Les interactions protéine-ligand interviennent dans de nombreux processus biochimiques et permettent notamment à certaines protéines sanguines d’assurer leur rôle de transport. Parmi ces protéines figurent notamment l’albumine, protéine la plus abondante du plasma, ou l’apolipoprotéine A1 (ApoA1), majoritaire au sein des lipoprotéines de haute densité (HDL). Dans un contexte diabétique, la glycation des protéines induit des modifications structurales affectant ainsi leur potentiel d’interaction.Le premier objectif de ce travail de thèse visait à déterminer l’impact de la glycation de l’albumine sur sa liaison au liraglutide, un médicament de plus en plus utilisé dans le traitement du diabète de type 2. Ensuite, la seconde partie de ce travail a consisté en la production d’une ApoA1 humaine recombinante fonctionnelle afin d’étudier ses propriétés d’interaction, sous forme libre ou associée aux phospholipides. La technique RMN (résonance magnétique nucléaire) a été utilisée sur les protéines préalablement marquées au fluor (pour le liraglutide) ou aux isotopes stables 13C/15N (pour l’ApoA1). La titration microcalorimétrique isotherme (ITC), méthode complémentaire à la RMN a été appliquée pour l’étude des interactions avec l’avantage de ne nécessiter aucun marquage. Différentes stratégies de clonage ont été explorées pour la surexpression de l’ApoA1 en bactérie Clearcoli.Les résultats obtenus démontrent une altération de l’affinité de l’albumine pour le liraglutide in vitro et in vivo, dépendante du degré de glycation. Ces résultats, enrichis d’une analyse lipidomique et peptidique, permettent d’expliquer les observations cliniques concernant la baisse de l’efficacité de médicaments liant l’albumine chez les patients ayant un diabète mal contrôlé. Concernant l’ApoA1, le choix de l’étiquette de fusion reste à optimiser, mais sa surexpression de manière soluble et abondante a été obtenue pour l’ApoA1 marquée et non marquée. / Protein-ligand interactions are involved in many biochemical processes. They are notably implicated in the role of transporter proteins in blood. Albumin, the most abundant plasma protein, and apolipoprotein A1 (ApoA1), which is the main component of high-density lipoprotein (HDL) belong to this class of proteins. In the context of diabetes, proteins are altered by glycation which leads to structural modifications and potentially affect their interactions.The first objective of this work was to determine the impact of albumin glycation on its binding to liraglutide, a drug increasingly used in the treatment of type 2 diabetes. Then, the second part of this work involved the production of recombinant functional human ApoA1 in order to study its interaction properties, in its lipid-free form or associated with phospholipids. The NMR (nuclear magnetic resonance) technique has been used on proteins previously labeled with fluorine (for liraglutide) or stable 13C/15N isotopes (for ApoA1). In addition, isothermal titration microcalorimetry (ITC), has been applied to the study of interactions with the advantage of not requiring any labeling. Various cloning strategies have been explored for the overexpression of ApoA1 in Clearcoli bacteria.The results demonstrate an alteration of the affinity of albumin for liraglutide in vitro and in vivo, depending on the degree of glycation. These results, supported by a lipidomic and peptide analysis, explain clinical observations concerning the decrease of efficacy of albumin-binding drugs in patients with poorly controlled diabetes. Regarding ApoA1, the choice of the fusion tag remains to be optimized, but both labeled and unlabeled ApoA1 were successfully overexpressed at high yields in a soluble form.

Glucagon-like peptide-1 (GLP-1) synthetic analog therapies are prescribed for type 2 diabetes due to their effects on insulin and glucagon secretion, and glycemic control. Recent studies also suggest that they may have cardiovascular benefits; however, the mechanism responsible for this is unknown. To examine this question, we evaluated the effects of GLP-1 and the GLP-1 synthetic analog, liraglutide on cell signaling and function in human aortic endothelial cells (HAECs). The results indicate that both agents inhibit TNFα and LPS induced cellular adhesion molecule expression and monocyte adhesion. They also show that incubation with 30pM GLP-1 and 100nM liraglutide stimulates an immediate increase in intracellular calcium, which activates calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). This in turn led to a 2.5 fold increase in the phosphorylation of both AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent protein kinase 1 (CaMK1) within 5 minutes. In addition both GLP-1 and liraglutide caused a 2-fold increase in the phosphorylation of the downstream AMPK/CaMK1 targets: endothelial nitric oxide synthase (eNOS) and cAMP response element-binding protein (CREB). Inhibition of CaMKKβ with STO-609 (0.5ug/mL) blocked the phosphorylation of both AMPK and CaMK1, confirming its pivotal role. Incubation of the HAECs for three hours with lipopolysaccharide (LPS, 2ug/mL) and TNFα (10ng/mL) increased the expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by 5 and 2 fold, respectively. Comparable increases in THP-1 monocyte adhesion to the HAECs, a putative initiating event in atherogenesis, also occurred. Pre-incubation for one hour with either GLP-1 or liraglutide inhibited these events. Likewise, pre-incubation with the CaMKK inhibitor STO-609, or use of lentivirus shRNA to knock down AMPK, blocked the inhibitory effects of both GLP-1 and liraglutide on monocyte adhesion. These results suggest that the recently observed cardiovascular benefits of GLP-1 and liraglutide could be mediated by their effects on CaMKKβ, AMPK and CaMK1 activation, which lead to decreased adhesion molecule expression and monocyte adhesion in endothelial cells. The finding that these effects occur at concentrations of GLP-1 (30pM) and liraglutide (100nM) observed in vivo also suggests they are physiologically relevant.

Analisamos a ação da liraglutida na flora intestinal e perda de peso de dois modelos de obesidade: por dieta hiperlipidica (HFD) e obesidade genética (ob/ob). Os modelos foram tratados com o fármaco durante duas semanas. Perfis metabólicos foram feitos por meio de testes glicêmicos e insulínicos, histologia do fígado, região cecal e coxins gordurosos, ingestão alimentar, peso corporal e metagenômica do conteúdo cecal. O tratamento induziu perda de peso com melhora dos níveis glicêmicos e redução da inflamação na região cecal e do fígado e foi capaz de reduzir o acúmulo de gordura hepática promovendo a redução da EHNA. A metagenômica mostrou mudança taxonômica geral, bem como a abundância relativa de bactérias envolvidas com peso e controle glicêmico:redução de Proteobacterias e aumento de Akkermansia muciniphila. Apresentamos evidências do fármaco revertendo DGHNA/EHNA e a perda de peso associados às mudanças da microbiota. Sugerimos uma lista de alvos bacterianos que podem interferir no metabolismo energético para o controle clinico de doenças metabólicas. / The study analyzed the effects of liraglutide on gut microbiota and weight-loss in two obesity model: induced by high fat diet (HFD) and genetic obese mice (ob/ob). Models were treated with liraglutide for two weeks. Metabolic profiles were measured by glycemic and insulin test, histological liver, cecal region and fat pad morphologies, food intake, body weight and metagenomic of cecal contents. The treatment induced weight-loss, improvement of glycemic parameters and reduction of inflammatory cells in the cecum and the liver and reduced fat accumulation in liver reverting NASH. The metagenomic showed a general changed in taxonomic structure as well the relative abundance of weight-relevant:reduction of Proteobacteria and increases of Akkermansia muciniphila. We showed evidences that liraglutide leads to improvement of NASH and weight loss associated with changes in microbiota. Moreover, by the profile of the gut microbiota, we present a bacterial target list that may affect energetic metabolism inducing a metabolic clinical controlled profile.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-06-05T17:25:05Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Daiane Didek.pdf: 2147992 bytes, checksum: fb8c4ca29516a68c37c8dc766dc39e8f (MD5) / Made available in DSpace on 2018-06-05T17:25:05Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Daiane Didek.pdf: 2147992 bytes, checksum: fb8c4ca29516a68c37c8dc766dc39e8f (MD5) Previous issue date: 2018-03-23 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / A liraglutida é um análogo do peptídeo semelhante ao glucagon-1 (GLP-1), já utilizada comercialmente para o tratamento da Diabetes mellitus tipo 2, que também mostra resultados na redução da ingestão alimentar e consequente redução do peso corporal. A associação do exercício físico com a liraglutida pode ser um importante meio de controle do metabolismo lipídico e ocasionalmente tratamento de alterações metabólicas como a obesidade. O objetivo do nosso trabalho foi avaliar o efeito da liraglutida, análogo do GLP-1 associado ao exercício físico nos parâmetros metabólicos, bioquímicos e antropométricos de ratos normais e obesos, induzidos por dieta de cafeteria. O experimento iniciou-se aos 21 dias de vida dos animais, estes foram divididos em oito grupos: Quatro controles (CON) recebendo ração padrão e agua ad libitum; quatro obesos (OBESO) recebendo a dieta de cafeteria ad libitum, adicionada a dieta padrão; subdivididos em (CON LIRA e OBESO LIRA) recebendo injeções subcutâneas de liraglutida dos 80 aos 90 dias de vida; (CON EXE LIRA e OBESO EXE LIRA) com intervenção da liraglutida e submetidos a natação por 15 minutos, três dias por semana e (CON EXE e OBESO EXE) somente com intervenção do exercício físico. Os resultados dos animais obesos demonstraram que a liraglutida reduziu, somente o consumo alimentar no final do experimento. O exercício físico mostrou melhores resultados na redução da gordura mesentérica, epididimal, retroperitoneal, níveis circulantes de glicose, índice de Lee, ganho de peso dos 80-90 dias de vida e aumentou o peso da glândula adrenal nos animais obesos, nos animais controle reduziu o peso do pâncreas, índice de Lee e colesterol total. A associação do exercício físico com a liraglutida apresentou melhores resultados na redução do peso corporal no final do experimento, redução do consumo dos 80-90 dias de vida, peso do fígado, níveis circulantes