Growth hormone (GH) has growth-stimulating effects on skeletal muscle and liver but a growth-inhibitory effect on adipose tissue. The mechanisms underlying these actions of GH are not fully understood. Two studies were conducted to achieve the following objectives: 1) to determine the cellular mechanism by which GH stimulates liver growth; 2) to determine the effects of GH on the commitment of mesenchymal stem cells (MSCs) to myogenic and adipogenic lineages. In the first study, the GH-deficient lit/lit male mice were injected (s.c.) daily with rbGH or vehicle for two weeks. GH-injected lit/lit mice tended to have a greater liver/body weight percentage than lit/lit control mice. GH injection did not alter the percentage of proliferating cells in the liver. However, GH-injected lit/lit mice had 18% larger hepatocytes and 16% less DNA per unit liver weight than those of lit/lit control mice. These data together indicate that GH stimulates liver growth in mice by increasing the size, not by increasing the number of hepatocytes. In the second study, we treated the MSC cell line C3H10T1/2 cells with or without 5'-azacytidine and rbGH for 4 days. We assessed the myogenic or adipogenic potential by determining the ability of these cells to differentiate into myotubes or adipocytes, respectively. C3H10T1/2 cells treated with 5'-azacytidine and GH formed more myotubes, myoblasts, and fewer adipocytes compared to cells treated with 5'-azacytidine alone. Taken together, these results suggest that GH enhances 5'-azacytidine-induced myogenic commitment but inhibits 5'-azacytidine-induced adipogenic commitment in C3H10T1/2 cells. / Master of Science

Obesity is a significant health care problem, affecting more than one third of the United States population and is an important risk factor for Type 2 Diabetes Mellitus (T2D). Adipose tissue expansion results in the recruitment and accumulation of macrophages, which secrete proinflammatory cytokines that impair insulin signaling. Adenosine regulates inflammation by signaling through G-protein coupled receptors (GPCRs), such as the A2b adenosine receptor (A2bAR). Recently a role for adenosine receptors has been described in the differentiation of osteoblasts and adipocytes. This thesis tests the hypothesis that the A2bAR regulates adipose tissue dynamics at the level of preadipocyte differentiation and macrophage inflammation. This thesis showed that activation of the A2bAR inhibited preadipocyte differentiation. A2bAR-induced adipocyte inhibition was dependent on the expression of Krüppel-like factor 4 (KLF4), which is important for stem cell maintenance and renewal. A2bAR knockdown enhanced adipogenesis in vitro and A2bAR knockout (KO) mice had more adipocytes as compared to wild type (WT) mice, suggesting enhanced adipogenesis in the absence of the A2bAR. The translational potential of this work is strengthened by the previous finding of elevated A2bAR expression in adipose tissue of obese individuals as well as our new finding of a close correlation between the expression of A2bAR and KLF4 in adipose tissue of obese individuals. A2bAR KO mice have impaired insulin resistance, in part due to reduced levels of insulin receptor substrate-2 (IRS-2). Proinflammatory cytokines have been shown to reduce IRS-2 levels. Given the role of the A2bAR in regulating inflammation, the contribution of A2bAR signaling in macrophages to insulin resistance was elucidated. Transgenic mice that express A2bAR only in macrophages were generated. Intriguingly, restoration of A2bAR signaling in macrophages ameliorated insulin resistance, glucose tolerance, and fat and liver tissue insulin signaling. As expected, tissue and plasma proinflammatory cytokine levels were reduced to that of WT mice. This suggested that the protective effect of A2bAR signaling on insulin resistance was due in large part to A2bAR control of macrophage cytokine expression. This thesis highlights the importance of A2bAR signaling in adipogenesis and in regulating inflammation in the setting of obesity and T2D.

No description available.

