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Background: NAFLD affects nearly 25% of the global population. Cardiovascular disease (CVD) is the most common cause of death among patients with NAFLD, in line with highly prevalent dyslipidemia in this population. Increased plasma triglyceride (TG)-rich lipoprotein (TRL) concentrations, an important risk factor for CVD, are closely linked with hepatic TG content. Therefore, it is of great interest to identify regulatory mechanisms of hepatic TRL production and remnant uptake in the setting of hepatic steatosis. Approach and Results: To identify liver-regulated pathways linking intrahepatic and plasma TG metabolism, we performed transcriptomic analysis of liver biopsies from two independent cohorts of obese patients. Hepatic encoding apolipoprotein F (APOF) expression showed the fourth-strongest negatively correlation with hepatic steatosis and the strongest negative correlation with plasma TG levels. The effects of adenoviral-mediated human ApoF (hApoF) overexpression on plasma and hepatic TG were assessed in C57BL6/J mice. Surprisingly, hApoF overexpression increased both hepatic very low density lipoprotein (VLDL)-TG secretion and hepatic lipoprotein remnant clearance, associated a similar to 25% reduction in plasma TG levels. Conversely, reducing endogenous ApoF expression reduced VLDL secretion in vivo, and reduced hepatocyte VLDL uptake by similar to 15% in vitro. Transcriptomic analysis of APOF-overexpressing mouse livers revealed a gene signature related to enhanced ApoB-lipoprotein clearance, including increased expression of Ldlr and Lrp1, among others. Conclusion: These data reveal a previously undescribed role for ApoF in the control of plasma and hepatic lipoprotein metabolism by favoring VLDL-TG secretion and hepatic lipoprotein remnant particle clearance.

The alimentary limb has been proposed to be a key driver of the weight-loss-independent metabolic improvements that occur upon bariatric surgery. However, the one anastomosis gastric bypass (OAGB) procedure, consisting of one long biliary limb and a short common limb, induces similar beneficial metabolic effects compared to Roux-en-Y Gastric Bypass (RYGB) in humans, despite the lack of an alimentary limb. The aim of this study was to assess the role of the length of biliary and common limbs in the weight loss and metabolic effects that occur upon OAGB. OAGB and sham surgery, with or without modifications of the length of either the biliary limb or the common limb, were performed in Gottingen minipigs. Weight loss, metabolic changes, and the effects on plasma and intestinal bile acids (BAs) were assessed 15 days after surgery. OAGB significantly decreased body weight, improved glucose homeostasis, increased postprandial GLP-1 and fasting plasma BAs, and qualitatively changed the intestinal BA species composition. Resection of the biliary limb prevented the body weight loss effects of OAGB and attenuated the postprandial GLP-1 increase. Improvements in glucose homeostasis along with changes in plasma and intestinal BAs occurred after OAGB regardless of the biliary limb length. Resection of only the common limb reproduced the glucose homeostasis effects and the changes in intestinal BAs. Our results suggest that the changes in glucose metabolism and BAs after OAGB are mainly mediated by the length of the common limb, whereas the length of the biliary limb contributes to body weight loss. NEW & NOTEWORTHY Common limb mediates postprandial glucose metabolism change after gastric bypass whereas biliary limb contributes to weight loss.

International audience; Background/Objectives: Innate lymphoid cells (ILCs) play an important role in the maintenance of immune and metabolic homeostasis in adipose tissue (AT). The crosstalk between AT ILCs and adipocytes and other immune cells coordinates adipocyte differentiation, beiging, glucose metabolism and inflammation. Although the metabolic and homeostatic functions of mouse ILCs have been extensively investigated, little is known about human adipose ILCs and their roles in obesity and insulin resistance (IR). Subjects/Methods: Here we characterized T and NK cell populations in omental AT (OAT) from women (n=18) with morbid obesity and varying levels of IR and performed an integrated analysis of metabolic parameters and adipose tissue transcriptomics. Results: In OAT, we found a distinct population of CD56-NKp46 + EOMES + NK cells characterized by expression of cytotoxic molecules, pro-inflammatory cytokines, and markers of cell activation. AT IFNγ + NK cells, but not CD4, CD8 or γδ T cells, were positively associated with glucose levels, glycated hemoglobin (HbA1c) and IR. AT NK cells were linked to a pro-inflammatory gene expression profile in AT and developed an effector phenotype in response to IL-12 and IL-15. Moreover, integrated transcriptomic analysis revealed a potential implication of AT IFNγ + NK cells in controlling adipose tissue inflammation, remodeling, and lipid metabolism. Conclusions: Our results suggest that a distinct IFNγ−producing NK cell subset is involved in metabolic homeostasis in visceral AT in humans with obesity and may be a potential target for therapy of IR.

