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In humans, glucocorticoids (GCs) are commonly prescribed because of their anti-inflammatory and immunosuppressive properties. However, high doses of GCs often lead to side effects, including diabetes and lipodystrophy. We recently reported that adipocyte glucocorticoid receptor (GR)-deficient (AdipoGR-KO) mice under corticosterone (CORT) treatment exhibited a massive adipose tissue (AT) expansion associated with a paradoxical improvement of metabolic health compared with control mice. However, whether GR may control adipose development remains unclear. Here, we show a specific induction of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic vascular endothelial growth factor A (VEGFA) expression in GR-deficient adipocytes of AdipoGR-KO mice compared with control mice, together with an increased adipose vascular network, as assessed by three-dimensional imaging. GR activation reduced HIF-1α recruitment to the Vegfa promoter resulting from Hif-1α downregulation at the transcriptional and posttranslational levels. Importantly, in CORT-treated AdipoGR-KO mice, the blockade of VEGFA by a soluble decoy receptor prevented AT expansion and the healthy metabolic phenotype. Finally, in subcutaneous AT from patients with Cushing syndrome, higher VEGFA expression was associated with a better metabolic profile. Collectively, these results highlight that adipocyte GR negatively controls AT expansion and metabolic health through the downregulation of the major angiogenic effector VEGFA and inhibition of vascular network development., (© 2024 by the American Diabetes Association.)

Objectives: Inflammatory bowel disease (IBD) results from a combination of genetic predisposition, dysbiosis of the gut microbiota and environmental factors, leading to alterations in the gastrointestinal immune response and chronic inflammation. Caspase recruitment domain 9 ( Card9 ), one of the IBD susceptibility genes, has been shown to protect against intestinal inflammation and fungal infection. However, the cell types and mechanisms involved in the CARD9 protective role against inflammation remain unknown., Design: We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of Card9 deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse)., Results: Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation., Conclusion: These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Nutrition during the early postnatal period can program the growth trajectory and adult size. Nutritionally regulated hormones are strongly suspected to be involved in this physiological regulation. Linear growth during the postnatal period is regulated by the neuroendocrine somatotropic axis, whose development is first controlled by GHRH neurons of the hypothalamus. Leptin that is secreted by adipocytes in proportion to fat mass is one of the most widely studied nutritional factors, with a programming effect in the hypothalamus. However, it remains unclear whether leptin stimulates the development of GHRH neurons directly. Using a Ghrh-eGFP mouse model, we show here that leptin can directly stimulate the axonal growth of GHRH neurons in vitro in arcuate explant cultures. Moreover, GHRH neurons in arcuate explants harvested from underfed pups were insensitive to the induction of axonal growth by leptin, whereas AgRP neurons in these explants were responsive to leptin treatment. This insensitivity was associated with altered activating capacities of the three JAK2, AKT and ERK signaling pathways. These results suggest that leptin may be a direct effector of linear growth programming by nutrition, and that the GHRH neuronal subpopulation may display a specific response to leptin in cases of underfeeding.

Abundance of Faecalibacterium prausnitzii, a dominant bacterium of the human microbiota that exhibits antiinflammatory effects, is decreased in patients with inflammatory bowel diseases (IBD). In humans, colonic lamina propria contains IL-10-secreting, Foxp3- Tregs characterized by a double expression of CD4 and CD8α (DP8α) and a specificity for F. prausnitzii. This Treg subset is decreased in IBD. The in vivo effect of DP8α cells has not been evaluated yet to our knowledge. Here, using a humanized model of a NSG immunodeficient mouse strain that expresses the HLA D-related allele HLA-DR*0401 but not murine class II (NSG-Ab° DR4) molecules, we demonstrated a protective effect of a HLA-DR*0401-restricted DP8α Treg clone combined with F. prausnitzii administration in a colitis model. In a cohort of patients with IBD, we showed an independent association between the frequency of circulating DP8α cells and disease activity. Finally, we pointed out a positive correlation between F. prausnitzii-specific DP8α Tregs and the amount of F. prausnitzii in fecal microbiota in healthy individuals and patients with ileal Crohn's disease.

