Cristina López-Vicario

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

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.

Resolution of inflammation in obesity-induced liver disease

Low-grade inflammation in adipose tissue is recognized as a critical event in the development of obesity-related co-morbidities. This chronic inflammation is powerfully augmented through the infiltration of macrophages, which together with adipocytes, perpetuate a vicious cycle of inflammatory cell recruitment and secretion of free fatty acids and deleterious adipokines that predispose to greater incidence of metabolic complications. In the last decade, many factors have been identified to contribute to mounting unresolved inflammation in obese adipose tissue. Among them, pro-inflammatory lipid mediators (i.e., leukotrienes) derived from the omega-6 polyunsaturated arachidonic acid have been shown to play a prominent role. Of note, the same lipid mediators that initially trigger the inflammatory response also signal its termination by stimulating the formation of anti-inflammatory signals. Resolvins and protectins derived from the omega-3 polyunsaturated docosahexaenoic and eicosapentaenoic acids have emerged as a representative family of this novel class of autacoids with dual anti-inflammatory and pro-resolving properties that act as “stop-signals” of the inflammatory response. This review discusses the participation of these endogenous autacoids in the resolution of adipose tissue inflammation, with a special emphasis in the amelioration of obesity-related metabolic dysfunctions, namely insulin resistance and non-alcoholic fatty liver disease.

Resolvin D1 primes the resolution process initiated by calorie restriction in obesity-induced steatohepatitis

Insulin resistance and nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis combined with inflammation, are major sequelae of obesity. Currently, lifestyle modification (i.e., weight loss) is the first‐line therapy for NASH. However, weight loss resolves steatosis but not inflammation. In this study, we tested the ability of resolvin D1 (RvD1), an anti‐inflammatory and proresolving molecule, to promote the resolution initiated by calorie restriction in obese mice with NASH. Calorie restriction reduced adipose and liver weight (–56 and –13%, respectively; P< 0.001), serum leptin and resistin levels, hepatic steatosis, and insulin resistance. In addition to these, mice receiving RvD1 during the dietary intervention showed increased adiponectin expression at both the mRNA and protein levels and reduced liver macrophage infiltration (–15%, P<0.01). Moreover, RvD1 skewed macrophages from an M1‐ to an M2‐like anti‐inflammatory phenotype, induced a specific hepatic miRNA signature (i.e., miR‐219‐5p and miR‐199a‐5p), and reduced inflammatory adipokine mRNA and protein expression and macrophage innate immune response. In precision‐cut liver slices (PCLSs), which override the influence of circulating factors, RvD1 attenuated hypoxia‐induced mRNA and protein expression of COX‐2, IL‐1β, IL‐6, and CCR7. Of note, RvD1 anti‐inflammatory actions were absent in macrophage‐depleted PCLSs. In summary, RvD1 acts as a facilitator of the hepatic resolution process by reducing the inflammatory component of obesity‐induced NASH.—Rius, B., Titos, E., Morán‐Salvador, E., López‐Vicario, C., García‐Alonso, V., González‐Périz, A., Arroyo, V., Claria, J. Resolvin D1 primes the resolution process initiated by calorie restriction in obesity‐induced steatohepatitis. FASEB J. 28, 836–848 (2014). www.fasebj.org

New Insights into the Role of Macrophages in Adipose Tissue Inflammation and Fatty Liver Disease: Modulation by Endogenous Omega-3 Fatty Acid-Derived Lipid Mediators

Obesity is causally linked to a chronic state of “low-grade” inflammation in adipose tissue. Prolonged, unremitting inflammation in this tissue has a direct impact on insulin-sensitive tissues (i.e., liver) and its timely resolution is a critical step toward reducing the prevalence of related co-morbidities such as insulin resistance and non-alcoholic fatty liver disease. This article describes the current state-of-the-art knowledge and novel insights into the role of macrophages in adipose tissue inflammation, with special emphasis on the progressive changes in macrophage polarization observed over the course of obesity. In addition, this article extends the discussion to the contribution of Kupffer cells, the liver resident macrophages, to metabolic liver disease. Special attention is given to the modulation of macrophage responses by omega-3-PUFAs, and more importantly by resolvins, which are potent anti-inflammatory and pro-resolving autacoids generated from docosahexaenoic and eicosapentaenoic acids. In fact, resolvins have been shown to work as endogenous “stop signals” in inflamed adipose tissue and to return this tissue to homeostasis by inducing a phenotypic switch in macrophage polarization toward a pro-resolving phenotype. Collectively, this article offers new views on the role of macrophages in metabolic disease and their modulation by endogenously generated omega-3-PUFA-derived lipid mediators.

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)

Cell-specific PPARγ deficiency establishes anti-inflammatory and anti-fibrogenic properties for this nuclear receptor in non-parenchymal liver cells.

BACKGROUND & AIMS PPARγ plays an essential role in the transcriptional regulation of genes involved in lipid and glucose metabolism, insulin sensitivity, and inflammation. We recently demonstrated that PPARγ plays a causative role in hepatocyte lipid deposition, contributing to the pathogenesis of hepatic steatosis. In this study, we investigated the role of PPARγ in the inflammatory and fibrogenic response of the liver. METHODS Heterozygous floxed/null Cre/LoxP mice with targeted deletion of PPARγ in either hepatocytes (Alb-Cre), macrophages (LysM-Cre) or hepatic stellate cells (HSCs) (aP2-Cre) were submitted to carbon tetrachloride (CCl4) liver injury. Further analyses were performed in precision-cut liver slices (PCLS) and primary cultures of hepatocytes, macrophages, and HSCs. RESULTS LysM-Cre mice displayed an exacerbated response to chronic CCl4 injury and showed higher necroinflammatory injury, lipid peroxidation, inflammatory infiltrate, cleaved-caspase-3 and caspase 3/7 activity, and COX-2, TNF-α, CXCL2, and IL-1β expression than Alb-Cre and control mice. The deleterious effects of PPARγ disruption in liver macrophages were confirmed in an acute model of CCl4 injury as well as in PCLS incubated with LPS. Moreover, LysM-Cre mice showed an aggravated fibrogenic response to CCl4, as revealed by more prominent Sirius Red and Massons trichrome staining, elevated hydroxyproline content and induced α-SMA and TIMP-1 expression. Importantly, aP2-Cre mice with specific disruption of PPARγ in HSCs, as confirmed by immunocytochemical analysis of individual liver cells, also showed exacerbated liver damage and fibrogenic response to CCl4. CONCLUSIONS These data unveil anti-inflammatory and anti-fibrogenic roles for PPARγ in non-parenchymal liver cells.

Role of bioactive lipid mediators in obese adipose tissue inflammation and endocrine dysfunction.

White adipose tissue is recognized as an active endocrine organ implicated in the maintenance of metabolic homeostasis. However, adipose tissue function, which has a crucial role in the development of obesity-related comorbidities including insulin resistance and non-alcoholic fatty liver disease, is dysregulated in obese individuals. This review explores the physiological functions and molecular actions of bioactive lipids biosynthesized in adipose tissue including sphingolipids and phospholipids, and in particular fatty acids derived from phospholipids of the cell membrane. Special emphasis is given to polyunsaturated fatty acids of the omega-6 and omega-3 families and their conversion to bioactive lipid mediators through the cyclooxygenase and lipoxygenase pathways. The participation of omega-3-derived lipid autacoids in the resolution of adipose tissue inflammation and in the prevention of obesity-associated hepatic complications is also thoroughly discussed.