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.

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.

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.

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