Glucagon-like peptide-1 (GLP-1) increases beta-cell function and growth through PKAand PI3-K/PKB, respectively. GLP-1 acts via a G-protein-coupled receptor, and PI3-Kγ is known to be activated by G βγ . The role of PI3-Kγ in the chronic effects of GLP-1 on the beta-cell was therefore investigated using PI3-Kγ knock-out (KO) mice treated with the GLP-1 receptor agonist, exendin-4 (Ex4; 1 nmol/kg s.c q24h for 14 d). In vivo, glucose and insulin responses were similar in PBS-and Ex4-treated KO and

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... wild-type (WT) mice. However, glucosestimulated insulin secretion was markedly impaired in islets from PBS-KO mice (P<0.05), and this was partially normalized by chronic Ex4 treatment (P<0.05). In contrast, insulin content was increased in PBS-KO islets, and this was paradoxically decreased by Ex4 treatment, as compared to the stimulatory effect of Ex4 on WT islets (P<0.05-0.01). Transfection of INS-1E beta-cells with siRNA for PI3-Kγ similarly decreased glucose-stimulated insulin secretion (P<0.01) and increased insulin content. Basal values for beta-cell mass, islet number and proliferation, GLUT2, glucokinase and IRS-2 were increased in PBS-KO mice (P<0.05-0.001) and, although they were increased by Ex4 treatment of WT animals (p<0.05), they were decreased in Ex4-KO mice (p<0.05-0.01). These findings indicate that PI3-Kγ deficiency impairs insulin secretion, resulting in compensatory islet growth to maintain normoglycemia. Chronic Ex4 treatment normalizes the secretory defect, thereby relieving the pressure for expansion of beta-cell mass. These studies reveal a new role for PI3-Kγ as a positive regulator of insulin secretion, and reinforce the importance of GLP-1 for the maintenance of normal beta-cell function. Glucagon-like peptide (GLP-1) is a 30 amino acid peptide secreted by the intestinal L cell in response to nutrient ingestion (1;2). GLP-1 is an incretin hormone that possesses a number of anti-diabetic actions, most notably, enhancement of beta-cell function through stimulation of glucose-dependent insulin synthesis and secretion (3-5), as well as inhibition of glucagon release (5;6), gastric emptying (6;7) and food intake (8). Importantly, these actions have been demonstrated in long-term studies in patients with type 2 diabetes, in whom treatment with GLP-1 or the long-acting GLP-1 receptor agonist, exendin-4 [Ex4; (9)] leads to reductions in glycemia, HbA1c and body weight (10;11). The stimulatory effects of GLP-1 on beta-cell function are mediated through the G αscoupled GLP-1 receptor, which increases cAMP levels upon ligand binding (12-14). Activation of both protein kinase (PK) A-dependent and PKA-independent pathways by cAMP then enhances glucose-stimulated insulin secretion, through modulation of ion channel activity as well as stimulation of granule exocytosis (15). However, a number of studies have now shown that both GLP-1 and Ex4 also increase beta-cell mass (BCM) in rodents, through mechanisms involving stimulation of both beta-cell proliferation and islet neogenesis (12;16-20). Interestingly, these growth effects of GLP-1 have recently been established to be dependent upon the pleiotropic signalling PKB/Akt pathway (16;21;22). Nonetheless, the mechanism by which GLP-1 receptor activation is coupled to the PKB signalling pathway remains unclear. Phosphatidylinositol-3-kinase (PI3-K) is the major upstream activator of PKB in most cells. The family of PI3-K's includes Class I -IV isoforms, with Class I being further subdivided into IA and IB (23-25). All of the Class I PI3-K isozymes are characterized by their ability to produce phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), essential for the activation of PKB. However, the Class IA enzymes are linked to tyrosine kinase receptor signalling, while the single Class IB isozyme, PI3Kγ, is activated by G-protein coupled receptor (GPCR) ligand binding. Although GPCRs are associated with several subunits of the G protein complex, only the βγ Immunological and morphometric analyses. Some mice were injected with 100 mg/kg (i.p.) 5bromo-2−deoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO) 6 hr before removal of pancreas (16). BCM was measured as described previously (16) . Briefly, dissected pancreata were weighed, cut into 6-8 pieces, fixed in Bouin's and paraffin-embedded, and 4 µm sections through all 6-8 pieces (to make n=1) were dewaxed, hydrated and incubated overnight at 4°C with guinea pig anti-insulin antibody (Dako Diagnostics, Mississauga, ON, Canada). The sections were then incubated for 1 hr with biotinylated anti-guinea pig antibody (Vector Laboratories, Burlington, ON, Canada), and subsequently treated for 1 hr with avidin/biotin complex (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA). Sections were then stained with 3,32-diaminobenzidine tetrahydrochloride (DAB; Sigma Chemical Co.) for 10 min, washed with tap water and counterstained with hematoxylin. Beta-cell and total pancreatic area per section were measured using a Zeiss Axioplan microscope with Axiovision software (Carl Zeiss Canada, Don Mills, ON, Canada). Total BCM for each pancreas was determined as the product of the total cross-sectional beta-cell area over total tissue area normalized for the weight of the pancreas before fixation. Pancreatic sections were also stained for BrdU using mouse anti-BrdU IgG (Amersham, Oakville, ON, Canada), followed by biotinylated anti-mouse IgG and DAB (3,32-diaminobenzidine, Sigma Chemical Co.), as previously described (16). PI3-Kγ was co-localized with insulin in fresh-frozen pancreatic sections fixed with formalin, using rabbit anti-human PI3-Kγ (p110γ, 1:50; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) and guinea pig anti-porcine insulin (1:100; DakoCytomation Inc., Mississauga, ON, Canada), respectively, followed by donkey anti-rabbit AlexaFluor-488-(Invitrogen Corp.) and donkey anti-guinea pig Cy2-(Jackson Immunoresearch Laboratories, Wet Grove, PA) coupled secondary antibodies, respectively. Isolated islets. Pancreatic islets of Langerhans were isolated by collagenase digestion as described (39), and groups of 5 islets were pre-incubated for 30 min in 200 µl of Krebs-Ringer 34. Harndahl L, Wierup N, Enerback S, Mulder H, Manganiello VC, Sundler F, Degerman E, Ahren B, Holst LS 2004 Beta-cell-targeted overexpression of phosphodiesterase 3B in mice causes impaired insulin secretion, glucose intolerance, and deranged islet morphology. J MP, Backx PH, Penninger JM 2002 Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways. Cell 110:737-749 36.