Raquel Horrillo

Obesity-induced insulin resistance and hepatic steatosis are alleviated by ω-3 fatty acids: a role for resolvins and protectins

Omega‐3‐polyunsaturated fatty acids (w‐3‐PUFAs) have well‐documented protective effects that are attributed not only to eicosanoid inhibition but also to the formation of novel biologically active lipid mediators (i.e., resolvins and protectins). In this study, we examined their effects on ob/ob mice, an obesity model of insulin resistance and fatty liver disease. Dietary intake ofw‐3‐PUFAs had insulin‐sensitizing actions in adipose tissue and liver and improved insulin tolerance in obese mice. Genes involved in insulin sensitivity (PPAR/γ), glucose transport (GLUT‐2/GLUT–4), and insulin receptor signaling (IRS‐1/IRS–2) were up‐regulated byw‐3‐PUFAs. Moreover,w‐3‐PUFAs increased adiponectin, an anti‐inflammatory and insulin‐sensitizing adipokine, and induced AMPK phosphorylation, a fuel‐sensing enzyme and a gatekeeper of the energy balance. Concomitantly, hepatic steatosis was alleviated byw‐3‐PUFAs. A lipidomic analysis with liquid chromatography/mass spectrome‐try/mass spectrometry revealed that w‐3‐PUFAs inhibited the formation of w‐6‐PUFA‐derived eicosanoids, while triggering the formation of w‐3‐PUFA‐derived resolvins and protectins. Moreover, representative members of these lipid mediators, namely resolvin E1 and protectin D1, mimicked the insulin‐sensitizing and antisteatotic effects of w‐3‐PUFAs and induced adiponectin expression to a similar extent that of rosigli‐tazone, a member of the thiazolidinedione family of antidiabetic drugs. Taken together, these findings uncover beneficial actions of w‐3‐PUFAs and their bioactive lipid autacoids in preventing obesity‐induced insulin resistance and hepatic steatosis.—Gonzalez‐Periz, A.,Horrillo, R., Ferre, N., Gronert, K., Dong, B., Moran‐Salvador, E.,Titos, E., Martinez‐Clemente, M.,Lopez‐Parra, M.,Arroyo, V., Claria, J. Obesity‐induced insulin resistance and hepatic steatosis are alleviated byw‐3 fatty acids: a role for resolvins and protectins. FASEB J. 23, 1946–1957 (2009)

Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA

Docosahexaenoic acid (DHA) is a ω‐3 essential fatty acid that reduces the incidence and severity of a number of diseases. Recently, a novel series of DHA‐derived lipid mediators with potent protective actions has been identified. In this study we demonstrate that dietary amplification of these DHA‐derived products protects the liver from necroinflammatory injury. In vitro, supplementation of hepatocytes with DHA significantly reduced hydrogen peroxide‐induced DNA damage, evaluated by the “comet assay,” and oxidative stress, determined by measurement of malondialdehyde levels. In vivo, dietary supplementation of mice with DHA ameliorated carbon tetrachloride‐induced necroinflammatory damage. In addition, hepatic cyclooxygenase‐2 expression and PGE2 levels were significantly reduced in mice fed DHA‐enriched diets. In these animals, increased hepatic formation of DHA‐derived lipid mediators (i.e., 17S‐hydroxy‐DHA (17S‐HDHA) and protectin D1) was detected by HPLC‐gas chromatography/mass spectrometry analysis. Consistent with these findings, synthetic 17‐HDHA abrogated genotoxic and oxidative damage in hepatocytes and decreased TNF‐α release and 5‐lipoxygenase expression in macrophages. In a transactivation assay, 17‐ HDHA acted in a concentration‐dependent manner as a PPARγ agonist. Taken together, these findings identify a potential role for DHA‐derived products, specifically 17S‐HDHA and protectin D1, in mediating the protective effects of dietary DHA in necroinflammatory liver injury.—González‐Périz, A., Planagumà, A., Gronert, K., Miquel, R., López‐Parra, M., Titos, E., Horrillo, R., Ferré, N., Deulofeu, R., Arroyo, V., Rodés, J., Clària, J. Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S‐hydroxy‐DHA. FASEB J. 20, E1844–E1855 (2006)

5-Lipoxygenase Activating Protein Signals Adipose Tissue Inflammation and Lipid Dysfunction in Experimental Obesity