Background & Aims: Cholangiopathies are chronic liver diseases in which damaged cholangiocytes trigger a proinflammatory and profibrotic reaction. The nuclear vitamin D receptor (VDR) is highly expressed in cholangiocytes and exerts immune-regulatory functions in these cells. In the present study, we examined the protective function of VDR and other vitamin D signaling pathways in chronic cholangiopathy and cholangiocytes., Methods: Vdr was invalidated in Abcb4 knockout mice, a widely used animal model of chronic cholangiopathy. The impact of vitamin D signaling on cholangiopathy features was examined in vivo and in cholangiocytes (primary and cell lines)., Results: Cholangiopathy features (i.e, cholestasis, ductular reaction and fibrosis) were aggravated in Vdr;Abcb4 double knockout mice compared to the Abcb4 simple knockout, and associated with an overexpression of proinflammatory factors. The proinflammatory phenotype of cholangiocytes was also exacerbated following VDR silencing in vitro. The expression of proinflammatory factors and the severity of cholangiopathy were reduced in the double knockout mice treated with the vitamin D analog calcipotriol or with vitamin D. In vitro, the inflammatory response to TNFα was significantly reduced by calcipotriol in biliary cells silenced for VDR, and this effect was abolished by co-silencing the plasma membrane receptor of vitamin D, protein disulfide-isomerase A3 (PDIA3)., Conclusions: Our results demonstrate an anti-inflammatory role of VDR signaling in cholangiocytes and cholangiopathy. They also provide evidence for PDIA3-mediated anti-inflammatory effects of vitamin D and vitamin D analog in these settings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background & Aims: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD., Methods: C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD., Results: When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity., Conclusion: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD., Lay Summary: There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Widely used for their anti-inflammatory and immunosuppressive properties, glucocorticoids are nonetheless responsible for the development of diabetes and lipodystrophy. Despite an increasing number of studies focused on the adipocyte glucocorticoid receptor (GR), its precise role in the molecular mechanisms of these complications has not been elucidated. In keeping with this goal, we generated a conditional adipocyte-specific murine model of GR invalidation (AdipoGR knockout [KO] mice). Interestingly, when administered a corticosterone treatment to mimic hypercorticism conditions, AdipoGR-KO mice exhibited an improved glucose tolerance and insulin sensitivity. This was related to the adipose-specific activation of the insulin-signaling pathway, which contributed to fat mass expansion, as well as a shift toward an anti-inflammatory macrophage polarization in adipose tissue of AdipoGR-KO animals. Moreover, these mice were protected against ectopic lipid accumulation in the liver and displayed an improved lipid profile, contributing to their overall healthier phenotype. Altogether, our results indicate that adipocyte GR is a key factor of adipose tissue expansion and glucose and lipid metabolism control, which should be taken into account in the further design of adipocyte GR-selective modulators., (© 2018 by the American Diabetes Association.)

Second-generation antipsychotics (SGAs) are well known for their metabolic side effects in humans, including obesity and diabetes. These compounds are maintained during pregnancy to prevent the relapse of psychoses, but they readily diffuse across the placenta to the fetus, as documented with the widely-prescribed drug olanzapine (OLZ). However, observational studies have provided conflicting results on the potential impact of SGAs on fetal growth and body weight, and their effects on metabolic regulation in the offspring. For this reason, our study has tested whether antenatal exposure of CD1 mice to OLZ influenced metabolic outcomes in the offspring of the first (F1) and second (F2) generations. In F1 mice, OLZ antenatal treatment caused a decrease in neonatal body weight in both genders, an effect that persisted throughout life only in male animals. Interestingly, F1 female mice also displayed altered glucose homoeostasis. F2 mice, generated by mating normal males with F1 female mice exposed to OLZ during antenatal life, exhibited higher neonatal body weights which persisted only in F2 female animals. This was associated with expansion of fat mass and a concordant pattern of adipose tissue gene expression. Moreover, male and female F2 mice were glucose-intolerant. Thus, our study has demonstrated that antenatal OLZ exposure induces multigenerational and gender-specific programming of glucose tolerance in the offspring mice as adults, and points to the need for careful monitoring of children exposed to SGAs during pregnancy., (Copyright © 2017. Published by Elsevier Masson SAS.)

