Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 22, 2007). Includes bibliographical references (p. 92-107).

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.

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.

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.

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

Background: Glucagon-like Peptide 1 (GLP-1) is a human incretin hormone that has been demonstrated to protect against non-lethal ischaemia reperfusion injury in the left ventricle in humans. It has been suggested from some animal research that this protection may be mediated through the pathway of ischaemic conditioning, of which the opening of the mKATP channel is a key step. Furthermore, it is uncertain whether the protection applies to the right ventricle. Finally, there is limited human evidence of a protective effect against lethal ischaemia reperfusion injury. Methods: Two studies use non-lethal ischaemia to test whether GLP-1 protection is maintained despite blockade of the mKATP channel with the sulfonylurea, glibenclamide. A demand ischaemia study uses dobutamine stress echo to compare LV function. The other uses transient coronary balloon occlusion to generate supply ischaemia during GLP-1 infusion, assessed by conductance catheter. A further transient balloon occlusion is also used to assess the effect of supply ischaemia on RV function. Finally, the GOLD PCI study assesses whether GLP-1 protects against periprocedural myocardial infarction when administered during elective PCI in a randomised, placebo controlled double blind trial. Results: Glibenclamide did not affect GLP-1 cardioprotection in either supply of demand ischaemia suggesting that GLP-1 protection is not mediated through the mKATP channel. The RV experienced stunning with RCA balloon occlusion but there was little evidence of cumulative ischaemic dysfunction with further occlusions. GOLD PCI is continuing to recruit patients. The nature of the study means results cannot be assessed until recruitment is complete. Conclusions: GLP-1 is an agent with potential for clinical use as a cardioprotective therapy. It’s mechanism of action in the heart remains uncertain.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22149号 / 医博第4540号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 上本 伸二, 教授 富樫 かおり / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM