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1.  Catalytic Site Inhibition of Insulin Degrading Enzyme by a Small Molecule Induces Glucose Intolerance in Mice 
Nature communications  2015;6:8250.
Insulin degrading enzyme (IDE) is a protease which cleaves insulin and other bioactive peptides such as amyloid-β. Knock-out and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin degrading protease, IDE is a candidate drug target in diabetes. Here we use kinetic Target-Guided Synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and SAXS analyses shows that it locks IDE in a closed conformation. Amongst a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signaling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.
doi:10.1038/ncomms9250
PMCID: PMC4580987  PMID: 26394692
2.  Structure-activity relationships of Imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, dual binders of human Insulin-Degrading Enzyme 
Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-β and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer’s disease, amyloid-β clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE’s role.
doi:10.1016/j.ejmech.2014.12.005
PMCID: PMC4325277  PMID: 25489670
enzymes; Structure-activity relationships; amyloid-beta peptides; inhibitors; X-ray diffraction; Docking
3.  The ubiquitous transcription factor CTCF promotes lineage-specific epigenomic remodeling and establishment of transcriptional networks driving cell differentiation 
Nucleus  2015;6(1):15-18.
Cell differentiation relies on tissue-specific transcription factors (TFs) that cooperate to establish unique transcriptomes and phenotypes. However, the role of ubiquitous TFs in these processes remains poorly defined. Recently, we have shown that the CCCTC-binding factor (CTCF) is required for adipocyte differentiation through epigenomic remodelling of adipose tissue-specific enhancers and transcriptional activation of Peroxisome proliferator-activated receptor gamma (PPARG), the main driver of the adipogenic program (PPARG), and its target genes. Here, we discuss how these findings, together with the recent literature, illuminate a functional role for ubiquitous TFs in lineage-determining transcriptional networks.
doi:10.1080/19491034.2015.1004258
PMCID: PMC4615151  PMID: 25565413
CCCTC-binding factor (CTCF); cell differentiation; cistrome; DNA hydroxymethylation; epigenome; enhancer; TET methylcytosine dioxygenase; transcriptome; ubiquitous transcription factor
4.  Farnesoid X Receptor Inhibits Glucagon-Like Peptide-1 Production by Enteroendocrine L-cells 
Nature communications  2015;6:7629.
Bile acids (BA) are signalling molecules which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex BA in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces Glucagon-Like Peptide-1 (GLP-1) production by L-cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L-cells and controls GLP-1 production is unknown. Here we show that FXR activation in L-cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR-deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes.
doi:10.1038/ncomms8629
PMCID: PMC4579574  PMID: 26134028
bile acids; glucose; glycolysis; intestine; pharmacology
5.  Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice 
Nature Communications  2015;6:8250.
Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.
Inhibiting insulin-degrading enzyme (IDE) has been proposed as a potential therapeutic strategy for the treatment of patients with diabetes. Here, the authors develop a novel IDE inhibitor but find that, surprisingly, IDE inhibition has negative effects on glucose tolerance in mice.
doi:10.1038/ncomms9250
PMCID: PMC4580987  PMID: 26394692
6.  MicroRNA-26a regulates insulin sensitivity and metabolism of glucose and lipids 
The Journal of Clinical Investigation  2015;125(6):2497-2509.
Type 2 diabetes (T2D) is characterized by insulin resistance and increased hepatic glucose production, yet the molecular mechanisms underlying these abnormalities are poorly understood. MicroRNAs (miRs) are a class of small, noncoding RNAs that have been implicated in the regulation of human diseases, including T2D. miR-26a is known to play a critical role in tumorigenesis; however, its function in cellular metabolism remains unknown. Here, we determined that miR-26a regulates insulin signaling and metabolism of glucose and lipids. Compared with lean individuals, overweight humans had decreased expression of miR-26a in the liver. Moreover, miR-26 was downregulated in 2 obese mouse models compared with control animals. Global or liver-specific overexpression of miR-26a in mice fed a high-fat diet improved insulin sensitivity, decreased hepatic glucose production, and decreased fatty acid synthesis, thereby preventing obesity-induced metabolic complications. Conversely, silencing of endogenous miR-26a in conventional diet–fed mice impaired insulin sensitivity, enhanced glucose production, and increased fatty acid synthesis. miR-26a targeted several key regulators of hepatic metabolism and insulin signaling. These findings reveal miR-26a as a regulator of liver metabolism and suggest miR-26a should be further explored as a potential target for the treatment of T2D.
doi:10.1172/JCI75438
PMCID: PMC4497741  PMID: 25961460
Endocrinology; Genetics; Metabolism
7.  SULF2 Strongly Prediposes to Fasting and Postprandial Triglycerides in Patients with Obesity and Type 2 Diabetes Mellitus 
Obesity (Silver Spring, Md.)  2014;22(5):1309-1316.
Objective
Hepatic overexpression of sulfatase-2 (SULF2), a heparan sulfate remodelling enzyme, strongly contributes to high triglyceride (TG) levels in obese, type 2 diabetic (T2DM) db/db mice. Nevertheless, data in humans are lacking. Here we sought to investigate the association of human hepatic SULF2 expression and SULF2 gene variants with TG metabolism in patients with obesity and/or T2DM.
