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Background: Adaptation of fat depots to change in fuel availability is critical for metabolic flexibility and cardiometabolic health. The mechanisms responsible for fat depot-specific lipid sensing and shuttling remain elusive. Adipose tissue microvascular endothelial cells (AT-EC) regulates bidirectional fatty acid fluxes depending on fed or fasted state. How AT-EC sense and adapt to metabolic changes according to AT location remains to be established. Methods: We combined transcriptional analysis of native human AT-EC together with in vitro approaches in primary human AT-EC and in vivo and ex vivo studies of mice under fed and fasted conditions. Results: Transcriptional large-scale analysis of human AT-EC isolated from gluteofemoral and abdominal subcutaneous AT revealed that the endothelium exhibits a fat depot–specific signature associated with lipid handling and Notch signaling enrichment. We uncovered a functional link between metabolic status and endothelial DLL4 (delta-like canonical notch ligand 4), which decreases with fasting. DLL4 regulates fatty acid uptake through nontranscriptional modulation of macropinocytosis-dependent long chain fatty acid uptake. Importantly, the changes in DLL4 expression, in response to energy transition state, is impaired under obesogenic conditions, an early alteration coinciding with a defect in systemic fatty acid fluxes adaptation and a resistance to weight loss. Conclusions: DLL4 is a major actor in the adaptive mechanisms of AT-EC to regulate lipid fluxes. It likely contributes to fat depot-dependent metabolism in response to energy transition states. AT-EC alteration with obesity may favor metabolic inflexibility and the development of cardiometabolic disorders.

International audience; Bladder cancer is the 10th most common cancer in the world and has a high risk of recurrence and metastasis. In order to sustain high energetic needs, cancer cells undergo complex metabolic adaptations, such as a switch toward aerobic glycolysis, that can be exploited therapeutically. Reactive oxygen species (ROS) act as key regulators of cancer metabolic reprogramming and tumorigenesis, but the sources of ROS remain unidentified. Monoamine oxidases (MAOs) are mitochondrial enzymes that generate H 2 O 2 during the breakdown of catecholamines and serotonin. These enzymes are particularly important in neurological disorders, but recently, a new link between MAOs and cancer has been uncovered, involving their production of ROS. At present, the putative role of MAOs in bladder cancer has never been evaluated. We observed that human urothelial tumor explants and the bladder cancer cell line AY27 expressed both MAO-A and MAO-B isoforms. Selective inhibition of MAO-A or MAO-B limited mitochondrial ROS accumulation, cell cycle progression and proliferation of bladder cancer cells, while only MAO-A inhibition prevented cell motility. To test whether ROS contributed to MAO-induced tumorigenesis, we used a mutated form of MAO-A which was unable to produce H 2 O 2. Adenoviral transduction of the WT MAO-A stimulated the proliferation and migration of AY27 cells while the Lys305Met MAO-A mutant was inactive. This was consistent with the fact that the antioxidant Trolox strongly impaired proliferation and cell cycle progression. Most interestingly, AY27 cells were highly dependent on glucose metabolism to sustain their growth, and MAO inhibitors potently reduced glycolysis and oxidative phosphorylation, due to pyruvate depletion. Accordingly, MAO inhibitors decreased the expression of proteins involved in glucose transport (GLUT1) and transformation (HK2). In conclusion, urothelial cancer cells are characterized by a metabolic shift toward glucose-dependent metabolism, which is important for cell growth and is under the regulation of MAO-dependent oxidative stress.

