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1.  CX3CL1 (fractalkine) and its receptor CX3CR1 regulate atopic dermatitis by controlling effector T cell retention in inflamed skin 
The Journal of Experimental Medicine  2014;211(6):1185-1196.
Fractalkine interactions with its receptor, CX3CR1, regulate CD4+ T cell retention in atopic dermatitis and offer a potential therapeutic target in allergic disease.
Atopic dermatitis (AD) is a chronic allergic dermatosis characterized by epidermal thickening and dermal inflammatory infiltrates with a dominant Th2 profile during the acute phase, whereas a Th1 profile is characteristic of the chronic stage. Among chemokines and chemokine receptors associated with inflammation, increased levels of CX3CL1 (fractalkine) and its unique receptor, CX3CR1, have been observed in human AD. We have thus investigated their role and mechanism of action in experimental models of AD and psoriasis. AD pathology and immune responses, but not psoriasis, were profoundly decreased in CX3CR1-deficient mice and upon blocking CX3CL1–CX3CR1 interactions in wild-type mice. CX3CR1 deficiency affected neither antigen presentation nor T cell proliferation in vivo upon skin sensitization, but CX3CR1 expression by both Th2 and Th1 cells was required to induce AD. Surprisingly, unlike in allergic asthma, where CX3CL1 and CX3CR1 regulate the pathology by controlling effector CD4+ T cell survival within inflamed tissues, adoptive transfer experiments established CX3CR1 as a key regulator of CD4+ T cell retention in inflamed skin, indicating a new function for this chemokine receptor. Therefore, although CX3CR1 and CX3CL1 act through distinct mechanisms in different pathologies, our results further indicate their interest as promising therapeutic targets in allergic diseases.
PMCID: PMC4042636  PMID: 24821910
2.  Soaping Up Type 2 Diabetes With Bile Acids? 
Diabetes  2013;62(12):3987-3989.
PMCID: PMC3837042  PMID: 24264396
3.  Dual Peroxisome Proliferator–Activated Receptor α/δ Agonist GFT505 Improves Hepatic and Peripheral Insulin Sensitivity in Abdominally Obese Subjects 
Diabetes Care  2013;36(10):2923-2930.
The development of new insulin sensitizers is an unmet need for the treatment of type 2 diabetes. We investigated the effect of GFT505, a dual peroxisome proliferator–activated receptor (PPAR)-α/δ agonist, on peripheral and hepatic insulin sensitivity.
Twenty-two abdominally obese insulin-resistant males (homeostasis model assessment of insulin resistance >3) were randomly assigned in a randomized crossover study to subsequent 8-week treatment periods with GFT505 (80 mg/day) or placebo, followed by a two-step hyperinsulinemic-euglycemic insulin clamp with a glucose tracer to calculate endogenous glucose production (EGP). The primary end point was the improvement in glucose infusion rate (GIR). Gene expression analysis was performed on skeletal muscle biopsy specimens.
GFT505 improved peripheral insulin sensitivity, with a 21% (P = 0.048) increase of the GIR at the second insulin infusion period. GFT505 also enhanced hepatic insulin sensitivity, with a 44% (P = 0.006) increase of insulin suppression of EGP at the first insulin infusion period. Insulin-suppressed plasma free fatty acid concentrations were significantly reduced on GFT505 treatment (0.21 ± 0.07 vs. 0.27 ± 0.11 mmol/L; P = 0.006). Neither PPARα nor PPARδ target genes were induced in skeletal muscle, suggesting a liver-targeted action of GFT505. GFT505 significantly reduced fasting plasma triglycerides (−21%; P = 0.003) and LDL cholesterol (−13%; P = 0.0006), as well as liver enzyme concentrations (γ-glutamyltranspeptidase: −30.4%, P = 0.003; alanine aminotransferase: −20.5%, P = 0.004). There was no safety concern or any indication of PPARγ activation with GFT505.
The dual PPARα/δ agonist GFT505 is a liver-targeted insulin-sensitizer that is a promising drug candidate for the treatment of type 2 diabetes and nonalcoholic fatty liver disease.