The presence of the so-called low-grade inflammatory state is recognized as a critical event in adipose tissue dysfunction, leading to altered secretion of adipokines and free fatty acids (FFAs), insulin resistance, and development of hepatic complications associated with obesity. This study was designed to investigate the potential contribution of the proinflammatory 5-lipoxygenase (5-LO) pathway to adipose tissue inflammation and lipid dysfunction in experimental obesity. Constitutive expression of key components of the 5-LO pathway, as well as leukotriene (LT) receptors, was detected in adipose tissue as well as in adipocyte and stromal vascular fractions. Adipose tissue from obese mice, compared with that from lean mice, exhibited increased 5-LO activating protein (FLAP) expression and LTB4 levels. Incubation of adipose tissue with 5-LO products resulted in NF-κB activation and augmented secretion of proinflammatory adipokines such as MCP-1, IL-6, and TNF-α. In addition, LTB4, but not LTD4, reduced FFA uptake in primary adipocytes, whereas 5-LO inhibition suppressed isoproterenol-induced adipose tissue lipolysis. In mice with dietary obesity, elevated FLAP expression in adipose tissue was paralleled with macrophage infiltration, increased circulating FFA levels, and hepatic steatosis, phenomena that were reversed by FLAP inhibition with Bay-X-1005. Interestingly, FLAP inhibition induced AMP-activated protein kinase phosphorylation in parallel with decreases in hormone-sensitive lipase activity and the expression and secretion of TNF-α and IL-6. Similar effects were observed in differentiated 3T3-L1 adipocytes incubated with either Bay-X-1005 or the selective LTB4 receptor antagonist U-75302. Taken together, these findings indicate that the 5-LO pathway signals the adipose tissue low-grade inflammatory state and steatogenic potential in experimental obesity.

5‐lipoxygenase deficiency reduces hepatic inflammation and tumor necrosis factor α–induced hepatocyte damage in hyperlipidemia‐prone ApoE‐null mice

The actual risk factors that drive hepatic inflammation during the transition from steatosis to steatohepatitis are unknown. We recently demonstrated that hyperlipidemia‐prone apolipoprotein E–deficient (ApoE−/−) mice exhibit hepatic steatosis and increased susceptibility to hepatic inflammation and advanced fibrosis. Because the proinflammatory 5‐lipoxygenase (5‐LO) pathway was found to be up‐regulated in these mice and given that 5‐LO deficiency confers cardiovascular protection to ApoE−/− mice, we determined the extent to which the absence of 5‐LO would alter liver injury in these mice. Compared with ApoE−/− mice, which showed expected hepatic steatosis and inflammation, ApoE/5‐LO double‐deficient (ApoE−/−/5‐LO−/−) mice exhibited reduced hepatic inflammation, macrophage infiltration, tumor necrosis factor α (TNF‐α), monocyte chemoattractant protein‐1 (MCP‐1) and interleukin (IL)‐18 expression, caspase‐3 and nuclear factor‐κB (NF‐κB) activities, and serum alanine aminotransferase levels in the absence of changes in hepatic steatosis. The lack of 5‐LO produced a remarkable insulin‐sensitizing effect in the adipose tissue because peroxisome proliferator‐activated receptor γ, insulin receptor substrate‐1, and adiponectin were up‐regulated, whereas c‐Jun amino‐terminal kinase phosphorylation and MCP‐1 and IL‐6 expression were down‐regulated. On the other hand, hepatocytes isolated from ApoE−/−/5‐LO−/− mice were more resistant to TNF‐α–induced apoptosis. The 5‐LO products leukotriene (LT) B4, LTD4, and 5‐HETE consistently triggered TNF‐α–induced apoptosis and compromised hepatocyte survival by suppressing NF‐κB activity in the presence of actinomycin D. Moreover, ApoE−/−/5‐LO−/− mice were protected against sustained high‐fat diet (HFD)‐induced liver injury and hepatic inflammation, macrophage infiltration and insulin resistance were significantly milder than those of ApoE−/− mice. Finally, pharmacological inhibition of 5‐LO significantly reduced hepatic inflammatory infiltrate in the HFD and ob/ob models of fatty liver disease. Conclusion: These combined data indicate that hyperlipidemic mice lacking 5‐LO are protected against hepatic inflammatory injury, suggesting that 5‐LO is involved in mounting hepatic inflammation in metabolic disease. (HEPATOLOGY 2010.)

Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential involvement of oxysterols

The contribution of metabolic factors to the severity of liver disease is not completely understood. In this study, apolipoprotein E-deficient (ApoE-/-) mice were evaluated to define potential effects of hypercholesterolemia on the severity of carbon tetrachloride (CCl4)-induced liver injury. Under baseline conditions, hypercholesterolemic ApoE-/- mice showed increased hepatic oxidative stress (SOD activity/4-hydroxy-2-nonenal immunostaining) and higher hepatic TGF-beta1, MCP-1, and TIMP-1 expression than wild-type control mice. After CCl4 challenge, ApoE-/- mice exhibited exacerbated steatosis (Oil Red O staining), necroinflammation (hematoxylin-eosin staining), macrophage infiltration (F4/80 immunohistochemistry), and fibrosis (Sirius red staining and alpha-smooth muscle actin immunohistochemistry) and more severe liver injury [alanine aminotransferase (ALT) and aspartate aminotransferase] than wild-type controls. Direct correlations were identified between serum cholesterol and hepatic steatosis, fibrosis, and ALT levels. These changes did not reflect the usual progression of the disease in ApoE-/- mice, since exacerbated liver injury was not present in untreated age-paired ApoE-/- mice. Moreover, hepatic cytochrome P-450 expression was unchanged in ApoE-/- mice. To explore potential mechanisms, cell types relevant to liver pathophysiology were exposed to selected cholesterol-oxidized products. Incubation of hepatocytes with a mixture of oxysterols representative of those detected by GC-MS in livers from ApoE-/- mice resulted in a concentration-dependent increase in total lipoperoxides and SOD activity. In hepatic stellate cells, oxysterols increased IL-8 secretion through a NF-kappaB-independent mechanism and upregulated TIMP-1 expression. In macrophages, oxysterols increased TGF-beta1 secretion and MCP-1 expression in a concentration-dependent manner. Oxysterols did not compromise cell viability. Taken together, these findings demonstrate that hypercholesterolemic mice are sensitized to liver injury and that cholesterol-derived products (i.e., oxysterols) are able to induce proinflammatory and profibrogenic mechanisms in liver cells.

Comparative Protection against Liver Inflammation and Fibrosis by a Selective Cyclooxygenase-2 Inhibitor and a Nonredox-Type 5-Lipoxygenase Inhibitor

In this study, we examined the relative contribution of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), two major proinflammatory pathways up-regulated in liver disease, to the progression of hepatic inflammation and fibrosis. Separate administration of 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236), a selective COX-2 inhibitor, and CJ-13,610, a 5-LO inhibitor, to carbon tetrachloride-treated mice significantly reduced fibrosis as revealed by the analysis of Sirius Red-stained liver sections without affecting necroinflammation. Conversely, combined administration of SC-236 and 4-[3-[4-(2-methylimidazol-1-yl)-phenylthio]]phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide (CJ-13,610) reduced both necroinflammation and fibrosis. These findings were confirmed in 5-LO-deficient mice receiving SC-236, which also showed reduced hepatic monocyte chemoattractant protein 1 expression. Interestingly, SC-236 and CJ-13,610 significantly increased the number of nonparenchymal liver cells with apoptotic nuclei (terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive). Additional pharmacological profiling of SC-236 and CJ-13,610 was performed in macrophages, the primary hepatic inflammatory cell type. In these cells, SC-236 inhibited prostaglandin (PG) E2 formation in a concentration-dependent manner, whereas CJ-13,610 blocked leukotriene B4 biosynthesis. Of note, the simultaneous addition of SC-236 and CJ-13,610 resulted in a higher inhibitory profile on PGE2 biosynthesis than the dual COX/5-LO inhibitor licofelone. These drugs differentially regulated interleukin-6 mRNA expression in macrophages. Taken together, these findings indicate that both COX-2 and 5-LO pathways are contributing factors to hepatic inflammation and fibrosis and that these two pathways of the arachidonic acid cascade represent potential targets for therapy.