Background The multifactorial nature of non-alcoholic fatty liver disease cannot be explained solely by genetic factors. Recent evidence revealed that DNA methylation changes take place at proximal promoters within susceptibility genes. This emphasizes the need for integrating multiple data types to provide a better understanding of the disease’s pathogenesis. One such candidate gene is paraoxonase-1 (PON1). Substantial interindividual differences in PON1 are apparent and could influence disease risk later in life. The aim of this study was therefore to determine the different regulatory aspects of PON1 variability and to examine them in relation to the predisposition to obesity-associated fatty liver disease. Results A targeted multi-omics approach was applied to investigate the interplay between PON1 genetic variants, promoter methylation, expression profile and enzymatic activity in an adult patient cohort with extensive metabolic and hepatic characterisation including liver biopsy. Alterations in PON1 status were shown to correlate with waist-to-hip ratio and relevant features of liver pathology. Particularly, the regulatory polymorphism rs705379:C > T was strongly associated with more severe liver disease. Multivariable data analysis furthermore indicated a significant association of combined genetic and epigenetic PON1 regulation. This identified relationship postulates a role for DNA methylation as a mediator between PON1 genetics and expression, which is believed to further influence liver disease progression via modifications in PON1 catalytic efficiency. Conclusions Our findings demonstrate that vertical data-integration of genetic and epigenetic regulatory mechanisms generated a more in-depth understanding of the molecular basis underlying the development of obesity-associated fatty liver disease. We gained novel insights into how NAFLD classification and outcome are orchestrated, which could not have been obtained by exclusively considering genetic variation.

Luisa Vonghia and David Dombrowicz equally contributed to the work.; International audience; Progression of fatty liver to non-alcoholic steatohepatitis (NASH) is a rapidly growing health problem. The presence of inflammatory infiltrates in the liver and hepatocyte damage distinguish NASH from simple steatosis. However, the underlying molecular mechanisms involved in the development of NASH remain to be fully understood. Here we perform transcriptional and immune profiling of patients with NASH before and after lifestyle intervention (LSI). Analysis of liver microarray data from a cohort of patients with histologically assessed non-alcoholic fatty liver disease (NAFLD) reveals a hepatic gene signature, which is associated with NASH and is sensitive to regression of NASH activity on LSI independently of body weight loss. Enrichment analysis reveals the presence of immune-associated genes linked to inflammatory responses, antigen presentation and cytotoxic cells in the NASH-linked gene signature. In an independent cohort, NASH is also associated with alterations in blood immune cell populations, including conventional dendritic cells (cDC) type 1 and 2, and cytotoxic CD8 T cells. Lobular inflammation and ballooning are associated with the accumulation of CD8 T cells in the liver. Progression from simple steatosis to NASH in a mouse model of diet-driven NASH results in a comparable immune-related hepatic expression signature and the accumulation of intrahepatic cDC and CD8 T cells. These results show that NASH, compared to normal liver or simple steatosis, is associated with a distinct hepatic immune-related gene signature, elevated hepatic CD8 T cells, and altered antigen-presenting and cytotoxic cells in blood. These findings expand our understanding of NASH and may identify potential targets for NASH therapy.

