aggravates cholestasis and impairs signaling pathways involved in liver repair as a consequence in c-Met misactivation.Work supported in part by CONACYT 131707, CD-2011–01166042, NNL and scholarship 234219.Peer Reviewe

Mitochondrial Molecular Pathophysiology of Nonalcoholic Fatty Liver Disease: A Proteomics Approach

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver condition that can progress to nonalcoholic steatohepatitis, cirrhosis and cancer. It is considered an emerging health problem due to malnourishment or a high-fat diet (HFD) intake, which is observed worldwide. It is well known that the hepatocytes’ apoptosis phenomenon is one of the most important features of NAFLD. Thus, this review focuses on revealing, through a proteomics approach, the complex network of protein interactions that promote fibrosis, liver cell stress, and apoptosis. According to different types of in vitro and murine models, it has been found that oxidative/nitrative protein stress leads to mitochondrial dysfunction, which plays a major role in stimulating NAFLD damage. Human studies have revealed the importance of novel biomarkers, such as retinol-binding protein 4, lumican, transgelin 2 and hemoglobin, which have a significant role in the disease. The post-genome era has brought proteomics technology, which allows the determination of molecular pathogenesis in NAFLD. This has led to the search for biomarkers which improve early diagnosis and optimal treatment and which may effectively prevent fatal consequences such as cirrhosis or cancer

Cholesterol enrichment in liver mitochondria impairs oxidative phosphorylation and disrupts the assembly of respiratory supercomplexes

Mitochondrial cholesterol accumulation is a hallmark of alcoholic and non-alcoholic fatty liver diseases and impairs the function of specific solute carriers through changes in membrane physical properties. However, its impact on mitochondrial respiration and organization of respiratory supercomplexes has not been determined so far. Here we fed mice a cholesterol-enriched diet (HC) supplemented with sodium cholate to examine the effect of cholesterol in mitochondrial function. HC feeding increased liver cholesterol content, which downregulated Srebp2 and Hmgcr expression, while sodium cholate administration decreased Cyp7a1 and Cyp8b1 mRNA levels, suggesting the downregulation of bile acid synthesis through the classical pathway. HC-fed mice exhibited increased expression of Stard1 and Mln64 and enhanced mitochondrial free cholesterol levels (2-3 fold), leading to decreased membrane fluidity. Mitochondria from HC-fed mice displayed increased cholesterol loading in both outer and inner mitochondrial membranes. Cholesterol loading decreased complex I and complex II-driven state 3 respiration and mitochondrial membrane potential. Decreased respiratory and uncoupling control ratio from complex I was also observed after in situ enrichment of mouse liver mitochondria with cholesterol or enantiomer cholesterol, the mirror image of natural cholesterol. Moreover, in vivo cholesterol loading decreased the level of complex III2 and the assembly of respiratory supercomplexes I1+III2+IV and I1+III2. Moreover, HC feeding caused oxidative stress and mitochondrial GSH (mGSH) depletion, which translated in hepatic steatosis and liver injury, effects that were rescued by replenishing mGSH with GSH ethyl ester. Overall, mitochondrial cholesterol accumulation disrupts mitochondrial functional performance and the organization of respiratory supercomplexes assembly, which can contribute to oxidative stress and liver injury

Association Between Serum Hemoglobin Levels and Non Alcoholic Fatty Liver Disease in a Mexican Population

Introduction and aim. Nonalcoholic fatty liver disease (NAFLD) is closely associated with overweight and obesity, becoming one of the most prevalent hepatic diseases nowadays. Circulating hemoglobin (Hb) concentration is significantly higher in people with NAFLD, compared to healthy patients. While liver biopsy remains the gold standard for NAFLD diagnosis, it is not the best technique due to adverse events that may occur. Therefore it is important to find less invasive and more sensitive markers. This study aimed to determine the association of serum Hb levels in patients with steatosis and fibrosis as a noninvasive marker.Material and methods. A 1,186 patient cross-sectional study nested in a randomized clinical trial (NCT01874249) was conducted. Patients were diagnosed by ultrasound for hepatic steatosis and fibroscan for fibrosis; blood test and anthropometric measurements were also assessed.Results. Serum Hb increased proportionally related to the steatosis level, being significantly higher in patients with severe steatosis than in patients with moderate and mild steatosis.Conclusion. Patients with non-alcoholic fatty liver disease showed elevated levels of circulating Hb, evidence that suggests that Hb exerts a protective role, as it may act as an antioxidant and may counteract the adverse effects of this disease

Acid sphingomyelinase is required for chronic alcohol-induced steatosis, ER stress and LPS susceptibility

Póster presentado en el 62nd Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), celebrada del 4 al 8 de noviembre de 2011 en San Francisco, California (Estados Unidos)The mechanisms of alcohol-induced liver disease (ALD) are still poorly understood, which has limited the availability of efficient therapy. The mechanisms contributing to the onset of fatty liver and damage following alcohol intake are multifactorial and include the sensitization to second hits (e.g. TNF, LPS), and ER stress. Since the role of acidic sphingomyelinase (ASMase), a specific source of ceramide generation, in alcohol-induced hepatic steatosis and injury is unknown, our aim was to investigate the contribution of ASMase in alcohol-induced steatosis, liver injury and susceptibility to LPS. Methods: Wild type and ASMase knockout mice were fed the Lieber DeCarli liquid diet containing alcohol (36% calories) for 4-5 weeks. Expression of ATF4, PDI, Grp78, CHOP, and lipogenic enzymes (SREBP-1, SREBP-2, DGAT and FAS) as well as the mitochondrial cholesterol transporting polypeptide StAR was examined by qPCR. Liver damage and susceptibility to LPS/concanavalin-A were examined by serum transaminases and H&E staining. Mitochondrial cholesterol trafficking was examined by confocal microscopy and fractionation studies. Although the intrinsic phenotype of ASMase knockout mice exhibit hepatic cholesterol and sphingomyelin accumulation, ASMase deficiency prevented alcohol-induced macrosteatosis and mitochondrial cholesterol accumulation compared to wild type mice. Indeed, ASMase was required for alcohol-induced activation of SREBP-1c, SREBP-2, DGAT-2, FAS and StAR (3-4 fold). Moreover, alcohol feeding to wild type resulted in expression of ATF4, PDI, Grp78 and CHOP, but these effects were blunted in ASMase knockout mice. Moreover, liver injury following alcohol feeding and alcohol-mediated sensitization to LPS were reduced in ASMase knockout mice. However, susceptibility to concanavalin A-mediated liver injury is preserved in ASMase null mice despite unchanged expression of TNF receptor 1 and TNF receptor 2. Interestingly, hepatic regeneration following hepatectomy in ASMase knockout mice was similar to that of wild type mice. Finally, we observed enhanced ASMase mRNA levels (3-4 fold) in liver samples from patients with acute alcoholic hepatitis compared to control subjects. In conclusion: Thus, these findings indicate a critical role for ASMase in the onset of steatosis and liver injury following alcohol intake, emerging as a potential novel target for therapeutic interventionPeer Reviewe