Function and mass adaptation of pancreatic beta cells in a model of glucocorticoids induced insulin-resistance

Les diabètes de type 1 et de type 2 sont caractérisés par une sécrétion insuffisante d’insuline et une diminution de la masse des cellules bêta. Pouvoir régénérer une masse de cellules bêta fonctionnelle est donc un enjeu thérapeutique dans le traitement du diabète. Dans cet optique, nous cherchons à identifier des facteurs et mécanismes permettant d’augmenter la masse de cellules bêta. Nous nous sommes intéressés aux mécanismes de plasticité des cellules bêta dans un contexte d’insulino résistance.Dans un modèle murin d’insulino-résistance provoquée par administration chronique de glucocorticoïdes, nous avons mis en évidence une adaptation de fonction des cellules bêta par hypersécrétion d’insuline. De manière intéressante une augmentation continue et progressive de la masse des cellules bêta par prolifération mais surtout par néogénèse de cellules bêta a pu être observée. Bien que la néogénèse de cellules bêta ait été décrite dans d’autres modèles murins comme un processus récapitulant le programme de différenciation fœtale c’est à dire dérivant de cellules canalaires marquées par l’expression de Sox9 et re-exprimant Ngn3, nos expériences de lignage endocrine ont révélé que les cellules bêta néoformées ne dérivent pas des cellules Sox9 ou Ngn3. L’invalidation du récepteur aux glucocorticoïdes (GR) dans le pancréas n’altère pas l’adaptation pancréatique par néogénèse dans notre modèle d’hypercorticisme, suggérant un effet indirect des GC sur la néogénèse de cellules bêta. Cette hypothèse a pu être confirmée par la mise en évidence de la présence dans le sérum des souris CORT d’un facteur capable de stimuler la néogénèse des cellules bêta in vitro. Enfin après déplétion totale des cellules bêta, l’administration de GC permet une restauration partielle de la masse de cellules béta par néogénèse.Nos résultats apportent la preuve d’une néogénèse active et induite de cellules bêta dans le pancréas adulte de souris insulino-résistantes. Cette adaptation pancréatique résulte d’une communication inter organe adaptative et l’identification du facteur pro-néogénique représente une piste thérapeutique pour les pathologies liées aux déficiences du pancréas endocrine.Type 1 and type 2 diabetes are characterized by an insufficient insulin secretion and a decrease of beta cell mass. Regenerate a functional beta cell mass is a therapeutic issue in the treatment of diabetes. In this context we search to identify factors and mechanisms for increasing beta cell mass. We investigated mechanisms of beta cell plasticity in a context of insulin resistance.In a mouse model of insulin resistance caused by chronic administration of glucocorticoids, we demonstrated an adaptation of beta cell function by an important increase of insulin secretion. Interestingly, a continuous and progressive increase in the mass of beta cells by proliferation but especially by neogenesis of beta cells was observed.Although beta cell neogenesis has been described in other mouse models as a process recapitulating the fetal differentiation program deriving from ductal cells labeled with Sox9 expression and re-expressing Ngn3, our endocrine lineage model revealed that neoformed beta cells do not derive from Sox9 or Ngn3 cells. Inactivation of the glucocorticoid (GR) receptor in the pancreas does not alter pancreatic adaptation by neogenesis in our model of hypercorticism, suggesting an indirect effect of GCs on beta cell neogenesis. This hypothesis could be confirmed by demonstrating the presence in the serum of CORT mice of a factor able to stimulate neogenesis of beta cells in vitro. Finally, after complete depletion of beta cells, GC administration allows a partial restoration of the beta cells mass by neogenesis.Our results provide evidence of an active and induced beta-cell neogenesis in the adult pancreas of insulin-resistant mice. This pancreatic adaptation results from an inter-organ adaptive communication and the identification of the pro-neogenic factor represents a therapeutic track for pathologies related to endocrine pancreas deficiencies

Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC

Predictive value of premature atrial complex characteristics in pulmonary vein isolation for patients with paroxysmal atrial fibrillation

