O diabetes mellitus do tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição seletiva de células pancreáticas produtoras de insulina. Existem diversas formas de tratamento do DM1, tais como administração de insulina, imunossupressores, transplantes de pâncreas ou de ilhotas pancreáticas, porém todos se mostram ineficientes em algum aspecto. Recentemente, uma nova classe de medicamentos, os inibidores da enzima dipeptidil peptidase 4 (iDPP-4), demonstrou eficiência terapêutica e segurança no tratamento de pacientes com diabetes mellitus do tipo 2 devido ao aumento do hormônio peptídeo-1 semelhante ao glucagon (GLP-1, do inglês glucagon-like peptide-1). Além disso, o uso de inibidores de DPP-4 em modelos experimentais de DM1 demonstrou proteção das células pancreáticas contra apoptose, estimulação de neogênese de ilhotas pancreáticas e melhora do controle homeostático da glicose. Esse presente projeto teve como objetivo avaliar o perfil imunológico e endocrinológico da administração do inibidor de DPP-4 (MK0431) em DM1 experimental quimicamente induzido por estreptozotocina em camundongos C57Bl/6. Os animais diabéticos foram tratados com ração controle ou ração contendo inibidor de DPP-4 (4g MK0431/Kg de ração) ad libitum durante 30 e 90 dias. Durante o tratamento os animais tiveram glicemia, peso e teste de tolerância oral à glicose avaliados. Ao final do tratamento, os animais foram eutanasiados e o sangue, baço, timo, linfonodos pancreáticos e pâncreas foram coletados. Após 30 dias de tratamento com inibidor, foi observado um aumento do hormônio GLP-1 no soro, além de um padrão imunológico favorável. Dentre os mecanismos imunológicos, foi possível observar um aumento de células T reguladoras (CD4+CD25+Foxp3+) no baço e uma diminuição da citocina IFN- no homogenato pancreático. Após 90 dias de tratamento com inibidor, também foi detectado um aumento de insulina e GLP-1 séricos e uma diminuição nos níveis glicêmicos dos animais tratados. Observou-se uma redução no padrão inflamatório no microambiente pancreático, caracterizado pela diminuição das citocinas TNF- e IFN- no homogenato pancreático e por uma redução da freqüência de macrófagos CD11b+ nos linfonodos pancreáticos. Os resultados obtidos neste projeto contribuíram para validar a eficácia terapêutica da administração de inibidor de DPP-4 no tratamento do DM1 experimental, bem como os mecanismos imunológicos e endocrinológicos envolvidos. Sem a ocorrência de efeitos tóxicos relevantes, o uso de inibidores de DPP-4 pode se tornar uma alternativa terapêutica para o tratamento do DM1 em humanos, que constitui uma doença crônica associada à baixa qualidade de vida em longo prazo e necessidade de tratamento de alto custo. / Davanso, M.R. Immunoendocrinological analyses after administration of dipeptidyl-peptidase-4 inhibitor on experimental type 1 diabetes. 2012. 105p. Thesis (Masters Degree) School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 2012. Type 1 Diabetes Mellitus (DM1) is an autoimmune disease characterized by the selective destruction of the insulin-producing pancreatic cells. Several forms of treatment for DM1 are current known such as insulin administration, immunosuppressors, pancreas or pancreatic islets transplantation, however, they all are inefficient in some aspect. Recently, a new class of drugs, the dipeptidyl-peptidase-4 inhibitors (iDPP-4) showed therapeutic efficacy and safety in the treatment with type 2 diabetes mellitus patients due to an increase in the glucagon-like peptide-1 (GLP-1). In addition, the use of DPP-4 inhibitors in experimental models of DM1 has demonstrated a protection of pancreatic cells against apoptosis, stimulation of pancreatic islets neogenesis and improvement in the glucose homeostatic control. This project evaluated the immunological and endocrinological profile of the DPP-4 (MK0431) inhibitor administration in experimental chemically induced DM1 by streptozotocin in C57BI/6 mice. The diabetic animals were treated with either a normal chow diet or diet containing DPP-4 inhibitor (4g MK0431/Kg of diet) ad libitum during 30 and 90 days. During the treatment the animals were evaluated regarding glycemia, weight, and oral glucose tolerance test. At the end of the treatment, the animals were killed and the blood, spleen, thymus, pancreatic lymph nodes and pancreas were collected. After 30 days of treatment with inhibitor, it was observed an increase in the hormone GLP-1 in the serum, besides a favorable immunological pattern. Among the immunologic mechanisms, it was possible to observe an increase in the regulator T cells (CD4+CD25+Foxp3+) of the spleen and a decrease in the cytokine IFN- in the pancreatic homogenate. After 90 days of treatment with inhibitor, it was also noticed an increase in the insulin and serum GLP-1 levels as well as a decrease in the glycemic levels in the treated animals. It was observed a reduction in the inflammatory pattern in the pancreatic microenvironment characterized by a decrease in the cytokines TNF- and IFN- in the pancreatic homogenate and by a reduction in the frequency of CD11b+ macrophages in the pancreatic lymph nodes. The results obtained in this project contributed to validate the therapeutic efficacy of the DPP-4 inhibitor administration in the treatment of experimental DM1, as well as the immunological and endocrinological mechanisms involved. Without the occurrence of relevant toxic effects, the use of DPP-4 inhibitors may become a therapeutic alternative for the treatment of DM1 in humans, which constitutes a chronic disease associated to low life quality and need for high cost treatment.