The objectives of this study were to determine: the effects of maternal melatonin (MEL) supplementation during late gestation on the histological and molecular regulation in the Longissimus dorsi (LM) muscle of fetal bovine offspring, composition and gene expression of fetal perirenal (PR) adipose tissue, and LM gene expression in postnatal offspring at birth and d 195 of age. Maternal supplementation of MEL during late gestation resulted in no difference in calf fetal body weight or birth weight. However, at d 195 of age, calves from MEL treated dams had an average body weight increase of 20 kg. Fetal LM weight and length tended to be increased in calves from MEL treated dams. Fetal gene expression of calves from MEL treated dams resulted in: increased LM adenosine monophosphate-activated protein kinase-α (AMPK) and decreased PR adiponectin (ADIPOQ), CCAAT enhancer binding protein alpha (CEBPA), proliferator activated receptor gamma (PPARg), and stearoyl-CoA desaturase (SCD). The improved metabolic status of LM coupled with the decrease in adipogenic gene expression, could result in calves from MEL treated dams having improved lean muscle accretion and reduced overall adiposity during postnatal development.

Visceral adipose tissue (AT) mass increases risk for cardiovascular disease and diabetes. Glucocorticoids (GCs) cause preferential expansion of visceral compared to subcutaneous AT through poorly understood mechanisms. GCs are necessary for adipogenesis, the differentiation of adipose stem cells (ASCs) to mature adipocytes. However, this process may be impaired in visceral depots. Insufficient adipogenesis can lead to excessive hypertrophy of existing adipocytes. This hypertrophic expansion increases cell death and inflammation, driving AT dysfunction. To better understand the genes and pathways by which high GCs cause preferential expansion of visceral fat we performed transcriptomic profiling (microarray) on paired samples of visceral (Omental, Om) and abdominal subcutaneous (Abdsc) AT explants cultured with the GC receptor agonist, dexamethasone (Dex), for 7 days. Gene set enrichment analysis showed the transforming growth factor beta (TGFβ) signaling pathway, most notably the secreted anti-adipogenic factors, TGFβ and activin A, was highly enriched in Om and suppressed less by Dex. We hypothesized that Om AT and ASCs secrete factors that inhibit adipogenesis in an autocrine/paracrine manner. Conditioned media (CM) from Om tissue and ASCs suppressed differentiation by 70-80% compared to control; Dex attenuated this anti-adipogenic effect. Both TGFβ and activin A levels were 4-5 fold higher in CM from Om compared to Abdsc ASCs. Both factors signal via cell surface receptors that increase SMAD2 phosphorylation (P-SMAD2), basal levels of which were 3-4 fold higher in Om ASCs. Additionally, CM from Om ASCs increased P-SMAD2. siRNA mediated knockdown of activin A improved differentiation of Om ASCs, but did not reach levels observed in Abdsc. Blocking TGFβ and activin A signaling using SB431542 robustly increased adipogenesis of Om ASCs and prevented the anti-adipogenic effect of CM. GCs decreased production of TGFβ and activin A, but both remained higher in OmCM. Overnight Dex treatment decreased P-SMAD2 and increased the expression of the TGFβ co-receptor, TGFBR3, which decreases TGFβ signaling, in Abdsc ASCs. GCs failed to decrease P-SMAD2 and increased TGFBR3 in Om ASCs only at high concentrations. Taken together, these data implicate GC-TGFβ crosstalk as a determinant of depot differences in adipogenic capacity and hypertrophic vs. healthy hyperplastic expansion of AT. / 2019-11-01T00:00:00Z

No description available.

No description available.

No description available.

