Pro-opiomelanocortin (POMC) is the precursor of many neuropeptides which includ adrenocorticotropin (ACTH). ACTH has a biological activity in regulating adrenocortical function. In the present study, we will investigate the effect of POMC gene transfer on adrenal cortex cells in cell cultures and animal models. The study included adrenal cortical H295R cells for adenovirus-mediated gene delivery. The effects of POMC gene on H295R cell steroidogenesis and cell proliferation were investigated. In addition, there were 32 SD rats dividing into three groups. 1) Control, injected with normal saline via tail vein (n = 8); 2) Ad-GFP, injected with adenovirus containing GFP (n=12); 3) Ad-POMC, injected with adenovirus containing recombinant POMC gene (n=12). Body weight (BW) was measured. Adrenals were collected, fixed and a series of sections were cut for stains for PCNA and MC2-R. The plasma cortisol and VEGF levels of rats were measured. The results showed that Ad-POMC delivery significantly increased the ACTH and cortisol levels by 50-100 fold and 20-100% in H295R cells, respectively. In addition, Ad-POMC delivery significantly inhibited the cell proliferation and increased the apoptotic cells. The expression of MC2-R protein of H295R cells was also suppressed after Ad-POMC delivery. In the study of SD rats, the Ad-POMC-treated rats exhibited reduced weight gain compared with other groups in the first 2 weeks; however, there was no significant change in BW between Ad-POMC and Ad-GFP groups during the experimental period. The weight of adrenal in Ad-POMC-treated rats was significantly higher than Ad-GFP group in the 8th week. Comparing the sequential adrenal weights in Ad-POMC group, those in 6th week were significantly higher than in 2nd and 4th weeks. The plasma VEFG levels of Ad-POMC-treated rats were higher than Ad-GFP group in the 8th week. The adrenal sections showed that Ad-POMC treated rats had moreanti-PCNA stained cells than Ad-GFP treated rats in 8th week. However, less anti-MC2R stained cells were found in Ad-POMC treated rats in 8th week. Ad-POMC treated rats had higher plasma cortisol levels than those in Ad-GFP treated rats, however, there were no statistical significances. In conclusion, POMC gene transfer modulates the morphology and function of the adrenal cortex. POMC gene inhibits the H295R cells proliferation by inducing MC2-R down-regulation and cells apoptosis. In SD rat adrenal, however, it stimulates adrenal cortex in biphasic pattern. The rapid growing pattern noted in the later phase may be due to the effect of VEGF. Besides, the physical regulation of cortisol synthesis is much stricter than that of ACTH.

<p>Single nucleotide polymorphisms (SNP) in leptin (LEP) and pro-opiomelanocortin (POMC) have been associated with beef carcass quality and yield respectively. Both hormones also play a role in immune performance. Since both of these genes are pleiotrophic, it was important to determine whether selection based on these SNPs would negatively affect immune cell numbers. A SNP in each of these hormones was assessed for effects on immune cell counts and antibody titres in twenty-seven beef cattle herds (n = 556). A commercial rabies vaccine was administered to these animals. Prior to being vaccinated, the types of lymphocytes evaluated included B cells, gamma delta cells, regular and activated CD4 and CD8 cells and numbers of lymphocytes as well as baseline serum antibody titres. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and lymphocyte types were again counted. Several cell types were significantly associated with the LEP genotype however, no consistent pattern of correlation was observed between LEP genotype (TT, CT or CC) and peripheral blood lymphocyte populations. The number of different lymphocytes significantly associated with LEP genotype increased from two on day 0 to four on day 42. Animals with CT and CC genotypes had significantly higher increased rabies antibody titres in the first 21 days after vaccination than those with TT genotypes. The POMC SNP also did not show a clear pattern of association between lymphocyte subtypes and genotype. There was no difference in response to the rabies vaccination associated with the POMC genotype. Our results suggested that selection at either of the SNPs examined in this research would not detrimentally impact immune function in beef cattle.</p>

