Background: Stem cells are a promising therapy for regeneration following myocardial infarction (MI). Another therapy currently under investigation for MI is glucagon-like peptide-1 (GLP-1), a natural incretin hormone that has cardio-protective properties, although a short half-life in vivo. GLP-1 CellBeads are a novel therapy, combining stem cells and GLP-1. Human mesenchymal stem cells (MSCs) were immortalised, engineered to secrete a fusion protein of GLP-1 and encapsulated in alginate. We have previously demonstrated that GLP-1 CellBeads significantly reduce infarct size and improve ejection fraction post-MI, but the underlying mechanisms are unclear. The therapy was assessed in an in vivo pig MI model and an in vitro cardiomyocyte ischaemia model. Methods: GLP-1 CellBeads were delivered to coronary artery branches in pigs, creating micro-infarcts, as determined by echocardiography. Cell-free beads (Beads) and CellBeads containing hMSCs without GLP-1 (Beads-MSC) were delivered as controls (n=3-5/group). Pigs were sacrificed one and four weeks post-MI. Tissue was analysed for: apoptosis, collagen, cardiomyocyte cross sectional area and myofibroblasts. The localised response around the beads was also measured using immunohistochemistry. Atomic force microscopy (AFM) was used to examine the ultra-structure of the collagen scar. The expression profiles of genes involved in collagen remodelling were measured using qRT-PCR. Viability of MSCs was measured using GFP-tagging and confirmed using qRT-PCR. To examine effects on apoptosis in vitro, human adult cardiomyocytes underwent ischaemia for 1 hour before incubation with: media conditioned with MSCs or MSC+GLP-1, GLP-1, Exendin-4 or media. Apoptosis and viability were measured at 24 and 48 hours respectively. Results: In the in vivo pig model, significant increases in apoptosis were observed in the infarct of all groups one week post-MI, with no differences between treatments. Despite decreased numbers of myofibroblasts, significantly more collagen was observed in MSC treated groups, with increased collagen fibril periodicity and a more organised collagen scar. The altered scar structure was reflected in differences in gene expression between groups, with an accelerated healing response in the MSC groups. However, significantly fewer myofibroblasts were observed in the MSC treated groups. Viability of MSCs was confirmed up to four weeks post-infusion, with GLP-1 secretion confirmed up to one week. In the in vitro ischaemia model, MSC+GLP-1 conditioned media significantly reduced cardiomyocyte apoptosis 24 hours post-ischaemia, compared to media alone. All agonists (GLP-1, MSC media and MSC+GLP-1 media) significantly improved viability compared to media alone 48 hours post-ischaemia. Conclusions GLP-1 CellBeads have a beneficial effect on healing following MI by significantly decreasing infarct size and improving ejection fraction post-MI. these benefits are associated with decreased cardiomyocyte apoptosis and altered collagen scar formation. The CellBeads act as local hubs for regeneration and are viable up to one month post-infusion. The effects observed are due to a combination of the GLP-1 and paracrine factors released from the hMSCs.

Le diabète est associé à une réduction de la masse des cellules β. Par conséquent, la régénération des cellules β est un champ d'étude important. Nous avons testé l'hypothèse selon laquelle le GLP-1, un médicament anti-diabète, stimule la differentiation de cellules progénitrices en cellules β matures (néogenèse) et étudié les mécanismes impliqués dans le processus. Nos résultats indiquent que le GLP-1 induit l'expression de marqueurs spécifiques à la cellule β (Pdxl, Glut2 et le gène de l'insuline) dans un modèle de cellules progénitrices (lignée AR42J). Cette action est précédée de l'expression de Ngn3, un gène contrôlant la differentiation des cellules endocrines du pancréas au cours du développement embryonnaire. De plus, notre étude suggère que la voie Pi3k et Hnf1-α/Hnf3-β participent à l'effet du GLP-1. Nos résultats pourraient mener à la découverte de cibles moléculaires permettant l'expansion de la masse de cellules β

