Analyse de deux gènes candidats physiologiques et positionnels de l'obésité humaine CART et PCSK1

L'obésité commune est une maladie multifactorielle, dont l'émergence récente comme maladie de masse, est liée à l'occidentalisation du mode de vie. Elle est la conséquence d'une diminution de l'activité physique et d'un accès illimité à une alimentation calorique. Néanmoins, les facteurs génétiques influencent la prédisposition individuelle, comme l'attestent des études familiales et l'identification de formes monogéniques d'obésité. Les déterminants des formes polygéniques fréquentes (95%) sont encore peu caractérisés, même si des études récentes ont montré notamment le rôle de certains gènes dans la signalisation de l'insuline (ENPP1) et les voies métaboliques de neurotransmetteurs (GABA, sérotonine), qui prédisposeraient à l'obésité dans un environnement peu favorable. L'implication des facteurs génétiques dans les maladies polygéniques tel que l'obésité est abordée par des approches directes d'analyses de gènes candidats physiologiques et indirectes par l'étude de gènes candidats positionnels localisés dans des régions chromosomiques de liaison à des traits phénotypiques. Deux études " génome entier " sur des familles caucasiennes françaises ont permis de souligner l'importance du locus 5cen-q comme région de susceptibilité à la variation du taux de leptine et à l'obésité. Parmi les nombreux gènes localisés dans cette région ; les gènes CART (5q12-q13) et PCSK1 (5q15-q21) sont exprimés au niveau du système nerveux central dans des régions impliquées dans le contrôle de la prise alimentaire et le maintien de l'homéostasie énergétique et sont des acteurs de la voie de la leptine. L'analyse de 5,4 kb du gène CART (Cocaine and Amphetamine Regulated Transcript), a permis l'identification d'un SNP de la région promotrice (SNP-3608T>C) qui pourrait être un composant des déterminants génétiques de l'obésité polygénique. Ce polymorphisme se révèle également être associé, dans une population générale, avec les sous-fractions de cholestérol et les apolipoprotéines plasmatiques suggérant une implication du gène CART dans l'athérogénèse. Ce SNP aurait également un effet dans le remodelage de la masse osseuse corticale dans une population de femmes danoises. Le gène PCSK1 (Proprotein Convertase Subtilisin/kexin type 1) code une endoprotéase à sérine de type subtilisine, impliquée dans la maturation de pro-hormones et de neuropeptides précurseurs tels que la proinsuline et POMC. Des mutations du gène PCSK1 responsables d'une forme monogénique rare d'obésité sévère de l'enfant ont été décrites. L'analyse de ce gène dans un contexte polygénique a permis l'identification de SNP fréquents (dont une mutation exonique non-synonyme) associés à l'obésité adulte et/ou infantile. L'approche génétique a validé des hypothèses physiologiques et a amélioré notre compréhension des voies métaboliques impliquées en suggérant l'effet de ces deux gènes dans l'obésité polygénique et le rôle pléiotropique du gène CART.Common obesity is a multifactorial disease, whose recent increase, is related to the modernization of life. This epidemic is the consequence of a physical inactivity and an unlimited access to over-nutrition and consumption of caloric food. Nevertheless, many familial studies and the identification of monogenic forms of obesity indicate that genetic factors are also involved. All determinants of the polygenic forms are still unknown, recent studies show the role of genes in the signalling of insulin (ENPP1) and metabolic pathways of neurotransmitters (GABA, serotonin) which would predispose to obesity in a sedentary, high calorie lifestyle. The identification of genetic factors in the polygenic diseases such as obesity is assessed by direct studies of physiological genes and by indirect analyses with positional candidate genes located in chromosomal regions of linkage to phenotype traits. Two genome wide-scans on French Caucasian families show the importance of the locus 5cen-q. Among many genes located in this region ; CART (5q12-q13) and PCSK1 (5q15-q21) genes are expressed in the central nervous system (principally in the hypothalamus) and are involved in the control of food intake and the regulation of energy homeostasis. The analysis of a 5,4 Kb region of the CART gene (Cocaine and Amphetamine Regulated Transcript), including the promoter, 3 exons, introns and the 3'UTR, resulted in the identification of a promoter SNP (SNP-3608T>C) which is associated with the polygenic obesity. In a general population, this polymorphism is also associated, with subfractions of plasma cholesterol and apolipoproteins which suggests that the CART gene maybe implicated in lipid metabolism and atherogenesis. Within a Danish study of menopausal women, the SNP-3608T>C was shown to effect remodelling of the bone mass (on arm BMD). PCSK1 (Proprotein Convertase Subtilisin/kexin type 1) Gene code for a neuroendocrine member of the family of subtilisin-like proprotein convertases and is important for the maturation of pro-hormones and neuropeptides precursors such as the proinsulin and POMC. PCSK1 gene mutations are responsible for a number of rare monogenic forms of severe obesity. The analysis of this gene in a polygenic context enabled the identification of frequent mutations including a non-synonymous exonic variant which is associated with adult and/or childhood polygenic obesity. The genetic approach validates physiological hypotheses and improves current understanding of metabolic pathways, and suggests a pleiotropic effect of the CART gene and that the CART and PCSK1 genes are implicated in polygenic obesity.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Activation of nicotinic acetylcholine receptors decreases apoptosis in human and female murine pancreatic islets

