Glucagon-like peptide-1 (GLP-1) is an anti-diabetic intestinal L cell hormone. The monounsaturated fatty acid, oleic acid (OA), is an effective GLP-1 secretagogue that crosses the cell membrane by an unknown mechanism. Immunoblotting demonstrated the presence of fatty acid transport proteins (CD36 and FATP1, 3 and 4) in the murine GLUTag L cell model. The cells demonstrated specific 3H-OA uptake, which was dose-dependently inhibited by unlabeled-OA. Phloretin and SSO, inhibitors of carrier-mediated transport and CD36, respectively, also significantly decreased 3H-OA uptake, as did knocking down FATP4 by transfection of siRNA. OA dose-dependently increased GLP-1 secretion in GLUTag cells, while phloretin and FATP4 knockdown, but not SSO, decreased this response. OA injected directly into the ileum of wild-type mice increased plasma GLP-1 levels; in contrast, preliminary findings suggest decreased GLP-1 levels in FATP4 null mice at 60 min. Collectively, these findings indicate a role for FATP4 in OA-induced GLP-1 secretion.

The gut hormone glucagon-like peptide-2 (GLP-2) has numerous beneficial effects on the intestinal epithelium, including increased mucosal growth and proliferation. GLP-2 is also necessary for the adaptive intestinal re-growth that occurs upon re-feeding after fasting. Although insulin-like growth factor (IGF)-1 and the IGF-1 receptor are known to be required for GLP-2-induced crypt-cell proliferation, the precise cellular localization of the IGF-1 receptor through which the intestinotrophic actions of GLP-2 are mediated remains unknown. I hypothesized that small intestinal growth responses to GLP-2 occur through an intestinal epithelial IGF-1 receptor-dependent pathway, through the use of an inducible, intestinal epithelial-specific IGF-1 receptor knockout (IE-igf1rKO) mouse. Intestinal growth and proliferative responses were examined in IE-igf1rKO and control mice following treatment with GLP-2, as well as in animals that were fasted and re-fed to induce GLP-2-dependent adaptation. In Chapter 3, it was demonstrated that IE-igf1rKO mice, as compared to control littermates, had normal small intestinal weight, morphometric parameters, proliferative index and differentiated epithelial cell lineage distribution. Administration of GLP-2 for 30 minutes increased nuclear translocation of !-catenin in non-Paneth crypt-cells, and stimulated the crypt-cell proliferative marker c-Myc 90 minutes following GLP-2 treatment, in control littermates but not in IE-igf1rKO mice. In Chapter 4, adaptive re-growth was studied by fasting IE-igf1rKO and control animals for 24 hours, or by fasting and then re-feeding mice for 24 hours. Small intestinal weight, crypt depth, villus height and crypt-cell proliferation were decreased in both control and IE-igf1rKO mice after 24 hour fasting. While re-feeding in control mice restored all of these parameters, re-fed IE-igf1rKO mice displayed abrogated adaptive re-growth of the crypt-villus axis as well as reduced crypt-cell proliferation. In Chapter 5, control mice responded to chronic GLP-2 with increased small intestinal weight, mucosal cross-sectional area, crypt depth, villus height and crypt-cell proliferation. However, the GLP-2-induced increase in crypt-cell proliferation was absent in IE-igf1rKO mice, in association with impaired growth of the crypt-villus axis. Taken together, these results indicate that the proliferative responses of the intestinal epithelium to exogenous GLP-2 administration and during conditions of GLP-2-dependent adaptive re-growth are dependent on the intestinal epithelial IGF-1 receptor.

Glucagon-like peptide-1 (GLP-1) is secreted from the enteroendocrine L cell following nutrient ingestion. Although GLP-1 regulates several aspects of nutrient homeostasis, one important function is to enhance glucose-dependent insulin secretion. In type 2 diabetes, post-prandial GLP-1 secretion is impaired. Insulin resistance, which is required for the pathogenesis of type 2 diabetes, is also associated with impaired GLP-1 secretion. I, therefore, hypothesized that insulin modulates GLP-1 secretion from the intestinal L cell and, furthermore, insulin resistance directly impairs the function of the endocrine L cell. In well-characterized L cell models, I established that insulin stimulates GLP-1 secretion through the MEK1/2-ERK1/2 pathway, and induction of insulin resistance in vitro attenuated insulin- and heterologous secretagogue-induced GLP-1 release. Furthermore, glucose-stimulated GLP-1 secretion was decreased in hyperinsulinemic-insulin resistant MKR mice, demonstrating that insulin resistance is associated with impaired L cell function. I next examined the role of the actin cytoskeleton in insulin-stimulated GLP-1 secretion. Insulin treatment transiently induced actin depolymerization, and depolymerization of the actin cytoskeleton potentiated insulin-stimulated GLP-1 release from the L cell, demonstrating that the cytoskeleton functions as a permissive barrier. Central to insulin’s effects on actin dynamics is the Rho GTPase, Cdc42, as siRNA-mediated knockdown and over-expression of a dominant-negative mutant, prevented insulin-stimulated actin remodeling and GLP-1 release. Insulin also promoted activation of PAK1, the downstream kinase of Cdc42, and over-expression of a kinase-dead PAK1 mutant attenuated insulin-stimulated GLP-1 release. In cells that expressed dominant-negative Cdc42 or kinase-dead PAK1, activation of ERK1/2 following insulin treatment was attenuated, demonstrating that the Cdc42-PAK1 axis regulates the activity of the canonical ERK1/2 pathway. In summary, this thesis demonstrates, for the first time, that insulin is a GLP-1 secretagogue, and this effect of insulin is mediated through the canonical ERK1/2 pathway and the Cdc42-PAK1 axis. Insulin resistance in the L cell impairs the responsiveness of the L cell to heterologous secretagogues. Collectively, these findings suggest that an alternative approach to treat type 2 diabetes and/or insulin resistance may be to directly improve the function of the L cell, thereby enhancing endogenous GLP-1 release.