Bakgrund: Typ-2 diabetes är en sjukdom som kännetecknas av nedsatt insulinkänslighet (insulinresistens) samtidigt minskar betacellsfunktionen gradvis med fördröjt och otillräckligt insulinsvar på stimuli. Produktionen av insulin i bukspottkörteln räcker inte för kroppens behov. Insulinet håller blodsockerhalten på en jämn nivå och vid brist på insulin blir blodsockerhalten i cellerna för hög. Riskfaktorer är exempelvis livsstilsfaktorer som kost, låg fysisk aktivitet, rökning och stress. Övervikt, hög ålder, hypertoni, dyslipidemi och nedsatt glukostolerans ökar också risken för diabetes. Metabola syndromet ger en ökad risk för sjukdom i kärlen och hjärtat. En vanlig dödsorsak hos diabetiker är  makrovaskulära komplikationer och det gäller framför allt kardiovaskulära komplikationer. Förekomsten är 2–4 gånger högre än hos patienter som inte har diabetes. Typ-2 diabetes behandlas först med kostintervention och ökad fysisk aktivitet eller med antidiabeteka läkemedel. Glukagonlik peptid 1(GLP-1) analoger är en ny läkemedelsgrupp. Behandling med GLP-1 analoger minskar risken för hjärt-kärlsjukdomar exempelvis arterioskleros och hjärtinfarkt genom att reducera flera riskfaktorer som fetma och diabetes.                                                                            Syfte: Examensarbetes syfte var att enligt kliniska studier undersöka om GLP-1 analoger är en säker och effektiv behandling av hjärtsjukdomar hos patienter med typ-2 diabetes.  Metod: Det här arbetet baserat på en undersökning i PubMed databas med sökorden” GLP-1 diabetes type-2 with cardiovascular risk” och därefter valdes fem artiklar som hade mål som var relevanta för arbetet. Resultat: Liraglutid visade en viktminsknings effekt  och förbättring  av HBA1c. Analysen i studien visade signifikant postprandial minskning av triglycerider, och  fastande och postprandiala apoCIII-koncentrationer minskade vilket kan vara kopplat  till minskad glykemi. Patienter som fick dulaglutid  hade en signifikant lägre värden på Body Mass Index, BMI, Pulsvågshastighet PWV, totalt serumkolesterol, LDL-kolesterol diastoliskt blodtryck. Resultat visade att Exenatid hade bättre effekt än  sitagliptin och insulin glargin för att uppnå de sammansatta målen såsom lågdensitetslipoprotein <2,59 mmol/L och systoliskt blodtryck <130 mm Hg. Resultatet visade en konsekvent minskning av kardiokomplikationer utan tecken på signifikant relation till blodtrycks-mätning eller stadium av hypertoni. Slutsats: GLP-1 analoger har visat hjärtskyddande effekt vid behandling av typ-2 diabetiker genom förbättring av flera faktorer som minskar den kardiovaskulära risken. Faktorer som viktminskning, prevalens av hypoglykemi, hypertoni och dyslipidemi spelar avgörande roll för att minska risk för hjärt-kärlsjukdom hos typ-2 diabetiker.

Glycemic control in patients with type 2 diabetes (T2D) can be achieved through potentiation of the signalling by glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Both incretin hormones have been traditionally characterized to be secreted by distinct enteroendocrine cells within the gut in response to nutrients. Signalling through the incretin receptors stimulates islet hormone release by potentiating glucose-stimulated insulin secretion from the β-cell and decreasing glucagon secretion from the α-cell. However, the bioactivity of GLP-1 and GIP is controlled by post-translational, N-terminal cleavage by the widely expressed serine protease dipeptidyl peptidase 4 (DPP4). As such, DPP4 inhibitors (DPP4i) have been successfully used to treat millions of patients with T2D. DPP4i target the catalytic active site of DPP4 and prevent the cleavage of the incretin hormones, thus prolonging their action. Recently, studies in genetically modified mice have demonstrated that GLP-1 is not solely an intestinally-derived peptide hormone and proposed that islet-derived GLP-1 is required for proper glucose homeostasis. Therefore, with the current study, we sought to assess whether β-cell-derived DPP4 is an important target for the regulation of glycemia. Treatment of Glp1r/Gipr^(β-cell-/-) mice with the DPP4 inhibitor sitagliptin demonstrated that β-cell incretin receptor signaling is required to mediate the beneficial effects of this class of drugs on glucose homeostasis. Additionally, Dpp4^(-/-) mice exhibited a significant reduction in hepatic glucose production during hyperinsulinemic-euglycemic clamps. Dpp4 mRNA, DPP4 protein and activity are present in isolated mouse islets, further supporting the islet as an important potential site of DPP4i action. In this study, we show that both DPP4i-treated wildtype islets and islets isolated from Dpp4^(β-cell-/-) mice exhibit increased glucose-stimulated insulin secretion (GSIS) during perifusion after a high-fat diet feeding. Genetic elimination of Dpp4 from islet β-cells also improved oral glucose tolerance and insulin sensitivity in female mice, but had no effects on circulating DPP4 or incretin levels. Finally, eliminating Dpp4 from β-cells or the whole pancreas did not improve whole-body glucose tolerance, response to DPP4i, insulin tolerance, or body weight in male mice fed chow or a high-fat diet. Therefore, we provide evidence for islet-derived DPP4 to have a role in local hormone responses to glucose; however, its role in systemic glucose metabolism is shown to be sex-dependent.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第18366号 / 人博第679号 / 新制||人||163(附属図書館) / 25||人博||679(吉田南総合図書館) / 31224 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦, 教授 津田 謹輔 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