Type 1 diabetes (T1DM) results from destruction of most insulin-secreting pancreatic beta-cells. The persistence of beta-cells decades after the onset of the disease indicates that the resistance of individual cells to the autoimmune insult is heterogeneous and might depend on the metabolic status of a cell at a given moment. The aim of this study is to investigate whether activation of nicotinic acetylcholine receptors (nACh-R) could increase beta-cell resistance against the adverse environment prevailing at the onset of T1DM. Here, we show that nACh-R activation by nicotine and choline, two agonists of the receptor, decreases murine and human beta-cell apoptosis induced by pro-inflammatory cytokines known to be present in the islet environment at the onset of T1DM. The protective mechanism activated by nicotine and choline involves attenuation of mitochondrial outer membrane permeabilization via modulation of endoplasmic reticulum stress, of the activity of Bcl-2 family proteins and cytoplasmic calcium levels. Local inflammation and endoplasmic reticulum stress being key determinants of beta-cell death in T1DM, we conclude that pharmacological activation of nACh-R could represent a valuable therapeutic option in the modulation of beta-cell death in T1DM

Foxa1 and Foxa2 regulate α-cell differentiation, glucagon biosynthesis, and secretion

The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse β-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes

Concomitant alpha7 and beta2 nicotinic AChR subunit deficiency leads to impaired energy homeostasis and increased physical activity in mice

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels well characterized in neuronal signal transmission. Moreover, recent studies have revealed nAChR expression in nonneuronal cell types throughout the body, including tissues involved in metabolism. In the present study, we screen gene expression of nAChR subunits in pancreatic islets and adipose tissues. Mice pancreatic islets present predominant expression of α7 and β2 nAChR subunits but at a lower level than in central structures. Characterization of glucose and energy homeostasis in α7β2nAChR(-/-) mice revealed no major defect in insulin secretion and sensitivity but decreased glycemia apparently unrelated to gluconeogenesis or glycogenolysis. α7β2nAChR(-/-) mice presented an increase in lean and bone body mass and a decrease in fat storage with normal body weight. These observations were associated with elevated spontaneous physical activity in α7β2nAChR(-/-) mice, mainly due to elevation in fine vertical (rearing) activity while their horizontal (ambulatory) activity remained unchanged. In contrast to α7nAChR(-/-) mice presenting glucose intolerance and insulin resistance associated to excessive inflammation of adipose tissue, the present metabolic phenotyping of α7β2nAChR(-/-) mice revealed a metabolic improvement possibly linked to the increase in spontaneous physical activity related to central β2nAChR deficiency

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

International audienceThe crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry

Mycobacterium lutetiense sp. nov., Mycobacterium montmartrense sp. nov. and Mycobacterium arcueilense sp. nov., members of a novel group of non-pigmented rapidly growing mycobacteria recovered from a water distribution system

From our recent survey of non-pigmented rapidly growing mycobacteria in the Parisian water system, three groups of isolates (taxons 1-3) corresponding to possible novel species were selected for taxonomic study. The three taxa each formed creamy white, rough colonies, had an optimal growth temperature of 30 °C, hydrolyzed Tween 80, were catalase-positive at 22 °C and expressed arylsulfatase activity. All three were susceptible to amikacin, ciprofloxacin and tigecycline. The three taxa produced specific sets of mycolic acids, including one family that has never previously been described, as determined by thin layer chromatography and nuclear magnetic resonance. The partial rpoB sequences (723 bp) showed 4-6 % divergence from each other and more than 5 % differences from the most similar species. Partial 16S rRNA gene sequences showed 99 % identity within each species. The most similar sequences for 16S rRNA genes (98-99 % identity over 1444-1461 bp) were found in the Mycobacterium fortuitum group, Mycobacterium septicum and Mycobacterium farcinogenes. The three taxa formed a new clade (bootstrap value, 99 %) on trees reconstructed from concatenated partial 16S rRNA, hsp65 and rpoB sequences. The above results led us to propose three novel species for the three groups of isolates, namely Mycobacterium lutetiense sp. nov. [type strain 071T=ParisRGMnew_1T (CIP 110656T=DSM 46713T)], Mycobacterium montmartrense sp. nov. [type strain 196T=ParisRGMnew_2T (CIP 110655T=DSM 46714T)] and Mycobacteriu marcueilense sp. nov. [type strain of 269T=ParisRGMnew_3T (CIP 110654T=DSM 46715T)]

Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus–HBGA Interaction

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles

Species-Specific <i>N</i>-Glycomes and Methylation Patterns of Oysters <i>Crassostrea gigas</i> and <i>Ostrea edulis</i> and Their Possible Consequences for the Norovirus–HBGA Interaction

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles