Esther Titos

The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARγ activation

The importance of inflammation in initiating the sequence of events that lead to liver fibrosis is increasingly recognized. In this study, we tested the effects of SC‐236, a selective cyclooxygenase (COX)‐2 inhibitor, in rats with carbon tetrachloride (CCl4)‐induced liver fibrosis. Livers from CCl4‐treated rats showed increased COX‐2 expression and 15‐deoxy‐prostaglandin (PG)J2 (15d‐PGJ2) formation, as well as decreased peroxisome proliferator‐activated receptor (PPAR)γ expression. In these animals, SC‐236 reduced liver fibrosis as revealed by histological analysis and by a reduction in hepatic hydroxyproline levels, metalloproteinase‐2 activity, and α‐smooth muscle actin expression. Interestingly, SC‐236 normalized 15d‐PGJ2 levels and restored PPARγ expression in the liver of CCl4‐treated rats. In isolated hepatic stellate cells (HSCs)—the major player in liver fibrogenesis—and Kupffer cells—the cell type primarily responsible for increased hepatic COX‐2—SC‐236 exhibited remarkable pro‐apoptotic and growth inhibitory properties. Of interest, SC‐236 decreased HSC viability to a similar extent than the PPARγ ligand rosiglitazone. Moreover, SC‐236 significantly induced PPARγ expression in HSCs and acted as a potent PPARγ agonist in a luciferase‐reporter trans‐activation assay. These data indicate that, by mechanisms involving non‐parenchymal cell apoptosis and PPARγ activation, the selective COX‐2 inhibitor SC‐236 might have therapeutic potential for prevention of liver fibrosis.

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)

Resolvin D1 and Its Precursor Docosahexaenoic Acid Promote Resolution of Adipose Tissue Inflammation by Eliciting Macrophage Polarization toward an M2-Like Phenotype

We recently demonstrated that ω-3-polyunsaturated fatty acids ameliorate obesity-induced adipose tissue inflammation and insulin resistance. In this study, we report novel mechanisms underlying ω-3-polyunsaturated fatty acid actions on adipose tissue, adipocytes, and stromal vascular cells (SVC). Inflamed adipose tissue from high-fat diet-induced obese mice showed increased F4/80 and CD11b double-positive macrophage staining and elevated IL-6 and MCP-1 levels. Docosahexaenoic acid (DHA; 4 μg/g) did not change the total number of macrophages but significantly reduced the percentage of high CD11b/high F4/80-expressing cells in parallel with the emergence of low-expressing CD11b/F4/80 macrophages in the adipose tissue. This effect was associated with downregulation of proinflammatory adipokines in parallel with increased expression of IL-10, CD206, arginase 1, resistin-like molecule α, and chitinase-3 like protein, indicating a phenotypic switch in macrophage polarization toward an M2-like phenotype. This shift was confined to the SVC fraction, in which secretion of Th1 cytokines (IL-6, MCP-1, and TNF-α) was blocked by DHA. Notably, resolvin D1, an anti-inflammatory and proresolving mediator biosynthesized from DHA, markedly attenuated IFN-γ/LPS-induced Th1 cytokines while upregulating arginase 1 expression in a concentration-dependent manner. Resolvin D1 also stimulated nonphlogistic phagocytosis in adipose SVC macrophages by increasing both the number of macrophages containing ingested particles and the number of phagocytosed particles and by reducing macrophage reactive oxygen species production. No changes in adipocyte area and the phosphorylation of hormone-sensitive lipase, a rate-limiting enzyme regulating adipocyte lipolysis, were observed. These findings illustrate novel mechanisms through which resolvin D1 and its precursor DHA confer anti-inflammatory and proresolving actions in inflamed adipose tissue.

Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts

Peroxisome proliferator‐activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ over‐expression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre‐loxP technology to generate hepatocyte (PPARγΔhep)‐ and macrophage (PPARγΔmac)‐specific PPARγ‐knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high‐fat diet‐induced hepatic steatosis. Down‐regulated expression of genes involved in lipogenesis (SCD1, SREBP‐1c, and ACC), lipid transport (CD36/FAT, L‐FABP, and MTP), and β‐oxidation (PPARα and ACO) was observed in PPARγΔhep mice. Moreover, PPARγΔhep mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision‐cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγΔhep mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγΔmac mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease.—Morán‐Salvador, E., López‐Parra, M., García‐Alonso, V., Titos, E., Martínez‐Clemente, M., González‐Périz, A., López‐Vicario, C., Barak, Y., Arroyo, V., Clària, J. Role for PPARγ in obesity‐induced hepatic steatosis as determined by hepatocyte‐ and macrophage‐specific conditional knockouts. FASEB J. 25, 2538–2550 (2011). www.fasebj.org

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.