International audienceBackgroundPremature atrial complexes from pulmonary veins are the main triggers for atrial fibrillation in the early stages. Thus, pulmonary vein isolation is the cornerstone of catheter ablation for paroxysmal atrial fibrillation. However, the success rate remains perfectible.AimTo assess whether premature atrial complex characteristics before catheter ablation can predict pulmonary vein isolation success in paroxysmal atrial fibrillation.MethodsWe investigated consecutive patients who underwent catheter ablation for paroxysmal atrial fibrillation from January 2013 to April 2017 in two French centres. Patients were included if they were treated with pulmonary vein isolation alone, and had 24-hour Holter electrocardiogram data before catheter ablation available and a follow-up of ≥ 6 months. Catheter ablation success was defined as freedom from any sustained atrial arrhythmia recurrence after a 3-month blanking period following catheter ablation.ResultsOne hundred and three patients were included; all had an acute successful pulmonary vein isolation procedure, and 34 (33%) had atrial arrhythmia recurrences during a mean follow-up of 30 ± 15 months (group 1). Patients in group 1 presented a longer history of atrial fibrillation (71.9 ± 65.8 vs. 42.9 ± 48.4 months; P = 0.008) compared with those who were “free from arrhythmia” (group 2). Importantly, the daily number of premature atrial complexes before catheter ablation was significantly lower in group 1 (498 ± 1413 vs. 1493 ± 3366 in group 2; P = 0.028). A daily premature atrial complex cut-off number of < 670 predicted recurrences after pulmonary vein isolation (41.1% vs. 13.3%; sensitivity 88.2%; specificity 37.7%; area under the curve 0.635; P = 0.017), and was the only independent predictive criterion in the multivariable analysis (4-fold increased risk).ConclusionPreprocedural premature atrial complex analysis on 24-hour Holter electrocardiogram in paroxysmal atrial fibrillation may improve patient selection for pulmonary vein isolation.ContexteL’isolation des veines pulmonaires (IVP) est la pierre angulaire du traitement invasif de la fibrillation atriale paroxystique (FAP). Cependant, le taux de succès de cette procédure reste perfectible.ObjectifIdentifier si certaines caractéristiques des extrasystoles atriales (ESA) enregistrées sur le Holter électrocardiogramme de 24 heures précédant l’ablation constituent un facteur prédictif de succès de procédure.MéthodesTous les patients ayant eu une IVP seule pour une FAP ont été analysés entre janvier 2013 et avril 2017 au sein de deux centres français. Les critères d’inclusion étaient : les patients adressés pour une primo-ablation de FAP, disposant d’un Holter électrocardiogramme avant l’ablation, dont le suivi post-ablation était ≥ 6 mois. Le critère de jugement principal était la récidive d’une arythmie atriale après une période de blanking de 3 mois post-ablation.RésultatsCent trois patients ont été inclus, et 34 (33 %) ont récidivé après un suivi moyen de 30 ± 15 mois. Ces patients présentaient une histoire de FA plus ancienne (71,9 ± 65,8 mois vs 42,9 ± 48,4 mois; p = 0,008). Le nombre d’ESA total sur 24 heures pré-ablation était significativement plus important dans le groupe « non récidive » que dans le groupe « récidive » (1493 ± 3366 vs 498 ± 1413, respectivement; p = 0,028). Un seuil d’ESA < 670/24 heures prédisait la récidive post-IVP (sensibilité 88,2 %; spécificité 37,7 %; AUC 0,635; p = 0,017).ConclusionL’étude des ESA en pré-ablation peut potentiellement améliorer la sélection des patients pour l’ablation de FAP par IVP seule

Impaired Glucose Homeostasis in a Tau Knock-In Mouse Model

International audienceAlzheimer’s disease (AD) is the leading cause of dementia. While impaired glucose homeostasis has been shown to increase AD risk and pathological loss of tau function, the latter has been suggested to contribute to the emergence of the glucose homeostasis alterations observed in AD patients. However, the links between tau impairments and glucose homeostasis, remain unclear. In this context, the present study aimed at investigating the metabolic phenotype of a new tau knock-in (KI) mouse model, expressing, at a physiological level, a human tau protein bearing the P301L mutation under the control of the endogenous mouse Mapt promoter. Metabolic investigations revealed that, while under chow diet tau KI mice do not exhibit significant metabolic impairments, male but not female tau KI animals under High-Fat Diet (HFD) exhibited higher insulinemia as well as glucose intolerance as compared to control littermates. Using immunofluorescence, tau protein was found colocalized with insulin in the β cells of pancreatic islets in both mouse (WT, KI) and human pancreas. Isolated islets from tau KI and tau knock-out mice exhibited impaired glucose-stimulated insulin secretion (GSIS), an effect recapitulated in the mouse pancreatic β-cell line (MIN6) following tau knock-down. Altogether, our data indicate that loss of tau function in tau KI mice and, particularly, dysfunction of pancreatic β cells might promote glucose homeostasis impairments and contribute to metabolic changes observed in AD

The transcription factor E2F1 controls the GLP-1 receptor pathway in pancreatic β cells

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PTN induces hypertrophic differentiation of hBMSC.

