Gotowa bibliografia na temat „GTP Homeostasis”

Obesity has grown to epidemic proportions, and in lack of efficient life-style and medical treatments, the bariatric surgeries are performed in rising numbers. The most common surgery is the Gastric Bypass (GBP) surgery, with the Biliopancreatic diversion with duodenal switch (DS) as an option for the most extreme cases with a BMI>50 kg/m2. In paper I 20 GBP-patients were examined during the first post-operative year regarding the natriuretic peptide, NT-ProBNP, which is secreted from the cardiac ventricles. Levels of NT-ProBNP quickly increased during the first post-surgery week, and later established itself on a higher level than pre-surgery. In paper II we report of 5 patient-cases after GBP-surgery with severe problems with postprandial hypoglycaemia that were successfully treated with GLP-1-analogs. The effect of treatment could be observed both symptomatically and in some cases using continuous glucose measuring systems (CGMS). In paper III three groups of subjects; 15 post-GBP patients, 15 post-DS, and 15 obese controls were examined for three days using CGMS during everyday life. The post-GBP group had high glucose variability as measured by MAGE and CONGA, whereas the post-DS group had low variability. Both post-operative groups exhibited significant time in hypoglycaemia, about 40 and 80 minutes per day <3.3mmol/l and 20 and 40 minutes < 2.8mmol/l, respectively, longer time for DS-group. Remarkably, only about 20% of these hypoglycaemic episodes were accompanied with symptoms. In Paper IV the hypoglycaemia counter regulatory system was investigated; 12 patients were examined before and after GBP-surgery with a stepped hypoglycaemic hyperinsulinemic clamp. The results show a downregulation of symptoms, counter regulatory hormones (glucagon, cortisol, epinephrine, norepinephrine, growth hormone), incretin hormones (GLP-1 and GIP), and sympathetic nervous response. In conclusion patients post bariatric surgery exhibit a downregulated counter regulatory response to hypoglycaemia, accompanied by frequent asymptomatic hypoglycaemic episodes in everyday life. Patients suffering from severe hypoglycaemic episodes can often be treated successfully with GLP-1-analogues.

L'épithélium intestinal, en constant renouvellement, assure de nombreuses fonctions vitales comme l'absorption des nutriments et le maintien d'une barrière entre le milieu extérieur et l'organisme. L'absorption intestinale des sucres est assurée par de nombreux transporteurs au niveau de l'intestin proximal. Parmi eux, GLUT2, localisé dans les entérocytes et les cellules endocrines de l'intestin, transporte le glucose, le fructose et le galactose. Les cellules L entéroendocrines produisent le GLP-1, un puissant stimulateur de la sécrétion d'insuline en réponse au glucose. L'objectif de ma thèse a été d'élucider le rôle de GLUT2 intestinal dans l'absorption des sucres et la fonction entéroendocrine grâce à l'étude d'un modèle murin spécifiquement invalidé pour ce transporteur dans les cellules épithéliales intestinales. La délétion intestinale de GLUT2 entraîne une malabsorption intestinale modérée des sucres associée à une distribution retardée du glucose aux tissus périphériques. Le retard spatial et temporel de l'absorption des sucres provoque une dysbiose intestinale au profit de bactéries ayant un rôle protecteur de l'homéostasie intestinale. De façon surprenante, l'invalidation de GLUT2 intestinal s'accompagne d'une chute de la densité de cellules L entéroendocrines, sans modification des niveaux plasmatiques de GLP-1. Cette étude met en exergue le rôle primordial de GLUT2 intestinal dans l'absorption des sucres et la fonction endocrine de l'intestin. Elle permet d'envisager le criblage de molécules capables d'inhiber l'activité de GLUT2 intestinal, pour atténuer la prise de poids et limiter les perturbations métaboliques induites par des régimes riches en sucres<br>The constantly renewing intestinal epithelium handles various essential functions including nutrient absorption and persistence of a barrier between our internal and external environments. Several transporters mediate sugar absorption in the proximal intestine. Among them, GLUT2, a very efficient glucose, fructose and galactose transporter and receptor, is located at the membranes of enterocytes and enteroendocrine cells. The enteroendocrine L-cells produce GLP-1, a strong activator of glucose-induced insulin secretion. This thesis aimed to further decipher the role of intestinal GLUT2 in sugar absorption and enteroendocrine cell function. To address this question, mice lacking GLUT2 specifically in intestinal epithelial cells have been generated and studied. Intestinal GLUT2 invalidation alters intestinal glucose absorption and delays glucose biodistribution to peripheral tissues. This spatial and temporal sugar absorption delay provokes intestinal dysbiosis, favoring gut microbiota having a protective impact on gut homeostasis. Surprisingly, intestinal GLUT2 deletion leads to a strong loss in enteroendocrine L cell density, with no impact on GLP-1 plasma levels. This study highlights critical roles for GLUT2 in sugar absorption and enteroendocrine cell function management. The use of specific GLUT2 inhibitors could be considered to limit body weight gain and metabolic disorders induced by sugar rich diets

