Pharmacologic IRE1/XBP1s activation promotes systemic adaptive remodeling in obesity.

In obesity, signaling through the IRE1 arm of the unfolded protein response exerts both protective and harmful effects. Overexpression of the IRE1-regulated transcription factor XBP1s in liver or fat protects against obesity-linked metabolic deterioration. However, hyperactivation of IRE1 engages regulated IRE1-dependent decay (RIDD) and TRAF2/JNK pro-inflammatory signaling, which accelerate metabolic dysfunction. These pathologic IRE1-regulated processes have hindered efforts to pharmacologically harness the protective benefits of IRE1/XBP1s signaling in obesity-linked conditions. Here, we report the effects of a XBP1s-selective pharmacological IRE1 activator, IXA4, in diet-induced obese (DIO) mice. IXA4 transiently activates protective IRE1/XBP1s signaling in liver without inducing RIDD or TRAF2/JNK signaling. IXA4 treatment improves systemic glucose metabolism and liver insulin action through IRE1-dependent remodeling of the hepatic transcriptome that reduces glucose production and steatosis. IXA4-stimulated IRE1 activation also enhances pancreatic function. Our findings indicate that systemic, transient activation of IRE1/XBP1s signaling engenders multi-tissue benefits that integrate to mitigate obesity-driven metabolic dysfunction.

Pharmacologic targeting of plasma cell endoplasmic reticulum proteostasis to reduce amyloidogenic light chain secretion.

Light chain (LC) amyloidosis (AL) involves the toxic aggregation of amyloidogenic immunoglobulin LCs secreted from a clonal expansion of diseased plasma cells. Current AL treatments use chemotherapeutics to ablate the AL plasma cell population. However, no treatments are available that directly reduce the toxic LC aggregation involved in AL pathogenesis. An attractive strategy to reduce toxic LC aggregation in AL involves enhancing endoplasmic reticulum (ER) proteostasis in plasma cells to reduce the secretion and subsequent aggregation of amyloidogenic LCs. Here, we show that the ER proteostasis regulator compound 147 reduces secretion of an amyloidogenic LC as aggregation-prone monomers and dimers in AL patient-derived plasma cells. Compound 147 was established to promote ER proteostasis remodeling by activating the ATF6 unfolded protein response signaling pathway through a mechanism involving covalent modification of ER protein disulfide isomerases (PDIs). However, we show that 147-dependent reductions in amyloidogenic LCs are independent of ATF6 activation. Instead, 147 reduces amyloidogenic LC secretion through the selective, on-target covalent modification of ER proteostasis factors, including PDIs, revealing an alternative mechanism by which this compound can influence ER proteostasis of amyloidogenic proteins. Importantly, compound 147 does not interfere with AL plasma cell toxicity induced by bortezomib, a standard chemotherapeutic used to ablate the underlying diseased plasma cells in AL. This shows that pharmacologic targeting of ER proteostasis through selective covalent modification of ER proteostasis factors is a strategy that can be used in combination with chemotherapeutics to reduce the LC toxicity associated with AL pathogenesis.

Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic Protein.

Activation of the unfolded protein response (UPR)-associated transcription factor ATF6 has emerged as a promising strategy to reduce the secretion and subsequent toxic aggregation of destabilized, amyloidogenic proteins implicated in systemic amyloid diseases. However, the molecular mechanism by which ATF6 activation reduces the secretion of amyloidogenic proteins remains poorly defined. We employ a quantitative interactomics platform to define how ATF6 activation reduces secretion of a destabilized, amyloidogenic immunoglobulin light chain (LC) associated with light-chain amyloidosis (AL). Using this platform, we show that ATF6 activation increases the targeting of this destabilized LC to a subset of pro-folding ER proteostasis factors that retains the amyloidogenic LC within the ER, preventing its secretion. Our results define a molecular basis for the ATF6-dependent reduction in destabilized LC secretion and highlight the advantage for targeting this UPR-associated transcription factor to reduce secretion of destabilized, amyloidogenic proteins implicated in AL and related systemic amyloid diseases.

Frontline Science: Specialized proresolving lipid mediators inhibit the priming and activation of the macrophage NLRP3 inflammasome.