Background & Aims: Although the role of inflammation to combat infection is known, the contribution of metabolic changes in response to sepsis is poorly understood. Sepsis induces the release of lipid mediators, many of which activate nuclear receptors such as the peroxisome proliferator-activated receptor (PPAR)alpha, which controls both lipid metabolism and inflammation. We aimed to elucidate the previously unknown role of hepatic PPAR alpha in the response to sepsis.Methods: Sepsis was induced by intraperitoneal injection of Escherichia coli in different models of cell-specific PPAR alpha-deficiency and their controls. The systemic and hepatic metabolic response was analyzed using biochemical, transcriptomic and functional assays. PPAR alpha expression was analyzed in livers from elective surgery and critically ill patients and correlated with hepatic gene expression and blood parameters.Results: Both whole body and non-hematopoietic PPAR alpha-deficiency in mice decreased survival upon bacterial infection. Livers of septic PPAR alpha-deficient mice displayed an impaired metabolic shift from glucose to lipid utilization resulting in more severe hypoglycemia, impaired induction of hyperketonemia and increased steatosis due to lower expression of genes involved in fatty acid catabolism and ketogenesis. Hepatocyte-specific deletion of PPAR alpha impaired the metabolic response to sepsis and was sufficient to decrease survival upon bacterial infection. Hepatic PPAR alpha expression was lower in critically ill patients and correlated positively with expression of lipid metabolism genes, but not with systemic inflammatory markers.Conclusion: During sepsis, PPAR alpha-deficiency in hepatocytes is deleterious as it impairs the adaptive metabolic shift from glucose to FA utilization. Metabolic control by PPAR alpha in hepatocytes plays a key role in the host defense against infection.Lay summary: As the main cause of death in critically ill patients, sepsis remains a major health issue lacking efficacious therapies. While current clinical literature suggests an important role for inflammation, metabolic aspects of sepsis have mostly been overlooked. Here, we show that mice with an impaired metabolic response, due to deficiency of the nuclear receptor PPAR alpha in the liver, exhibit enhanced mortality upon bacterial infection despite a similar inflammatory response, suggesting that metabolic interventions may be a viable strategy for improving sepsis outcomes. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Macrophages display heterogeneous phenotypes, including the classical M1 proinflammatory and the alternative M2 anti-inflammatory polarization states. The transducin-like enhancer of split-1 (TLE1) is a transcriptional corepressor whose functions in macrophages have not been studied yet. We report that TLE1 is highly expressed in human alternative macrophages in vitro and in atherosclerotic plaques as well as in adipose tissue M1/M2 mixed macrophages. TLE1 silencing in alternative macrophages decreases the expression of the M2 markers IL-1Ra and IL-10, while it exacerbates TNFα and CCL3 induction by lipopolysaccharide. Hence, TLE1 is expressed in human macrophages where it has potential anti-inflammatory and alternative phenotype promoting properties.

International audience; Adipocyte differentiation and function relies on a network of transcription factors, which is disrupted in obesity-associated low grade, chronic inflammation leading to adipose tissue dysfunction. In this context, there is a need for a thorough understanding of the transcriptional regulatory network involved in adipose tissue pathophysiology. Recent advances in the functional annotation of the genome has highlighted the role of non-coding RNAs in cellular differentiation processes in coordination with transcription factors. Using an unbiased genome-wide approach, we identified and characterized a novel long intergenic non-coding RNA (lincRNA) strongly induced during adipocyte differentiation. This lincRNA favors adipocyte differentiation and coactivates the master adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARγ) through interaction with the paraspeckle component and hnRNP-like RNA binding protein 14 (RBM14/NCoAA), and was therefore called PPARγ-activator RBM14-associated lncRNA (Paral1). Paral1 expression is restricted to adipocytes and decreased in humans with increasing body mass index. A decreased expression was also observed in diet-induced or genetic mouse models of obesity and this down-regulation was mimicked in vitro by TNF treatment. In conclusion, we have identified a novel component of the adipogenic transcriptional regulatory network defining the lincRNA Paral1 as an obesity-sensitive regulator of adipocyte differentiation and function.

The genomic CDKN2A/B locus, encoding p16(INK4a) among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16(INK4a) is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16(INK4a) knock-down in 3T3/L1 preadipocytes or p16(INK4a) deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16(INK4a) in adipose tissue formation. p16(INK4a)-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16(INK4a)-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16(INK4a)-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16(INK4a) gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16(INK4a) modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

Background & Aims Peroxisome proliferator-activated receptors (PPARs) have been implicated in non-alcoholic steatohepatitis (NASH) pathogenesis, mainly based on animal data. Gene expression data in NASH patients are scarce. We studied liver PPARα, β/δ, and γ expression in a large cohort of obese patients assessed for presence of NAFLD at baseline and 1year follow-up. Methods Patients presented to the obesity clinic underwent a hepatic work-up. If NAFLD was suspected, liver biopsy was performed. Gene expression was studied by mRNA quantification. Patients were reassessed after 1year. Results 125 patients were consecutively included in the study, of which 85 patients had paired liver biopsy taken at 1year of follow-up. Liver PPARα expression negatively correlated with the presence of NASH ( p =0.001) and with severity of steatosis ( p =0.003), ballooning ( p =0.001), NASH activity score ( p =0.008) and fibrosis ( p =0.003). PPARα expression was positively correlated to adiponectin (R 2 =0.345, p =0.010) and inversely correlated to visceral fat (R 2 =−0.343, p 2 =−0.411, p 2 =−0.233, p =0.012). Liver PPARβ/δ and PPARγ expression did not correlate with any histological feature nor with glucose metabolism or serum lipids. At 1year, correlation of PPARα expression with liver histology was confirmed. In longitudinal analysis, an increase in expression of PPARα and its target genes was significantly associated with histological improvement ( p =0.008). Conclusion Human liver PPARα gene expression negatively correlates with NASH severity, visceral adiposity and insulin resistance and positively with adiponectin. Histological improvement is associated with an increase in expression of PPARα and its target genes. These data might suggest that PPARα is a potential therapeutic target in NASH.