<p>Therapeutic peptides have become a clinically and commercially important drug class providing novel treatment options in variety of disease areas. Today, more than 80 peptide drugs are marketed worldwide and hundreds more are in development. However, the development of peptide drugs can be hindered by their tendency to self-associate to form fibrils, an impurity that can affect potency and increase the potential for adverse immune responses in patients. Fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. From a pharmaceutical development perspective, early detection of instabilities can inform the development of mitigation strategies to minimize the risk of product failure and avoid costly delays in clinical development. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and formulations.</p> <p>The objective of this dissertation was to develop structurally modified fibrillation-resistant peptides based on a mechanistic understanding of the fibrillation process. The therapeutic peptides studied were human calcitonin (hCT), a glucagon/GLP-1 analog, and human insulin B-chain (INSB). Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) and other biophysical methods were used to provide mechanistic understanding of the intermolecular interactions and structural transitions during peptide fibrillation. Coupled with proteolytic digestion, pulsed HDX-MS of fibrillating peptides enabled identification of the residues involved in the early interactions leading to fibrillation based on their differential deuterium exchange rates. The high-resolution residue level information was used to make site-specific modifications to hCT, with phosphorylation in the central region resulting in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog under the stress conditions employed. Reversible ‘prodrug’ modifications such as phosphorylation can aid the rational design of fibrillation-resistant therapeutic peptides. Furthermore, the effects of structural modifications on peptide fibrillation were evaluated by reducing the Cys1-Cys7 disulfide bond in hCT, and by C-terminal amidation or substitution with a helix-stabilizing residue (α-aminoisobutyric acid, Aib) in the glucagon/GLP-1 analog peptide. Finally, studies of insulin B-chain probed fibrillation mechanisms of this therapeutically important peptide, contributing to our understanding of the mechanisms of insulin fibrillation with the broad goal of developing fibrillation-resistant, rapid-acting, monomeric insulin analogs. Overall, the results demonstrate that small structural changes can have significant effects on peptide fibrillation, that pulsed HDX-MS can be used to probe these effects, and that an understanding of these effects can inform the rational development of fibrillation-resistant peptide drugs. </p>

Indiana University-Purdue University Indianapolis (IUPUI) / Glucagon-like peptide 1 (GLP-1) receptor agonists are a class of incretin based therapeutics which aid in blood glucose management in Type II diabetes mellitus (T2DM). Recent studies have demonstrated direct cardiovascular benefits conferred by these agents including protection in ischemia and heart failure. Despite these observations, human clinical trials fail to support improvements in cardiovascular outcomes independent of glucose lowering effects in the T2DM populations. Prior data from our lab demonstrate that obesity impairs GLP-1 associated increases in myocardial glucose uptake. However, the reasons for this impairment/resistance to cardiac effects of GLP-1 in the setting of obesity remain ill defined. This investigation tested the hypothesis that underlying differences in the cardiac proteome and microRNA (miR) transcriptome could contribute to distinct cardiac responses to ischemia and activation of GLP-1 signaling in the setting of obesity. To identify whether obesity modulated cardiac functional responses to GLP 1 related drugs, we first examined the effects of obesity on cardiac function, miR transcriptome, and proteome in response to short duration ischemia-reperfusion (I/R). We observed divergent physiologic responses (e.g. increased diastolic volume and systolic pressure in lean, decreased diastolic volumes in obese) to regional I/R in obese vs lean hearts that were associated with significant molecular changes as detected by protein mass spectrometry and miR microarray. Molecular changes were related to myocardial calcium handling (SERCA2a, histidine-rich Ca2+ binding protein), myocardial structure and function (titin), and miRs relating to cardiac metabolism, hypertrophy, and cell death, including miR-15, miR-30, miR-199a, miR-214. Importantly, these effects were modified differently by GLP-1 agonism in lean vs obese swine. Additional studies investigated the functional effects of 30 days of treatment with the GLP-1 analogue liraglutide on a model of slowly-developing, unrelieved coronary ischemia. Liraglutide failed to reduce infarct size or collagen deposition. However, analysis of left ventricular pressure-volume relationships support that liraglutide improved diastolic relaxation/filling, load-dependent indices of cardiac function, and cardiac efficiency in response to sympathetic stimulation in obese swine. Taken together, these findings support that miR and proteomic differences underlie distinct changes in functional cardiac responses to I/R and pharmacologic activation of GLP-1 signaling in the setting of obesity.

Ce travail porte sur des approches translationnelles dans les synucléinopathies notamment l’atrophie multisystématisée (AMS). Au-delà de leur rôle dans la régulation du glucose, l’insulin et l’insulin like growth factor-1 (IGF-1) ont des propriétés neurotrophiques. Des études ont montrées que la signalisation de l’insuline/IGF-1 est altérée dans la maladie d'Alzheimer et des données suggèrent l’altération de l’insuline/IGF-1 dans la maladie de Parkinson (MP) et l’AMS. Nous avons mis en évidence une résistance à l’insuline dans les neurones des patients MP et AMS ainsi que dans les oligodendrocytes chez les patients AMS.Mon travail a également consisté à cibler la troncation de l’α-synuclein (α-syn) comme cible thérapeutique. Nous avons démontré dans un modèle murin d’AMS que la diminution de l’α-syn tronquée permettait de réduire l’agrégation d’α-syn et la dégénérescence des neurones dopaminergiques.Enfin, nous avons étudié l’implication dans l’AMS des métalloprotéinases matricielles (MMP), des enzymes impliquées dans remodelage de la matrice, la démyélinisation, la troncation de l’α-syn et la perméabilité de la barrière hémato-encéphalique. Ce travail nous a permis de montrer une augmentation de l’expression et de l’activité de MMPs chez les patients AMS. Nous avons également montré que les cellules gliales sont la source de cette augmentation et que la MMP-2 est retrouvée dans les agrégats des patients AMS.Nous montrons ici de caractéristiques distinctes de l’AMS comme des altérations qui se produisent dans les oligodendrocytes. Nous présentons aussi VX-765 comme un candidat prometteur pour ralentir la progression de la pathologie dans un contexte de synucléinopathie. / This work focused on translational approaches in synucleinopathies and more specifically in multiple system atrophy (MSA). Beyond their role in glucose homeostasis, insulin/IGF-1 are neurotrophic factors in the brain. Studies have shown altered insulin/IGF-1 signalling in Alzheimer’s disease and data suggest impaired insulin signaling/IGF-1 in Parkinson's disease (PD) and MSA. The aim of my work was to characterize insulin/IGF-1 signalling in MSA and PD brain tissue. Both groups showed neuronal insulin resistance. Oligodendrocytes in MSA patients were also insulin resistant.In line with the translational approach, we also targeted α-synuclein (α-syn) truncation pharmacologically in MSA transgenic mice, which led to reduced α-syn aggregation and the protection of dopaminergic neurons.We also assessed the activity and distribution of matrix metalloproteinases (MMPs) in the brain of MSA patients compared to healthy controls. MMPs are involved in the remodelling of the extracellular matrix, demyelination, α-syn truncation and blood brain barrier permeability. We showed altered expression and activity of MMPs in two distinct structures in MSA brains. We were also able to show that glial cells were the source of increased MMPs and show a unique expression of MMPs in α-syn aggregates of MSA patients compared to PD, evidence that might hint at a mechanism that is differently altered between PD and MSA.We here show distinct pathological features of MSA such as key alterations occurring in oligodendrocytes, further supporting MSA as a primary oligodendrogliopathy. We also present VX-765 as a candidate drug for disease modification in synucleinopathies.