de triglicerídeos e insulina, índice HOMA-IR, nos animais obesos, porém aumentou o TNF-α nos animais obesos e controles. Concluímos que a intervenção com o exercício físico foi eficaz na redução de alguns parâmetros relacionados ao desenvolvimento da obesidade, porém a sua associação com a liraglutida por 10 dias mostra melhores resultados na redução do peso corporal, consumo alimentar e parâmetros bioquímicos, em animais obesos obtidos por dieta de cafeteria. / Liraglutide is an analog of the Glucagon-like peptide-1 (GLP-1), already commercially used for the treatment of Type 2 Diabetes Mellitus, which also shows results in the reduction of food intake and consequent reduction of body weight. The association of physical exercise with liraglutide may be an important means of controlling lipid metabolism and, occasionally, the treatment of metabolic disorders such as obesity. The objective of our study was to evaluate the effect of liraglutide, GLP-1 analog associated with physical exercise on metabolic, biochemical and anthropometric parameters of normal and obese rats, induced by the cafeteria diet. The experiment started at the 21 days of life of the animals, which these divided into eight groups: Four controls (CON) receiving standard chow and water ad libitum; four obese (OBESO) receiving the cafeteria diet ad libitum, added to the standard diet. The animals were further subdivided into (CON LIRA and OBESO LIRA) receiving subcutaneous injections of liraglutide from 80 to 90 days of life; (CON EXE LIRA and OBESO EXE LIRA) with intervention of liraglutide and submitted to swimming for 15 minutes, three days a week and (CON EXE and OBESO EXE) only with physical exercise intervention. The results of obese animals show that liraglutide reduced only food intake at the end of experiment. The physical exercise show better results in the reduction of the mesenteric, epididymal, retroperitoneal fat pad, circulating levels of glucose, Lee index, weight gain from 80-90 days of life and increased adrenal gland weight in obese animals, in control animals reduced the weight of the pancreas, Lee index and total cholesterol. The association of exercise with liraglutide show better results in reducing body weight at the end of the experiment, food intake of the 80-90 days of life, liver weight, circulating levels of triglycerides and insulin, HOMA-IR index, in obese animals, but increased the TNF-α in obese and control animals. We conclude that the intervention with physical exercise was effective in reducing some parameters related to the development of obesity, but its association with liraglutide for 10 days show that better results in reducing body weight, food intake and biochemical parameters in obese animals obtained by cafeteria diet.

OBJECTIVE: To review the evidence surrounding a potential association between liraglutide and pancreatitis. DATA SOURCES: A literature search was conducted in MEDLINE (1948-July 12, 2012) and EMBASE (1974-week 27, 2012) using the search terms pancreatitis, liraglutide, and glucagon-like peptide 1/adverse effects. Reference citations from identified publications were reviewed. The manufacturer was contacted and regulatory documents from the Food and Drug Administration website were reviewed for unpublished data related to cases of pancreatitis associated with liraglutide use. STUDY SELECTION AND DATA EXTRACTION: All identified sources that were published in English were considered for inclusion. DATA SYNTHESIS: Eleven cases of pancreatitis have been reported in patients taking liraglutide. Seven were from the LEAD (Liraglutide Effect and Action in Diabetes) studies, 1 was reported in the extension of a clinical trial, and 1 was in an unpublished obesity trial. Two were published postmarketing case reports. Nine of the cases reported were diagnosed as acute pancreatitis, while 2 were classified as chronic pancreatitis. The mean age of the patients was 57.5 years and mean body mass index was 33.92 kg/m2. Six of the 11 cases occurred in male patients. Nine of the patients were white and 1 was African American. In 7 of the cases, onset occurred at liraglutide doses at or above 1.8 mg daily. Common comorbidities included history of pancreatitis, cholelithiasis, and diabetes. One case was fatal. CONCLUSIONS: Pancreatitis is a potential complication with liraglutide therapy. Liraglutide should be used cautiously in patients at risk of pancreatitis (eg, alcohol abuse, history of pancreatitis, cholelithiasis).