The genomic CDKN2A/B locus, encoding p16INK4a among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16INK4a is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16INK4a knock-down in 3T3/L1 preadipocytes or p16INK4a deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16INK4a in adipose tissue formation. p16INK4a-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16INK4a-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16INK4a-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16INK4a gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16INK4a modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

Control of gene transcription relies on concomitant regulation by multiple transcriptional regulators (TRs). However, how recruitment of a myriad of TRs is orchestrated at cis-regulatory modules (CRMs) to account for coregulation of specific biological pathways is only partially understood. Here, we have used mouse liver CRMs involved in regulatory activities of the hepatic TR, NR1H4 (FXR; farnesoid X receptor), as our model system to tackle this question. Using integrative cistromic, epigenomic, transcriptomic, and interactomic analyses, we reveal a logical organization where trans-regulatory modules (TRMs), which consist of subsets of preferentially and coordinately corecruited TRs, assemble into hierarchical combinations at hepatic CRMs. Different combinations of TRMs add to a core TRM, broadly found across the whole landscape of CRMs, to discriminate promoters from enhancers. These combinations also specify distinct sets of CRM differentially organized along the genome and involved in regulation of either housekeeping/cellular maintenance genes or liver-specific functions. In addition to these TRMs which we define as obligatory, we show that facultative TRMs, such as one comprising core circadian TRs, are further recruited to selective subsets of CRMs to modulate their activities. TRMs transcend TR classification into ubiquitous versus liver-identity factors, as well as TR grouping into functional families. Hence, hierarchical superimpositions of obligatory and facultative TRMs bring about independent transcriptional regulatory inputs defining different sets of CRMs with logical connection to regulation of specific gene sets and biological pathways. Altogether, our study reveals novel principles of concerted transcriptional regulation by multiple TRs at CRMs.

Objective Hepatic overexpression of sulfatase-2 (SULF2), a heparan sulfate remodeling enzyme, strongly contributes to high triglyceride (TG) levels in obese, type 2 diabetic (T2DM) db/db mice. Nevertheless, data in humans are lacking. Here, the association of human hepatic SULF2 expression and SULF2 gene variants with TG metabolism in patients with obesity and/or T2DM was investigated. Methods Liver biopsies from 121 obese subjects were analyzed for relations between hepatic SULF2 mRNA levels and plasma TG. Associations between seven SULF2 tagSNPs and TG levels were assessed in 210 obese T2DM subjects with dyslipidemia. Replication of positive findings was performed in 1,316 independent obese T2DM patients. Postprandial TRL clearance was evaluated in 29 obese T2DM subjects stratified by SULF2 genotype. Results Liver SULF2 expression was significantly associated with fasting plasma TG (r = 0.271; P = 0.003) in obese subjects. The SULF2 rs2281279(A>G) SNP was reproducibly associated with lower fasting plasma TG levels in obese T2DM subjects (P < 0.05). Carriership of the minor G allele was associated with lower levels of postprandial plasma TG (P < 0.05) and retinyl esters levels (P < 0.001). Conclusions These findings implicate SULF2 as potential therapeutic target in the atherogenic dyslipidemia of obesity and T2DM.

Rationale: Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions. Objective: To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. Methods and Results: Macrophages near calcium deposits display an alternative phenotype being both CD68 and mannose receptor–positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/c-fos–mediated downstream signaling resulting in impaired bone resorption capacity. Conclusions: These results indicate that macrophages surrounding calcium deposits in human atherosclerotic plaques are phenotypically defective being unable to resorb calcification.

Control of gene transcription relies on concomitant regulation by multiple transcriptional regulators (TRs). However, how recruitment of a myriad of TRs is orchestrated at cis-regulatory modules (CRMs) to account for coregulation of specific biological pathways is only partially understood. Here, we have used mouse liver CRMs involved in regulatory activities of the hepatic TR, NR1H4 (FXR; farnesoid X receptor), as our model system to tackle this question. Using integrative cistromic, epigenomic, transcriptomic, and interactomic analyses, we reveal a logical organization where trans-regulatory modules (TRMs), which consist of subsets of preferentially and coordinately corecruited TRs, assemble into hierarchical combinations at hepatic CRMs. Different combinations of TRMs add to a core TRM, broadly found across the whole landscape of CRMs, to discriminate promoters from enhancers. These combinations also specify distinct sets of CRM differentially organized along the genome and involved in regulation of either housekeeping/cellular maintenance genes or liver-specific functions. In addition to these TRMs which we define as obligatory, we show that facultative TRMs, such as one comprising core circadian TRs, are further recruited to selective subsets of CRMs to modulate their activities. TRMs transcend TR classification into ubiquitous versus liver-identity factors, as well as TR grouping into functional families. Hence, hierarchical superimpositions of obligatory and facultative TRMs bring about independent transcriptional regulatory inputs defining different sets of CRMs with logical connection to regulation of specific gene sets and biological pathways. Altogether, our study reveals novel principles of concerted transcriptional regulation by multiple TRs at CRMs.