L’hyperglycémie de stress et la variabilité glycémique, consécutives à la réaction inflammatoire péri opératoire, sont associées à une morbidité et une mortalité accrues en chirurgie cardiaque. L’insulinothérapie intraveineuse administrée à l’aide de protocoles complexes, dits « dynamiques », constitue à l’heure actuelle le traitement de référence de l’hyperglycémie de stress. L’intérêt du contrôle glycémique péri-opératoire est admis par tous, sans qu’il existe de consensus véritable quant aux objectifs à atteindre, et reste très exigeant en termes de charge de soins. Dans la 1ère partie de ce travail, nous avons voulu vérifier si, 7 ans après sa mise en place, l’observance du protocole d’insulinothérapie utilisé dans notre Unité de Soins Intensifs de Chirurgie Cardiaque était conforme à celle mesurée lors de son implantation. Nous avons constaté des dérives majeures dans l’application du protocole qui ont pu être corrigées par la mise en place de mesures correctrices simples. Dans une 2ème partie du travail, nous avons cherché à évaluer si, à l’instar de la chirurgie cardiaque classique, une variabilité glycémique accrue était associée à une altération du pronostic des patients bénéficiant d’une procédure moins invasive (remplacement valvulaire aortique percutané ou TAVI). Nous avons ainsi analysé les données des patients ayant bénéficié d’un TAVI dans notre centre, et inclus dans les registres multicentriques français France et France-2. Nos résultats suggèrent une association entre une augmentation de la variabilité glycémique et un risque accru de complications cardiovasculaires majeures dans les 30 premiers jours, indépendamment de la qualité du contrôle glycémique obtenu. Enfin, dans une 3ème partie nous avons voulu savoir si exenatide, analogue de synthèse de GLP-1, permettait d’améliorer le contrôle glycémique péri opératoire en chirurgie cardiaque. Nous avons conduit un essai randomisé contrôlé de phase II/III montrant que l’administration intraveineuse (IV) d’exenatide, ne permettait pas d’améliorer la qualité du contrôle glycémique ou de réduire la variabilité glycémique par rapport à l’insuline IV, mais permettait de retarder l’administration d’insuline et de diminuer la quantité d’insuline administrée. Notre étude suggère également une diminution de la charge en soins. Du fait des données rapportées chez l’animal et dans l’infarctus du myocarde, nous avons également conduit une étude ancillaire suggérant l’absence d’effets cardioprotecteurs majeurs d’exenatide sur les lésions d’ischémie-reperfusion myocardiques, ne permettant pas d’améliorer la fonction cardiaque gauche à court et à moyen terme. L’optimisation du contrôle glycémique en chirurgie cardiaque nécessite ainsi la recherche de stratégies visant à améliorer l’observance des protocoles de soins et à réduire la variabilité glycémique. La place des analogues du GLP-1 reste à définir dans cette indication. / Stress hyperglycemia and glycemic variability are associated with increased morbidity and mortality in cardiac surgery patients. Intravenous (IV) insulin therapy using complex dynamic protocols is the gold standard treatment for stress hyperglycemia. If the optimal blood glucose target range remains a matter of debate, blood glucose control using IV insulin therapy protocols has become part of the good clinical practices during the postoperative period, but implies a significant increase in nurse workload. In the 1st part of the thesis, we aimed at checking the nurse-compliance to the insulin therapy protocol used in our Cardiac Surgery Intensive Care Unit 7 years after its implementation. Major deviations have been observed and simple corrective measures have restored a high level of nurse compliance. In the 2nd part of this thesis, we aimed at assessing whether blood glucose variability could be related to poor outcome in transcatheter aortic valve implantation (TAVI) patients, as reported in more invasive cardiac surgery procedures. The analysis of data from patients who undergone TAVI in our institution and included in the multicenter France and France-2 registries suggested that increased glycemic variability is associated with a higher rate of major adverse events occurring between the 3rd and the 30th day after TAVI, regardless of hyperglycemia. In the 3rd part if this thesis, we conducted a randomized controlled phase II/III trial to investigate the clinical effectiveness of IV exenatide in perioperative blood glucose control after coronary artery bypass graft surgery. Intravenous exenatide failed to improve blood glucose control and to decrease glycemic variability, but allowed to delay the start in insulin infusion and to lower the insulin dose required. Moreover, IV exenatide could allow a significant decrease in nurse workload. The ancillary analysis of this trial suggested that IV exenatide did neither provide cardio protective effect against myocardial ischemia-reperfusion injuries nor improve the left ventricular function by using IV exenatide. Strategies aiming at improving nurse compliance to insulin therapy protocols and at reducing blood glucose variability could be suitable to improve blood glucose control in cardiac surgery patients. The use of the analogues of GLP-1 in cardiac surgery patients needs to be investigated otherwise.

