| Background:Nowadays, China has become the first largest country of diabetes. Recently, Diabetes epidemiological survey with Chinese Diabetes Society (CDS) demonstrated that over the age of20in population, the age-standardized diabetes prevalence was9.7%, while the proportion of prediabetes as much as15.5%. Prediabetes is diabetes high-risk groups, about60%of diabetic patients were from this populations.The pathophysiology of T2DM is multifaceted, In2008, Professor Defrozon summarized the progression of research of diabetes, then to definite eight factors attributing to the pathophysiology of T2DM, which includes defects of (3-cell’s function, decreased glucose uptake of muscle, increased glucagon, increased hepatic glycogen, lipotoxicity, deficience of incretin, increased glucose by tubular reabsorption, decreased of the inhibition of central nervous system to intake of glucose. In fact, the islet β cells mass decreased as early as in IGT, In onset T2DM, the islet β cells mass decreased to60%, and the function of β cells decreased to80%. Therefore, study for proliferation, apoptosis and insulin secretion of β cell is virtual important.A large number of experiments have been confirmed that ACh is released from nerve terminals and acts upon muscarinic cholinergic receptors in the beta cell plasma membrane, then to induce the amplification of insulin secretion via acting on phosphatidylinositol protein kinase C pathway. However, the insulinontropic effect of ACh was inhibited in PMA pretreatment.HCNP is an11amino acid peptides which was cleaved from the amino terminal of HCNP-pp by chymotrypsin-like thiol protease in the hippocampus. HCNP has been implicated in acetylcholine synthesis and secretion by enhancing activity of choline acetyltransferase (ChAT) in the central nervous system. However, HCNP whether have the above effects to induce the amplification of insulin secretion in islet β cells, there is no reported in the literature.As a multifunctional protein, HCNP-pp has high homologous with phosphatidyl-ethanolamine-binding protein (PEBP) and Raf-1kinase inhibitor protein(RKIP), which was especially interference with phosphorylation of Raf-1kinase and activation, further to prevent cell proliferation via inhibition of mitogen-activated protein kinase (MAPK) pathway. Previous studies have demonstrated MAPK signaling pathway plays an important role in β-cells proliferation and differentiation. HCNP-pp was expressed only islet β-cells in pancreas, which inhibits β-cell proliferation. However, there is no reported in the literature for the changes of HCNP-pp in diabetes state, and whether is correlated with the the decreased proliferation of β-cells.Glucagon-like peptide-1(GLP-1) plays an important role in the intestine pancreatic islet axis, which attributes to the specific G protein-coupled receptor via cAMP-protein kinase A on β cells, then to act on the roles of the "incretin". Moreover, GLP-1has the cooperative effect with acetylcholine to activate the insulinotropic action of insulin. The existing cell culture and animal experiments have confirmed that the glucagon-like peptide may activate and promote the mitogen-activated protein kinase signal transduction pathways to improve β-cell proliferation. Thus, GLP-1may be the ideal medication for prediabetes. As a human glucagon like peptide-1analogue, Liraglutide has a significant roles in promoting insulin secretion and β-cell proliferation. However, whether the effects of Liraglutide to prohibit the progression of prediabetes was correlated with HCNP and its precursor protein, there is no reported in the literature.In summary, the subject of the β-cell line INS-1cells and OLETF rats from the following aspects:1, To observe the insulitropic action of HCNP in INS-1cells, then to explore the relative pathways of HCNP on the role of the INS-1cells via detection of ChAT, M3R, and HCNP-pp gene expression.2, To observe the proliferation of islet cell and secretion of insulin of Liraglutide in IGT stage of OLETF rats. In order to explore the possible mechanism of Liraglutide, we will measure the levels of HCNP-pp, HCNP, GLP-1R, ChAT and M3R.Part1The insulitropic action of HCNP in INS-1cells.Objective:To explore the impact of the HCNP INS-1cells insulin secretion and the possible pathwaysMethods:(1) HCNP synthesis and purity detection:HCNP was applicated by solid phase chemical synthesis, and the sequence of HCNP is deemed to be H-Ala-Ala-Ile-Asp-Ser-Gln-Trp-Ala-Gly-Pro-Leu-OH. The products were isolated and purified using reverse high performance liquid chromatography and electrospray ionization mass spectrometry.