A possível variabilidade na resposta fisiológica hormonal após a alimentação, relacionada à elevação do GIP pós-prandial entre indivíduos, despertou o interesse para avaliar a influência do polimorfismo no gene para o receptor do polipeptídeo insulinotrópico dependente de glicose (GIPR) na homeostase glicêmica e na função da célula beta pancreática. Foram estudados 25 adultos (12 mulheres, 13 homens) com IMC<30 e sem doenças relacionadas à síndrome metabólica. Foram coletadas amostras de sangue nos tempos basal (T0) e após refeição contendo 58g de carboidratos e 28g de lipídeos a cada 30 minutos até o T150, para dosagem de insulinemia e glicemia. Foi realizada pesquisa da variante Glu354Gln para o GIPR (SNPGIPR, rs1800437) em amostra de sangue periférico pela técnica de PCRRFLP. Foi avaliada a homeostase glicêmica de jejum e pós-prandial através dos cálculos de HOMA-IR, HOMA-B, área sob a curva (AUC) de insulina e glicemia e sua razão (AUCi/AUCg), índice insulinogênico (IGI) em cada tempo relativo ao basal e razão IGI/HOMA-IR. Na curva pós-prandial, 72% dos indivíduos apresentaram picos de glicose e 64% de insulina até o T60, com média de 107 mg/dl e 42,1 UI/mL, respectivamente. A pesquisa do SNP-GIPR revelou alelo C presente (C+, polimórfico) em 7 indivíduos, com 72% dos genótipos GG, 24% GC e 4% CC. Houve diferença significativa entre C+ e o grupo com alelo C ausente (C-) em relação ao HOMA-B (100+/-26 vs 160+/- 189%, respectivamente; p=0,04). Não houve diferença estatisticamente significativa quanto aos seguintes parâmetros: AUCg, AUCi, HOMA-IR, IGI e razões AUCi/AUCg e IGI/HOMA-IR. Porém, em todos os tempos, o IGI foi superior no grupo C+ (T30, 189%; T60, 147%; T90, 1066%; T120, 29% e T150, 93%), enquanto que uma tendência semelhante ocorreu para a razão IGI/HOMA-IR exceto no T150. O indivíduo portador do SNP-GIPR em homozigose (CC) apresentou resposta precoce de insulina. Além disso, 42,9% dos indivíduos C+ apresentaram história familiar de diabetes em comparação com 22,2% do grupo C- (p=0,29). Em conclusão, estudamos o perfil de glicemia e insulina de jejum e na curva pós-prandial incluindo 1ª e 2ª fases de secreção insulínica de uma amostra de indivíduos sadios com distribuição genotípica do SNP-GIPR semelhante à população reportada na literatura. Indivíduos euglicêmicos C+ e C- apresentaram semelhanças em diversos dos parâmetros utilizados para avaliar a função da célula beta pancreática e resistência insulínica. Por outro lado, foi encontrada variação no HOMA-B entre os genótipos estudados, sugerindo redução na função da célula beta relacionada ao polimorfismo do GIPR, sem alteração na sensibilidade à insulina. A redução do HOMA-B em indivíduos sadios, independente do histórico familiar de diabetes, sugere aprofundar o estudo deste SNP-GIPR como possível marcador precoce para o risco de falência pancreática associada ao diabetes tipo 2.<br>The potential variability in hormonal physiological response after feeding, related to the elevation of postprandial GIP between individuals, arouse interest to evaluate the influence of polymorphisms in the gene for glucose-dependent insulinotropic polypeptide receptor (GIPR) in glucose homeostasis and in pancreatic beta cell function. Altogether 25 adults (12 women, 13 men) with BMI <30 and without metabolic syndrome related diseases were studied. After diet containing 58g of carbohydrates and 28g of lipids, blood samples at baseline (T0) and every 30 minutes until the T150 were collected for measurement of insulin and glucose. The polymorphic GIPR variant Glu354Gln, rs1800437 (SNP-GIPR) was studied in peripheral blood sample through PCR-RFLP. Glucose homeostasis in fasting and postprandial was assessed by calculating HOMA-IR, HOMA-B, area under the curve (AUC) of insulin and glucose, AUCi/AUCg ratio, insulinogenic index (IGI) at each time relative to baseline and IGI/HOMA-IR ratio. In the postprandial curve, 72% of subjects showed glucose peaks and 64% insulin until the T60, with mean of 107 mg/dl and 42.1 IU/mL, respectively. The SNP-GIPR study revealed presence of C allele (C+) in 7 individuals, with 72% of the genotypes GG, 24% GC and 4%CC. There was a significant difference between C + or the