No tratamento do diabetes mellitus tipo 2, busca-se a otimização do controle glicêmico para evitar as complicações da doença. Quando a combinação mais utilizada (sulfoniluréia e biguanida) falha em manter o controle glicêmico, um terceiro agente é associado, podendo ser a insulina ou outro hipoglicemiante oral. O objetivo deste estudo foi comparar os efeitos da sitagliptina (droga inibidora da enzima dipeptidil-peptidase-4) com os da insulina NPH ao deitar, como terceira droga no tratamento do diabetes mellitus tipo 2, em pacientes inadequadamente controlados com glibenclamida e metformina. Trinta e cinco pacientes, com dados clínicos, demográficos e laboratoriais semelhantes, foram randomizados para receber sitagliptina (grupo Sitagliptina, n= 18) ou insulina NPH ao deitar (grupo Insulina, n= 17) e reavaliados após 24 semanas. Amostras sanguíneas foram coletadas para dosagens de hemoglobina glicada (HbA1c), colesterol total e frações, ácido úrico, transaminases, proteína C reativa (PCR) e grelina em jejum. No teste de tolerância à dieta de 500 calorias, foram determinadas as concentrações de glicose, triglicérides, ácidos graxos livres (AGL), peptídeo glucagon-símile tipo 1 ativo (GLP-1a), glucagon, peptídeo C, pró-insulina e insulina. Monitorização ambulatorial da pressão arterial e ecocardiografia com Doppler tecidual também foram realizados. Decorridas 24 semanas, ambos os tratamentos promoveram reduções semelhantes das concentrações de HbA1c. A insulina NPH ao deitar foi superior à sitagliptina, ao promover redução da glicemia de jejum e das concentrações de triglicérides após a refeição. Diminuição das concentrações de glucagon pós-prandiais e aumento das concentrações de GLP-1 ativo de jejum e após a refeição foram observados somente no grupo Sitagliptina. Concentrações de peptídeo C não se alteraram após o tratamento com sitagliptina, porém diminuíram após a terapia com insulina. Os dois tratamentos reduziram as concentrações de pró-insulina pós-prandiais e de grelina de jejum. Nenhum deles alterou as concentrações de PCR, colesterol, ácido úrico e enzimas hepáticas, exceto a de fosfatase alcalina, que diminuiu em ambos os grupos. Peso, índice de massa corpórea, relação cintura/quadril e pressões arteriais sistólicas e diastólicas permaneceram inalterados. Avaliação cardíaca, realizada em 29 pacientes, mostrou disfunção diastólica do ventrículo esquerdo (DDVE), sinal pré-clínico da cardiomiopatia diabética, em 53 % e 64 % dos pacientes que foram incluídos nos grupos Sitagliptina e Insulina, respectivamente. Melhora da função diastólica de VE foi observada em 40,0% (6/15) dos pacientes do grupo Sitagliptina e em 7,0% (1/14) dos pacientes do grupo Insulina (p=0,049). Esta melhora da função cardíaca pareceu não depender do controle glicêmico, da PA e do status inflamatório. Concluindo, ambos os tratamentos foram efetivos, promovendo redução semelhante da HbA1c. Ambas as drogas parecem melhorar a função das células b, observada pela diminuição das concentrações de pró-insulina. A insulina NPH ao deitar foi mais efetiva na redução da glicemia de jejum e das concentrações de triglicérides após a refeição. Entretanto, melhora na função diastólica de VE foi superior no grupo Sitagliptina. Assim, o inibidor da DPP-4 parece ser uma droga promissora para a prevenção da cardiomiopatia diabética / The main goal of the treatment of type 2 diabetes mellitus is to achieve normal glucose levels in order to prevent diabetic complications. When two oral agents (sulfonylurea plus biguanide) fail to maintain target-level control, a third oral agent must be added or insulin must be started. The aim of this study was to compare sitagliptin, a dipeptidyl-peptidase-4 enzyme inhibitor, with bedtime NPH insulin as a third-line agent in type 2 diabetes mellitus patients inadequately controlled with metformin plus glyburide combined therapy. Thirty-five patients with similar clinical, demographic and basal laboratory characteristics were randomized to receive sitagliptin (Sitagliptin group, n=18) or bedtime NPH insulin (Insulin group, n=17) and reassessed after 24 weeks. Fasting blood samples were collected for determinations of glycated hemoglobin (HbA1c), total- LDL- and HDL-cholesterol, uric acid, liver enzymes, C-reactive protein and ghrelin levels. Determinations of blood glucose, triglycerides, free fatty acids, active glucagon-like peptide 1 (GLP-1), glucagon, C-peptide, pro-insulin and insulin levels was made during the meal tolerance test. Ambulatory blood pressure monitoring and tissue Doppler echocardiography were also performed. After 24 weeks, both treatments resulted in similar HbA1c levels. Bedtime NPH insulin was superior to sitagliptin in reducing fasting plasma glucose and postprandial triglyceride levels. Decreased postprandial glucagon and increased active GLP-1 levels were only observed in the Sitagliptin group. C-peptide levels did not change after treatment with sitagliptin, but decreased following insulin therapy. Both treatments reduced postprandial pro-insulin and fasting ghrelin levels. Neither treatment changed C-reactive protein, cholesterol, uric acid or liver enzymes, except alkaline phosphatase, which decreased in both. Weight, body mass index, waist-hip ratio and systolic and diastolic blood pressures remained unchanged. Cardiac evaluation was performed in 29 patients and showed basal left ventricular diastolic dysfunction, the pre-clinical signal of diabetic cardiomyopathy, in 53% and in 64% of patients in the Sitagliptin and Insulin group, respectively. Left ventricular diastolic function improved in 40% (6/15) of patients receiving sitagliptin and in 7% (1/14) of patients receiving bedtime NPH insulin (p= 0.049). This improvement did not seem to be influenced by glycemic control, blood pressure or inflammatory status. In conclusion, both treatments were effective in reducing HbA1c and seemed to similarly improve b-cell function, as shown by reduced pro-insulin levels. Bedtime NPH insulin was more effective in reducing fasting plasma glucose and postprandial triglyceride levels. However, improvement in left ventricular diastolic function was higher in the Sitagliptin group. Therefore, sitagliptin seems to be a promising drug for the prevention of diabetic cardiomyopathy