<p>Single nucleotide polymorphisms (SNP) in leptin (LEP) and pro-opiomelanocortin (POMC) have been associated with beef carcass quality and yield respectively. Both hormones also play a role in immune performance. Since both of these genes are pleiotrophic, it was important to determine whether selection based on these SNPs would negatively affect immune cell numbers. A SNP in each of these hormones was assessed for effects on immune cell counts and antibody titres in twenty-seven beef cattle herds (n = 556). A commercial rabies vaccine was administered to these animals. Prior to being vaccinated, the types of lymphocytes evaluated included B cells, gamma delta cells, regular and activated CD4 and CD8 cells and numbers of lymphocytes as well as baseline serum antibody titres. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and lymphocyte types were again counted. Several cell types were significantly associated with the LEP genotype however, no consistent pattern of correlation was observed between LEP genotype (TT, CT or CC) and peripheral blood lymphocyte populations. The number of different lymphocytes significantly associated with LEP genotype increased from two on day 0 to four on day 42. Animals with CT and CC genotypes had significantly higher increased rabies antibody titres in the first 21 days after vaccination than those with TT genotypes. The POMC SNP also did not show a clear pattern of association between lymphocyte subtypes and genotype. There was no difference in response to the rabies vaccination associated with the POMC genotype. Our results suggested that selection at either of the SNPs examined in this research would not detrimentally impact immune function in beef cattle.</p>

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 148-168).

The mechanism of weight gain due to treatment with olanzapine, a serotonin receptor antagonist, has not been fully understood. Weight gain and food intake are under the control of neuropeptides/hormones, POMC (proopiomelanocortin), CART (cocaine and amphetamine regulated transcript), AgRP (Agouti-related peptide) and NPY (neuropeptide Y) that are synthesized and secreted from the arcuate nucleus (ARC) of hypothalamus. In this study, the altereration of the ARC neuropeptide/hormone levels both in humans and rats were determined as one of the weight gain mechanism. To examine olanzapine&rsquo / s weight gain effects, male first attack psychotic patients (pre-treatment), were hospitalized and treated for 4 -weeks (post-treatment), (n = 22), and healthy control group (n = 26) were included to the study. Case-control association design was used to analyze the changes in body mass index (BMI), peripheral leptin and the ARC neuropeptides levels. In patients, after 4-weeks of the olanzapine treatment / BMI and the waist circumference were significantly increased with average weight gain of 4.33 kg. In pre-treatment group, NPY levels were significantly lower while &alpha / -MSH, the anorexigenic product of POMC levels were significantly higher vs. control. At post-treatment, both leptin and NPY levels were significantly increased but the CART levels did not change. To further understand the underlying mechanism of olanzapine induced weight gain, the drug was orally administrated to 10 healthy male Wistar rats to analyze both the hypotalamic gene expression and peripheral levels of those candidate neuropeptides. In rats food consumption was increased and hypotalamic mRNA levels of NPY, AgRP and POMC were decreased while CART levels did not show any alteration. Consistent with the expression data, circulating levels of NPY, AgRP and &alpha / -MSH decreased significantly but CART levels were also reduced unexpectedly. In conclusion, it may be presumed that the antagonistic effect of olanzapine on the ARC neurons might be the basis for a disregulation of the neurohormones secretion which may cause weight gain in the treated psychotic patients.

Epidemiological studies show that offspring subjected to maternal undernutrition during early pregnancy are prone to developing obesity and other diseases in adulthood. The hypothalamic energy regulating pathway may be altered in these offspring, with epigenetic changes as a core mechanism. Therefore, this thesis aimed to determine if epigenetic changes are present in this pathway in the hypothalami from offspring subjected to maternal undernutrition. The investigations are focused on the glucocorticoid receptor (GR) as an inhibitor of the anorexigenic neuropeptide pro-opiomelanocortin (POMC), with potential modifications leading to increased food intake and the development of obesity. To achieve this, an established sheep model developed by our collaborators was used, during which maternal ewes were undernourished periconceptionally to produce a 10-15% decrease in body weight. We found that hypothalami from fetal offspring had greater epigenetic modifications when this reduction in maternal body weight was maintained from 60 days before conception until 30 days into pregnancy, with lower levels of POMC and GR promoter methylation. This was associated with increased GR mRNA expression. Other regions of the brain that also express POMC and GR, did not exhibit these epigenetic modifications. This study revealed that maternal undernutrition induces tissue specific epigenetic changes in fetal hypothalami which may contribute to disease in later life. Twins have been shown to have similar phenotypic characteristics as maternally undernourished offspring and therefore it has been suggested that they may also be programmed, but by intrauterine growth restriction. Consequently, extensive methylation and histone analysis of GR and POMC promoter regions was carried out in twin fetal hypothalami and compared to maternally undernourished groups. Interestingly, the decreased POMC and GR methylation of our amplicons in the maternally undernourished fetal hypothalami was also observed in twin fetal hypothalamic. This was concomitant with histone modifications and alterations in overall DNA methyltransferase activity. However, it was found that there were no changes in the POMC and GR mRNA expression levels in twin fetuses, but we postulate that this may occur later in life. To determine if changes in the fetal epigenetic status of hypothalamic GR and POMC impacted the adult progeny, tissues were obtained from adult offspring of maternally undernourished ewes. Epigenetic changes in the hypothalamic GR promoter observed in the fetal group persisted into adulthood, with concurrent increases in GR mRNA and GR protein expression. Of these groups the undernourished adult male offspring had decreased hypothalamic POMC expression and increased fat mass, changes that are consistent with an obese phenotype. The epigenetic and expression status of GR in the hippocampus and pituitary were modified, but in a tissue and sex specific manner. POMC epigenetic changes in the brain were complex, with various levels of epigenetic and expression changes. Overall periconceptional undernutrition induces hypothalamic specific changes in the epigenetic status of the GR gene which is known to regulate energy balance. Hypothalamic changes were persistent from the fetal stage into adulthood, with modifications in other tissues occurring after birth. These adaptations have the potential to increase the offspring’s propensity to develop obesity and altered stress regulation in later life.