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Type 2 diabetes (T2D) is a chronic metabolic disease characterized by hyperglycemia, insulin resistance, and the dysfunction of β-cells. While there are several therapies for T2D, there is no effective treatment that can reverse the functional decline of pancreatic β cells in T2D patients. Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by human intestinal L cells, which can stimulate the proliferation and differentiation of β cells and promote glucose-stimulated insulin secretion (GSIS), thereby playing a critical role in maintaining glycemic homeostasis. Recently, GLP-1-based medications have been developed for treating T2D. However, most of the GLP-1-based drugs are expensive and have significant adverse effects. Therefore, development of safer and more convenient agents that can mimic the physiologically fed state to promote endogenous GLP-1 secretory function of intestinal L-cells to improve glucose homeostasis holds great potential for the prevention and treatment of T2D. This project aimed to examine whether natural compound genipin promotes intestinal GLP-1 secretion and exerts anti-diabetic effects. I found that genipin rapidly increased GLP-1 secretion from intestinal L-cells, with 10 and 100 μM concentration inducing significant incretin hormone release. L-cells exposed to genipin displayed a rapid increase in intracellular [Ca²⁺]i and the activity of phospholipase C (PLC). Inhibition of PLC ablated genipin-stimulated Ca²⁺] increase and GLP-1 secretion, suggesting that genipin-induced GLP-1 release from the cells depends on the PLC/Ca²⁺ pathway. In vivo, genipin reduced the non-fasting and fasting blood glucose levels, improved insulin resistance, and protected again high fat diet-induced liver damage. All together, these data indicate that genipin is a naturally occurring anti-diabetic agent, which could be a pharmaceutical lead for developing anti-diabetic drugs. / M.S. / More than 34 million Americans are suffering from diabetes, with over 90% of these cases being type 2 diabetes (T2D). Loss of β-cell mass and function is central to the deterioration of glycemic control over time in T2D. Therefore, preservation or improvement of β-cell mass and its insulin secretory function could prevent and treat T2D. While there are several pharmaceutical therapies for T2D, no effective treatment is available for reversing functional decline of pancreatic β-cells in T2D patients. It has been well recognized that glucagon-like peptide-1 (GLP-1), which is an incretin hormone secreted from intestinal L-cells, plays a critical role in maintaining glycemic homeostasis via potentiating glucose-stimulated insulin secretion and promoting β-cell proliferation. This present work is to determine whether natural compound genipin promotes intestinal GLP-1 secretion and thus exerts anti-diabetic effect.

Type 2 diabetes (T2D) is one of the fasting growing chronic diseases, caused by insulin resistance and pancreatic β-cell dysfunction. While over thirty medications were approved to treat T2D in the United States, less than one in four patients treated with anti-diabetic drugs achieved the glycemic target. Thus, identifying more effective anti-diabetic drugs is still needed for improving glycemic control in T2D patients. Incretins are gut hormones that possess potent insulinotropic action, which have drawn considerable attention in research and developing treatment strategy for T2D. Specifically, glucagon like peptide 1 (GLP-1), the most important incretin that is secreted from enteroendocrine L-cells in response to food ingestion, plays a vital role in maintaining glycemic homeostasis via potentiating glucose stimulated insulin secretion (GSIS) and promoting pancreatic β-cell proliferation and survival. Therefore, targeting L-cells to induce GLP-1 secretion would be an alternative strategy for treating T2D. The goal of this research was to identify low-cost and safe naturally occurring agents as a primary or adjuvant treatment for T2D. Here, I found that a small molecule, elenolic acid (EA), which was generated in our lab but is also present in mature olive and extra virgin olive oil, dose-dependently stimulated GLP-1 secretion in mouse clonal L-cells and isolated mouse ileum crypts. EA induced a rapid increase in intracellular [Ca2+]i and the production of inositol trisphosphate in L-cells, indicating that EA activates phospholipase C (PLC)-mediated signaling. Consistently, inhibition of (PLC) ablated EA-stimulated increase of [Ca2+]i and