Introdução: A sitagliptina, inibidor da dipeptidil-peptidase IV, impede a degradação do GLP-1 (peptídeo-1 semelhante ao glucagon), um dos principais hormônios incretínicos. A dieta interfere na secreção de GLP-1, no entanto, a interação das drogas que aumentam o GLP-1 e os macronutrientes da dieta é pouco estudada. Objetivo e Métodos: Determinar o efeito da sitagliptina, na secreção de GLP-1, glucagon, insulina, peptídeo-C, ácidos graxos livres e na glicemia após três dietas, isocalóricas, de diferentes composições nutricionais em pacientes diabéticos tipo 2, recém-diagnosticados, virgens de tratamento, quando comparado a uso de placebo. Dezesseis indivíduos nessas condições foram submetidos a dietas hiperglicídica, hiperprotêica e hiperlipídica, isocalóricas entre si. Dosaram-se nos tempos 0, 30, 60, 120 e 180 minutos os parâmetros: glicose, insulina, peptídeo C, GLP-1, glucagon e AGL. Foi calculada média de área sob a curva e cálculo da área incremental, além de análise de variância para medidas repetidas. Resultados: Durante o teste de dieta hiperglicídica a glicemia foi maior em todos os tempos quando comparado aos testes com PTN e LPD independentemente do uso de sitagliptina (p<0,05). Sitagliptina diminuiu a glicemia em todos os tempos, quando comparado ao uso de placebo (p<0,05). Durante a dieta CHO, a secreção de glucagon foi menor que nas dietas LPD e PTN (p<0,05). Já a concentração de insulina foi maior com a dieta CHO em relação à dieta LPD (p<0,05). A concentração de insulina e peptídeo C foi maior em todos os tempos na dieta CHO (p<0,05). A concentração de GLP-1 foi significativamente maior durante o teste hiperlipídico em relação à dieta CHO. Durante a dieta LPD, a medida de GLP-1 foi maior em todos os tempos. A dieta CHO apresentou medida de GLP-1 menor em todos os tempos do que as outras dietas (p<0,05). A medida de GLP-1 no tempo foi maior (até 120\') com o uso de sitagliptina do que com o uso do placebo, apesar de não estatisticamente significativa. Os níveis de AGL no tempo foram maiores com o uso do placebo do que com o uso da sitagliptina, apesar de não estatisticamente significativo. Conclusão: Houve diminuição da glicemia em todos os tempos com sitagliptina, independentemente da dieta testada. Houve diminuição do efeito da sitagliptina durante o uso da dieta hiperglicídica / Background: Sitagliptin, a dipeptidil-peptidase IV inhibitor, prevents the degradation of GLP-1 (glucagon-like peptide 1), one of the incretin hormones. It is well-known that diet interferes in the GLP-1 secretion; however, the interaction between drugs that stimulates the release of GLP-1 and the macronutrients from diet is hardly studied. Objective and Methods: To demonstrate the effect of sitagliptin on glycemia, and on the secretion of GLP-1, glucagon, insulin, C-peptide, and free fatty acids after three isocaloric diets with different nutritional compositions, in drug-naïve patients, newly diagnosed with type 2 diabetes, when compared to the use of placebo. Sixteen individuals were subjected to a high-carbohydrate diet, a high-protein diet, and a high-fat diet, all of which with similar caloric values. At 0, 30, 60, 120 and180 minutes after the diet, glucose, insulin, C-peptide, GLP-1, glucagon, and AGL were measured. The mean area under the curve, the incremental area, and the variance for repeated measures were calculated. Results: During high-carbohydrate diet, glycemia was higher for all time points, when compared to the PTN and LPD diets, independently of sitagliptin (p<0,05). Sitagliptin reduced glycemia during three diets when compared to placebo (p<0,05). During CHO diet, secretion of glucagon was smaller than it was during the LDP and PTN diets (p<0,05). On the other hand, insulin concentration was higher than during the LPD diet (p<0,05). Concentrations of insulin and C-peptide were higher for all the time points during the CHO diet (p<0,05). GLP-1 concentration was significantly higher during the high-fat diet than during the high-carbohydrate diet. During the LPD diet, the quantity of the GLP-1 was larger for all time points. The CHO diet presented lower GLP-1 level, for all the time points, than the other diets (p<0,05). The GLP-1 level (up to 120min) with the use of sitagliptin was higher with LPD and PTN diet than it was with the CHO diet. The AGL levels for all time points were higher with placebo than with sitagliptin, although not statistically significant. Conclusion: There was a reduction in glycemia with sitagliptin, independently of the diet tested, for all time points. There was a reduction in sitagliptin effect during the use of the high-carbohydrate diet