PMCID: PMC3781493  PMID: 23715754
4.  A dynamic CTCF chromatin binding landscape promotes DNA hydroxymethylation and transcriptional induction of adipocyte differentiation 
Nucleic Acids Research  2014;42(17):10943-10959.
CCCTC-binding factor (CTCF) is a ubiquitously expressed multifunctional transcription factor characterized by chromatin binding patterns often described as largely invariant. In this context, how CTCF chromatin recruitment and functionalities are used to promote cell type-specific gene expression remains poorly defined. Here, we show that, in addition to constitutively bound CTCF binding sites (CTS), the CTCF cistrome comprises a large proportion of sites showing highly dynamic binding patterns during the course of adipogenesis. Interestingly, dynamic CTCF chromatin binding is positively linked with changes in expression of genes involved in biological functions defining the different stages of adipogenesis. Importantly, a subset of these dynamic CTS are gained at cell type-specific regulatory regions, in line with a requirement for CTCF in transcriptional induction of adipocyte differentiation. This relates to, at least in part, CTCF requirement for transcriptional activation of both the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) and its target genes. Functionally, we show that CTCF interacts with TET methylcytosine dioxygenase (TET) enzymes and promotes adipogenic transcriptional enhancer DNA hydroxymethylation. Our study reveals a dynamic CTCF chromatin binding landscape required for epigenomic remodeling of enhancers and transcriptional activation driving cell differentiation.
PMCID: PMC4176165  PMID: 25183525
5.  Sox17 Regulates Liver Lipid Metabolism and Adaptation to Fasting 
PLoS ONE  2014;9(8):e104925.
Liver is a major regulator of lipid metabolism and adaptation to fasting, a process involving PPARalpha activation. We recently showed that the Vnn1 gene is a PPARalpha target gene in liver and that release of the Vanin-1 pantetheinase in serum is a biomarker of PPARalpha activation. Here we set up a screen to identify new regulators of adaptation to fasting using the serum Vanin-1 as a marker of PPARalpha activation. Mutagenized mice were screened for low serum Vanin-1 expression. Functional interactions with PPARalpha were investigated by combining transcriptomic, biochemical and metabolic approaches. We characterized a new mutant mouse in which hepatic and serum expression of Vanin-1 is depressed. This mouse carries a mutation in the HMG domain of the Sox17 transcription factor. Mutant mice display a metabolic phenotype featuring lipid abnormalities and inefficient adaptation to fasting. Upon fasting, a fraction of the PPARα-driven transcriptional program is no longer induced and associated with impaired fatty acid oxidation. The transcriptional phenotype is partially observed in heterozygous Sox17+/− mice. In mutant mice, the fasting phenotype but not all transcriptomic signature is rescued by the administration of the PPARalpha agonist fenofibrate. These results identify a novel role for Sox17 in adult liver as a modulator of the metabolic adaptation to fasting.
PMCID: PMC4139292  PMID: 25141153
6.  Imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, substrate-dependent modulators of insulin-degrading enzyme in amyloid-β hydrolysis 
Substrates of Insulin-Degrading Enzyme are numerous and share little homology, like amyloid-beta and insulin. Small molecules binding both at the permanent exosite and at the discontinuous, conformational catalytic site, were discovered and co-crystallized with Insulin-Degrading Enzyme. Selective inhibition of amyloid-beta degradation over insulin hydrolysis was possible. Neuroblastoma cells treated with the optimized compound display a dose-dependent increase in amyloid-beta levels.
PMCID: PMC4128174  PMID: 24735644
enzymes; medicinal chemistry; Structure-activity relationships; amyloid-beta peptides; inhibitors; X-ray diffraction
7.  Giardia muris Infection in Mice Is Associated with a Protective Interleukin 17A Response and Induction of Peroxisome Proliferator-Activated Receptor Alpha 
Infection and Immunity  2014;82(8):3333-3340.
