Type 1 diabetes mellitus (T1D) is an autoimmune disorder that targets the insulin-producing β-cells. The gut may play a role in the pathogenesis of T1D, as genetically-susceptible individuals and animal models of T1D exhibit increased intestinal permeability and improving gut barrier function can interfere with the onset of diabetes. Moreover gut-derived peptides are capable of modifying barrier function and regulate β-cell mass via effects on proliferation and apoptosis. I tested whether chronic administration of glucagon-like peptide-2 (GLP-2), a peptide which potently improves gut barrier function, modifies diabetes onset in a mouse model of T1D, the non obese diabetic (NOD) mouse. Although chronic treatment with a long-acting GLP-2 analogue was associated with improved intestinal barrier function, it failed to delay the onset of T1D. Once the autoimmune attack is initiated, pathogenic T-cells infiltrate the islets and trigger the death of β-cells. Studies in animal models have revealed that β-cells exhibit a compensatory response in the initial stages of the immune attack, which eventually fails, resulting in β-cell mass deficiency and onset of T1D. Glucagon-like peptide-1 (GLP-1) exerts both proliferative and anti-apoptotic actions on β-cells. I hypothesized that chronic activation of the GLP-1 receptor (GLP-1R) would delay or prevent the loss of functional β-cell mass in the NOD mouse. I have shown that chronic administration of the GLP-1R agonist exendin-4 significantly delayed the onset of diabetes and enhanced β-cell mass. Furthermore, GLP-1R activation was associated with a reduction of islet-infiltrating immune cells, as well as changes in lymphocyte subpopulations. Consequently, I addressed whether the GLP-1R has a role in the immune system of NOD and C57Bl/6 mice. GLP-1R mRNA transcripts were detectable in several immune subpopulations, and GLP-1R activation was associated with cAMP production in primary splenocytes and thymocytes. Furthermore I demonstrated that GLP-1R signaling controls proliferation of thymocytes and lymphocytes, and is required for maintaining peripheral regulatory T-cells. In summary, these studies establish that while GLP-2R activation is not sufficient to modify disease onset in a murine model of T1D, GLP-1R activation reduces the extent of diabetes development by exerting actions on β-cells and the immune system.

Diabetes Mellitus (DM) is a metabolic disorder in which the body fails to achieve glucose homeostasis, due to either insulin resistance or reduced insulin secretion or both. This inadequate glucose control leads to hyperglycemia which, if left unchecked, leads to secondary complications like nephropathy, neuropathy, retinal degeneration and other serious conditions. In non-disease state, normal glucose level in the blood is maintained by pancreatic β-cells, which secrete insulin. However, during diabetes development, there is loss of β-cell mass and function; resulting in decreased insulin secretion which is the ultimate cause of hyperglycemia. The ability to non-invasively monitor changes in the β-cell mass during the development or treatment of diabetes would be a significant advance in diabetes management. However, a primary limitation for analysis of β-cell mass and developing dysfunction is the lack of specificity of β-cell targeting agents. Our novel approach for achieving the required specificity for a usable β-cell targeted contrast agent is to target a set of receptors on the cell surface that, as a combination, are unique to that cell. Through genetic screening, Glucagon Like Peptide-1 Receptor (GLP-1R) and α2Adrenergic Receptor (α2AR) were chosen as a potential molecular barcode for β-cells since their combination expression is relatively unique to the β-cells. GLP-1R and α2AR are both G-protein couple receptors (GPCRs) that, apart from being a β-cell specific combination, play an important role in regulating fundamental downstream signaling pathways in β-cells. To target these receptors effectively, we synthesized a multivalent ligand composed of Yohimbine (Yhb), an α2 adrenergic receptor (α2AR) antagonist, linked to an active Glucagon-like Peptide 1 analog (GLP-1₇₋₃₆). In this manuscript, I describe the synthesis and characterization of binding selectivity and signaling ability of GLP-1/Yhb at the cellular level. Using high throughput binding assays, we observed high affinity binding of GLP-1/Yhb to βTC3 cells, a β-cell mimetic line expressing both receptors, at a Kd of ~3 nM. Using microscopy, we observed significant Cy5-tagged GLP-1/Yhb binding and rapid internalization in cells expressing the complementary receptor pair at low concentrations, as low as 1 nM and 5 nM. When one of the receptors was made inaccessible due to presence of saturating quantities of a single unlabeled monomer, GLP-1/Yhb-Cy5 failed to bind to the cells at low concentrations (<10 nM). Similarly, in cells where either GLP-1R or α2AR were knocked down (using shRNA), binding of GLP-1/Yhb was significantly reduced (≤half of cells with both receptors), indicating strong selectivity of the ligand to cells expressing the combination of receptors. We also observed that GLP-1/Yhb construct modulates downstream signaling inβ TC3 cells resulting in enhanced Glucose Stimulated Insulin Secretion (GSIS). In presence of stimulatory glucose, GLP-1/Yhb significantly potentiated GSIS with a half-maximal effective dose of 2.6 nM. Compared to GLP-1₇₋₃₆ alone or GLP-1₇₋₃₆ and Yhb monomers added together, only GLP-1/Yhb could significantly potentiate GSIS at 1 nM, demonstrating that GLP-1/Yhb could translate high affinity binding to increased efficacy for GSIS potentiation. Unlike for insulin secretion, high affinity divalent binding did not translate to increased cAMP production at low concentrations, with significant increases above baseline seen only at 10 nM and higher. Nevertheless, these data show that GLP-1/Yhb binds selectively to β-cells and affects signaling, demonstrating its potential for targeted β-cell imaging and therapy. Overall, our work indicates that synthetic heterobivalent ligands, such as GLP-1/Yhb can be developed to increase cellular specificity and sensitivity making them a strong candidate for both noninvasive imaging and targeted therapy.