group without C allele (C-) in HOMA-B (100 +/- 26 vs. 160 +/- 89 % respectively; p = 0.04).There was no significant difference in the following parameters: AUCg, AUCi, HOMA-IR, IGI, AUCi/AUCg and IGI/HOMA-IR. However, the IGI was higher in the C+ allele group at all time-points (T30, 189%; T60, 147%; T90, 1066%; T120, 29% and T150, 93%), while a similar trend occurred for the IGI/HOMA-R ratio, except in T150. Furthermore, family history of diabetes was positive in 42.9% of C+ subjects, compared with 22.2% from C- group (p = 0.29). In conclusion, we have studied the fasting and postprandial variation of glucose and insulin, including insulin secretion’s 1st and 2nd phases of healthy subjects; our sample presented genotype distribution similar to the reported population in the literature. C+ and C- euglycemic subjects had similar parameters of beta cell function and insulin sensitivity. In other way, we found variation in HOMA-B according to the genotype, suggesting a reduction in beta cell function related to GIPR polymorphism, without change in insulin sensitivity. The reduction in HOMA-B in healthy individuals, regardless of family history of diabetes, suggests further study of this SNP-GIPR as a possible early marker for the risk of pancreatic failure associated with type 2 diabetes.

L’obésité et le diabète de type 2 (DT2) sont des pathologies métaboliques associées à des perturbations de l’homéostasie glucidique et énergétique. Les enterohormones sont des acteurs importants de la regulation des mécanismes perturbés lors de ces pathologies. Parmi ces enterohormones, le GLP-1, sécrété par les cellules entéroendocrines de type L suite à un repas, permet d’amplifier la sécrétion d’insuline par les cellules β-pancréatiques et de diminuer la prise alimentaire. L’objectif de ma thèse a été de caractériser le rôle du récepteur nucléaire HNF-4γ dans l’homéostasie énergétique et la fonction endocrine de l’intestin.A l’aide d’un modèle murin d’invalidation totale et constitutive du facteur de transcription HNF-4γ, notre équipe a mis en évidence que l’absence de HNF-4γ induit une amélioration de la tolérance au glucose grâce à une augmentation du nombre de cellules L et de la quantité plasmatique de GLP-1 en réponse au glucose. L’ensemble de ces données démontre pour la première fois un rôle de HNF-4γ dans l’homéostasie glucidique via une modulation du lignage enteroendocrine spécifique du GLP-1 et suggère que son absence pourrait protéger les souris de l’établissement d’un DT2.Par ailleurs, la perte d’expression de HNF-4γ confère une protection vis-à-vis de la prise de poids et de l’intolérance au glucose normalement induites par six semaines d’un régime riche en lipides et en fructose grâce une perte énergétique accrue dans les fécès essentiellement due à une malabsorption des acides gras.En conclusion, cette étude met en exergue le rôle du récepteur nucléaire intestinal HNF-4γ dans la fonction enteroendocrine et la susceptibilité à l’obésité et au DT2<br>Obesity and type 2 diabetes (T2D) are metabolic pathologies associated with glucose and energy homeostasis perturbations. Enterohormones are important players in the regulation of the mechanisms disturbed during these pathologies. Among these enterohormones, GLP-1, secreted by enteroendocrine L cells in response to a meal, potentiates insulin secretion by pancreatic β cells and inhibits food intake. The aim of my thesis was to characterize the role of the nuclear receptor HNF-4γ in the energy homeostasis and the endocrine function of the intestine.By using a total and constitutive HNF-4γ knock-out mouse model, our team has highlighted that the loss of hnf-4γ induces an improved glucose tolerance. This effect is due to an increased GLP-1 cell number and GLP-1 plasma levels in response to glucose. All together these data demonstrate for the first time a role of HNF-4γ in glucose homeostasis through a modulation of the enteroendocrine lineage specific for GLP-1 and suggest that its absence could protect mice from the T2D establishment.The loss of HNF-4γ protects mice from body weight gain and glucose intolerance normally induced by six weeks of a high-fat/high-fructose diet demonstrating its involvement in obesity and T2D. HNF-4γ -/- mice are protected from obesity by a greater energy loss in faeces mainly due to lipid malabsorption. These results demonstrate that HNF-4γ is necessary for the intestinal fatty acids uptake.In conclusion, this study highlights the role of the intestinal nuclear receptor HNF-4γ in enteroendocrine function and susceptibility to obesity and T2D