The protozoan parasite Giardia duodenalis (Giardia lamblia) is one of the most commonly found intestinal pathogens in mammals, including humans. In the current study, a Giardia muris-mouse model was used to analyze cytokine transcription patterns and histological changes in intestinal tissue at different time points during infection in C57BL/6 mice. Since earlier work revealed the upregulation of peroxisome proliferator-activated receptors (PPARs) in Giardia-infected calves, a second aim was to investigate the potential activation of PPARs in the intestines of infected mice. The most important observation in all mice was a strong upregulation of il17a starting around 1 week postinfection. The significance of interleukin 17A (IL-17A) in orchestrating a protective immune response was further demonstrated in an infection trial or experiment using IL-17 receptor A (IL-17RA) knockout (KO) mice: whereas in wild-type (WT) mice, cyst secretion dropped significantly after 3 weeks of infection, the IL-17RA KO mice were unable to clear the infection. Analysis of the intestinal response further indicated peroxisome proliferator-activated receptor alpha (PPARα) induction soon after the initial contact with the parasite, as characterized by the transcriptional upregulation of ppara itself and several downstream target genes such as pltp and cpt1. Overall, PPARα did not seem to have any influence on the immune response against G. muris, since PPARα KO animals expressed il-17a and could clear the infection similar to WT controls. In conclusion, this study shows for the first time the importance of IL-17 production in the clearance of a G. muris infection together with an early induction of PPARα. The effect of the latter, however, is still unclear.
PMCID: PMC4136230  PMID: 24866800
8.  miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages 
Biochimica et biophysica acta  2014;1841(6):827-835.
Liver X receptors (LXRα and LXRβ) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRβ. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity.
PMCID: PMC3996726  PMID: 24603323
Micro-RNA; ox-LDL; LXR target gene; ABC; ApoA-I; HDL
9.  miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages 
Biochimica et Biophysica Acta  2014;1841(6):827-835.
Liver X receptors (LXRα and LXRβ) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRβ. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity.
Graphical abstract
•Functional differences of miR-206 in the liver and macrophages•In the liver, miR-206 suppresses LXRα expression and signaling.•In macrophages, miR-206 increases LXRα abundance and promotes cholesterol efflux.•In macrophages, LXRα activation represses miR-206 expression.•In macrophages, pro-inflammatory stimuli increase miR-206 expression.
PMCID: PMC3996726  PMID: 24603323
miR, Micro-RNA; LXRs, liver X receptors; ApoE, apolipoprotein E; ABCs, ATP-binding cassette transporters; KO, knockout; SREBP, sterol regulatory element-binding protein; Micro-RNA; ox-LDL; LXR target gene; ABC; ApoA-I; HDL
10.  Downregulation of the tumour suppressor p16INK4A contributes to the polarisation of human macrophages toward an adipose tissue macrophage (ATM)-like phenotype 
Diabetologia  2011;54(12):3150-3156.
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 p16INK4A, with the development of type 2 diabetes. In the present study, p16INK4A levels in human ATMs and the role of p16INK4A in acquiring the ATM phenotype were assessed.
Gene expression of p16INK4A 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, p16INK4A levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16INK4A in MDMs from healthy donors was investigated by small interfering (si)RNA-mediated silencing or adenovirus-mediated overproduction of p16INK4A.
Compared with MDMs and AMs, ATMs from obese patients expressed lower levels of p16INK4A. In vitro, IL-4-induced M2 polarisation resulted in lower p16INK4A protein levels after differentiation of monocytes from healthy donors in macrophages. Silencing of p16INK4A 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 p16INK4A in MDMs diminished M2 marker gene expression and the response to LPS. Western blot analysis revealed that p16INK4A 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 p16INK4A inhibits the acquisition of the ATM phenotype. The age-related increase in p16INK4A level may thus influence normal ATM function and contribute to type 2 diabetes risk.