Nutrition plays a critical role in programming and shaping linear growth during early postnatal life through direct action on the development of the neuroendocrine somatotropic (GH/IGF-1) axis. IGF-1 is a key factor in modulating the programming of linear growth during this period. Notably, IGF-1 preferentially stimulates axonal growth of GHRH neurons in the arcuate nucleus of the hypothalamus (Arc), which is crucial for the proliferation of somatotroph progenitors in the pituitary, thus influencing later GH secretory capacity. However, other nutrition-related hormones may also be involved. Among them, insulin shares several structural and functional similarities with IGF-1, as well as downstream signaling effectors. We investigated the role of insulin in the control of Arc axonal growth using an in vitro model of arcuate explants culture and a cell-type specific approach (GHRH-eGFP mice) under both physiological conditions (normally fed pups) and those of dietary restriction (underfed pups). Our data suggest that insulin failed to directly control axonal growth of Arc neurons or influence specific IGF-1-mediated effects on GHRH neurons. Insulin may act on neuronal welfare, which appears to be dependent on neuronal sub-populations and is influenced by the nutritional status of pups in which Arc neurons develop.

Aims: This study aimed to assess the safety and efficacy at midterm follow-up of left atrial appendage occlusion (LAAO) using different devices, in real life in Belgium., Methods and Results: Between June 2009 and November 2016, 457 consecutive patients (63% male, 75±12 yrs, CHA2DS2-VASc 4±0.6, HAS-BLED 3.5±0.7) undergoing LAAO were included. Technical success was 97.1%. There were 19 periprocedural major adverse events (4.1%) including three deaths (0.6%), nine tamponades (1.9%), four major bleedings (0.8%) and two device embolisations (0.4%). Among patients successfully implanted having a complete follow-up (672 patient-years, median follow-up 370 days), the actual annual stroke rate was 1.2%, lower than the expected stroke risk of 4% (70% reduction). The observed bleeding rate was 2%, while the calculated risk was 3.7% (46% reduction). Kaplan-Meier analysis showed a similar overall survival (93±2% and 87±3% versus 91±3% and 87±4%; p=0.35) and event-free survival (92±2% and 84±3% versus 88±3% and 80±5%; p=0.17) at one and two years, for the ACP/Amulet versus the WATCHMAN groups of patients, respectively., Conclusions: The data from the Belgian left atrial appendage occlusion registry suggest that the procedure is effective and relatively safe in a real-world setting, using either the WATCHMAN or the ACP/Amulet device.

Nutrition during the perinatal period programs body growth. Growth hormone (GH) secretion from the pituitary regulates body growth and is controlled by Growth Hormone Releasing Hormone (GHRH) neurons located in the arcuate nucleus of the hypothalamus. We observed that dietary restriction during the early postnatal period (i.e. lactation) in mice influences postnatal growth by permanently altering the development of the somatotropic axis in the pituitary gland. This alteration may be due to a lack of GHRH signaling during this critical developmental period. Indeed, underfed pups showed decreased insulin-like growth factor I (IGF-I) plasma levels, which are associated with lower innervation of the median eminence by GHRH axons at 10 days of age relative to normally fed pups. IGF-I preferentially stimulated axon elongation of GHRH neurons in in vitro arcuate explant cultures from 7 day-old normally fed pups. This IGF-I stimulating effect was selective since other arcuate neurons visualized concomitantly by neurofilament labeling, or AgRP immunochemistry, did not significantly respond to IGF-I stimulation. Moreover, GHRH neurons in explants from age-matched underfed pups lost the capacity to respond to IGF-I stimulation. Molecular analyses indicated that nutritional restriction was associated with impaired activation of AKT. These results highlight a role for IGF-I in axon elongation that appears to be cell selective and participates in the complex cellular mechanisms that link underfeeding during the early postnatal period with programming of the growth trajectory., Competing Interests: The authors have declared that no competing interests exist.