The prototypic proinflammatory cytokine IL-1ß plays a central role in innate immunity and inflammatory disorders. The formation of mature IL-1ß from an inactive pro-IL-1ß precursor is produced via nonconventional multiprotein complexes called the inflammasomes, of which the most common is the nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3) inflammasome composed by NLRP3, (ASC) apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD), and caspase-1. Specialized proresolving mediators (SPMs) promote resolution of inflammation, which is an essential process to maintain host health. SPMs prevent excessive inflammation by terminating the inflammatory response and returning to tissue homeostasis without immunosupression. This study tested the hypothesis that modulation of the NLRP3 inflammasome in macrophages is one mechanism involved in the SPM-regulated processes during resolution. Our findings demonstrate that the SPM resolvin D2 (RvD2) suppressed the expression of pro-IL-1ß and reduced the secretion of mature IL-1ß in bone marrow-derived macrophages challenged with LPS+ATP (classical NLRP3 inflammasome model) or LPS+palmitate (lipotoxic model). Similar findings were observed in thioglycolate-elicited peritoneal macrophages, in which RvD2 remarkably reduced ASC oligomerization, inflammasome assembly, and caspase-1 activity. In vivo, in a self-resolving zymosan A-induced peritonitis model, RvD2 blocked the NLRP3 inflammasome leading to reduced release of IL-1ß into the exudates, repression of osteopontin, and MCP-1 expression and induction of M2 markers of resolution (i.e., CD206 and arginase-1) in peritoneal macrophages. RvD2 inhibitory actions were receptor mediated and were abrogated by a selective GPR18 antagonist. Together, these findings support the hypothesis that SPMs have the ability to inhibit the priming and to expedite the deactivation of the NLRP3 inflammasome in macrophages during the resolution process.

The soluble guanylate cyclase stimulator IW-1973 prevents inflammation and fibrosis in experimental non-alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation and fibrosis. Soluble guanylate cyclase (sGC) stimulation reduces inflammation and fibrosis in experimental models of lung, kidney and heart disease. Here, we tested whether sGC stimulation is also effective in experimental NASH. EXPERIMENTAL APPROACH: NASH was induced in mice by feeding a choline-deficient, l-amino acid-defined, high-fat diet. These mice received either placebo or the sGC stimulator IW-1973 at two different doses (1 and 3 mg·kg-1 ·day-1 ) for 9 weeks. IW-1973 was also tested in high-fat diet (HFD)-induced obese mice. Steatosis, inflammation and fibrosis were assessed by Oil Red O, haematoxylin-eosin, Masson's trichrome, Sirius Red, F4/80 and α-smooth muscle actin staining. mRNA expression was assessed by quantitative PCR. Levels of IW-1973, cytokines and cGMP were determined by LC-MS/MS, Luminex and enzyme immunoassay respectively. KEY RESULTS: Mice with NASH showed reduced cGMP levels and sGC expression, increased steatosis, inflammation, fibrosis, TNF-α and MCP-1 levels and up-regulated collagen types I α1 and α2, MMP2, TGF-ß1 and tissue metallopeptidase inhibitor 1 expression. IW-1973 restored hepatic cGMP levels and sGC expression resulting in a dose-dependent reduction of hepatic inflammation and fibrosis. IW-1973 levels were ≈40-fold higher in liver tissue than in plasma. IW-1973 also reduced hepatic steatosis and adipocyte hypertrophy secondary to enhanced autophagy in HFD-induced obese mice. CONCLUSIONS AND IMPLICATIONS: Our data indicate that sGC stimulation prevents hepatic steatosis, inflammation and fibrosis in experimental NASH. These findings warrant further evaluation of IW-1973 in the clinical setting.

Association of a variant in the gene encoding for ERV1/ChemR23 with reduced inflammation in visceral adipose tissue from morbidly obese individuals.