PMCID: PMC4020795  PMID: 21968977
Adipose Tissue; metabolism; Cell Polarity; Cyclin-Dependent Kinase Inhibitor p16; biosynthesis; genetics; Diabetes Mellitus, Type 2; metabolism; Down-Regulation; Female; Gene Silencing; Humans; Macrophages; metabolism; Male; NF-kappa B; metabolism; Obesity; metabolism; Plaque, Atherosclerotic; metabolism; RNA, Small Interfering; metabolism; Toll-Like Receptor 4; metabolism; Adipose tissue macrophages; CDKN2A; Inflammation; Macrophage polarisation; Senescence; Type 2 diabetes
11.  Detrimental Effects of Diet-Induced Obesity on τ Pathology Are Independent of Insulin Resistance in τ Transgenic Mice 
Diabetes  2013;62(5):1681-1688.
The τ pathology found in Alzheimer disease (AD) is crucial in cognitive decline. Midlife development of obesity, a major risk factor of insulin resistance and type 2 diabetes, increases the risk of dementia and AD later in life. The impact of obesity on AD risk has been suggested to be related to central insulin resistance, secondary to peripheral insulin resistance. The effects of diet-induced obesity (DIO) on τ pathology remain unknown. In this study, we evaluated effects of a high-fat diet, given at an early pathological stage, in the THY-Tau22 transgenic mouse model of progressive AD-like τ pathology. We found that early and progressive obesity potentiated spatial learning deficits as well as hippocampal τ pathology at a later stage. Surprisingly, THY-Tau22 mice did not exhibit peripheral insulin resistance. Further, pathological worsening occurred while hippocampal insulin signaling was upregulated. Together, our data demonstrate that DIO worsens τ phosphorylation and learning abilities in τ transgenic mice independently from peripheral/central insulin resistance.
PMCID: PMC3636620  PMID: 23250356
12.  Activation of intestinal peroxisome proliferator-activated receptor-α increases high-density lipoprotein production 
European Heart Journal  2012;34(32):2566-2574.
Peroxisome Proliferator-Activated Receptor (PPAR) α is a transcription factor controlling lipid metabolism in liver, heart, muscle and macrophages. PPARα-activation increases plasma HDL-cholesterol and exerts hypotriglyceridemic actions via the liver. However, the intestine expresses PPARα, produces HDL and chylomicrons and is exposed to diet-derived PPARα ligands. Therefore, we examined the effects of PPARα-activation on intestinal lipid and lipoprotein metabolism.
Methods and Results
The impact of PPARα-activation was evaluated in term of HDL-related gene expression in mice, ex-vivo in human jejunal biopsies and in Caco-2/TC7 cells. ApoAI/HDL secretion, cholesterol esterification and trafficking were also studied in-vitro.
In parallel to improving plasma lipid profiles and increasing liver and intestinal expression of fatty-acid-oxidation genes, treatment with the dual PPARα/δ-ligand GFT505 resulted in a more pronounced increase of plasma HDL compared to fenofibrate in mice. GFT505, but not fenofibrate, increased the expression of HDL-production genes such as apolipoprotein-AI and ATP-Binding-Cassette-A1 transporter in murine intestines. A similar increase was observed upon PPARα-activation of human biopsies and Caco-2/TC7 cells. Additionally, HDL secretion by Caco-2/TC7 cells increased. Moreover, PPARα-activation decreased the cholesterol-esterification capacity of Caco-2/TC7 cells, modified cholesterol trafficking and reduced apolipoprotein-B secretion.
PPARα-activation reduces cholesterol esterification, suppresses chylomicron- and increases HDL-secretion by enterocytes. These results identify the intestine as a target organ of PPARα-ligands with entero-hepatic tropism to reduce atherogenic dyslipidemia.