Identification of new adipokines that potentially link obesity to insulin resistance represents a major challenge. We recently showed that NOV/CCN3, a multifunctional matricellular protein, is synthesized and secreted by adipose tissue, with plasma levels highly correlated with BMI. NOV involvement in tissue repair, fibrotic and inflammatory diseases, and cancer has been previously reported. However, its role in energy homeostasis remains unknown. We investigated the metabolic phenotype of NOV(-/-) mice fed a standard or high-fat diet (HFD). Strikingly, the weight of NOV(-/-) mice was markedly lower than that of wild-type mice but only on an HFD. This was related to a significant decrease in fat mass associated with an increased proportion of smaller adipocytes and to a higher expression of genes involved in energy expenditure. NOV(-/-) mice fed an HFD displayed improved glucose tolerance and insulin sensitivity. Interestingly, the absence of NOV was associated with a change in macrophages profile (M1-like to M2-like), in a marked decrease in adipose tissue expression of several proinflammatory cytokines and chemokines, and in enhanced insulin signaling. Conversely, NOV treatment of adipocytes increased chemokine expression. Altogether, these results show that NOV is a new adipocytokine that could be involved in obesity-associated insulin-resistance., (© 2016 by the American Diabetes Association.)

Mismatch-repair (MMR)-deficient cells show increased in vitro tolerance to thiopurines because they escape apoptosis resulting from MMR-dependent signaling of drug-induced DNA damage. Prolonged treatment with immunosuppressants including azathioprine (Aza), a thiopurine prodrug, has been suggested as a risk factor for the development of late onset leukemias/lymphomas displaying a microsatellite instability (MSI) phenotype, the hallmark of a defective MMR system. We performed a dose effect study in mice to investigate the development of MSI lymphomas associated with long term Aza treatment. Over two years, Aza was administered to mice that were wild type, null or heterozygous for the MMR gene Msh2. Ciclosporin A, an immunosuppressant with an MMR-independent signaling, was also administered to Msh2(wt) mice as controls. Survival, lymphoma incidence and MSI tumor phenotype were investigated. Msh2(+/-) mice were found more tolerant than Msh2(wt) mice to the cytotoxicity of Aza. In Msh2(+/-) mice, Aza induced a high incidence of MSI lymphomas in a dose-dependent manner. In Msh2(wt) mice, a substantial lifespan was only observed at the lowest Aza dose. It was associated with the development of lymphomas, one of which displayed the MSI phenotype, unlike the CsA-induced lymphomas. Our findings define Aza as a risk factor for an MSI-driven lymphomagenesis process.

Circadian rhythms have an essential role in feeding behavior and metabolism. RORα is a nuclear receptor involved in the interface of the circadian system and metabolism. The adipocyte glyceroneogenesis pathway derives free fatty acids (FFA) liberated by lipolysis to reesterification into triglycerides, thus regulating FFA homeostasis and fat mass. Glyceroneogenesis shares with hepatic gluconeogenesis the key enzyme phosphoenolpyruvate carboxykinase c (PEPCKc), whose gene is a RORα target in the liver. RORα-deficient mice (staggerer, ROR(sg/sg)) have been shown to exhibit a lean phenotype and fasting hypoglycemia for unsolved reasons. In the present study, we investigated whether adipocyte glyceroneogenesis might also be a target pathway of RORα, and we further evaluated the role of RORα in hepatocyte gluconeogenesis. In vivo investigations comparing ROR(sg/sg) mice with their wild-type (WT) littermates under fasting conditions demonstrated that, in the absence of RORα, the release of FFA into the bloodstream was altered and the rise in glycemia in response to pyruvate reduced. The functional analysis of each pathway, performed in adipose tissue or liver explants, confirmed the impairment of adipocyte glyceroneogenesis and liver gluconeogenesis in the ROR(sg/sg) mice; these reductions of FFA reesterification or glucose production were associated with decreases in PEPCKc mRNA and protein levels. Treatment of explants with RORα agonist or antagonist enhanced or inhibited these pathways, respectively, in tissues isolated from WT but not ROR(sg/sg) mice. Our results indicated that both adipocyte glyceroneogenesis and hepatocyte gluconeogenesis were regulated by RORα. This study demonstrates the physiological function of RORα in regulating both glucose and FFA homeostasis., (Copyright © 2015 the American Physiological Society.)

The use of high dose baclofen for alcohol-dependence emerged in France from 2008 based on empirical findings, and is still off-label. However, due to the rapid increase in this prescribing practice, the French health authorities have decided to frame it using an extraordinary regulatory measure named "temporary recommendation for use" (TRU). Baclofen prescribers from CAMTEA, a regional team-based off-label system for supervising baclofen prescribing, which was developed much prior to the TRU, discuss herein the pros and cons of this measure and the applicability of its different aspects in the daily clinical practice., (© 2014 Société Française de Pharmacologie et de Thérapeutique.)