No description available.

No description available.

Yes / Seasonal rhythms in energy balance are well documented across temperate and equatorial zones animals. The long-term regulated changes in seasonal physiology consists of a rheostatic system that is essential to successful time annual cycles in reproduction, hibernation, torpor, and migration. Most animals use the annual change in photoperiod as a reliable and robust environmental cue to entrain endogenous (i.e. circannual) rhythms. Research over the past few decades has predominantly examined the role of first order neuroendocrine peptides for the rheostatic changes in energy balance. These anorexigenic and orexigenic neuropeptides in the arcuate nucleus include neuropeptide y (Npy), agouti-related peptide (Agrp), cocaine and amphetamine related transcript (Cart) and pro-opiomelanocortin (Pomc). Recent studies also indicate that VGF nerve growth factor inducible (Vgf) in the arcuate nucleus is involved in the seasonal regulation of energy balance. In situ hybridization, qPCR and RNA-sequencing studies have identified that Pomc expression across fish, avian and mammalian species, is a neuroendocrine marker that reflects seasonal energetic states. Here we highlight that long-term changes in arcuate Pomc and Vgf expression is conserved across species and may provide rheostatic regulation of seasonal energy balance. / Academy of Medical Sciences, Leverhulme Trust

Durant le développement embryonnaire, une seule cellule, le zygote, est à l’origine de la formation de tous les types de cellules de l’organisme. L’information génétique nécessaire au destin d’une cellule y est gardé dans la chromatine condensée, d’abord inaccessible, et se déploie progressivement au moment opportun de la différenciation cellulaire grâce à des facteurs pionniers. Les facteurs de transcription pionniers sont des acteurs clés des cascades génétiques et épigénétiques déterminant le destin cellulaire. Les facteurs pionniers ont la propriété unique de reconnaître leurs cibles dans la chromatine fermée. Leur action permet d’augmenter l’accessibilité à un répertoire d’enhancers spécifique à un destin cellulaire, ouvrant la voie aux autres facteurs de transcription. Les aspects entourant la reconnaissance initiale des facteurs pionniers à leurs cibles dans la chromatine fermée sont mal compris. Dans l’hypophyse, le facteur pionnier Pax7 est spécifique aux cellules du lobe intermédiaire et met en place le programme génétique mélanotrope. Pour se faire, Pax7 repère les régions régulatrices clé de l’identité mélanotrope dans la chromatine fermée, afin d’accroître l’accessibilité à l’ADN permettant la liaison d’autres facteurs non-pionniers. Pax7 possède deux domaines de liaison à l’ADN, le domaine paired (PD) et l’homéodomaine (HD), chacun liant un motif de séquence caractéristique. De plus, une séquence cible composite, formé de la juxtaposition des motifs reconnus par PD et HD, est enrichie aux sites pionniers de Pax7. Dans le but de définir les propriétés de liaison à l’ADN de Pax7, nous avons caractérisé les interactions de Pax7 avec ses différentes séquences cibles. Nous avons démontré par des expériences de liaison à l’ADN in vitro (retard sur gel) que l’interaction de Pax7 avec le motif composite est d’affinité supérieure et sous forme de monomère où le PD est principalement impliqué. Comme les sites cibles de Pax7 dans l’hétérochromatine sont marqués par la méthylation de l’ADN, nous avons montré que la méthylcytosine dans le motif de liaison n’interfère pas avec la liaison à l’ADN par Pax7. De plus, des mutations ponctuelles aux domaines de liaison à l’ADN de Pax7 ont montré que les deux domaines de liaison à l’ADN sont nécessaires pour le recrutement aux sites dans la chromatine fermée, a fortiori pour l’ouverture de la chromatine. Des études antérieures ayant identifié des transcrits alternatifs de Pax7, nous avons étudié l’impact fonctionnel des variants Pax7. Les quatre isoformes Pax7 comportent des résidus d’acides aminés alternatifs au niveau du PD : elles ont des propriétés de liaison à l’ADN in vitro ainsi qu’un potentiel de transactivation très similaires. Bien que les isoformes de Pax7 aient des activités pionnières variables, leur action différentielle n’est pas dû à