PMCID: PMC3984589  PMID: 22843443
Animals; Apolipoproteins B; metabolism; Butyrates; pharmacology; Caco-2 Cells; Cells, Cultured; Chalcones; pharmacology; Enterocytes; metabolism; Esterification; physiology; Fatty Acids; metabolism; Female; Humans; Jejunum; metabolism; Lipoproteins, HDL; metabolism; Mice; Mice, Knockout; PPAR alpha; antagonists & inhibitors; physiology; Phenylurea Compounds; pharmacology; Propionates; pharmacology; PPARα; intestine; HDL; dyslipidemia
13.  Beneficial Metabolic Effects of Rapamycin Are Associated with Enhanced Regulatory Cells in Diet-Induced Obese Mice 
PLoS ONE  2014;9(4):e92684.
The “mechanistic target of rapamycin” (mTOR) is a central controller of growth, proliferation and/or motility of various cell-types ranging from adipocytes to immune cells, thereby linking metabolism and immunity. mTOR signaling is overactivated in obesity, promoting inflammation and insulin resistance. Therefore, great interest exists in the development of mTOR inhibitors as therapeutic drugs for obesity or diabetes. However, despite a plethora of studies characterizing the metabolic consequences of mTOR inhibition in rodent models, its impact on immune changes associated with the obese condition has never been questioned so far. To address this, we used a mouse model of high-fat diet (HFD)-fed mice with and without pharmacologic mTOR inhibition by rapamycin. Rapamycin was weekly administrated to HFD-fed C57BL/6 mice for 22 weeks. Metabolic effects were determined by glucose and insulin tolerance tests and by indirect calorimetry measures of energy expenditure. Inflammatory response and immune cell populations were characterized in blood, adipose tissue and liver. In parallel, the activities of both mTOR complexes (e. g. mTORC1 and mTORC2) were determined in adipose tissue, muscle and liver. We show that rapamycin-treated mice are leaner, have enhanced energy expenditure and are protected against insulin resistance. These beneficial metabolic effects of rapamycin were associated to significant changes of the inflammatory profiles of both adipose tissue and liver. Importantly, immune cells with regulatory functions such as regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were increased in adipose tissue. These rapamycin-triggered metabolic and immune effects resulted from mTORC1 inhibition whilst mTORC2 activity was intact. Taken together, our results reinforce the notion that controlling immune regulatory cells in metabolic tissues is crucial to maintain a proper metabolic status and, more generally, comfort the need to search for novel pharmacological inhibitors of the mTOR signaling pathway to prevent and/or treat metabolic diseases.
PMCID: PMC3977858  PMID: 24710396
14.  Metformin interferes with bile acid homeostasis through AMPK-FXR crosstalk 
The Journal of Clinical Investigation  2014;124(3):1037-1051.
The nuclear bile acid receptor farnesoid X receptor (FXR) is an important transcriptional regulator of bile acid, lipid, and glucose metabolism. FXR is highly expressed in the liver and intestine and controls the synthesis and enterohepatic circulation of bile acids. However, little is known about FXR-associated proteins that contribute to metabolic regulation. Here, we performed a mass spectrometry–based search for FXR-interacting proteins in human hepatoma cells and identified AMPK as a coregulator of FXR. FXR interacted with the nutrient-sensitive kinase AMPK in the cytoplasm of target cells and was phosphorylated in its hinge domain. In cultured human and murine hepatocytes and enterocytes, pharmacological activation of AMPK inhibited FXR transcriptional activity and prevented FXR coactivator recruitment to promoters of FXR-regulated genes. Furthermore, treatment with AMPK activators, including the antidiabetic biguanide metformin, inhibited FXR agonist induction of FXR target genes in mouse liver and intestine. In a mouse model of intrahepatic cholestasis, metformin treatment induced FXR phosphorylation, perturbed bile acid homeostasis, and worsened liver injury. Together, our data indicate that AMPK directly phosphorylates and regulates FXR transcriptional activity to precipitate liver injury under conditions favoring cholestasis.
PMCID: PMC3938262  PMID: 24531544
15.  Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy 
Nature medicine  2013;19(8):1039-1046.