BACKGROUND AND AIMS Acute kidney injury (AKI) accounts for 13.3 million cases per year worldwide and further responsible for 1.7 million deaths per year [1]. Increasing incidence of AKI, progression toward CKD or end-stage renal disease (ESRD), affect on long term health, high mortality, morbidity and high costs are all accountable for the need to analyze AKI at a closer level [2]. Rhabdomyolysis (RM) accounting for 10% of AKI cases is due to damage of the skeletal muscle causing a release of muscle cell contents like myoglobin and toxins into the bloodstream leading to AKI, renal failure and possibly death. RM-induced AKI (RIAKI) occurs due to a variety of causes like fall risk, exposure to drugs, infections, muscle hypoxia, infections, body-temperature changes and metabolic and electrolyte disorders [3]. The immune system has been reported to play a role in the pathogenesis of AKI through inflammatory pathways. Macrophages are known to be involved in AKI to CKD transition [4]. Macrophages play an important role in the pathogenesis of RIAKI. While macrophage subtypes (inflammatory type) have been reported to drive RIAKI lesions and prognosis [5], the role of the other immune cells needs to be explored. The aim of this study was to analyze the recruitment of the whole immune system during RIAKI through single-cell RNA sequencing. METHOD Two month old C57BL/6J male mice were used for this study. Mice were administered saline (control) or glycerol i.m (7.5 mL/kg 50% glycerol diluted in saline) for induction of RM. Mouse tail vein blood was collected at 6, 24 and 48 h and serum was obtained after centrifugation (5 min at 2000 rpm). Biochemical analysis tests were used to evaluate RM intensity (creatinine phosphokinase, CPK) and renal function (blood urea nitrogen, BUN) using a Pentra 400 analyzer. To analyze the immune cell recruitment in kidney, kidneys from control and glycerol treated mice were decapsulated, minced and incubated with collagenase and DNase. The dissociated cells were then incubated in red cell lysis buffer and passed through a 40 µm filter. The dissociated cells were further incubated with CD45 antibodies and a viability marker. Kidney CD45 live cells were sorted using BD Influx cell sorter. Preparation of single cell libraries was done and single-cell RNA sequencing was performed according to 10× Genomics protocol. RESULTS Eight clusters (macrophages, monocytes, endothelial cells, neutrophils, natural killer T cells, B cells, T cells and dendritic cells) were revealed after single-cell RNA sequencing of CD45+ renal cells. Recruitment of macrophages, monocytes and neutrophils were revealed to be modified by RIAKI. As expected, NF-kB signaling pathway was the major pathway upregulated in multiple clusters. Pathway analysis in T cell cluster showcased T cell selection and differentiation and T cell receptor signaling pathway. AA467197 (a long non-coding RNA which has been reported in renal ischemia–reperfusion) was upregulated through differential expression analysis in multiple clusters. Macrophage subclustering revealed a high macrophage diversity in the seven macrophage subclusters that shared markers for macrophage subtypes (M1, M2) and pro-inflammatory markers. KLf9 was upregulated in cluster 7, which has been reported to be a stress-responsive gene associated having pro-inflammatory effect. NF-kB and IL-2 STAT5 signaling pathways were the major pathways upregulated in multiple clusters. Drug repositioning studies with connectivity map showcased SRC inhibitions as potent pharmacological intervention strategy. CONCLUSION This study revealed the immune landscaping and pathways associated in RIAKI. Single-cell approach illustrated macrophages diversity in pathological context and suggested candidates for further therapeutic approaches.

Estrogen receptor alpha (ER alpha) regulates gene transcription through two activation functions (ER alpha-AF1 and ER alpha-AF2). We recently found that the protection conferred by 17 beta-estradiol against obesity and insulin resistance requires ER alpha-AF2 but not ER alpha-AF1. However, the interplay between the two ER alpha-AFs is poorly understood in vivo and the metabolic influence of a specific ER alpha-AF1 action remains to be explored. To this end, wild-type, ER alpha-deficient, or ER alpha-AF1 deficient ovariectomized female mice were fed a high fat diet and concomitantly administered with vehicle or tamoxifen, a selective ER modulator that acts as a ER alpha-AF1 agonist/ER alpha-AF2 antagonist. In ovariectomized wild-type mice, tamoxifen significantly reduced food intake and totally prevented adiposity, insulin resistance, and steatosis. These effects were abolished in ER alpha-deficient and ER alpha-AF1 deficient mice, revealing the specific role of ER alpha-AF1 activation. Finally, hepatic gene expression changes elicited by tamoxifen in wild-type mice were abrogated in ER alpha-AF1 deficient mice. The combination of pharmacologic and transgenic approaches thus indicates that selective ER alpha-AF1 activation by tamoxifen is sufficient to elicit metabolic protection, contrasting with the specific requirement of ER alpha-AF2 in the metabolic actions of 17 beta-estradiol. This redundancy in the ability of the two ER alpha-AFs to separately mediate metabolic prevention strikingly contrasts with the contribution of both ER alpha-AFs in breast cancer proliferation, shedding new light on the therapeutic potential of selective ER modulation.