leurs propriétés intrinsèques de liaison à l’ADN ou de transactivation. Nos travaux ont identifié les particularités de Pax7 par rapport à d’autres facteurs pionniers dans les mécanismes de reconnaissance des sites dans l’hétérochromatine. Ainsi, les enhancers sujet à l’action pionnière de Pax7 contiennent typiquement plus de motifs cibles que les cibles d’action transcriptionnelle et l’action pionnière de Pax7 un recrutement fort aux sites pionniers ; de plus, les deux domaines de liaison à l’ADN intacts sont essentiels pour ce recrutement et l’action pionnière. Nos travaux ont défini les paramètres requis pour le reprogrammage cellulaire par un pionnier, Pax7, tout particulièrement en regard des sites de recrutement dans la chromatine fermée. La reprogrammation de cellules souches pour la production de cellules différenciées présente un potentiel thérapeutique unique en médecine régénérative et nos travaux supportent le rôle critique des pionniers dans ce contexte. / During embryonic development, a single cell, the zygote, is responsible for the formation of all cell types in the body. The genetic information necessary for cell fates is stored there in condensed chromatin, initially inaccessible, and is gradually deployed at the opportune moment of cell differentiation by the pioneer factors. Pioneer transcription factors are key determinants in the genetic and epigenetic cascades leading to cell fate. Pioneer factors have the unique property of recognizing their DNA targets in closed chromatin. Their action provides accessibility to a repertoire of enhancers specific to a cell fate, opening the way for other transcription factors. We poorly understand initial recognition of pioneer factors at their targets in closed chromatin. In the pituitary gland, the pioneer factor Pax7 is specific to the intermediate lobe and implements the melanotrope genetic program. To do so, Pax7 identifies key regulatory regions of melanotrope identity in closed chromatin to provide DNA accessibility allowing the binding of other non-pioneer factors. Pax7 has two DNA binding domains (DBD), the paired domain (PD) and the homeodomain (HD), each binding a characteristic sequence motif. In addition, a composite target sequence, formed from the juxtaposition of PD and HD motifs, is enriched at the pioneer sites of Pax7. In order to define the DNA binding properties of the pioneer factor Pax7, we characterized the interactions of Pax7 with its different target sequences. We have demonstrated by in vitro DNA binding experiments (gel shift) that the interaction of Pax7 with the composite motif is of higher affinity and as a monomeric form in which the PD is primarily involved. As Pax7 target sites in heterochromatin are marked by DNA methylation, we showed that methylcytosine within the binding motif does not interfere with Pax7 DNA binding. Using point mutations in the Pax7 DBDs, we showed that both DBDs are required for the recruitment to sites in closed chromatin, furthermore for chromatin opening. Since previous studies identified alternative Pax7 transcripts, we investigated the functional impact of Pax7 variants. The four Pax7 isoforms contain alternative amino acid residues in the PD: they have similar in vitro DNA binding properties and transactivation potential. Although Pax7 isoforms have varying pioneering activities, their differential action is not due to their intrinsic DNA-binding or transactivation properties. Our work has identified the particularities of Pax7 compared to other pioneering factors in the mechanisms of site recognition in heterochromatin. Thus, the enhancers subject to the pioneer action of Pax7 typically contain more target motifs than the targets of transcriptional action and the pioneer action of Pax7 a strong recruitment to the pioneer sites; moreover, the two intact DNA-binding domains are essential for this recruitment and pioneering action. Our work has defined the parameters required for cellular reprogramming by a pioneer, Pax7, particularly with regard to recruitment to sites in closed chromatin. The reprogramming of stem cells for the production of differentiated cells has unique therapeutic potential in regenerative medicine and our work supports the critical role of pioneers in this context.