Design and 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 1316 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 (RE) levels (P<0.001).
Conclusions
These findings implicate SULF2 as potential therapeutic target in the atherogenic dyslipidemia of obesity and T2DM.
doi:10.1002/oby.20682
PMCID: PMC4008695  PMID: 24339435
Triglycerides; SULF2; obesity; diabetes; postprandial
8.  Adventitial Tertiary Lymphoid Organs as Potential Source of MicroRNA Biomarkers for Abdominal Aortic Aneurysm 
Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology.
doi:10.3390/ijms160511276
PMCID: PMC4463700  PMID: 25993295
abdominal aortic aneurysm; adventitial tertiary lymphoid organs; microRNA; laser microdissection; quantitative RT-PCR
9.  Effects of the PPAR-α agonist fenofibrate on acute and short-term consequences of brain ischemia 
In stroke, there is an imperative need to develop disease-modifying drugs able to (1) induce neuroprotection and vasculoprotection, (2) modulate recovery and brain plasticity, and (3) limit the short-term motor and cognitive consequences. We hypothesized that fenofibrate, a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, could exert a beneficial effect on immediate and short-term poststroke consequences related to its pleiotropic mechanisms. Rats or mice were subjected to focal ischemia to determine the effects of acute treatment by fenofibrate on (i) motor and memory impairment, (2) both cerebral and vascular compartments, (3) inflammation, (4) neurogenesis, and (5) amyloid cascade. We show that fenofibrate administration results in both neuronal and vascular protection and prevents the short-term motor and cognitive poststroke consequences by interaction with several mechanisms. Modulation of PPAR-α generates beneficial effects in the immediate poststroke consequences by mechanisms involving the interactions between polynuclear neutrophils and the vessel wall, and microglial activation. Fenofibrate modulates mechanisms involved in neurorepair and amyloid cascade. Our results suggest that PPAR-α agonists could check the key points of a potential disease-modifying effect in stroke.
doi:10.1038/jcbfm.2013.233
PMCID: PMC3948136  PMID: 24398933
brain ischemia; inflammation; neuroprotection; peroxisome proliferator-activated receptor-alpha
10.  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.
doi:10.1084/jem.20121350
PMCID: PMC4042636  PMID: 24821910
11.  Soaping Up Type 2 Diabetes With Bile Acids? 
Diabetes  2013;62(12):3987-3989.
doi:10.2337/db13-1278
PMCID: PMC3837042  PMID: 24264396
12.  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.
OBJECTIVE
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.
RESEARCH DESIGN AND METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
doi:10.2337/dc12-2012
PMCID: PMC3781493  PMID: 23715754
13.  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.
doi:10.1093/nar/gku780
PMCID: PMC4176165  PMID: 25183525
14.  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.
doi:10.1371/journal.pone.0104925
PMCID: PMC4139292  PMID: 25141153
15.  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.
doi:10.1016/j.ejmech.2014.04.009
PMCID: PMC4128174  PMID: 24735644
enzymes; medicinal chemistry; Structure-activity relationships; amyloid-beta peptides; inhibitors; X-ray diffraction
16.  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.
doi:10.1128/IAI.01536-14
PMCID: PMC4136230  PMID: 24866800
17.  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.
doi:10.1016/j.bbalip.2014.02.006
PMCID: PMC3996726  PMID: 24603323
Micro-RNA; ox-LDL; LXR target gene; ABC; ApoA-I; HDL
18.  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
Highlights
•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.
doi:10.1016/j.bbalip.2014.02.006
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
19.  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.
Aims/hypothesis
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.
Methods
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.
Results
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.
Conclusions/interpretation
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.
doi:10.1007/s00125-011-2324-0
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
20.  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.
doi:10.2337/db12-0866
PMCID: PMC3636620  PMID: 23250356
21.  Activation of intestinal peroxisome proliferator-activated receptor-α increases high-density lipoprotein production 
European Heart Journal  2012;34(32):2566-2574.
Aims
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.
Conclusions
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.
doi:10.1093/eurheartj/ehs227
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
22.  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.
doi:10.1371/journal.pone.0092684
PMCID: PMC3977858  PMID: 24710396
23.  The Sirt1 activator SRT3025 provides atheroprotection in Apoe−/− mice by reducing hepatic Pcsk9 secretion and enhancing Ldlr expression 
European Heart Journal  2014;36(1):51-59.
Aims
The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture.
Methods and results
Apolipoprotein E-deficient (Apoe−/−) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg−1 diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr−/− mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis.
Conclusion
We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis.
doi:10.1093/eurheartj/ehu095
PMCID: PMC4286317  PMID: 24603306
Sirt1; LDL-cholesterol; Pcsk9; LDL receptor; Atherogenesis
24.  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.
doi:10.1172/JCI68815
PMCID: PMC3938262  PMID: 24531544
25.  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.
doi:10.1038/nm.3213
PMCID: PMC3737409  PMID: 23852339
Rev-erb-α; skeletal muscle; oxidative capacity; mitochondrial biogenesis; autophagy

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