Os praguicidas organofosforados (OPs) são amplamente utilizados na indústria química e na agricultura em todo o mundo. Muitos deles são potenciais causadores da \"Neuropatia Retardada Induzida por Organofosforados\" (NRIOP), caracterizada pela degeneração distal de axônios do sistema nervoso central e periférico (degeneração do tipo Walleriana). O praguicida triclorfom (dimetil 2,2,2-tricloro-1-hidroxietil fosfonato) tem sido utilizado em larga escala na produção agrícola e também no controle de vetores transmissores de várias doenças. Há relatos de efeitos neuropáticos em seres humanos expostos ao triclorfom, mas apesar disso, o seu potencial neuropático e seus mecanismos de neurotoxicidade ainda não foram elucidados. Assim, no presente estudo foram avaliados os mecanismos de toxicidade do praguicida OP triclorfom utilizando linhagem SH-SY5Y de neuroblastoma humano como modelo, e também foram avaliados possíveis agentes neuroprotetores em células tratadas com o bem estabelecido indutor da neuropatia mipafox. Foram utilizados como possíveis neuroprotetores: a amilorida e nimodipino (bloqueadores de canais de cálcio tipo T e L respectivamente), MDL 28170 (inibidor (III) de calpaína) e liraglutida (um agonista do \"glucagon like peptide\" -GLP-1). Foram usados o mipafox, como modelo de indução da NRIOP e o paraoxon, como modelo não indutor da NRIOP. Os ensaios de inibição e reativação da esterase susceptível à neuropatia (ESNp) e de citotoxicidade mostraram que somente o mipafox e o triclorfom apresentaram inibição e envelhecimento da ESNp superiores a 70% em concentrações com baixa toxicidade, condição compatível com a indução da NRIOP. A ativação das calpaínas foi observada apenas no tratamento com mipafox, efeito este inibido pela nimodipina, amilorida e pelo MDL 28170. Triclorfom e o mipafox causaram elevação significativa nos níveis de cálcio intracelular e da caspase-3, além de inibir significativamente o crescimento de neuritos. Os três OPs avaliados foram capazes de diminuir a captação da glicose, que foi aumentada nos grupos tratados com mipafox associado com neuroprotetores. As quatro substâncias utilizadas como possíveis neuroprotetoras reduziram significativamente o dano causado pelo mipafox sobre os neuritos. O modelo usado no estudo mostrou-se apropriado para a caracterização dos OP neuropáticos, pois permitiu a diferenciação dos efeitos do mipafox (neuropático) e do paraoxon (não-neuropático). Os dados obtidos indicam que o triclorfom tem potencial indutor de NRIOP, e a amilorida, nimodipino liraglutida e MDL apresentaram potencial neuroprotetor / Organophosphorus (OP) pesticides are widely used in the chemical industry and agriculture around the world. \"Organophosphate Induced Delayed Neuropathy\" (OPIDN) is characterized by distal degeneration of axons of the central and peripheral nervous system (Wallerian-type degeneration). The OP trichlorfom (dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate) has been used in large scale in agricultural production and also for the control of vectors that are transmitter of diseases. There are reports of neuropathic effects in humans exposed to trichlorfon, but despite this, its neuropathic potential and mechanisms of neurotoxicity have not yet been elucidated. Thus, in the present study we evaluated the mechanisms of toxicity of trichlorfom and possible neuroprotective agents in SH-SY5Y human neuroblastoma cells treated with the well-established neuropathy inducer mipafox. The following neuroprotective agents were used: amyloride and nimodipine (T and L-type calcium channel blockers, respectively), MDL 28170 (calpain inhibitor (III) and liraglutide (a \"glucagon-like peptide\" - GLP-1 agonist). Mipafox was used as neuropathic OP and paraoxon was used as a non-neuropathic OP. Inhibition and reactivation assays of neurpathy target esterase (NTE) and cytotoxicity showed that only mipafox and trichlorfon showed inhibition and aging of 70% of the NTE in concentrations that presented low toxicity, consistent with development of OPIDN. Activation of calpain was observed only in the treatment with mipafox, an effect inhibited by nimodipine, amyloride and MDL 28170. Triclorfom and mipafox caused significant increase in the levels of intracellular calcium, caspase-3 and significantly inhibiting neurite growth. All three OPs assessed in this work caused a decrease in glucose uptake, which was increased in groups treated whith mipafox plus neuroprotectors. Substances used as possible neuroprotective agents significantly reduced the inhibitory effect of mipafox on neurite outgrowth. The model used in the study proved to be appropriate for characterizing neuropathic OPs, because it allowed a differentiation of the effects of mipafox (neuropathic) and paraoxon (non-neuropathic). The data obtained indicate that trichlorfonm has potential in cause OPIDN. Amiloride, nimodipine, MDL and liraglutide have presented potential neuroprotective effects