The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-α is highly expressed in oxidative skeletal muscle and plays a role in mitochondrial biogenesis and oxidative function, in gain- and loss-of function studies. Rev-erb-α-deficiency in skeletal muscle leads to reduced mitochondrial content and oxidative function, resulting in compromised exercise capacity. This phenotype was recapitulated in isolated fibers and in muscle cells upon Rev-erbα knock-down, while Rev-erb-α over-expression increased the number of mitochondria with improved respiratory capacity. Rev-erb-α-deficiency resulted in deactivation of the Stk11–Ampk–Sirt1–Ppargc1-α signaling pathway, whereas autophagy was up-regulated, resulting in both impaired mitochondrial biogenesis and increased clearance. Muscle over-expression or pharmacological activation of Rev-erb-α increased respiration and exercise capacity. This study identifies Rev-erb-α as a pharmacological target which improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function.
PMCID: PMC3737409  PMID: 23852339
Rev-erb-α; skeletal muscle; oxidative capacity; mitochondrial biogenesis; autophagy
16.  O-GlcNAcylation Links ChREBP and FXR to Glucose-Sensing 
Accumulating evidence suggests that O-GlcNAc transferase, an enzyme responsible for O-GlcNAc post-translational modification acts as a nutrient sensor that links glucose and the hexosamine biosynthetic pathway to the regulation of transcriptional factors involved in energy homeostasis. In liver, glucose signaling is mediated by carbohydrate response element-binding protein (ChREBP), which stimulates glycolytic and lipogenic gene expression through its binding on a specific ChoRE DNA sequence. Modulation of ChREBP by O-GlcNAcylation increases its DNA binding affinity and its activity. ChREBP transcriptional activity also depends on the presence of several other co-factors and transcriptional factors. Among them, the nuclear Farnesoid X Receptor (FXR), a key transcription factor of bile acid metabolism involved in the gut–liver axis homeostasis was recently shown to directly interact with ChREBP, acting as a repressor on the ChoRE of glycolytic genes. Interestingly, similarly to ChREBP, FXR is O-GlcNAcylated in response to glucose. This review discusses the importance of ChREBP and FXR modifications through O-GlcNAcylation in liver and how glucose can modify their mutual affinity and transcriptional activity.
PMCID: PMC4292737  PMID: 25628602
ChREBP; FXR; glucose-sensing; O-GlcNAcylation; liver metabolism
17.  Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes 
Molecular and Cellular Biology  2013;33(11):2202-2211.
The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes.
PMCID: PMC3648076  PMID: 23530060
18.  Human adipose tissue macrophages display activation of cancer-related pathways 
The Journal of Biological Chemistry  2012;287(26):21904-21913.
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 to 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 over-expression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signalling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumour-associated macrophages (TAM) showed that ATM, but not MDM resemble TAM. Indirect co-culture experiments demonstrated that factors secreted by pre-adipocytes, 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.
PMCID: PMC3381151  PMID: 22511784
Adipocytes; cytology; Adipose Tissue; metabolism; Azo Compounds; pharmacology; Cell Line, Tumor; Chemokines; metabolism; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; methods; Inflammation; Macrophages; cytology; metabolism; Neoplasms; metabolism; Obesity; metabolism; Oligonucleotide Array Sequence Analysis; Phenotype; macrophages; obesity; cancer; adipose tissue; chemokines
19.  Increased Atherosclerotic Lesions in LDL Receptor Deficient Mice With Hematopoietic Nuclear Receptor Rev‐erbα Knock‐ Down 
Nuclear receptor Rev‐erbα plays important roles in circadian clock timing, lipid metabolism, adipogenesis, and vascular inflammation. However, the role of Rev‐erbα in atherosclerotic lesion development has not been assessed in vivo.