The gut hormone glucagon-like peptide-2 (GLP-2) has numerous beneficial effects on the intestinal epithelium, including increased mucosal growth and proliferation. GLP-2 is also necessary for the adaptive intestinal re-growth that occurs upon re-feeding after fasting. Although insulin-like growth factor (IGF)-1 and the IGF-1 receptor are known to be required for GLP-2-induced crypt-cell proliferation, the precise cellular localization of the IGF-1 receptor through which the intestinotrophic actions of GLP-2 are mediated remains unknown. I hypothesized that small intestinal growth responses to GLP-2 occur through an intestinal epithelial IGF-1 receptor-dependent pathway, through the use of an inducible, intestinal epithelial-specific IGF-1 receptor knockout (IE-igf1rKO) mouse. Intestinal growth and proliferative responses were examined in IE-igf1rKO and control mice following treatment with GLP-2, as well as in animals that were fasted and re-fed to induce GLP-2-dependent adaptation. In Chapter 3, it was demonstrated that IE-igf1rKO mice, as compared to control littermates, had normal small intestinal weight, morphometric parameters, proliferative index and differentiated epithelial cell lineage distribution. Administration of GLP-2 for 30 minutes increased nuclear translocation of !-catenin in non-Paneth crypt-cells, and stimulated the crypt-cell proliferative marker c-Myc 90 minutes following GLP-2 treatment, in control littermates but not in IE-igf1rKO mice. In Chapter 4, adaptive re-growth was studied by fasting IE-igf1rKO and control animals for 24 hours, or by fasting and then re-feeding mice for 24 hours. Small intestinal weight, crypt depth, villus height and crypt-cell proliferation were decreased in both control and IE-igf1rKO mice after 24 hour fasting. While re-feeding in control mice restored all of these parameters, re-fed IE-igf1rKO mice displayed abrogated adaptive re-growth of the crypt-villus axis as well as reduced crypt-cell proliferation. In Chapter 5, control mice responded to chronic GLP-2 with increased small intestinal weight, mucosal cross-sectional area, crypt depth, villus height and crypt-cell proliferation. However, the GLP-2-induced increase in crypt-cell proliferation was absent in IE-igf1rKO mice, in association with impaired growth of the crypt-villus axis. Taken together, these results indicate that the proliferative responses of the intestinal epithelium to exogenous GLP-2 administration and during conditions of GLP-2-dependent adaptive re-growth are dependent on the intestinal epithelial IGF-1 receptor.

Les travaux de Melançon (Melançon 2005) ont montré que le récepteur au glucagon (RG) peut être modulé rapidement par 180 minutes d'exercice ou 24 heures de jeûne. Cette modulation provient en partie de la hausse de la densité des récepteurs au glucagon membranaire. Plus récemment, les travaux de Merlen (Merlen, Fabrega et al., 2006) ont montré l'endocytose du récepteur au glucagon suite à un bolus de glucagon. Le présent projet vise à établir une cinétique de protéines reliées à la signalisation et à l'internalisation du RG. Spécifiquement, nous avons effectué une cinétique sur 24 heures suivant un exercice intense et un jeûne prolongé pour analyser par immunobuvardage l'AMPK, la LKB1, la GRK, la PKC et la β-arrestine. Des rats mâles Sprague-Dawley ont été aléatoirement assignés à des groupes contrôle, d'exercice (90 min et 180 min d'exercice, 180 min + 1 h, + 3 h, + 12 h et + 24 h de récupération au repos post-exercice) ou de jeûne (6 h, 12 h et 24 h de jeûne, 24 h jeûne + 6 h, + 12 h et + 24 h de récupération nourrie post-jeûne). Aucun changement statistiquement significatif n'est observé au niveau de la glycémie. Cependant, les niveaux de glycogène hépatique sont grandement diminués dans les deux cas, 98% de déplétion après 24 heures de jeûne et 74% après 3 heures d'exercice en comparaison à leur groupe contrôle respectif. Une période d'exercice induit, chez des rats sains, une augmentation significative de 244% des récepteurs membranaires au glucagon alors qu'il n'y a aucune variation significative de l'expression des récepteurs totaux. Ces données permettent de formuler l'hypothèse d'un mécanisme de sensibilisation/désensibilisation des récepteurs au glucagon expliquant cette grande mobilité lors de l'exercice. Le mécanisme de sensibilisation apparait en même temps qu'une augmentation des quantités d'AMPK et de GRK de l'ordre de 183 et 158% respectivement. Le mécanisme de désensibilisation en récupération s'opère simultanément avec une augmentation de l'expression de PKC de 207% et une diminution d'AMPK de l'ordre de 60%. Une période de jeûne prolongé induit chez des rats sains une augmentation significative de 165% des récepteurs membranaires au glucagon. Cette augmentation n'est accompagnée d'aucun changement significatif dans l'expression des protéines étudiées ouvrant la voie à la synthèse de l'ARNm pour contribuer à l'augmentation des RG membranaires lors du jeûne. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Récepteur au glucagon, AMPK, LKB1, GRK, PKC, β-arrestine, jeûne, exercice

Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists would reduce the development of atherosclerosis in diabetic Apoe-/- mice. Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85), and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks (0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless, taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or macrophage infiltration (P=0.45, sinus; P=0.26, arch). Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not significantly modify the development of atherosclerosis.

No description available.

Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists would reduce the development of atherosclerosis in diabetic Apoe-/- mice. Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85), and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks (0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless, taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or macrophage infiltration (P=0.45, sinus; P=0.26, arch). Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not significantly modify the development of atherosclerosis.

<p>Peptide drugs are an exciting class of pharmaceuticals currently in development for the treatment of a variety of diseases; however, their main drawback is a short half-life, which dictates multiple and frequent injections. We have developed two novel peptide delivery approaches -Protease Operated Depots (PODs) and GLP-1-ELP depots- to provide sustained and tunable release of a peptide drug from an injectable s.c. depot. </p><p>We demonstrate proof-of-concept of these delivery systems, by fusion of monomer or protease cleavable oligomers of glucagon-like peptide-1 (GLP-1), a type-2 diabetes peptide drug, and a thermally responsive, depot-forming elastin-like-polypeptide (ELP) that undergoes thermally triggered inverse phase transition below body temperature, thereby forming an injectable depot. Utilizing a novel system we designed for repetitive gene synthesis, various GLP-1 polymers were designed and tested as potential therapeutic payload for PODs. By attachment to various ELPs, designed to transition above or below body temperature, we created both depot forming GLP-ELP fusions and soluble control. All fusion constructs maintained alpha helical content and were shown to be resistant to proteolytic degradation. In vitro activated PODs and GLP-ELP fusions were able to activate the GLP-1 receptor and remarkably, a single injection of both GLP-1 PODs and GLP-ELP fusions were able to reduce blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide drug. These findings suggest that ELP based peptide depots may offer a modular, genetically encoded alternative to various synthetic peptide delivery schemes for sustained delivery of peptide therapeutics.</p> / Dissertation

Glucose metabolism in pancreatic α- and β-cells is believed to regulate secretion of glucagon and insulin, respectively. In β-cells, ATP links glucose metabolism to electrical activity and insulin secretion. In α-cells, ATP has been attributed various roles in glucose-regulated glucagon release, but the underlying mechanisms are poorly understood. Despite its importance in insulin and glucagon secretion little is known about ATP kinetics in α- and β-cells. In this thesis, the novel fluorescent ATP biosensor Perceval was used to monitor physiologically relevant ATP concentrations with little influence of ADP. Glucose stimulation of β-cells within mouse and human pancreatic islets induced pronounced rise of ATP with superimposed oscillations. Simultaneous measurements of the sub-plasma membrane ATP and Ca2+ concentrations revealed glucose-induced oscillations in opposite phase. ATP increased further and the oscillations ceased when voltage-dependent Ca2+ influx was prevented. In contrast, ATP promptly decreased in response to K+-depolarization-induced elevation of Ca2+. Also mobilization of Ca2+ from intracellular stores lowered ATP, but the negative effect was not due to increased ATP consumption by the sarco/endoplasmic reticulum Ca2+-ATPase. Store-operated Ca2+ entry alone had little effect but markedly elevated ATP when combined with muscarinic receptor activation. When comparing ATP and Ca2+ responses in α- and β-cells within the same islet, glucose-induced ATP generation was much less pronounced and the dose-response relationship left-shifted in the α-cells. At basal glucose, individual α-cells showed Ca2+ and concomitant ATP oscillations in opposite-phase with variable frequency. These oscillations largely cancelled out when averaging data from several α-cells. At high glucose, the Ca2+ and ATP oscillations in α-cells tended to synchronize with the corresponding β-cell oscillations. Since β-cell Ca2+ oscillations drive pulsatile insulin secretion, which is antiparallel to pulsatile glucagon secretion, there seems to be an inverse relationship between changes in α-cell Ca2+ and glucagon release. This paradox is attributed to paracrine inhibition overriding Ca2+ stimulation, since somatostatin receptor blockade potently stimulated glucagon release with little effect on α-cell Ca2+ signalling. The data indicate that complex ATP-Ca2+ interactions in α- and β-cells underlie cell-intrinsic regulation of glucagon and insulin secretion and that paracrine inhibition of glucagon release becomes important in hyperglycaemia.