Dados recentes do nosso laboratório sugerem que a enzima dipeptidil peptidase IV (DPPIV), uma serino-protease que pode ser encontrada ancorada na membrana celular de diversos tipos celulares ou na forma solúvel no plasma, possui um papel importante na fisiopatologia da insuficiência cardíaca (IC). Mais especificamente, demonstramos que a atividade da DPPIV circulante está associada com piores desfechos cardiovasculares em modelo experimental e pacientes com IC. Ademais, observamos que a inibição crônica da DPPIV atenua o desenvolvimento e/ou a progressão da IC em ratos submetidos à injúria do miocárdio. Entretanto, não é sabido se a inibição desta peptidase é capaz de reverter a disfunção cardiorrenal em ratos com IC estabelecida. Assim, este trabalho teve como objetivo testar a hipótese que a inibição da DPPIV exerce efeitos terapêuticos em ratos com IC. Para tal, ratos com IC foram tratados diariamente com o inibidor da DPPIV Vildagliptina (80 ou 120 mg/kg/dia) ou veículo (HF) durante quatro semanas. Ratos Sham não-tratados foram utilizados como controle. Análises ecocardiográficas demonstraram que ratos HF exibiram área fracional (FAC) menor e tempo de relaxamento isovolumétrico (TRIV) maior que ratos Sham. Por sua vez, o tratamento com a dose maior de Vildagliptina foi capaz de aumentar a FAC e diminuir o TRIV. Esta melhora funcional foi acompanhada por melhoras estruturais, visto que a inibição da DPPIV foi capaz de reduzir a hipertrofia cardíaca e a deposição de colágeno intersticial no miocárdio remanescente de ratos tratados com Vildagliptina em comparação aos ratos HF. Adicionalmente, ratos com IC exibiram maior teor de água nos pulmões, menor excreção urinária de sódio, menor fluxo urinário e menor ritmo de filtração glomerular em comparação ao grupo Sham. Por sua vez, o manuseio renal de sal e água foi completamente restaurado pelo tratamento crônico com 120 mg/kg/dia Vildagliptina. A normalização da função renal induzida pela inibição crônica da DPPIV foi associada com um aumento da expressão do receptor do peptídeo-1 semelhante ao glucagon (GLP-1) e maior ativação da proteína cinase A em córtex renal, isto é, da via de sinalização deflagrada pela ligação GLP-1/GLP-1R. Além disso, os níveis pós-prandiais do GLP-1, principal substrato da DPPIV que exerce ações insulinotrópicas, cardio e renoprotetoras, estavam mais baixos em ratos HF que em ratos Sham. Esta diminuição dos níveis circulantes de GLP-1 (ativo e total) em ratos HF foi acompanhada de intolerância à glicose bem como de maiores níveis plasmáticos de insulina. A inibição da DPPIV com Vildagliptina melhorou a biodisponibilidade e a secreção de GLP-1 após carga oral de glicose. Em conjunto, estes resultados sugerem que a inibição da DPPIV melhora a função cardiorrenal e metabólica de ratos com IC. Além disso, a secreção e a biodisponibilidade do GLP-1 encontram-se prejudicadas em ratos com IC e o tratamento com Vildagliptina é capaz de restaurar a sinalização mediada por este peptídeo. Assim, os inibidores da DPPIV podem ser eficazes não apenas para a prevenção, mas também para o tratamento da insuficiência cardíaca em ratos / Recent data from our laboratory suggest that the enzyme dipeptidyl peptidase IV (DPPIV), a serine protease that can be found anchored in the cell membrane of different cell types or in the soluble form in plasma, plays an important role in the pathophysiology of heart failure (HF). More specifically, we have demonstrated that the activity of circulating DPPIV is associated with poorer cardiovascular outcomes in an experimental model and patients with HF. In addition, we have found that chronic inhibition of DPPIV attenuates the development and/or progression of HF in rats with myocardial injury. However, it is unknown whether the inhibition of this peptidase is able to reverse the cardiorenal dysfunction in rats with established HF. Therefore, this study aimed to test the hypothesis that inhibition of DPPIV exerts therapeutic effects in rats with HF. To this end, HF rats were treated daily with the DPPIV inhibitor vildagliptin (80 or 120 mg/kg/day) or vehicle (HF) for four weeks. Untreated Sham rats were used as controls. Echocardiographic analysis demonstrated that HF rats exhibit lower fractional area change (FAC) and higher isovolumetric relaxation time (IVRT) than Sham rats. On the other hand, treatment with the highest dose of vildagliptin was able to increase FAC and decrease IVRT. These functional improvements were accompanied by structural improvements, since inhibition of DPPIV was also able to reduce cardiac hypertrophy and interstitial collagen deposition in the remaining myocardium of rats treated with vildagliptin rats compared to HF. In addition, HF rats exhibited higher water content in the lungs, lower urinary sodium excretion, lower urinary flow and lower glomerular filtration rate compared to the Sham group. In turn, the renal handling of salt and water was completely restored by chronic treatment with vildagliptin 120 mg/kg/day. Normalization of the renal function induced by chronic inhibition of DPPIV was associated with an increase in the expression of the glucagon like peptide-1 receptor (GLP-1R) and enhanced protein kinase A activation in the renal cortex, the signaling pathway triggered by bind between GLP-1/GLP-1R. In addition, the postprandial levels of GLP-1, the main substrate of DPPIV that exerts insulinotropic, cardio and renoprotective actions, were lower in HF rats than in Sham. This decrease in circulating levels of GLP-1 (active and total) in HF rats was accompanied by impaired glucose tolerance and higher plasma insulin levels. The inhibition of the DPPIV with vildagliptin improved the bioavailability and secretion after an oral glucose load. Taken together, these results suggest that the inhibition of DPPIV ameliorates the cardiorenal and metabolic function of rats with HF. Furthermore, bioavailability and secretion of GLP-1 are impaired in HF rats and vildagliptin is able to restore the signaling mediated by this peptide. Therefore, DPPIV inhibitors can be effective not only in preventing but also for the treatment of HF in rats