Mestrado em Biologia Molecular e Celular<br>A síntese proteica é um processo essencial para que todos os organismos mantenham a homeostasia celular. Os tRNAs são elementos cruciais na síntese proteica, uma vez que codificam a informação genética presente no mRNA. A linha celular HeLa, utilizada neste estudo, foi primeiramente isolada de uma mulher com cancro do colo do útero e desde então tem sido bastante usada na investigação, sendo muito importante no estudo das bases moleculares de muitas doenças. De modo a monitorizar a agregação proteica nesta linha celular, um sistema repórter foi desenvolvido utilizando uma fusão entre HspB1 (Hsp27) e a GFP. HspB1 é um chaperone molecular com capacidade de recrutar outros chaperones e restabelecer a conformação ideal das proteínas em situações de stress. A GFP é uma proteína fluorescente que marca certas condições biológicas de interesse. Para perceber o impacto dos erros da tradução na agregação de proteínas e no surgimento das doenças, o principal objetivo deste estudo foi desenvolver uma linha celular estável (HeLa) expressando um sistema repórter HspB1-GFP, de modo a monitorizar os erros no enovelamento das proteínas em resposta ao stress proteotóxico. Ao longo deste estudo o sistema repórter expressando HspB1-GFP foi desenvolvido com sucesso, permitindo assim a sua utilização para identificar situações fisiológicas e patológicas em que a agregação de proteínas ocorre em células de mamífero.<br>Protein synthesis is essential for all organisms to maintain cell homeostasis. tRNAs are crucial elements in protein synthesis as they decode the genetic information organized in the mRNA codons. A HeLa cell line, used in this study, was first isolated from a woman with cervical cancer and since then was highly used in biological studies, being extremely important in the study of the molecular basis of several diseases. In order to monitor protein aggregation in this cell line, a reporter system was developed using an HspB1 (Hsp27) and a GFP fusion. HspB1 is a small heat shock protein that, in stress situations, recruits other proteins in order to restore the conformation of the proteins. GFP is a biosensor that reports several cellular conditions of interest. To understand the impact of translation errors on protein aggregation and on the disease arising, the main goal of this study was to develop a stable cell line (HeLa) expressing a reporter system HspB1-GFP to monitor the protein misfolding in response to proteotoxic stress. During this study, the reporter system expressing HspB1-GFP was developed successfully, allowing the identification of physiological and pathological situations where protein aggregation occurs in mammalian cells.

Nucleotide based second messengers are known to regulate wide variety of processes in all domains of life. Two such bacterial second messengers are (p)ppGpp (guanosine tetra- or pentaphosphate) and c-di-GMP (cyclic dimeric guanosine monophosphate). The alarmone (p)ppGpp is synthesized by bacteria to face any kind of stress; while the signalling nucleotide c-di-GMP is synthesized principally to switch from motile (planktonic) to sessile (biofilm) life style. Apart from mediating the said functions, these nucleotides also regulate transcription, translation, replication, virulence and pathogenicity of the several bacterial species. In this work, we have tried to uncover novel functions or phenotypes that are governed by the second messengers (p)ppGpp and c-di-GMP in Mycobacterium smegmatis. In M. smegmatis, (p)ppGpp and c-di-GMP are synthesized and degraded by the bifunctional proteins RelMsm and DcpA, respectively. The architecture of both the proteins is similar; the synthesis and hydrolysis domains for the second messengers occur in tandem. The knockout mutants of relMsm and dcpA genes, ∆relMsm and ∆dcpA, have been used in this study to uncover the novel functions of these second messengers in mycobacterial physiology. Chapter 1 provides is an overview of the current literature pertaining to (p)ppGpp and c-di-GMP. An historical perspective with regard to the discovery of the (p)ppGpp and c-di-GMP is given. The metabolism of these second messengers has been discussed. This has been followed by the description of various functions governed by the second messengers. Finally, the scope of the current work has been outlined. Chapter 2 investigates the effect of disrupting (p)ppGpp and c-di-GMP signalling on the antibiotic sensitivity in M. smegmatis. Using Phenotype Microarray (PM) technology, the growth of ∆relMsm and ∆dcpA knock out strains was compared to those of the wild-type and respective complemented strains in 240 different antimicrobials. It was found that the knockout mutants displayed enhanced survival in the presence of multiple antibiotics. The PM data was corroborated by the independent determination of minimum inhibitory concentrations of seven different antibiotics. Finally, the plausible reasons for the multidrug resistance of ∆relMsm