L'acide ascorbic acid (AA) a été considéré, pendant longtempss, comme une molecule devantêtre absorbée dans la nutrition, et prévenant le scorbut. Notre hypothèse, fondé sur desrésulats de notre groupe, suggèrent de nouvelles fonctions.Parmi celles-ci, nous nous sommes posé la question de l'AA molècule de signalling, durantl'embryogenèse et chez l'adulte, commme l'acide rétinoique (principe actif de la vitamine A)l'est. A cet effet, nous avons utilisé deux modèles cellulaires : des cellules souchesembryonnaires murines et des lignées de cellules souches/progénétrices adultes. Nous avonsainsi montré que l'AA stimule la différentiation de ces cellules en cellules musculairessquelettiques et en osteoblastes et inhibe l'adipogenèse et la neurogenèse. Cet effet passe parle transporteur de l'AA SVCT2 et implique la voie p38/MAPK. D'autre part, nous avonsdemontré que l'AA agit en compétition avec le RA, sur la neurogenèse et la myogenèse.Enfin, dans des cellules mésenchymateuses adultes, nous avons montré que l'AA inhibel'adipogenèse et stimule l'ostéogenèse. Cette action, comme chez l'embryon implique SVCT2et une modulation du pool du cAMP.En conclusion, l'AA pousse les cellules à se différencier en cellule musculaire squelettique eten ostéoblste et inhibie l'adipogenèse et la neurogenèse. / AA has been considered for a long time as a molecule involved in nutrition, to prevent scurvy. Our hypothesis is that AA could also be involved in development during embryogenesis, as well as in cell differentiation in adults. The aim of this study is to evaluate the potential implication of AA in cell differentiation, especially of mesenchyme cells, and to propose potential pathways that could be involved in these processes. Using murine ESCs we observed that AA markedly enhance the differentiation of ESCs toward muscle cells. Furthermore, we demonstrated that induction of myocytes by AA involves p38MAPK pathway and p-CREB. Moreover, we demonstrated that AA acts in mirror with retinoic acid. ESCs treated with RA mainly differentiate into neuronal cells, but AA compete, in a dosage dependent way to this differentiation. AA induces differentiation of ESCs into cardiac myocytes and could probably acts through p38MAPK pathway. Regarding adipocyte we revealed that SVCT2 expression significantly decreased as preadipocytes cells differentiate to adipocytes. This data suggests that mature adipocytes could not receive signals from AA. In addition, our results show that the expression of SVCT2 is increased in cells treated with AA and without IBMX. Moreover, we demonstrated that AA evolves in decreasing of cells containing lipids. Finally, we demonstrated that AA is not only involved in muscle differentiation of mesenchyme but is also involved in adipose tissue as a negative inducer. In conclusion, AA drives differentiation of ESCs toward muscle cells and osteoblast, incompetition with RA, and has a negative effect on adipogenesis and neurogenesis differentiation.