In the version of this article initially published, ANR grant ANR-16-RHUS-0006 to author Joel T. Haas was not included in the Acknowledgements. The error has been corrected in the HTML and PDF versions of the article.

Peroxisome Proliferator-Activated Receptors (PPAR) are transcription factors suggested to be involved in inflammatory lesions of autoimmune encephalomyelitis and multiple sclerosis (MS). Our objective was to assess whether Natalizumab (NTZ) therapy is associated with alterations of PPAR expression in MS patients. We analyzed gene expression of PPAR in peripheral blood mononuclear cells (PBMC) as well as blood inflammatory markers in women with MS previously medicated with first-line immunomodulators (baseline) and after NTZ therapy. No differences in PPARα, PPARβ/δ, PPARγ, and CD36 mRNA expression were found in PBMC between patients under baseline and healthy controls. At three months, NTZ increased PPARβ/δmRNA (p=0.009) in comparison to baseline, while mRNA expression of PPARγand CD36 (a well-known PPAR target gene) was lower in comparison to healthy controls (p=0.026andp=0.028, resp.). Although these trends of alterations remain after six months of therapy, the results were not statistically significant. Osteopontin levels were elevated in patients (p=0.002) and did not change during the follow-up period of NTZ treatment. These results suggest that PPAR-mediated processes may contribute to the mechanisms of action of NTZ therapy.

International audience; Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

That promising neuroprotectants failed to demonstrate benefit against stroke highlights the great difficulties to translate preclinical pharmacological effects in clinical outcomes. Part of this hurdle implies the complex response to injury of the neurovascular unit increasing the cerebrovascular permeability at the level of the blood-brain barrier (BBB). Previous studies reported neuroprotection in animal models upon activation of the nuclear receptor PPARα (peroxisome proliferator-activated receptor) α, but the cellular targets at the BBB level remain largely unexplored. Here, to study whether PPAR-α activation acts on BBB permeability, we adapted a mouse BBB cell model to ischaemic conditions at the stage of occlusion defined in vitro as oxygen-glucose deprivation (OGD). This model consists of a co-culture of brain capillary endothelial cells (ECs) on a filter insert placed upon a rat glial cell culture. The EC monolayer permeability increase induced by 4 h of OGD was significantly restricted after treatment with the PPAR-α agonist fenofibric acid (FA) 24 h before or at the onset of OGD. Treatments of separated ECs or glial cells showed that this protective effect was conferred by BBB ECs but not glial cells. Furthermore, co-cultures with ECs from PPAR-α-deficient mice revealed that FA had no effect on OGD-induced hyperpermeability. No transcriptional modulation of classical PPAR-α target genes such as SOD, ICAM-1, VCAM-1, ACO, CPT-1, PDK-4 or ET-1 was observed in wild type mouse ECs. In conclusion, these results suggest that part of the preventive PPAR-α-mediated protection may occur via BBB ECs by limiting hyperpermeability.

Coronary artery disease (CAD) is a major cause of morbidity and mortality. Mutations in C6ORF105, associated with decreased gene expression, positively correlate with the risk of CAD in Chinese populations. Moreover, the C6ORF105-encoded protein may play a role in coagulation. Here, we report that C6ORF105 gene expression is lower in circulating mononuclear cells from obese diabetic than lean subjects. Moreover, C6ORF105 is expressed in human macrophages and atherosclerotic lesions, where its expression positively correlates with expression of the transcription factor Peroxisome Proliferator-Activated Receptor (PPAR)γ. Activation of PPARγ increases, in a PPARγ-dependent manner, the expression of C6ORF105 in human macrophages and atherosclerotic lesions.

Rationale: In atherosclerotic plaques, iron preferentially accumulates in macrophages where it can exert pro-oxidant activities. Objective: The objective of this study was, first, to better characterize the iron distribution and metabolism in macrophage subpopulations in human atherosclerotic plaques and, second, to determine whether iron homeostasis is under the control of nuclear receptors, such as the liver X receptors (LXRs). Methods and Results: Here we report that iron depots accumulate in human atherosclerotic plaque areas enriched in CD68 and mannose receptor (MR)-positive (CD68 + MR + ) alternative M2 macrophages. In vitro IL-4 polarization of human monocytes into M2 macrophages also resulted in a gene expression profile and phenotype favoring iron accumulation. However, M2 macrophages on iron exposure acquire a phenotype favoring iron release, through a strong increase in ferroportin expression, illustrated by a more avid oxidation of extracellular low-density lipoprotein by iron-loaded M2 macrophages. In line, in human atherosclerotic plaques, CD68 + MR + macrophages accumulate oxidized lipids, which activate LXRα and LXRβ, resulting in the induction of ABCA1, ABCG1, and apolipoprotein E expression. Moreover, in iron-loaded M2 macrophages, LXR activation induces nuclear factor erythroid 2-like 2 expression, thereby increasing ferroportin expression, which, together with a decrease of hepcidin mRNA levels, promotes iron export. Conclusions: These data identify a role for M2 macrophages in iron handling, a process regulated by LXR activation.

Fibrates and glitazones are two classes of drugs currently used in the treatment of dyslipidemia and insulin resistance (IR), respectively. Whereas glitazones are insulin sensitizers acting via activation of the peroxisome proliferator-activated receptor (PPAR) gamma subtype, fibrates exert their lipid-lowering activity via PPARalpha. To determine whether PPARalpha activators also improve insulin sensitivity, we measured the capacity of three PPARalpha-selective agonists, fenofibrate, ciprofibrate, and the new compound GW9578, in two rodent models of high fat diet-induced (C57BL/6 mice) or genetic (obese Zucker rats) IR. At doses yielding serum concentrations shown to activate selectively PPARalpha, these compounds markedly lowered hyperinsulinemia and, when present, hyperglycemia in both animal models. This effect relied on the improvement of insulin action on glucose utilization, as indicated by a lower insulin peak in response to intraperitoneal glucose in ciprofibrate-treated IR obese Zucker rats. In addition, fenofibrate treatment prevented high fat diet-induced increase of body weight and adipose tissue mass without influencing caloric intake. The specificity for PPARalpha activation in vivo was demonstrated by marked alterations in the expression of PPARalpha target genes, whereas PPARgamma target gene mRNA levels did not change in treated animals. These results indicate that compounds with a selective PPARalpha activation profile reduce insulin resistance without having adverse effects on body weight and adipose tissue mass in animal models of IR.

Plasma fibrinogen levels have been identified as an important risk factor for cardiovascular diseases. Among the few compounds known to lower circulating fibrinogen levels in humans are certain fibrates. We have studied the regulation of fibrinogen gene expression by fibrates in rodents. Treatment of adult male rats with fenofibrate (0.5% [wt/wt] in the diet) for 7 days decreased hepatic A-, Bβ-, and γ-chain mRNA levels to 52% ± 7%, 46% ± 8%, and 81% ± 19% of control values, respectively. In parallel, plasma fibrinogen concentrations were decreased to 63% ± 7% of controls. The suppression of fibrinogen expression was dose-dependent and was already evident after 1 day at the highest dose of fenofibrate tested (0.5% [wt/wt]). Nuclear run-on experiments showed that the decrease in fibrinogen expression after fenofibrate occurred at the transcriptional level, as exemplified for the gene for the A-chain. Other fibrates tested showed similar effects on fibrinogen expression and transcription. The effect of fibrates is specific for peroxisome proliferator-activated receptor- (PPAR) because a high-affinity ligand for PPARγ, the thiazolidinedione BRL 49653, lowered triglyceride levels, but was unable to suppress fibrinogen expression. Direct evidence for the involvement of PPAR in the suppression of fibrinogen by fibrates was obtained using PPAR-null (−/−) mice. Compared with (+/+) mice, plasma fibrinogen levels in (−/−) mice were significantly higher (3.20 ± 0.48 v 2.67 ± 0.42 g/L). Also, hepatic fibrinogen A-chain mRNA levels were 25% ± 11% higher in the (−/−) mice. On treatment with 0.2% (wt/wt) fenofibrate, a significant decrease in plasma fibrinogen to 77% ± 10% of control levels and in hepatic fibrinogen A-chain mRNA levels to 65% ± 12% of control levels was seen in (+/+) mice, but not in (−/−) mice. These studies show that PPAR regulates basal levels of plasma fibrinogen and establish that fibrate-suppressed expression of fibrinogen in rodents is mediated through PPAR.