and ∆dcpA strains have been discussed. Chapter 3 explores how the impairment of (p)ppGpp and c-di-GMP alters the cell wall of M. smegmatis. Thin layer chromatography analysis of cell wall fractions such as glycopeptidolipids (GPLs), mycolic acids, polar and apolar lipids was carried out. It was found that the amount of GPLs and polar lipids were reduced in the ∆relMsm and ∆dcpA knockout strains. Chapter 4 explores the effect of (p)ppGpp and c-di-GMP on the growth, cell morphology and cell division in M. smegmatis. It was found that the ∆relMsm and ∆dcpA knockout strains have slow growth compared to those of the wild type and respective complemented strain. The overproduction of (p)ppGpp and c-di-GMP, achieved through overexpression of Rel and DcpA proteins, encased the overexpression strains relOE and dcpAOE in a biofilm like matrix. The higher levels of (p)ppGpp and c-di-GMP caused M. smegmatis assume coccoid morphology. Microscopy analyses revealed that the ∆relMsm and ∆dcpA strains are elongated, multinucleate and multiseptate. Chapter 5 explores effects of (p)ppGpp and c-di-GMP on the global gene expression profile in M. smegmatis. Many genes were shown to be differentially expressed in the ∆relMsm and ∆dcpA knockout strains. Genes regulating cell division, cell wall biosynthesis, superoxide metabolism or reactive oxygen species metabolism and genes encoding transporters were differentially expressed in the ∆relMsm and ∆dcpA knockout mutants. The microarray data were corroborated by quantitative real-time PCR. Gene expression data explained the multidrug resistance, the reduction in the level of GPLs and polar lipids, slow growth, changes in cell morphology and defective cell division exhibited by the ∆relMsm and ∆dcpA knockout mutants. Chapter 6 summarizes the entire work embodied in the thesis. Appendix 1 lists the 240 antimicrobials compounds and their mode of action for which antibiotic sensitivity of the ∆relMsm and ∆dcpA knockout mutants was tested. Appendix 2 lists the growth differences among the knockout, wild type and complemented strains in the form of area under curve values. Appendix 3 lists the genes that were differentially expressed in the ∆relMsm and ∆dcpA knockout strains. Appendix 4 is a comprehensive review on the kinetic and thermodynamic parameters governing the sigma factor competition in Escherichia coli and how (p)ppGpp and anti-sigma factors regulate this competition among sigma factors for the limited pool of core RNA polymerase in E. coli.

Mechanisms to augment the cellular function and mass of beta-cells may be effective means of treating type 2 diabetes. Important in the physiological control of beta-cell function and nutrient disposal are factors released from gut enteroendocrine cells during nutrient digestion. In enteroendocrine L-cells, post-translational processing of proglucagon gives rise to a number of proglucagon-derived peptides. One such peptide, glucagon-like peptide-1 (GLP-1), acts via its own receptor (GLP-1R) to stimulate beta-cell insulin secretion, proliferation and survival. Another, oxyntomodulin (OXM), weakly activates the GLP-1R and inhibits food intake in a GLP-1R-dependent manner in rodents, which led us to hypothesize that OXM modulates GLP-1R-dependent glucoregulation. While OXM did not mimic the inhibitory effect of GLP-1 on gastric emptying in mice, OXM stimulated insulin secretion, beta-cell survival and improved glucose tolerance in a GLP-1R-dependent manner. In a similar manner to GLP-1, glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K-cells, physiologically stimulates insulin secretion via a distinct GIP receptor (GIPR) in beta-cells. Beyond the beta-cell, GIP and GLP-1 appear to exert divergent actions for the control of glucose homeostasis. Moreover, I illustrate that physiological and pharmacological GLP-1R signalling may be comparatively more important for the preservation of beta-cell mass and glucose homeostasis in murine streptozotocin-induced diabetes. Lastly, studies in rodents and humans have showed that metformin increases circulating levels of GLP-1, leading us to hypothesize that GIP and GLP-1 may be involved in the glucoregulatory effects of metformin. Interestingly, transcripts for the Glp1r and Gipr were significantly increased within islets of metformin-treated mice, and metformin treatment enhanced the sensitivity of cultured beta-cells to GIP and GLP-1. In summary, these studies illustrate mechanisms by which enteroendocrine peptides compare and contrast with respect to beta-cell survival and function and the control of glucose homeostasis.

Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut non- endocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and co-localized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.