N. to measure, by radioimmunoassay (RIA), the plasma levels of insulin, C-peptide (Linco Study, Billerica, MA), somatostatin (Phoenix Pharmaceuticals, Belmont, CA), and total GLP-1 (catalog no. GLP1T-36HK, Millipore, Billerica, MA). RIA (Linco) was used to measure basal plasma glucagon levels in mice fasted for 4 h (from 07:00 to 11:00). Glucose Tolerance Test Male mice ( 5/genotype, 5 weeks old) were fasted over night from 17:00 to 08:00 the following day before becoming anesthetized and given glucose via oral gavage (3.0 g/kg BWT). Blood was drawn from the retro-orbital sinus to measure blood glucose and plasma insulin levels at 0C120 min post-glucose administration. In parallel experiments, anesthetized mice were injected intraperitoneally with 5 g of the GLP-1 receptor antagonist, exendin (9C39) (American Peptide Co. Inc., Sunnyvale, CA), 20 min before glucose administration. Hyperglycemic Clamp Analysis in Awake Mice To assess -cell function = 7/genotype, 7 weeks of age) were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg BWT) and xylazine (10 mg/kg BWT), and an indwelling catheter was put into the right internal jugular vein. Four-to-five days later, mice were fasted over night before being subjected to hyperglycemic clamp analysis with a continuous infusion of 20% glucose to raise and maintain plasma glucose levels Resiquimod at 300 mg/dl for 2 h. Blood samples (40 l) were collected at 15C20-min intervals over a period of 120 min to measure plasma glucose and insulin concentrations. Hyperinsulinemia-Euglycemic Clamp Analysis A 2-h hyperinsulinemic-euglycemic clamp was performed in overnight-fasted awake mice (= 11/genotype, 7C8 weeks older) with primed and continuous infusion of human being regular insulin (Humulin; Lilly) at a rate of 2.5 milliunitskg?1min?1, while described (6). Glucose metabolism was estimated with a continuous infusion of [3-3H]glucose (PerkinElmer Existence Sciences) for 2 h prior to (1850 Bq/min) and throughout the clamps (3700 Bq/min). Plasma GLP-1 Measurement in Response to Dental Glucose Mice (6/genotype) were fasted overnight, subjected to an oral glucose administration (3 g/kg BWT), and anesthetized with pentobarbital immediately after glucose administration before their retro-orbital blood was drawn 30 min later on to measure blood glucose and plasma insulin levels. For GLP-1 measurement, plasma from each genotype was pooled to determine GLP-1 levels in triplicate in 300-l aliquots, as above. Immunohistochemical Analysis Six-month-old male mice were anesthetized with pentobarbital, and their pancreases cautiously dissected, cleared of extra fat and spleen, weighed, and fixed over night in 4% paraformaldehyde. Cells were inlayed in paraffin and consecutive 7-m sections were mounted on slides. Sections were then stained with antibodies against insulin (Dako, Carpinteria, CA), glucagon (Sigma), somatostatin (Chemicon, Temecula, CA), pancreatic polypeptide (PP) (Linco), or a mixture of glucagon, somatostatin, and polypeptide (3Ab) and visualized utilizing 3,3-diaminobenzidine tetrahydrochloride reaction, as explained (19). Immunofluorescence Staining As explained previously (9), small intestinal tissues were fixed over night in Bouin’s remedy, inlayed in paraffin, and slice into 4-m consecutive sections. Following deparaffinization, sections were exposed to antigen retrieval by cautiously boiling inside a microwave oven in 10 Resiquimod mm citrate buffer, pH 6.0. Sections were then incubated at 4 C over night with -CEACAM2 custom-made anti-peptide polyclonal antibody raised in rabbit against the keyhole limpet Resiquimod hemocyanin-conjugated HPLC-purified peptide CNAEIVRFVTGTNKTIKGPVH in CEACAM2 (Bethyl Laboratories, Montgomery, TX) (final dilution SEB 1:50) (6). Subsequently, sections were incubated having a goat Resiquimod -rabbit Cy5 (final dilution 1:400, Dianova 111-175-144) for 1 h to visualize with epifluorescence (Keyence BZ9000) equipped with a Plan Apo objective (Nikon). DAPI (1:9000) was used to visualize nuclei. Fluorescence-activated Cell Sorter Purification of Isolated Islets Islets were isolated from the intraductal collagenase digestion method (20). After PBS wash, the suspension was approved through a 35-m filter before fluorescence-activated cell sorter (FACS) analysis, based on autofluorescence and size (21). Cells were sorted directly into TRIzol Resiquimod reagent, and the purity of the sorted fractions was determined by real time PCR for insulin and glucagon in each portion. Insulin Secretion from Isolated Islets Purification of islets was carried out on pancreases of 4-month-old mice by collagenase digestion (22). Islets were then resuspended in RPMI 1640 medium comprising 10% newborn calf serum and 5.5 mm glucose.