International audience; The beneficial metabolic actions of estrogen-based therapies are mainly mediated by estrogen receptor α (ERα), a nuclear receptor that regulates gene transcription through two activation functions (AFs): AF-1 and AF-2. Using mouse models deleted electively for ERαAF-1 (ERαAF-1°) or ERαAF-2 (ERαAF-2°), we determined their respective roles in the actions of estrogens on body composition and glucose homeostasis in response to either a normal diet or a high-fat diet (HFD). ERαAF-2° males and females developed accelerated weight gain, massive adiposity, severe insulin resistance, and glucose intolerance--quite reminiscent of the phenotype observed in mice deleted for the entire ERα protein (ERα(-/-)). In striking contrast, ERαAF-1° and wild-type (wt) mice shared a similar metabolic phenotype. Accordingly, 17β-estradiol administration regulated key metabolic genes in insulin-sensitive tissues and conferred a strong protection against HFD-induced metabolic disturbances in wt and ERαAF-1° ovariectomized mice, whereas these actions were totally abrogated in ERαAF-2° and ERα(-/-) mice. Thus, whereas both AFs have been previously shown to contribute to endometrial and breast cancer cell proliferation, the protective effect of estrogens against obesity and insulin resistance depends on ERαAF-2 but not ERαAF-1, thereby delineating new options for selective modulation of ERα.

Introduction et objectifs Le recepteur des œstrogenes alpha (ERα) hepatocytaire pourrait representer une cible pertinente pour prevenir les hepatopathies metaboliques. Dans ce contexte, l’objectif de notre etude etait de caracteriser l’interet d’une activation selective de la fonction d’activation ERα-AF1 en combinant une approche pharmacologique (administration de tamoxifene, TAM, agoniste ERα-AF1 et antagoniste ERαAF2), et l’utilisation de modeles murins transgeniques. Materiels et methodes Des souris femelles sauvages (WT), ou genetiquement invalidees pour ERα (ERα-/-), ERα-AF1 (ERα-AF1), ou specifiquement pour ERα dans les hepatocytes (ERα-Hep), ont ete ovariectomisees, puis traitees pendant 12 semaines par placebo, TAM (1,2 mg/kg/j) ou E2 (80 μg/kg/j) et simultanement soumises a un regime gras. Resultats Chez les souris WT, le TAM et l’E2 previennent l’obesite, l’insulino-resistance et la steatose induites par le regime, les effets protecteurs du TAM etant abolis chez les souris ERα-/- et ERα-AF1°. Chez les souris ERα-Hep, les effets du TAM sont egalement totalement abolis, tandis que l’E2 exerce toujours une protection complete vis-a-vis de l’obesite et de l’hyperglycemie, l’effet anti-steatosique etant seulement attenue (p

BACKGROUND: Fibroblast growth factor 2 (FGF2) is a major angiogenic factor involved in angiogenesis and arteriogenesis, however the regulation of its expression during these processes is poorly documented. FGF2 mRNA contains an internal ribosome entry site (IRES), a translational regulator expected to allow mRNA expression during cellular stress. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have developed a skin ischemia model in transgenic mice expressing a reporter transgene under the control of the FGF2 IRES. The results reveal that FGF2 is induced at the protein level during ischemia, concomitant with HIF-1alpha induction and a decrease in FGF2 mRNA. In addition, the FGF2 IRES is strongly activated under these ischemic conditions associated with hypoxia, whereas cap-dependent translation is repressed by 4E-BP hypophosphorylation. We also show that up-regulation of FGF2 protein expression in response to hypoxia correlates with the increase of FGF2 IRES activity in vitro, in human retinoblasts 911. The use of siRNAs targeting HIF or FGF2 indicates that FGF2 and HIF-1alpha reciprocally regulate their expression/accumulation, by a negative feedback loop in early hypoxia, followed by a positive feedback loop in late hypoxia. CONCLUSION/SIGNIFICANCE: FGF2 expression is up-regulated in vivo and in vitro in response to hypoxia. Strikingly, this up-regulation is not transcriptional. It seems to occur by an IRES-dependent mechanism, revealing new mechanistic aspects of the hypoxic response. In addition, our data show that FGF2 interacts with HIF-1alpha in a unique crosstalk, with distinct stages in early and late hypoxia. These data reveal the physiological importance of IRES-dependent translation during hypoxic stress and underline the complexity of the cellular response to hypoxia, suggesting a novel role of FGF2 in the regulation of HIF-1alpha during the induction of angiogenesis.

