| Purposes: Enhancement of insulin secretion from the islet B cells is a principal goal for treatment of patients with type 2 diabetes. Some gastrointestinal hormones are associated with insulin secretion. The glucagon-like peptide-l(GLP-l) is a kind of gastrointestinal hormone formed by alternative tissue-specific cleavages in the L cells of the intestine, the A cells of the endocrine pancreas and neurons in the brain. GLP-1 is based on the proglucagon gene sequence. It is secreted mainly as "truncated" GLP-l(7-36 )amide with more activities. Though GLP-1(7-36)NH2 can increase insulin secretion, but how does GLP-1(7-36)NH2 stimulate insulin secretion? The answer to this question is fundamental for the assessment of the therapeutic potential of the gut hormone. In this experiment, radio-immunoassay and hybridization in situ were applied to observe the insulinotropic activities of GLP-1 (7-36)NH2 and reveal the mechanisms underlying this process.Methods: Rat pancreases were removed from 3-5 day-old Sprague-Dawley rats and dissected into 0.5mm3 segments and islets were isolated by the collagenase digestion method of Wangling et al. Thoroughly washed islets and suspended in modified RPMI-1640 medium supplemented with 10% fetal bovine serum, and added to 50ml cell culture flasks. The islets were incubated for 20 hours at 37癈 with 95% air and 5% CO2. After 24 hours of incubation, the medium containing the suspended islets were carefully removed and washed with serum-free midium, then resuspended in modified RPMI-1640 medium. The islets were added into 96-well tissue cultureplates(containing 20? islets per well) for 24 hours. The effects of GLP-1(7-36)NH2 with different concentrations on insulin secretion were measured by radioimmuno-assay(RIA). Effects of 8-bromo-cyclic 3',5'-adenosine monophosphate (8-Br-cAMP) on insulin secretion and effects of GLP-1(7-36)NH2 on intracellular cAMP were determined by RIA. Influences of GLP-1(7-36)NH2 (20nmol/L) on insulin mRNA expression were determined by hybridizaiton in situ. The mean optical densities (MOD) of B cells stained by DAB were analyzed by high pathological image analysis system (HPIAS).The data were showed as Means盨D and analyzed using the Student's t-test and one-way ANOVA. P<0.05 is considered as significant. Results:1. The effects of GLP-1(7-36)NH2 on insulin secretionGLP-1(7-36)NH2 with the concentration of 2.5nmol/L, 5..0nmol/L, l0.0nmol/L, 20.0nmol/L, 40.0nmol/L respectively were added to the medium as different experimental groups , 24 hours later, insulin amount are 68.76?1.71 72.30?3.13 104.16?5.57 110.98?.29 111.58?0.65mIU/L respectively, and the insulin account is 55.53?.63mIU/Lin the control group. There was no significant difference between the groups with 2.5nmol/Land 5.0nmol/L GLP-1(7-36)NH2 respectively; and there was not significant difference among the groups with lO.Onmol/L, 20.0nmol/L and 40.0nmol/L GLP-1(7-36)NH2 respectively. But the difference is significant between experimental groups and control group(P<0.05).The data show that with the rising concentration of GLP-1(7-36)NH2, there is an increasing amount of insulin.2. The actions of 8-Br-cAMP on insulin secretionInsulin concentration of control group is 52.59 ?9.77mIU/L, and that of the experiment group is 117.03?0.04mIU/L, there is significant difference between them(P<0.05).3. The effects of GLP-1(7-36)NH2 on intracellular cAMPThe amount of cAMP of control group is 28.11 ?.41nmol/L, and that of the experiment group is 173.88?.62nmol/L, there is significant difference between them (P<0.05).4. The effects of GLP-1(7-36)NH2 on insulin mRNA expressionThe B cell cytoplasma color of the experiment group is brown stained by DAB, but that of control group is weak. The mean optical densities(MOD) of the control group are 0.07436+0.01794, the maximum is 0.1943 and the minimum is 0.03798. The MOD of the experiment group are 0.2404+0.03368, the maximum is 0.3428 and the minimum is 0.1911. The difference is significant between two groups (F...
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