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.

Introduction: Stroke is caused from the occlusion of any cerebral artery leading to cerebral ischemia, brain damage and consequent neurological impairments and disability. The primary causes of mortality in western populations is stroke. Diabetes type 2 is a high risk factor for stroke. Stroke leads to an observable increase of neural stem cell proliferation in the subventricular zone and enhances neurogenesis in the adult rodent and human brain which suggest a mechanism contributing to stroke recovery. Neurogenesis in type 2 diabetes patients is impaired. However, whether stroke-induced neurogenesis is impaired in diabetes has not been studied. Exendin-4 is a drug for clinical treatment of type 2 diabetes which has been shown to have neuroprotective properties in animal studies. However whether Exendine-4 leads to increased neurogenesis after  stroke in the diabetic brain has not been previously studied.  Aims: The specific aims of this project were to determine whether stroke-induced stem cell proliferation is impacted by diabetes in the mouse, and if Exendine-4 regulates stroke-induced stem cell proliferation in normal and diabetic mice. Material and Methods: Aged obese/type 2 diabetic mice were subjected to stroke. The Exendin-4 treatment was started 1.5 hours thereafter. Treatment was continued for one week before animals were sacrificed. Brains were isolated and the neurons were immunostained using the specific proliferation marker Ki67. Neural stem cell proliferation was quantified by counting Ki67+ cells in the ipsilateral (subventricular zone in stroke hemisphere).The estimation was assessed by stereological counts of proliferating stem cell in the subventricular zone.  Results: The number of proliferating stem cell after stroke was statistically significantly higher in the normal mice versus diabetic mice. The effect was present in both sides (control and stroke) of the subventricular zone. Exendine-4 treatment induced statistically significant increased of  stem cell proliferation in normal mice but not in diabetic mice.   Conclusions: The result of this study shows that type 2 diabetes decreased the proliferation of neural stem cell in the subventricular zone and that Exendin-4 enhanced the subventricular proliferation in a preclinical model of clinical relevance. The data suggest that the Exendin-4 treatment could be administered to normal patients suffering from stroke in the ambulance or in the emergency room although more studies are needed.

A scourge of Type I and Type II diabetes impacts the health of hundreds of millions worldwide. The number and prevalence of diabetics are expected to rise dramatically in the next two decades. Diabetes is defined by chronic hyperglycemia which can result in a number of detrimental and costly metabolic, renal, cardiovascular and neurological disorders. Identification of at risk individuals and effective blood glucose management are critical to improving diabetic outcomes and preventing hyperglycemic complications. Diabetes prevention and treatment is limited by the understanding of islet function and mass in the diabetogenic and diabetic state. The islets of Langerhans are dispersed throughout the pancreas and comprise <2% of the pancreatic mass. The reclusive nature of islet cells presents unique challenges understanding disease development. No agent capable of exclusively targeting pancreatic β-cells within the islet has been discovered and the lack of targeting agent specificity impedes efforts to: quantify β-cell mass and develop novel therapeutics. We propose β-cell targeting can be improved by targeting unique combinations of receptors simultaneously with multivalent ligands. A synthetic multivalent agent composed of two β-cell specific diabetic therapeutics, glucagon-like peptide-1 (GLP-1) and glibenclamide (Glb), targeted against the GLP-1R and the sulfonylurea-1 receptor (SUR1) is a lead compound for the development of specific bi-functional islet cell targeting agents for use in the in vivo detection and treatment of β -cells. Herein, we describe the synthesis and initial characterization of a heterobivalent ligand composed of GLP-1 coupled to Glb. The heterobivalent ligand binds to an unaltered β-cell line with increased specificity relative to a human pancreatic exocrine cell line. Additionally, receptor cross-linking modifies β-cell signaling. Exposure of β-cells to the heterobivalent ligand results in antagonism of SUR1-Ca²⁺ signaling and equipotent agonism of GLP-1R-cAMP signaling, in comparison to the cognate monomeric ligands (Glb and GLP-1). Perturbations in intracellular signaling modifies β-cell insulin secretion resulting in decreased basal insulin secretion and with maintained yet reduced ability to potentiate β-cell glucose stimulated insulin secretion. GLP-1/Glb β-cell specificity and functional modulation suggests combinatorial receptor targeting is an effective strategy for the development of bi-functional cell-specific targeting agents, warranting further investigation and optimization.