Dados recentes do nosso laboratório sugerem que a enzima dipeptidil peptidase IV (DPPIV), uma serino-protease que pode ser encontrada ancorada na membrana celular de diversos tipos celulares ou na forma solúvel no plasma, possui um papel importante na fisiopatologia da insuficiência cardíaca (IC). Mais especificamente, demonstramos que a atividade da DPPIV circulante está associada com piores desfechos cardiovasculares em modelo experimental e pacientes com IC. Ademais, observamos que a inibição crônica da DPPIV atenua o desenvolvimento e/ou a progressão da IC em ratos submetidos à injúria do miocárdio. Entretanto, não é sabido se a inibição desta peptidase é capaz de reverter a disfunção cardiorrenal em ratos com IC estabelecida. Assim, este trabalho teve como objetivo testar a hipótese que a inibição da DPPIV exerce efeitos terapêuticos em ratos com IC. Para tal, ratos com IC foram tratados diariamente com o inibidor da DPPIV Vildagliptina (80 ou 120 mg/kg/dia) ou veículo (HF) durante quatro semanas. Ratos Sham não-tratados foram utilizados como controle. Análises ecocardiográficas demonstraram que ratos HF exibiram área fracional (FAC) menor e tempo de relaxamento isovolumétrico (TRIV) maior que ratos Sham. Por sua vez, o tratamento com a dose maior de Vildagliptina foi capaz de aumentar a FAC e diminuir o TRIV. Esta melhora funcional foi acompanhada por melhoras estruturais, visto que a inibição da DPPIV foi capaz de reduzir a hipertrofia cardíaca e a deposição de colágeno intersticial no miocárdio remanescente de ratos tratados com Vildagliptina em comparação aos ratos HF. Adicionalmente, ratos com IC exibiram maior teor de água nos pulmões, menor excreção urinária de sódio, menor fluxo urinário e menor ritmo de filtração glomerular em comparação ao grupo Sham. Por sua vez, o manuseio renal de sal e água foi completamente restaurado pelo tratamento crônico com 120 mg/kg/dia Vildagliptina. A normalização da função renal induzida pela inibição crônica da DPPIV foi associada com um aumento da expressão do receptor do peptídeo-1 semelhante ao glucagon (GLP-1) e maior ativação da proteína cinase A em córtex renal, isto é, da via de sinalização deflagrada pela ligação GLP-1/GLP-1R. Além disso, os níveis pós-prandiais do GLP-1, principal substrato da DPPIV que exerce ações insulinotrópicas, cardio e renoprotetoras, estavam mais baixos em ratos HF que em ratos Sham. Esta diminuição dos níveis circulantes de GLP-1 (ativo e total) em ratos HF foi acompanhada de intolerância à glicose bem como de maiores níveis plasmáticos de insulina. A inibição da DPPIV com Vildagliptina melhorou a biodisponibilidade e a secreção de GLP-1 após carga oral de glicose. Em conjunto, estes resultados sugerem que a inibição da DPPIV melhora a função cardiorrenal e metabólica de ratos com IC. Além disso, a secreção e a biodisponibilidade do GLP-1 encontram-se prejudicadas em ratos com IC e o tratamento com Vildagliptina é capaz de restaurar a sinalização mediada por este peptídeo. Assim, os inibidores da DPPIV podem ser eficazes não apenas para a prevenção, mas também para o tratamento da insuficiência cardíaca em ratos / Recent data from our laboratory suggest that the enzyme dipeptidyl peptidase IV (DPPIV), a serine protease that can be found anchored in the cell membrane of different cell types or in the soluble form in plasma, plays an important role in the pathophysiology of heart failure (HF). More specifically, we have demonstrated that the activity of circulating DPPIV is associated with poorer cardiovascular outcomes in an experimental model and patients with HF. In addition, we have found that chronic inhibition of DPPIV attenuates the development and/or progression of HF in rats with myocardial injury. However, it is unknown whether the inhibition of this peptidase is able to reverse the cardiorenal dysfunction in rats with established HF. Therefore, this study aimed to test the hypothesis that inhibition of DPPIV exerts therapeutic effects in rats with HF. To this end, HF rats were treated daily with the DPPIV inhibitor vildagliptin (80 or 120 mg/kg/day) or vehicle (HF) for four weeks. Untreated Sham rats were used as controls. Echocardiographic analysis demonstrated that HF rats exhibit lower fractional area change (FAC) and higher isovolumetric relaxation time (IVRT) than Sham rats. On the other hand, treatment with the highest dose of vildagliptin was able to increase FAC and decrease IVRT. These functional improvements were accompanied by structural improvements, since inhibition of DPPIV was also able to reduce cardiac hypertrophy and interstitial collagen deposition in the remaining myocardium of rats treated with vildagliptin rats compared to HF. In addition, HF rats exhibited higher water content in the lungs, lower urinary sodium excretion, lower urinary flow and lower glomerular filtration rate compared to the Sham group. In turn, the renal handling of salt and water was completely restored by chronic treatment with vildagliptin 120 mg/kg/day. Normalization of the renal function induced by chronic inhibition of DPPIV was associated with an increase in the expression of the glucagon like peptide-1 receptor (GLP-1R) and enhanced protein kinase A activation in the renal cortex, the signaling pathway triggered by bind between GLP-1/GLP-1R. In addition, the postprandial levels of GLP-1, the main substrate of DPPIV that exerts insulinotropic, cardio and renoprotective actions, were lower in HF rats than in Sham. This decrease in circulating levels of GLP-1 (active and total) in HF rats was accompanied by impaired glucose tolerance and higher plasma insulin levels. The inhibition of the DPPIV with vildagliptin improved the bioavailability and secretion after an oral glucose load. Taken together, these results suggest that the inhibition of DPPIV ameliorates the cardiorenal and metabolic function of rats with HF. Furthermore, bioavailability and secretion of GLP-1 are impaired in HF rats and vildagliptin is able to restore the signaling mediated by this peptide. Therefore, DPPIV inhibitors can be effective not only in preventing but also for the treatment of HF in rats