Methods and Results
The nuclear receptor Rev‐erbα was knocked down in mouse haematopoietic cells by means of shRNA‐lentiviral transduction, followed by bone marrow transplantation into LDL receptor knockout mice. The Rev‐erbα protein in peripheral macrophage was reduced by 70% as compared to control mice injected with nontargeting shRNA lentivirus‐transduced bone marrow. A significant increase in atherosclerotic lesions was observed around the aorta valves as well as upon en face aorta analysis of Rev‐erbα knock‐down bone marrow recipients (P<0.01) as compared to the control mice, while plasma cholesterol, phospholipid, and triacylglycerol levels were not affected. Overexpression of Rev‐erbα in bone marrow mononuclear cells decreased inflammatory M1 while increasing M2 macrophage markers, while Rev‐erbα knock down increased the macrophage inflammatory phenotype in vitro and in vivo. Furthermore, treatment of differentiating macrophages with the Rev‐erbα ligand heme promoted expression of antiinflammatory M2 markers.
These observations identify hematopoietic cell Rev‐erbα as a new modulator of atherogenesis in mice.
PMCID: PMC3828791  PMID: 23963755
atherosclerosis; macrophages; Rev‐erbα
20.  p16INK4a deficiency promotes IL-4-induced polarization and inhibits proinflammatory signaling in macrophages 
Blood  2011;118(9):2556-2566.
The CDKN2A locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize towards classically (CAMφ) or alternatively (AAMφ) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined.
Here, we show that p16INK4a-deficiency (p16−/−) modulates the macrophage phenotype. Transcriptome analysis revealed that p16−/− bone marrow-derived macrophages (BMDM) exhibit a phenotype resembling interleukin (IL)-4-induced macrophage polarization. In line with this observation, p16−/− BMDM displayed a decreased response to classically polarizing IFNγ and LPS and an increased sensitivity to alternative polarization by IL-4. Furthermore, mice transplanted with p16−/− bone marrow displayed higher hepatic AAMφ marker expression levels upon Schistosoma mansoni infection, an in vivo model of AAMφ phenotype-skewing. Surprisingly, p16−/− BMDM did not display increased IL-4-induced STAT6 signaling, but decreased IFNγ-induced STAT1 and LPS-induced IKKα,β phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,β. These findings identify p16INK4a as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases.
PMCID: PMC3677739  PMID: 21636855
Animals; Bone Marrow Transplantation; Cyclin-Dependent Kinase Inhibitor p16; deficiency; physiology; Cytokines; biosynthesis; Genes, p16; I-kappa B Kinase; physiology; Inflammation; genetics; Interferon-gamma; pharmacology; Interleukin-4; pharmacology; Janus Kinase 2; physiology; Lipopolysaccharides; pharmacology; Liver; metabolism; pathology; Macrophage Activation; drug effects; Macrophages; drug effects; physiology; Mice; Mice, Inbred C57BL; Phosphorylation; Protein Processing, Post-Translational; Radiation Chimera; STAT1 Transcription Factor; physiology; STAT6 Transcription Factor; physiology; Schistosomiasis; immunology; Signal Transduction
21.  The Elongation Complex Components BRD4 and MLLT3/AF9 Are Transcriptional Coactivators of Nuclear Retinoid Receptors 
PLoS ONE  2013;8(6):e64880.
Nuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation.
PMCID: PMC3677938  PMID: 23762261
22.  Impaired alternative macrophage differentiation of peripheral blood mononuclear cells from obese subjects 
Visceral obesity, a chronic, low-grade inflammatory disease, predisposes 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 sub-populations 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 (PBMC). We hypothesized 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 PBMC isolated from lean and obese subjects with or without alterations in glucose homeostasis. Our results indicate that PBMC from obese subjects have an altered expression of M2 markers and that their monocytes are less susceptible to differentiate toward an alternative phenotype. Thus PBMC in obesity are programmed, which may contribute to the inflammatory dysregulation and increased susceptibility to inflammatory diseases in these patients.
PMCID: PMC3655375  PMID: 22192929
Adipose Tissue; metabolism; Adult; Cell Differentiation; Diabetes Mellitus, Type 2; complications; metabolism; Female; Gene Expression Profiling; Humans; Inflammation; etiology; metabolism; Inflammation Mediators; metabolism; Leukocytes, Mononuclear; metabolism; Macrophages; cytology; metabolism; Middle Aged; Obesity; complications; metabolism; Phenotype; obesity; macrophages; polarization; inflammation; gene expression
23.  11β-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis 
The FASEB Journal  2013;27(4):1519-1531.