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.

Type 1 diabetes mellitus (T1D) is an autoimmune disorder that targets the insulin-producing β-cells. The gut may play a role in the pathogenesis of T1D, as genetically-susceptible individuals and animal models of T1D exhibit increased intestinal permeability and improving gut barrier function can interfere with the onset of diabetes. Moreover gut-derived peptides are capable of modifying barrier function and regulate β-cell mass via effects on proliferation and apoptosis. I tested whether chronic administration of glucagon-like peptide-2 (GLP-2), a peptide which potently improves gut barrier function, modifies diabetes onset in a mouse model of T1D, the non obese diabetic (NOD) mouse. Although chronic treatment with a long-acting GLP-2 analogue was associated with improved intestinal barrier function, it failed to delay the onset of T1D. Once the autoimmune attack is initiated, pathogenic T-cells infiltrate the islets and trigger the death of β-cells. Studies in animal models have revealed that β-cells exhibit a compensatory response in the initial stages of the immune attack, which eventually fails, resulting in β-cell mass deficiency and onset of T1D. Glucagon-like peptide-1 (GLP-1) exerts both proliferative and anti-apoptotic actions on β-cells. I hypothesized that chronic activation of the GLP-1 receptor (GLP-1R) would delay or prevent the loss of functional β-cell mass in the NOD mouse. I have shown that chronic administration of the GLP-1R agonist exendin-4 significantly delayed the onset of diabetes and enhanced β-cell mass. Furthermore, GLP-1R activation was associated with a reduction of islet-infiltrating immune cells, as well as changes in lymphocyte subpopulations. Consequently, I addressed whether the GLP-1R has a role in the immune system of NOD and C57Bl/6 mice. GLP-1R mRNA transcripts were detectable in several immune subpopulations, and GLP-1R activation was associated with cAMP production in primary splenocytes and thymocytes. Furthermore I demonstrated that GLP-1R signaling controls proliferation of thymocytes and lymphocytes, and is required for maintaining peripheral regulatory T-cells. In summary, these studies establish that while GLP-2R activation is not sufficient to modify disease onset in a murine model of T1D, GLP-1R activation reduces the extent of diabetes development by exerting actions on β-cells and the immune system.

The syndrome known as Gastric Dilation Air Sacculitis (GDAS) has recently been described by Lumsden et al. (2002) for Chinook salmon (Oncorhynchus tshawytscha, Walbaum), in seawater (SW) culture in New Zealand. The syndrome is characterised by distended abdomens, gastric dilation and air sacculitis, increased feed conversion ratios (FCR) and mortality. Consequently, financial returns on affected stocks are greatly reduced. A study into the epidemiology and physiology of the syndrome was initiated, working with the major aquaculture company, The New Zealand King Salmon Company (NZKS). The study revealed causative factors of GDAS. GDAS was experimentally induced only in saltwater by feeding a commercially manufactured low-cohesion pelleted diet. Control groups were fed a different diet with high physical cohesion. Low-cohesion pellets have previously been associated with a high incidence of GDAS in commercial sea cages. These data implicated osmoregulatory stress and physical properties of the feed in GDAS development. In addition, gastrointestinal (GI) physiology in GDAS -affected and -control fish was characterised. The process of GDAS development in O. tshawytscha is characterised by a loss of smooth muscle tone of the stomach as it distends. Laplace's law (P= 2T/r, where P is the distending pressure, T is the tension in the wall and r is the radius of the cylinder) predicts that unless muscle mass increases, the ability of the stomach wall to contract will be lost and consequently a loss of GI motor function will result. Therefore, GI circular smooth muscle integrity in terms of (1) stimulated and maximal contractility, (2) osmoregulatory ability of the intestine and the (3) control of the GI system was studied in pathologically affected (+ve) and unaffected (-ve) smolt. Affected fish showed changes in GI circular smooth muscle function and osmoregulatory dysfunction. Feeding different diets induced distinct gastric evacuation patterns. The intestinal brake hypothesis is presented and argued to be the probable mechanism for GDAS development. GDAS (+ve) serum showed the presence of factors capable of contracting gut smooth muscle. In addition, potential humoral mediators of the intestinal brake in fish were investigated.