Systemic inflammation in decompensated cirrhosis: Characterization and role in acute-on-chronic liver failure

Acute‐on‐chronic liver failure (ACLF) in cirrhosis is characterized by acute decompensation (AD), organ failure(s), and high short‐term mortality. Recently, we have proposed (systemic inflammation [SI] hypothesis) that ACLF is the expression of an acute exacerbation of the SI already present in decompensated cirrhosis. This study was aimed at testing this hypothesis and included 522 patients with decompensated cirrhosis (237 with ACLF) and 40 healthy subjects. SI was assessed by measuring 29 cytokines and the redox state of circulating albumin (HNA2), a marker of systemic oxidative stress. Systemic circulatory dysfunction (SCD) was estimated by plasma renin (PRC) and copeptin (PCC) concentrations. Measurements were performed at enrollment (baseline) in all patients and sequentially during hospitalization in 255. The main findings of this study were: (1) Patients with AD without ACLF showed very high baseline levels of inflammatory cytokines, HNA2, PRC, and PCC. Patients with ACLF showed significantly higher levels of these markers than those without ACLF; (2) different cytokine profiles were identified according to the type of ACLF precipitating event (active alcoholism/acute alcoholic hepatitis, bacterial infection, and others); (3) severity of SI and frequency and severity of ACLF at enrollment were strongly associated. The course of SI and the course of ACLF (improvement, no change, or worsening) during hospitalization and short‐term mortality were also strongly associated; and (4) the strength of association of ACLF with SI was higher than with SCD. Conclusion: These data support SI as the primary driver of ACLF in cirrhosis. (Hepatology 2016;64:1249‐1264).

Inhibition of soluble epoxide hydrolase modulates inflammation and autophagy in obese adipose tissue and liver: Role for omega-3 epoxides

Significance Our study demonstrates that stabilization of cytochrome P-450 epoxides derived from omega-3 polyunsaturated fatty acids through inhibition of the inactivating enzyme soluble epoxide hydrolase (sEH) exerts beneficial actions in counteracting metabolic disorders associated with obesity. In addition, our study sheds more light on the role of sEH in cellular homeostasis by providing evidence that omega-3 epoxides and sEH inhibition regulate autophagy and endoplasmic reticulum stress in insulin-sensitive tissues, especially the liver. Therefore, administration of a sEH inhibitor is a promising strategy to prevent obesity-related comorbidities. Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in a number of diseases that have inflammation as a common underlying cause. sEH limits tissue levels of cytochrome P450 (CYP) epoxides derived from omega-6 and omega-3 polyunsaturated fatty acids (PUFA) by converting these antiinflammatory mediators into their less active diols. Here, we explored the metabolic effects of a sEH inhibitor (t-TUCB) in fat-1 mice with transgenic expression of an omega-3 desaturase capable of enriching tissues with endogenous omega-3 PUFA. These mice exhibited increased CYP1A1, CYP2E1, and CYP2U1 expression and abundant levels of the omega-3–derived epoxides 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic (19,20-EDP) in insulin-sensitive tissues, especially liver, as determined by LC-ESI-MS/MS. In obese fat-1 mice, t-TUCB raised hepatic 17,18-EEQ and 19,20-EDP levels and reinforced the omega-3–dependent reduction observed in tissue inflammation and lipid peroxidation. t-TUCB also produced a more intense antisteatotic action in obese fat-1 mice, as revealed by magnetic resonance spectroscopy. Notably, t-TUCB skewed macrophage polarization toward an antiinflammatory M2 phenotype and expanded the interscapular brown adipose tissue volume. Moreover, t-TUCB restored hepatic levels of Atg12-Atg5 and LC3-II conjugates and reduced p62 expression, indicating up-regulation of hepatic autophagy. t-TUCB consistently reduced endoplasmic reticulum stress demonstrated by the attenuation of IRE-1α and eIF2α phosphorylation. These actions were recapitulated in vitro in palmitate-primed hepatocytes and adipocytes incubated with 19,20-EDP or 17,18-EEQ. Relatively similar but less pronounced actions were observed with the omega-6 epoxide, 14,15-EET, and nonoxidized DHA. Together, these findings identify omega-3 epoxides as important regulators of inflammation and autophagy in insulin-sensitive tissues and postulate sEH as a druggable target in metabolic diseases.