<p>hBMSC from 3 independent patients were cultured in micromass with chondrogenic medium in absence or with increasing doses of PTN (0 pg/ml white boxes, 50 pg/ml grey boxes and 500 pg/ml black boxes) for 21 days. All conditions were performed in triplicate per patient. (<b>A</b>): Alizarin red staining of chondrogenic pellets. (<b>B</b>): Collagen10 immunostaining of chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100 µm. (<b>C</b>): Real-time polymerase chain reaction analysis of hypertrophic related genes expression. RNA were purified from hBMSC at day 0 and cultured without or with PTN at day 7 and 14. Expression levels of hypertrophic genes Matrix Metalloprotease 13 (MMP13), Collagen 10A1 (Col10) and Alkaline Phosphatase (ALP) are related to Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Values are the mean±SEM. The gene expression values at day 0 are represented by straightened lines when they are different of 0. Statistical analysis were performed, with a one way anova Kruskal-Wallis test, between values from free-PTN conditions (white boxes) at day 0, 7 and 14 (##: p<0.01; ###: p<0.001) and between values from increasing PTN doses from the same day (*: p<0.05; **: p<0.01).</p

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PTN increases glycosaminoglycans content of chondrogenic induced hBMSC.

<p>hBMSC from 3 independent patients were cultured in micromass with chondrogenic medium in the absence or with increasing doses of PTN (0 pg/ml white boxes, 50 pg/ml grey boxes and 500 pg/ml black boxes) for 21 days. All conditions were performed in triplicate per patient. (<b>A</b>): Alcian blue staining of sulfated GAGS in chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100 µm. (<b>B</b>): Total sulfated GAGs quantification. After 14 days in micromass culture, sulfated GAGs were extracted from pellets and quantified as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088287#pone.0088287-Barbosa1" target="_blank">[27]</a>. GAG amount was normalized according to pellet volume and reported as µg of total GAGs per mm³. (<b>C</b>): Real-time polymerase chain reaction analysis of proteoglycan protein core-gene expression. RNA were purified from hBMSC at day 0 and after 7 and 14 days of culture without or with PTN. Expression levels of proteoglycans core expression Aggrecan (ACAN), Biglycan (BGN), Decorin (DCN), Versican (VCAN) are related to Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Values are the mean±SEM. The gene expression values at day 0 are represented by straightened lines. Statistical analysis were performed, with a one way anova Kruskal-Wallis test, between values from free-PTN conditions (white boxes) at day 0, 7 and 14 (###: p<0.001), and between values from increasing PTN doses from the same day (*: p<0.05; **: p<0.01; ***: p<0.001).</p

PTN increases cartilage specific protein and gene expression during hBMSC chondrogenic differentiation.

<p>hBMSC from 3 independent patients were cultured in micromass with chondrogenic medium in absence or with increasing doses of PTN (0 pg/ml white boxes, 50 pg/ml grey boxes and 500 pg/ml black boxes) for 21 days. All conditions were performed in triplicate per patient. (<b>A</b>): Collagen 2 immunostaining of chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100 µm. (<b>B</b>): Real-time polymerase chain reaction analysis of cartilage related genes expression. RNA were purified from hBMSC at day 0 and after 7 and 14 days of culture without or with PTN. Expression levels of cartilage genes: SRY-box9 (Sox9), Cartilage Oligomeric Matrix Protein (COMP) and Collagen9A1 (Col9) are normalized to Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Values are the mean±SEM. The gene expression values at day 0 are represented by straightened lines. Statistical analysis were performed, with a one way anova Kruskal-Wallis test, between values from free-PTN conditions (white boxes) at day 0, 7 and 14 (##: p<0.01; ###: p<0.001), and between values from increasing PTN doses from the same day (**: p<0.01; ***: p<0.001).</p

PTN chondroinductive effects are inhibited by inhibitors of PTN receptors and Pi3K.

<p>hBMSC were cultured in micromass with chondrogenic medium with 0/ml or 500 pg/ml of PTN with or without Ly294002 (15 µM) or p111-136 peptide (100 ng/ml). All conditions were performed in triplicate. (<b>A</b>): Real-time polymerase chain reaction analysis of late chondrogenic marker genes after 14 days of treatment. Expression levels of Matrix Metalloprotease 13 (MMP13), Collagen 10A1 (Col10) and Alkaline Phosphatase (ALP) are related to Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Values are the mean±SEM. Statistical analysis were performed, with a one way anova Kruskal-Wallis test between DMSO treated and Ly294002 or P111-136 treated hBMSC (*: p<0.05; ***: p<0.001). (<b>B</b>): Collagen10 immunostaining of chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100µm.</p