Disruption of the 12/15‐lipoxygenase gene (Alox15) protects hyperlipidemic mice from nonalcoholic fatty liver disease

We have shown that Alox15, the gene encoding for 12/15‐lipoxygenase (12/15‐LO), is markedly up‐regulated in livers from apolipoprotein E‐deficient (ApoE−/−) mice, which spontaneously develop nonalcoholic fatty liver disease secondary to hyperlipidemia. In the current study, we used ApoE−/− mice with a targeted disruption of the Alox15 gene to assess the role of 12/15‐LO in the development and progression of hepatic steatosis and inflammation. Compared with ApoE−/− mice, which exhibited extensive hepatic lipid accumulation and exacerbated inflammatory injury, ApoE/12/15‐LO double‐knockout (ApoE−/−/12/15‐LO−/−) mice showed reduced serum alanine aminotransferase levels; decreased hepatic steatosis, inflammation, and macrophage infiltration; and decreased fatty acid synthase, tumor necrosis factor α (TNFα), monocyte chemoattractant protein‐1 (MCP‐1), interleukin (IL)‐18, and IL‐6 expression. Remarkably, disruption of Alox15 attenuated glucose intolerance and high‐fat diet‐induced insulin resistance, up‐regulated insulin receptor substrate‐2, and exerted opposite effects on hepatic c‐Jun amino‐terminal kinase and adenosine monophosphate–activated protein kinase phosphorylation, known negative and positive regulators of insulin signaling, respectively. In adipose tissue, the absence of Alox15 induced significant reductions in the expression of the proinflammatory and insulin‐resistant adipokines MCP‐1, TNFα, and resistin while increasing the expression of glucose transporter‐4. Interestingly, compared with ApoE−/− mice, which exhibited increased hepatic caspase‐3 staining, ApoE−/−/12/15‐LO−/− mice showed attenuated hepatocellular injury. Consistent with this finding, hepatocytes isolated from ApoE−/− mice were more vulnerable to TNFα‐induced programmed cell death, an effect that was not observed in hepatocytes carrying a targeted disruption of the Alox15 gene. Conclusion: Collectively, our data suggest a potentially relevant mechanism linking 12/15‐LO to the promotion of hepatic steatosis, insulin resistance, and inflammation in experimental liver disease of metabolic origin. (HEPATOLOGY 2010)

Protection from hepatic lipid accumulation and inflammation by genetic ablation of 5-lipoxygenase.

Five-lipoxygenase (5-LO) has been postulated as a pathogenic factor in liver injury. Indeed, Alox5, the gene coding for 5-LO, is heavily over-expressed in experimental liver disease, in which 5-LO inhibition consistently ameliorates hepatic steatosis, inflammation and fibrosis. Herein, we report the findings in mice with targeted deletion of Alox5 as a proof of concept of the role of 5-LO in liver injury. Our findings demonstrate that ablation of Alox5 in mice confers protection against carbon tetrachloride-induced liver injury since hepatic necroinflammation, inflammatory infiltrate, hepatocyte ballooning and serum ALT levels were significantly reduced in Alox5-deficient mice. These mice also showed a lower degree of hepatic steatosis, which affected micro- and macrosteatosis to a similar extent. Moreover, microarray analysis revealed a differential profile of hepatic gene expression in Alox5-deficient mice, with a total of 117 genes differentially expressed in these animals. Functional grouping of these genes revealed that 28 (approximately 24% of total changes) were related to the category of lipid metabolism, including the lipogenic factors Lpin1, C/EBP, Fasn, Acly and Elovl6. Moreover, Ingenuity Pathway Analysis revealed lipid metabolism as the molecular/cellular function most affected by the loss of Alox5. These findings confirm at a genetic level that Alox5 plays a pathogenic role in the response of the liver to injury.

Mecanismos básicos de lesión hepatocelular. Papel de los mediadores lipídicos de inflamación

Resumen La presencia de lesion en el parenquima celular es comun a un gran numero de enfermedades cronicas del higado, como por ejemplo las hepatitis virales, la hepatitis alcoholica, las colestasis cronicas o la esteatohepatitis. Aunque la patogenia puede variar segun el agente etiologico, hay una serie de mecanismos comunes a todas ellas. Entre estos mecanismos destacan la activacion de las celulas de Kupffer y el reclutamiento de celulas inflamatorias, la formacion de radicales libres del oxigeno y la aparicion de estres oxidativo, la produccion de citocinas, principalmente del factor de necrosis tumoral alfa y el factor de crecimiento transformante beta, y la liberacion de mediadores de inflamacion derivados de la oxidacion del acido araquidonico a traves de la ciclooxigenasa 2 y la 5-lipooxigenasa.