Hepatic overexpression of sulfatase-2 (SULF2), a heparan sulfate remodeling enzyme, strongly contributes to high triglyceride (TG) levels in obese, type 2 diabetic (T2DM) db/db mice. Nevertheless, data in humans are lacking. Here, the association of human hepatic SULF2 expression and SULF2 gene variants with TG metabolism in patients with obesity and/or T2DM was investigated.Liver biopsies from 121 obese subjects were analyzed for relations between hepatic SULF2 mRNA levels and plasma TG. Associations between seven SULF2 tagSNPs and TG levels were assessed in 210 obese T2DM subjects with dyslipidemia. Replication of positive findings was performed in 1,316 independent obese T2DM patients. Postprandial TRL clearance was evaluated in 29 obese T2DM subjects stratified by SULF2 genotype.Liver SULF2 expression was significantly associated with fasting plasma TG (r = 0.271; P = 0.003) in obese subjects. The SULF2 rs2281279(AG) SNP was reproducibly associated with lower fasting plasma TG levels in obese T2DM subjects (P0.05). Carriership of the minor G allele was associated with lower levels of postprandial plasma TG (P0.05) and retinyl esters levels (P0.001).These findings implicate SULF2 as potential therapeutic target in the atherogenic dyslipidemia of obesity and T2DM.

Objective— 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome proliferator– activated receptor-γ (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism, and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPARγ therein. Methods and Results— 11β-HSD1 gene expression is higher in proinflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages, whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages but not in resting macrophages or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPARγ induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Conclusion— Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity.

International audience; Visceral obesity is a chronic, low-grade inflammatory disease that predisposes people to the metabolic syndrome, type 2 diabetes and its cardiovascular complications. Adipose tissue is not a passive storehouse for fat, but an endocrine organ synthesizing and releasing a variety of bioactive molecules, some of which are produced by infiltrated immune-inflammatory cells including macrophages. Two different subpopulations of macrophages have been identified in adipose tissue: pro-inflammatory 'classical' M1 and anti-inflammatory 'alternative' M2 macrophages, and their ratio is suggested to influence the metabolic complications of obesity. These macrophages derive primarily from peripheral blood mononuclear cells (PBMCs). We hypothesised that obesity and the metabolic syndrome modulate PBMC functions. Therefore, alteration of the monocyte response, and more specifically their ability to differentiate toward alternative anti-inflammatory macrophages, was assessed in PBMCs isolated from lean and obese subjects with or without alterations in glucose homeostasis. Our results indicate that PBMCs from obese subjects have an altered expression of M2 markers and that their monocytes are less susceptible to differentiate toward an alternative phenotype. Thus PBMCs in obesity are programmed, which may contribute to the inflammatory dysregulation and increased susceptibility to inflammatory diseases in these patients.

Human adipose tissue macrophages (ATMs) display an alternatively activated (M2) phenotype, but are still able to produce excessive inflammatory mediators. However, the processes driving this particular ATM phenotype are not understood. Genome-wide association studies associated the CDKN2A locus, encoding the tumour suppressor p16(INK4A), with the development of type 2 diabetes. In the present study, p16(INK4A) levels in human ATMs and the role of p16(INK4A) in acquiring the ATM phenotype were assessed.Gene expression of p16 ( INK4A ) in ATMs was analysed and compared with that in monocyte-derived macrophages (MDMs) from obese patients or with macrophages from human atherosclerotic plaques (AMs). Additionally, p16(INK4A) levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16(INK4A) in MDMs from healthy donors was investigated by small interfering (si)RNA-mediated silencing or adenovirus-mediated overproduction of p16(INK4A).Compared with MDMs and AMs, ATMs from obese patients expressed lower levels of p16 ( INK4A ). In vitro, IL-4-induced M2 polarisation resulted in lower p16(INK4A) protein levels after differentiation of monocytes from healthy donors in macrophages. Silencing of p16(INK4A) in MDMs mediated by siRNA increased the expression of M2 marker genes and enhanced the response to lipopolysaccharide (LPS), to give a phenotype resembling that of ATM. By contrast, adenovirus-mediated overproduction of p16(INK4A) in MDMs diminished M2 marker gene expression and the response to LPS. Western blot analysis revealed that p16(INK4A) overproduction inhibits LPS- and palmitate-induced Toll-like receptor 4 (TLR4)-nuclear factor of κ light polypeptide gene enhancer in B cells (NF-κB) signalling.These results show that p16(INK4A) inhibits the acquisition of the ATM phenotype. The age-related increase in p16(INK4A) level may thus influence normal ATM function and contribute to type 2 diabetes risk.