Molecular interplay between Δ5/Δ6 desaturases and long-chain fatty acids in the pathogenesis of non-alcoholic steatohepatitis

Objective The mechanisms underlying non-alcoholic steatohepatitis (NASH) are not completely elucidated. In the current study we integrated gene expression profiling of liver biopsies from NASH patients with translational studies in mouse models of steatohepatitis and pharmacological interventions in isolated hepatocytes to identify new molecular targets in NASH. Design and results Using oligonucleotide microarray analysis we identified a significant enrichment of genes involved in the multi-step catalysis of long-chain polyunsaturated fatty acids, namely, Δ-5 desaturase (Δ5D) and Δ6D in NASH. Increased expression of Δ5D and Δ6D at both mRNA and protein level were confirmed in livers from mice with high-fat diet-induced obesity and NASH. Gas chromatography analysis revealed impaired desaturation fluxes toward the ω-6 and ω-3 pathways resulting in increased ω-6 to ω-3 ratio and reduced ω-3 index in human and mouse fatty livers. Restoration of hepatic ω-3 content in transgenic fat-1 mice expressing an ω-3 desaturase, which allows the endogenous conversion of ω-6 into ω-3 fatty acids, produced a significant reduction in hepatic insulin resistance, steatosis, macrophage infiltration, necroinflammation and lipid peroxidation, accompanied by attenuated expression of genes involved in inflammation, fatty acid uptake and lipogenesis. These results were mostly reproduced by feeding obese mice with an exogenous ω-3-enriched diet. A combined Δ5D/Δ6D inhibitor, CP-24879, significantly reduced intracellular lipid accumulation and inflammatory injury in hepatocytes. Interestingly, CP-24879 exhibited superior antisteatotic and anti-inflammatory actions in fat-1 and ω-3-treated hepatocytes. Conclusions These findings indicate that impaired hepatic fatty acid desaturation and unbalanced ω-6 to ω-3 ratio play a role in the pathogenesis of NASH.

Aspirin (ASA) regulates 5-lipoxygenase activity and peroxisome proliferator-activated receptor α-mediated CINC-1 release in rat liver cells: novel actions of lipoxin A4 (LXA4) and ASA-triggered 15-epi-LXA4

The mechanism of action of aspirin (ASA) is related to cyclooxygenase (COX) inhibition, but additional actions cannot be excluded for their antiinflammatory properties and antithrombotic activity. In the current investigation, we examined the effects of ASA on COX and 5‐lipoxygenase (5‐LO) pathways and its impact on peroxisome proliferator‐activated receptor α (PPARα) and cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1) levels in rat liver cells. In Kupffer cells, the liver resident macrophages, ASA switched eicosanoid biosynthesis from prostaglandin E2 (PGE2) to leukotriene B4 (LTB4) and 15‐epi‐lipoxin A4 (15‐epi‐LXA4) formation. In hepatocytes, ASA significantly inhibited PPARα protein expression and CINC‐1 secretion, effects that were also observed in hepatocytes exposed to the selective PPARα agonist Wy‐14643. In contrast, treatment of hepatocytes with PGE2 in association with LTB4 had no significant effect on PPARα but stimulated CINC‐1 release. Interestingly, the endogenous antiinflammatory eicosanoids LXA4 and ASA‐triggered 15‐epi‐LXA4, in addition to inhibiting macrophage 5‐LO activity to a similar extent as PGE2, significantly reduced PPARα and CINC‐1 levels in hepatocytes. Taken together and because arachidonic acid‐derived products, PPARα levels, and CINC‐1 secretion are involved in the extent and duration of an inflammatory response, these findings provide additional molecular mechanisms for the pharmacological properties of ASA.