Obesity comorbidities are closely associated with chronic low-grade adipose tissue inflammation. A number of SNPs associated with inflammation has been identified, underscoring the impact of genetic determinants on this process. Here, we screened SNPs in genes with pro-inflammatory (IL-1ß, IL-6, STAT3 and JAK2), anti-inflammatory (IL-10 and SOCS3) and pro-resolving (ERV1/ChemR23) properties in 101 obese and 99 non-obese individuals. Among the SNPs analyzed, we identified that individuals carrying a C allele in the rs1878022 polymorphism of the ERV1/ChemR23 gene, which encodes for the receptor of the pro-resolving mediator RvE1, had increased ERV1/ChemR23 protein expression and reduced levels of the inflammatory cytokine IL-6 in adipose tissue. Moreover, patients carrying the C allele in homozygosity had lower plasma levels of IL-6, IFN-α2, IL-15, IL-1ra, IL-10, GM-CSF, G-CSF and VEGF and enhanced leukocyte responsiveness to RvE1. C-carriers also exhibited decreased TAG to HDL ratio, a surrogate marker of insulin resistance and a predictor of incident fatty liver. Finally, we confirmed in vivo that the ERV1/ChemR23 receptor regulates systemic and tissue inflammation since mice lacking ERV1/ChemR23 expression showed increased IL-6 levels in adipose tissue and peritoneal macrophages. Together, our study identified an ERV1/ChemR23 variant that protects patients with obesity from excessive inflammatory burden.

The specialized proresolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are hallmarks of nonalcoholic fatty liver disease (NAFLD), which is the hepatic manifestation of the metabolic syndrome associated with obesity. The specialized proresolving lipid mediator maresin 1 (MaR1) preserves tissue homeostasis by exerting cytoprotective actions, dampening inflammation, and expediting its timely resolution. Here, we explored whether MaR1 protects liver cells from lipotoxic and hypoxia-induced ER stress. Mice were rendered obese by high-fat diet feeding, and experiments were performed in primary hepatocytes, Kupffer cells, and precision-cut liver slices (PCLSs). Palmitate-induced lipotoxicity increased ER stress and altered autophagy in hepatocytes, effects that were prevented by MaR1. MaR1 protected hepatocytes against lipotoxicity-induced apoptosis by activating the UPR prosurvival mechanisms and preventing the excessive up-regulation of proapoptotic pathways. Protective MaR1 effects were also seen in hepatocytes challenged with hypoxia and TNF-α-induced cell death. High-throughput microRNA (miRNA) sequencing revealed that MaR1 actions were associated with specific miRNA signatures targeting both protein folding and apoptosis. MaR1 also prevented lipotoxic-triggered ER stress and hypoxia-induced inflammation in PCLSs and enhanced Kupffer cell phagocytic capacity. Together, these findings describe the ability of MaR1 to oppose ER stress in liver cells under conditions frequently encountered in NAFLD.-Rius, B., Duran-Güell, M., Flores-Costa, R., López-Vicario, C., Lopategi, A., Alcaraz-Quiles, J., Casulleras, M., Lozano, J. J., Titos, E., Clària, J. The specialized proresolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress.

Pro-resolving actions of SPM in adipose tissue biology.

White adipose tissue plays an important endocrine role in balancing metabolic homeostasis. During conditions of nutrient excess, as occurs in obesity, there is an expansion of adipose tissue mass associated with a state of "low-grade" inflammation in this tissue. This chronic, unresolved inflammation of adipose tissue is deleterious and leads to many pathological sequelae associated with obesity including insulin resistance, type 2 diabetes and non-alcoholic fatty liver disease. Recently, a novel genus of anti-inflammatory and pro-resolving lipid mediators endogenously generated from membrane phospholipid-derived polyunsaturated fatty acids has been identified during the resolution phase of acute inflammation. These molecules have been termed specialized pro-resolving mediators and act not only as "stop-signals" of inflammatory response but also as facilitators of timely resolution of inflammation. In this review, we provide a comprehensive description of the role of these specialized pro-resolving mediators as endogenous counter-regulators of the persistent inflammatory status present in white adipose tissue of obese individuals. In addition, we discuss the potentiality of these molecules as a novel therapy for the prevention of metabolic co-morbidities associated with obesity.

Polymorphisms in the IL-1 gene cluster influence systemic inflammation in patients at risk for acute-on-chronic liver failure.