GLP-1 secretion in L-cells. In addition, EA-triggered GLP-1 secretion from L-cells was blocked by YM-254890, a Gαq inhibitor. Consistent with our in vitro study, a single dose of EA acutely stimulated GLP-1 secretion in mice, accompanied with an improved oral glucose tolerance. Chronic administration of EA restored the impaired glucose and lipid homeostasis in DIO mice, which may be partially due to promoting GLP-1 secretion and reduced hepatic gluconeogenesis. In addition, EA suppressed appetite, reduced food intake and gastric emptying rate, as well as promoted weight loss in obese mice, demonstrating that it is also an anti-obesity agent. Further, EA treatment reduced lipid absorption, and promoted hepatic fatty acid oxidation, and reversed abnormal plasma lipid profiles in DIO mice. Consistently, EA exerted potent anti-diabetic action in db/db mice, and its blood glucose-lowering effect is comparable with that of liraglutide in blood glycemic control but is better than that of metformin in this overt diabetic model. Collectively, I have identified for the first time, as to the best of our knowledge, that EA could be a dual-action compound that exerts anti-diabetic effects via activation of the GLP-1 mediated metabolic pathway and suppression of hepatic gluconeogenesis, leading to effective control on food intake, body weight gain, and glycemia in T2D mice. / Doctor of Philosophy / Type 2 diabetes (T2D) is one of the fasting growing chronic diseases, which results from insulin resistance and pancreatic β-cell dysfunction. Even though there have been over thirty drugs approved to treat T2D in the United States, less than 25% of patients treated with anti-diabetic drugs achieved the glycemic target. Thus, more effective anti-diabetic drugs are still needed for improving glycemic control in patients with T2D. Incretins are a group of gut hormones and responsible for over 50% postprandial insulin secretion in humans, which have drawn considerable attention in research and developing a treatment strategy for T2D. Specifically, glucagon-like peptide 1 (GLP-1), the most important incretin that is secreted from enteroendocrine L-cells in response to food ingestion, plays a vital role in controlling blood glucose via potentiating glucose-stimulated insulin secretion (GSIS) and promoting pancreatic β-cell proliferation and survival. Therefore, targeting L-cells to induce GLP-1 secretion would be an alternative strategy for treating T2D. The goal of this research was to identify low-cost and safe naturally occurring agents as a primary or adjuvant treatment for T2D. Here, I found that a small molecule, elenolic acid (EA), which was synthesized in our lab but is also present in mature olive and extra virgin olive oil, dose-dependently stimulated GLP-1 secretion in mouse clonal L-cells and isolated mouse ileum crypts (containing L-cells). Further experiments showed that EA induced a rapid increase in intracellular [Ca2+]i and the production of inositol trisphosphate (IP3) in L-cells, indicating that EA activates phospholipase C (PLC)-mediated signaling, as IP3 is a direct product of PLC. Consistently, inhibition of PLC ablated EA-stimulated increase of [Ca2+]i and GLP-1 secretion in L-cells. In addition, EA-triggered GLP-1 secretion from L-cells was blocked by YM-254890, a Gαq inhibitor. In line with the in vitro study, a single dose of EA acutely elevated plasma GLP-1 concentration in mice, accompanied by improved oral glucose tolerance. Chronic administration of EA restored the impaired glucose and lipid homeostasis in diet-induced obese (DIO) mice, which may be partially due to promoting GLP-1 secretion and reduced hepatic gluconeogenesis. In addition, EA suppressed appetite, reduced food intake, and gastric emptying rate, as well as promoted weight loss in the DIO mice, demonstrating that it is also an anti-obesity agent. Further, EA treatment reduced lipid absorption and promoted hepatic fatty acid oxidation, as well as reversed abnormal plasma lipid profiles in the DIO mice. Consistently, EA exerted potent anti-diabetic action in predisposed diabetic mice (db/db), and its blood glucose-lowering effect is comparable with that of liraglutide, a commercial GLP-1 receptor agonist, in blood glycemic control but is better than that of metformin, a widely used first-line anti-diabetic drug, in this overt diabetic model. Collectively, I have identified for the first time, as to the best of our knowledge, that EA could be a dual-action compound that exerts anti-diabetic effects via activation of the GLP-1 mediated metabolic pathway and suppression of hepatic gluconeogenesis, leading to effective control on food intake, body weight gain, and glycemia in T2D mice.

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.