11β-Hydroxysteroid dehydrogenase type-1 (11β-HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11β-HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11β-HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice were treated with a selective 11β-HSD1 inhibitor or crossed with 11β-HSD1-KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11β-HSD1 inhibition or deficiency attenuated atherosclerosis (74–76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T-cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein-1 (MCP-1) and 36% lower lesional vascular cell adhesion molecule-1 (VCAM-1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE-KO mice reduced atherosclerosis by 51%. 11β-HSD1-null macrophages show 76% enhanced cholesterol ester export. Thus, 11β-HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11β-HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.—Kipari, T., Hadoke, P. W. F., Iqbal, J., Man, T. Y., Miller, E., Coutinho, A. E., Zhang, Z., Sullivan, K. M., Mitic, T., Livingstone, D. E. W., Schrecker, C., Samuel, K., White, C. I., Bouhlel, M. A., Chinetti-Gbaguidi, G., Staels, B., Andrew, R., Walker, B. R., Savill, J. S., Chapman, K. E., Seckl, J. R. 11β-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis.
PMCID: PMC3606528  PMID: 23303209
atherogenesis; glucocorticoids; inflammation
24.  Hepatic glucose sensing is required to preserve β cell glucose competence 
The Journal of Clinical Investigation  2013;123(4):1662-1676.
Liver glucose metabolism plays a central role in glucose homeostasis and may also regulate feeding and energy expenditure. Here we assessed the impact of glucose transporter 2 (Glut2) gene inactivation in adult mouse liver (LG2KO mice). Loss of Glut2 suppressed hepatic glucose uptake but not glucose output. In the fasted state, expression of carbohydrate-responsive element-binding protein (ChREBP) and its glycolytic and lipogenic target genes was abnormally elevated. Feeding, energy expenditure, and insulin sensitivity were identical in LG2KO and control mice. Glucose tolerance was initially normal after Glut2 inactivation, but LG2KO mice exhibited progressive impairment of glucose-stimulated insulin secretion even though β cell mass and insulin content remained normal. Liver transcript profiling revealed a coordinated downregulation of cholesterol biosynthesis genes in LG2KO mice that was associated with reduced hepatic cholesterol in fasted mice and reduced bile acids (BAs) in feces, with a similar trend in plasma. We showed that chronic BAs or farnesoid X receptor (FXR) agonist treatment of primary islets increases glucose-stimulated insulin secretion, an effect not seen in islets from Fxr–/– mice. Collectively, our data show that glucose sensing by the liver controls β cell glucose competence and suggest BAs as a potential mechanistic link.
PMCID: PMC3613916  PMID: 23549084
25.  Macrophage polarization in metabolic disorders: functions and regulation 
Current Opinion in Lipidology  2011;22(5):365-372.
Purpose of review
To discuss recent findings on the role and regulation of macrophage polarization in obesity and atherosclerosis.
Recent findings
Macrophages infiltrate the vascular wall during atherosclerosis and adipose tissue during obesity. At least two distinct sub-populations with different functions, the classically (M1) and the alternatively (M2) activated macrophages, have been found in these tissues. Reciprocal skewing of macrophage polarization between the M1 and M2 states is a process modulated by diet, humoral and transcription factors, such as the nuclear receptor Peroxisome Proliferator-Activated Receptor gamma (PPARγ).
Recent literature highlights the importance not only of the number of infiltrated macrophages, but also their activation in the maintenance of the inflammation state. Identifying mechanisms and molecules able to modify the balance between M1 and M2 represents a promising field of research.
PMCID: PMC3565956  PMID: 21825981
Adipose Tissue; immunology; Animals; Humans; Inflammation; immunology; Macrophages; cytology; metabolism; Metabolic Diseases; immunology; macrophages; obesity; atherosclerosis; nuclear receptors

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