Acute-on-chronic liver failure (ACLF) in cirrhosis is an increasingly recognized syndrome characterized by acute decompensation, organ failure(s) and high short-term mortality. Recent findings suggest that an overexuberant systemic inflammation plays a primary role in ACLF progression. In this study, we examined whether genetic factors shape systemic immune responses in patients with decompensated cirrhosis. Six single-nucleotide polymorphisms (SNPs) in inflammation-related genes (interleukin [IL]-1 beta [IL-1ß], rs1143623; IL-1 receptor antagonist [IL-1ra], rs4251961; IL-10, rs1800871; suppressor of cytokine signaling-3, rs4969170; nucleotide-binding oligomerization domain-containing protein 2, rs3135500; and chemerin chemokine-like receptor 1, rs1878022) were genotyped in 279 patients with cirrhosis with (n = 178) and without (n = 101) ACLF from the CANONIC study of the CLIF consortium. Among these SNPs, we identified two polymorphisms belonging to the IL-1 gene cluster (IL-1ß and IL-1ra) in strong association with ACLF. Both SNPs were protective against ACLF; IL-1ß (odds ratio [OR], 0.34, 95% confidence interval [CI], 0.13-0.89; P < 0.05) and IL-1ra (OR, 0.58; 95% CI, 0.35-0.95; P < 0.05) under the recessive and overdominant inheritance models, respectively. These protective SNPs translated into reduced circulating levels of IL-1ß, IL-1α, IL-6, granulocyte-colony stimulating factor, granulocyte-macrophage colony-stimulating factor, and C-reactive protein at enrollment as well as after 7-14 days of admission. These findings were confirmed in vitro in leukocytes incubated with plasma from patients with decompensated cirrhosis carrying the protective SNP genotypes. Notably, a higher frequency of the protective genotypes was observed in patients without (80%) than in those with (20%) ACLF. Consistently, patients carrying the combined protective genotypes showed a lower 28-day mortality rate. CONCLUSION: These data identify two common functional polymorphisms in the IL-1 gene cluster, which are associated with the inflammatory process related to development of ACLF. (Hepatology 2017;65:202-216).

Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein Secreted from Mammalian Cells.

Transthyretin (TTR) is a tetrameric serum protein associated with multiple systemic amyloid diseases. In these disorders, TTR aggregates in extracellular environments through a mechanism involving rate-limiting dissociation of the tetramer to monomers, which then misfold and aggregate into soluble oligomers and amyloid fibrils that induce toxicity in distal tissues. Using an assay established herein, we show that highly destabilized, aggregation-prone TTR variants are secreted as both native tetramers and non-native conformations that accumulate as high-molecular-weight oligomers. Pharmacologic chaperones that promote endoplasmic reticulum (ER) proteostasis of destabilized TTR variants increase their fraction secreted as a tetramer and reduce extracellular aggregate populations. In contrast, disrupting ER proteostasis reduces the fraction of destabilized TTR secreted as a tetramer and increases extracellular aggregates. These results identify ER proteostasis as a factor that can affect conformational integrity and thus toxic aggregation of secreted amyloidogenic proteins associated with the pathology of protein aggregation diseases.

Signaling and Immunoresolving Actions of Resolvin D1 in Inflamed Human Visceral Adipose Tissue.

Persistent activation of the innate immune system greatly influences the risk for developing metabolic complications associated with obesity. In this study, we explored the therapeutic potential of the specialized proresolving mediator (SPM) resolvin D1 (RvD1) to actively promote the resolution of inflammation in human visceral adipose tissue from obese (Ob) patients. Using liquid chromatography-tandem mass spectrometry-based metabololipidomic analysis, we identified unbalanced production of SPMs (i.e., D- and E-series resolvins, protectin D1, maresin 1, and lipoxins) with respect to inflammatory lipid mediators (i.e., leukotriene B4 and PGs) in omental adipose tissue from Ob patients. In parallel, high-throughput transcriptomic analysis revealed a unique signature in this tissue that was characterized by overactivation of the IL-10 signaling pathway. Incubation of inflamed Ob visceral adipose tissues and human macrophages with RvD1 limited excessive activation of the IL-10 pathway by reducing phosphorylation of STAT proteins. Of interest, RvD1 blocked STAT-1 and its target inflammatory genes (i.e., CXCL9), as well as persistent STAT3 activation, without affecting the IL-10 anti-inflammatory response characterized by inhibition of IL-6, IL-1ß, IL-8, and TNF-α. Furthermore, RvD1 promoted resolution by enhancing expression of the IL-10 target gene heme oxygenase-1 by mechanisms dependent on p38 MAPK activity. Together, our data show that RvD1 can tailor the quantitative and qualitative responses of human inflamed adipose tissue to IL-10 and provide a mechanistic basis for the immunoresolving actions of RvD1 in this tissue. These findings may have potential therapeutic implications in obesity-related insulin resistance and other metabolic complications.