No tratamento do diabetes mellitus tipo 2, busca-se a otimização do controle glicêmico para evitar as complicações da doença. Quando a combinação mais utilizada (sulfoniluréia e biguanida) falha em manter o controle glicêmico, um terceiro agente é associado, podendo ser a insulina ou outro hipoglicemiante oral. O objetivo deste estudo foi comparar os efeitos da sitagliptina (droga inibidora da enzima dipeptidil-peptidase-4) com os da insulina NPH ao deitar, como terceira droga no tratamento do diabetes mellitus tipo 2, em pacientes inadequadamente controlados com glibenclamida e metformina. Trinta e cinco pacientes, com dados clínicos, demográficos e laboratoriais semelhantes, foram randomizados para receber sitagliptina (grupo Sitagliptina, n= 18) ou insulina NPH ao deitar (grupo Insulina, n= 17) e reavaliados após 24 semanas. Amostras sanguíneas foram coletadas para dosagens de hemoglobina glicada (HbA1c), colesterol total e frações, ácido úrico, transaminases, proteína C reativa (PCR) e grelina em jejum. No teste de tolerância à dieta de 500 calorias, foram determinadas as concentrações de glicose, triglicérides, ácidos graxos livres (AGL), peptídeo glucagon-símile tipo 1 ativo (GLP-1a), glucagon, peptídeo C, pró-insulina e insulina. Monitorização ambulatorial da pressão arterial e ecocardiografia com Doppler tecidual também foram realizados. Decorridas 24 semanas, ambos os tratamentos promoveram reduções semelhantes das concentrações de HbA1c. A insulina NPH ao deitar foi superior à sitagliptina, ao promover redução da glicemia de jejum e das concentrações de triglicérides após a refeição. Diminuição das concentrações de glucagon pós-prandiais e aumento das concentrações de GLP-1 ativo de jejum e após a refeição foram observados somente no grupo Sitagliptina. Concentrações de peptídeo C não se alteraram após o tratamento com sitagliptina, porém diminuíram após a terapia com insulina. Os dois tratamentos reduziram as concentrações de pró-insulina pós-prandiais e de grelina de jejum. Nenhum deles alterou as concentrações de PCR, colesterol, ácido úrico e enzimas hepáticas, exceto a de fosfatase alcalina, que diminuiu em ambos os grupos. Peso, índice de massa corpórea, relação cintura/quadril e pressões arteriais sistólicas e diastólicas permaneceram inalterados. Avaliação cardíaca, realizada em 29 pacientes, mostrou disfunção diastólica do ventrículo esquerdo (DDVE), sinal pré-clínico da cardiomiopatia diabética, em 53 % e 64 % dos pacientes que foram incluídos nos grupos Sitagliptina e Insulina, respectivamente. Melhora da função diastólica de VE foi observada em 40,0% (6/15) dos pacientes do grupo Sitagliptina e em 7,0% (1/14) dos pacientes do grupo Insulina (p=0,049). Esta melhora da função cardíaca pareceu não depender do controle glicêmico, da PA e do status inflamatório. Concluindo, ambos os tratamentos foram efetivos, promovendo redução semelhante da HbA1c. Ambas as drogas parecem melhorar a função das células b, observada pela diminuição das concentrações de pró-insulina. A insulina NPH ao deitar foi mais efetiva na redução da glicemia de jejum e das concentrações de triglicérides após a refeição. Entretanto, melhora na função diastólica de VE foi superior no grupo Sitagliptina. Assim, o inibidor da DPP-4 parece ser uma droga promissora para a prevenção da cardiomiopatia diabética / The main goal of the treatment of type 2 diabetes mellitus is to achieve normal glucose levels in order to prevent diabetic complications. When two oral agents (sulfonylurea plus biguanide) fail to maintain target-level control, a third oral agent must be added or insulin must be started. The aim of this study was to compare sitagliptin, a dipeptidyl-peptidase-4 enzyme inhibitor, with bedtime NPH insulin as a third-line agent in type 2 diabetes mellitus patients inadequately controlled with metformin plus glyburide combined therapy. Thirty-five patients with similar clinical, demographic and basal laboratory characteristics were randomized to receive sitagliptin (Sitagliptin group, n=18) or bedtime NPH insulin (Insulin group, n=17) and reassessed after 24 weeks. Fasting blood samples were collected for determinations of glycated hemoglobin (HbA1c), total- LDL- and HDL-cholesterol, uric acid, liver enzymes, C-reactive protein and ghrelin levels. Determinations of blood glucose, triglycerides, free fatty acids, active glucagon-like peptide 1 (GLP-1), glucagon, C-peptide, pro-insulin and insulin levels was made during the meal tolerance test. Ambulatory blood pressure monitoring and tissue Doppler echocardiography were also performed. After 24 weeks, both treatments resulted in similar HbA1c levels. Bedtime NPH insulin was superior to sitagliptin in reducing fasting plasma glucose and postprandial triglyceride levels. Decreased postprandial glucagon and increased active GLP-1 levels were only observed in the Sitagliptin group. C-peptide levels did not change after treatment with sitagliptin, but decreased following insulin therapy. Both treatments reduced postprandial pro-insulin and fasting ghrelin levels. Neither treatment changed C-reactive protein, cholesterol, uric acid or liver enzymes, except alkaline phosphatase, which decreased in both. Weight, body mass index, waist-hip ratio and systolic and diastolic blood pressures remained unchanged. Cardiac evaluation was performed in 29 patients and showed basal left ventricular diastolic dysfunction, the pre-clinical signal of diabetic cardiomyopathy, in 53% and in 64% of patients in the Sitagliptin and Insulin group, respectively. Left ventricular diastolic function improved in 40% (6/15) of patients receiving sitagliptin and in 7% (1/14) of patients receiving bedtime NPH insulin (p= 0.049). This improvement did not seem to be influenced by glycemic control, blood pressure or inflammatory status. In conclusion, both treatments were effective in reducing HbA1c and seemed to similarly improve b-cell function, as shown by reduced pro-insulin levels. Bedtime NPH insulin was more effective in reducing fasting plasma glucose and postprandial triglyceride levels. However, improvement in left ventricular diastolic function was higher in the Sitagliptin group. Therefore, sitagliptin seems to be a promising drug for the prevention of diabetic cardiomyopathy