Les aldose réductases (AKR1B) sont des oxydoréductases dépendantes du NADPH initialement décrites pour leurs fonctions de détoxication cellulaire et de réduction du glucose. La découverte de l’expression d’Akr1b7 dans le tissu adipeux murin ainsi que l’activité prostaglandine F2α synthase (PGFS) spécifique de certaines isoformes suggèrent des rôles biologiques inédits pour ces enzymes. La prostaglandine F2α (PGF2α) inhibant l’adipogenèse, cette fonction PGFS met en avant l’implication des AKR1B dans la physiologie du tissu adipeux blanc (TAB). L’objectif de ces travaux était de caractériser l’expression de l’ensemble des AKR1B au sein des TAB murins et humains et de comprendre leur impact sur l’homéostasie du tissu adipeux et en particulier sur l’adipogenèse et la lipolyse. Nous avons montré que l’ensemble des AKR1B était exprimé dans le TAB murin. Akr1b3, Akr1b8 et Akr1b16 sont exprimées à la fois dans les fractions stroma‑vasculaires (contenant des cellules immunitaires, vasculaires, progénitrices…) et adipocytaires. A l’inverse, Akr1b7 n’est pas exprimé par les adipocytes. Les analyses réalisées in vitro indiquent qu’à l’exception d’Akr1b16, les isoformes murines des AKR1B voient leur expression augmenter précocement et transitoirement au cours de l’adipogenèse. Chez l’homme, l’isoforme AKR1B1 est exprimée dans le TAB sous‑cutané de patients obèses alors qu’AKR1B10 est difficilement détectable (western blot, RT‑qPCR). In vitro, l’expression d’AKR1B1 augmente tout au long de la différenciation adipocytaire contrairement à AKR1B10 qui est préférentiellement exprimé dans les cellules indifférenciées. L’utilisation d’un inhibiteur spécifique des AKR1B montre que l’activité PGFS d’AKR1B1 constitue un frein à l’adipogenèse. Nous montrons aussi que les mécanismes régulant l’action de la PGF2α diffèrent en fonction des espèces. Chez l’homme, l’expression du récepteur FP est régulée dans le temps alors que dans les cellules murines, c’est l’expression des PGFS et donc la synthèse de PGF2α qui définit, au cours de l’adipogenèse, la fenêtre d’action de cette prostaglandine. Les souris invalidées pour la PGFS Akr1b7 présentent une diminution des quantités intra‑tissulaires en PGF2α associée à une expansion accrue de leurs tissus adipeux due à une augmentation de l’adipogenèse et à une hypertrophie adipocytaire sans modification de l’expression des enzymes impliquées dans la lipogenèse (Volat et al., 2012). Ces données en accord avec le rôle anti‑adipogénique de la PGF2α suggèrent aussi une action sur la lipolyse. Nous démontrons ici que la perte d’Akr1b7 entraîne une diminution de l’activité lipolytique du TAB. L’utilisation de cellules murines (3T3‑L1) et humaines (hMADS) différenciées en adipocytes, nous a permis de montrer que la stimulation de l’activité lipolytique suite à l’activation du récepteur FP résultait en partie d’une augmentation de la phosphorylation de HSL (forme active) et de l’accumulation de la lipase ATGL. Le troisième volet de ce travail de thèse a consisté à caractériser un modèle de souris transgénique surexprimant AKR1B1 dans le TAB (souris aP2‑AKR1B1) afin d’étudier le rôle biologique de cette isoforme humaine. / Aldose reductases are NADPH-dependent oxydoreductases described for their involvement in cellular detoxification and glucose reduction. The discovery of Akr1b7 expression in murine adipose tissue together with the prostaglandin F2α Synthase (PGFS) activity of some isoforms suggest unreleased biological roles for these enzymes. Prostaglandin F2α (PGF2α) inhibiting adipogenesis, this PGFS function highlights AKR1B potential involvement in white adipose tissue (WAT) physiology. This work aimed at characterising the expression of all AKR1B in both murine and human WAT and understanding their impact on adipose tissue homeostasis and especially on adipogenesis and lipolysis. We showed that all AKR1B were expressed in murine WAT. Akr1b3, Akr1b8 and Akr1b16 were both expressed in the stromal vascular fraction (containing immune cells, vascular cells, progenitors…) and in the adipose fraction. In contrast, Akr1b7 was not expressed in adipocytes. In vitro analyses indicated that, except for Akr1b16, murine AKR1B isoform expression increased early and transiently during adipogenesis. In human, AKR1B1 was expressed in human subcutaneous WAT from obese patients whereas AKR1B10 was hardly detectable (western blot, RT‑qPCR). In vitro, AKR1B1 expression increased throughout adipocyte differentiation unlike AKR1B10, which was preferentially expressed in undifferentiated cells. Using an AKR1B specific inhibitor, we demonstrated that AKR1B1 PGFS activity was a dampen to adipogenesis. We also showed that mechanisms regulating PGF2α action differed according to the species. In human cells, the expression of FP receptor was time-regulated whereas, in murine cells, PGFS expression and thus, PGF2α synthesis, limited PGF2α activity during adipogenesis. Akr1b7 knockout mice have decreased PGF2α intratissular levels associated with an expansion of adipose tissue resulting from an increase of adipogenesis and an adipocyte hypertrophia without any modification of lipogenic enzymes expression (Volat et al., 2012). These data, in agreement with PGF2α anti-adipogenic action, suggest an impact on lipolysis. We demonstrated that loss of Akr1b7 led to a decrease of WAT lipolytic activity. The use of murine (3T3‑L1) and human (hMADS) differentiated cells allowed us to show that the stimulation of lipolysis in response to FP activation was, in part, due to an increase of HSL phosphorylation (active form) and an increase of ATGL accumulation. The third part of this work consisted in characterizing the phenotype of transgenic mice overexpressing AKR1B1 in WAT (aP2‑AKR1B1 mice) in order to study the biological role of this human isoform.