Prostaglandin E2 Exerts Multiple Regulatory Actions on Human Obese Adipose Tissue Remodeling, Inflammation, Adaptive Thermogenesis and Lipolysis.

Obesity induces white adipose tissue (WAT) dysfunction characterized by unremitting inflammation and fibrosis, impaired adaptive thermogenesis and increased lipolysis. Prostaglandins (PGs) are powerful lipid mediators that influence the homeostasis of several organs and tissues. The aim of the current study was to explore the regulatory actions of PGs in human omental WAT collected from obese patients undergoing laparoscopic bariatric surgery. In addition to adipocyte hypertrophy, obese WAT showed remarkable inflammation and total and pericellular fibrosis. In this tissue, a unique molecular signature characterized by altered expression of genes involved in inflammation, fibrosis and WAT browning was identified by microarray analysis. Targeted LC-MS/MS lipidomic analysis identified increased PGE2 levels in obese fat in the context of a remarkable COX-2 induction and in the absence of changes in the expression of terminal prostaglandin E synthases (i.e. mPGES-1, mPGES-2 and cPGES). IPA analysis established PGE2 as a common top regulator of the fibrogenic/inflammatory process present in this tissue. Exogenous addition of PGE2 significantly reduced the expression of fibrogenic genes in human WAT explants and significantly down-regulated Col1α1, Col1α2 and αSMA in differentiated 3T3 adipocytes exposed to TGF-ß. In addition, PGE2 inhibited the expression of inflammatory genes (i.e. IL-6 and MCP-1) in WAT explants as well as in adipocytes challenged with LPS. PGE2 anti-inflammatory actions were confirmed by microarray analysis of human pre-adipocytes incubated with this prostanoid. Moreover, PGE2 induced expression of brown markers (UCP1 and PRDM16) in WAT and adipocytes, but not in pre-adipocytes, suggesting that PGE2 might induce the trans-differentiation of adipocytes towards beige/brite cells. Finally, PGE2 inhibited isoproterenol-induced adipocyte lipolysis. Taken together, these findings identify PGE2 as a regulator of the complex network of interactions driving uncontrolled inflammation and fibrosis and impaired adaptive thermogenesis and lipolysis in human obese visceral WAT.

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.

Pro-resolving mediators produced from EPA and DHA: Overview of the pathways involved and their mechanisms in metabolic syndrome and related liver diseases.

A novel genus of pro-resolving lipid mediators endogenously generated from omega-3 polyunsaturated fatty acids has been identified in exudates obtained during the resolution phase of acute inflammation. The term specialized pro-resolving mediators (SPM) has been coined for these lipid mediators, comprising four novel chemical mediator families designated resolvins of the E series (if derived from eicosapentaenoic acid) and resolvins of the D series, protectins and maresins (if generated from docosahexaenoic acid). These SPM act not only as "stop-signals" of inflammatory response, but also as facilitators of the ability of macrophages to clear apoptotic cells (efferocytosis) and migrate to peripheral lymph nodes (efflux), thus, expediting their removal from sites of inflammation. In this review, we provide an overview of the current efforts to elucidate the structure-function, biosynthesis and actions of these omega-3-derived SPM in the context of inflammatory diseases. We specifically highlight the role of these SPM as endogenous counter-regulators of the persistent inflammatory status present in adipose tissue of obese individuals and describe the potential therapeutic impact of these bioactive lipid autacoids on the prevention of hepatic co-morbidities associated with obesity and the metabolic syndrome.

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

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.

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-1ß, 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.

Coordinate functional regulation between microsomal prostaglandin E synthase-1 (mPGES-1) and peroxisome proliferator-activated receptor γ (PPARγ) in the conversion of white-to-brown adipocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.

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-1ß 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 Masson's 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.

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.