Rationale: A crucial step in atherogenesis is the infiltration of the subendothelial space of large arteries by monocytes where they differentiate into macrophages and transform into lipid-loaded foam cells. Macrophages are heterogeneous cells that adapt their response to environmental cytokines. Th1 cytokines promote monocyte differentiation into M1 macrophages, whereas Th2 cytokines trigger an “alternative” M2 phenotype. Objective: We previously reported the presence of CD68 + mannose receptor (MR) + M2 macrophages in human atherosclerotic plaques. However, the function of these plaque CD68 + MR + macrophages is still unknown. Methods and Results: Histological analysis revealed that CD68 + MR + macrophages locate far from the lipid core of the plaque and contain smaller lipid droplets compared to CD68 + MR − macrophages. Interleukin (IL)-4–polarized CD68 + MR + macrophages display a reduced capacity to handle and efflux cellular cholesterol because of low expression levels of the nuclear receptor liver x receptor (LXR)α and its target genes, ABCA1 and apolipoprotein E, attributable to the high 15-lipoxygenase activity in CD68 + MR + macrophages. By contrast, CD68 + MR + macrophages highly express opsonins and receptors involved in phagocytosis, resulting in high phagocytic activity. In M2 macrophages, peroxisome proliferator-activated receptor (PPAR)γ activation enhances the phagocytic but not the cholesterol trafficking pathways. Conclusions: These data identify a distinct macrophage subpopulation with a low susceptibility to become foam cells but high phagocytic activity resulting from different regulatory activities of the PPARγ-LXRα pathways.

International audience; Farnesoid X receptor (FXR) is highly expressed in liver and intestine where it controls bile acid (BA), lipid and glucose homeostasis. Here we show that FXR is expressed and functional, as assessed by target gene expression analysis, in human islets and beta-cell lines. FXR is predominantly cytosolic-localized in the islets of lean mice, but nuclear in obese mice. Compared to FXR+/+ mice, FXR-/- mice display a normal architecture and beta-cell mass but the expression of certain islet-specific genes is altered. Moreover, glucose-stimulated insulin secretion (GSIS) is impaired in the islets of FXR-/- mice. Finally, FXR activation protects human islets from lipotoxicity and ameliorates their secretory index.

International audience; The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator of genes implicated in lipid homeostasis and inflammation. PPARalpha trans-activity is enhanced by recruitment of coactivators such as SRC1 and CBP/p300 and is inhibited by binding of corepressors such as NCoR and SMRT. In addition to ligand binding, PPARalpha activity is regulated by post-translational modifications such as phosphorylation and ubiquitination. In this report, we demonstrate that hPPARalpha is SUMOylated by SUMO-1 on lysine 185 in the hinge region. The E2-conjugating enzyme Ubc9 and the SUMO E3- ligase PIASy are implicated in this process. In addition, ligand treatment decreases the SUMOylation rate of hPPARalpha. Finally, our results demonstrate that SUMO-1 modification of hPPARalpha down-regulates its trans-activity through the specific recruitment of corepressor NCoR but not SMRT leading to the differential expression of a subset of PPARalpha target genes. In conclusion, hPPARalpha SUMOylation on lysine 185 down-regulates its trans-activity through the selective recruitment of NCoR.

Background & Aims: Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erb alpha is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erb alpha in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. Methods: Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erb alpha. Results: Rev-erb alpha-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erb alpha-deficient mice, whereas adenovirus-mediated hepatic Rev-erb alpha overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erb alpha-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erb alpha-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erb alpha target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erb alpha-deficient mice. Conclusions: These data identify a role for Rev-erb alpha in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression.

Th1 cytokines promote monocyte differentiation into proatherogenic M1 macrophages, while Th2 cytokines lead to an "alternative" anti-inflammatory M2 macrophage phenotype. Here we show that in human atherosclerotic lesions, the expression of M2 markers and PPARgamma, a nuclear receptor controlling macrophage inflammation, correlate positively. Moreover, PPARgamma activation primes primary human monocytes into M2 differentiation, resulting in a more pronounced anti-inflammatory activity in M1 macrophages. However, PPARgamma activation does not influence M2 marker expression in resting or M1 macrophages, nor does PPARgamma agonist treatment influence the expression of M2 markers in atherosclerotic lesions, indicating that only native monocytes can be primed by PPARgamma activation to an enhanced M2 phenotype. Furthermore, PPARgamma activation significantly increases expression of the M2 marker MR in circulating peripheral blood mononuclear cells. These data demonstrate that PPARgamma activation skews human monocytes toward an anti-inflammatory M2 phenotype.