Introduction Les œstrogenes previennent l’accumulation de masse grasse et l’installation d’une insulinoresistance via leur recepteur α (ERα). Ce recepteur nucleaire est connu pour reguler la transcription de nombreux genes grâce a deux fonctions de transactivation, AF-1 et AF-2, et nous avons recemment montre que, en activant le recepteur independamment de la fonction AF-1, il est possible de conserver l’integralite des effets protecteurs des œstrogenes sur le metabolisme glucidique et la paroi vasculaire, sans effet deletere sur l’uterus. Materiels et methodes Pour determiner l’implication de la fonction AF-2 dans l’action benefique des œstrogenes sur la composition corporelle, la sensibilite a l’insuline et l’homeostasie glucidique, des souris deficientes en ERα (ERα−/−) ou specifiquement en AF-2 (AF-2°), ont ete etudiees en conditions de regime standard ou hyperlipidique (mâles et femelles, n = 8–10 par sexe et par genotype). Resultats Par comparaison a leurs controles sauvages, les souris ERα−/− et AF-2° sont caracterisees par une prise de poids acceleree avec accumulation massive de tissu adipeux, une insulinoresistance et une intolerance au glucose. Pour s’affranchir de l’effet potentiel des steroides sexuels endogenes, des souris femelles ovariectomisees ont ensuite recu une administration chronique de 17β- estradiol a doses physiologiques. Comme attendu, ce traitement previent l’exces d’adiposite, ainsi que l’alteration de la sensibilite a l’insuline (clamps hyperinsulinemiques, voie de signalisation de l’insuline dans les tissus peripheriques) et de la tolerance au glucose chez les souris sauvages, mais ces effets sont totalement abolis chez les souris ERα−/− et AF-2°. Conclusion Comme precedemment demontre concernant leurs effets vasculoprotecteurs, l’action benefique des œstrogenes sur l’adiposite, la sensibilite a l’insuline et la tolerance au glucose s’avere independante de la fonction AF-1, mais dependante de la fonction AF-2 du ERα. Ces resultats ouvrent la voie a une modulation selective du recepteur activant specifiquement AF-2 dans une perspective de prevention cardiovasculaire et metabolique.

Rationnel Les œstrogenes previennent l’accumulation de masse grasse et la survenue d’un diabete de type 2 via leur recepteur α(ERα), mais leurs effets indesirables limitent leur interet therapeutique. ER_regule la transcription de nombreux genes grâce a ses deux fonctions de transactivation, AF-1 et AF-2, et nous avons montre que, en activant le recepteur independamment de la fonction AF-1, il est possible de conserver l’integralite des actions vasculoprotectrices des oestrogenes sans effet deletere sur l’uterus. Materiels et methodes Pour etudier l’implication des fonctions de transactivation de ER_dans l’action benefique des oestrogenes sur la composition corporelle et l’homeostasie glucidique, des souris deficientes en ERα(ERα-/-), en AF-1 (AF-1-/-) ou AF-2 (AF-2-/-), ont ete suivies jusqu’a l’âge de 7 mois (regime standard, mâles et femelles, n = 8–10 par sexe et par genotype). Resultats Par comparaison a leurs controles sauvages, les souris ERα-/- ont ete caracterisees par une prise de poids acceleree avec accumulation massive de tissu adipeux, une insulinoresistance et une intolerance au glucose. Les souris AF-2-/- ont developpe un profil phenotypique et metabolique identique. En revanche, nous n’avons pas observe d’acceleration du gain ponderal et du developpement de la masse grasse chez les animaux AF-1-/-. De plus, la tolerance au glucose etait conservee chez les mâles, et alteree uniquement tardivement chez les femelles AF-1-/-. De facon concordante, l’effet protecteur de l’administration chronique de 17β-estradiol vis-a-vis de l’accumulation de tissu adipeux chez des femelles ovariectomisees etait aboli chez les souris ERα-/- et AF-2-/-, mais totalement conserve chez les souris AF-1-/-. Conclusion Comme pour leurs effets vasculoprotecteurs, l’action favorable des oestrogenes sur la masse grasse et la tolerance au glucose est en grande partie independante de la fonction AF-1 du ERα. Ces resultats ouvrent la voie a une modulation selective du recepteur n’activant pas AF-1 dans une perspective de prevention cardiovasculaire et metabolique.