(2) The culture and activity identification of INS-1cells:INS-1cells were cultured with RPMI-1640containing10%fetal bovine serum at37℃,5%CO2incubator. Inverted phase contrast microscope to observe the growth of cells.(3) Acute tests for insulin secretion:Insulin release was determined using monolayers of INS-1cells. The cells were harvested with0.1%trypsin and0.1%EDTA, then seeded into6-well plates at a density of6x105cells per well. The cells were randomly divided into three groups: Control group, which were given the RPMI1640fluid culture; HCNP group were given the RPMI-1640liquid culture containing synthetic HCNP(50pg/ml); HCNP+PMA group, which were put phorbol-12-myristate-13-acetate(PMA) before the stage of insulin release, then incubated for18hours. INS-1cells were cultured in5%CO2incubator after5days, studies were precede by40-min preincubation at37℃in KRBH supplemented with3.3mM of glucose. Using the same buffer supplemented with glucose of5.6mM,16.7mM,20μM of neostigmine,100μM of chloride choline, After40-min incubation, the buffer was removed from each well and aliquots for measurement of insulin by radio-immunoassay.(4) The determnation of genes expression of ChAT and M3R in INS-1cells: The expression of genes were determined using monolayers of INS-1cells. The cells were harvested with0.1%trypsin and0.1%EDTA., then seeded into6-well plates at a density of6x105cells per well. The cells were randomly divided into control group and HCNP group; INS-1cells were treated with Trizol solution, total RNA extracted according to the instructions of kits and stored at-80℃. The mRNA contents (ChAT, M3R) were measured by real-time quantitative PCR(qRT-PCR). Results:(1) Radioimmunoassay methods for the determination of insulin in cell culture supernatant glucose concentration3.3mmol/L,5.6mmol/L and16.7mmol/L. In different concentrations of glucose(5.6mmol/L and16.7mmol/L), HCNP group insulin levels was greater than the Control and HCNP+PMA groups.(5.6mmol/L:10.09±1.41,8.48±0.92,8.36±1.05μIU/ml),(F=6.340, P=0.006);(16.7mmol/L:10.82±1.32,8.73±0.75,8.88±1.01μIU/ml),(F=10.836, P<0.001), the difference was statistically significant. In3.3mmol/L glucose, the insulin levels in three groups have no significant differences (3.3mmol/L:8.36±0.97、8.27±0.79,7.94±0.97μIU/ml),(F=0.535, P=0.592).(2) Compared with control group, the mRNA content of ChAT and M3R were significantly increased in HCNP group, control group(ChAT1.01±0.30, M3R0.72±0.15×105copies), HCNP group(ChAT3.01±0.47, M3R1.06±0.18); the difference was statistically significant, ChAT(t=-8.837, P<0.001), M3R(t=-3.546, P=0.005).Conclusions:(1) In the same cell culture environment, HCNP may promote the release of insulin, and pretreatment with PMA greatly reduced the effect, indicating that PKC pathway plays a vital role in the insulinotropic action of HCNP.(2) The mRNA content of ChAT and M3R were significantly increased in HCNP group, suggesting the insulinotropic action of HCNP maybe is corrected with the higher expression of ChAT and M3R. Part2The impacts of HCNP-pp and HCNP in islet cells of prediabetes rats which were treated with liraglutide on the functions and proliferation of β cellsObjective:To measure the impacts of liraglutide on OGTT, index of early-phase insulin secretion (Ins30/ΔGlu30)and insulin-positive cell density(Ins-PCD) in prediabetes rats, further to explore the correlaiton between Liraglutide’s effections and the changes of HCNP-pp, HCNP.Methods:(1) OGTT experiment, Ins30/AGlu30and insulin-positive cells density(Ins-PCD) test:Four-week-old male OLETF rats were fed with a standard chow diet and water ad libitum. At the age of14weeks, for determinating the state of IGT, all OLETF rats were conducted OGTT. After15h fasting, glucose solution (2g/kg) was orally administered, and blood sample were drawn at0,30,60, and120min from the tail vein with glucose oxidase detecting.The diagnosis of DM and IGT, was confirmed using international standards. Briefly, Rats with a peak level of blood glucose>16.7mmol/L and the120-minute blood glucose>11.1mmol/L were determined the state of DM; If rats met either of the above-mentioned conditions were regarded as impaired glucose tolerant (IGT). The IGT-OLETF rats were randomly divided into3groups (n=8for each group)were given100μg/kg,200μg/kg Liraglutide and saline(0.9%,1.0ml/kg), twice daily intraperitoneal injection, respectively. After12weeks of administration, to measure OGTT experiment again. The index of early-phase insulin secretion using the ratio of insulin incremental value to glucose incremental value at30min after the meal Ins3O/ΔGlu30=(Ins30-InsO)/(Glu30-Glu0), to determine Ins3O/AGlu3O by ELSIA, and to measure Ins-PCD by RIA methods.(2) Islet cells HCNP-pp, ChAT and M3R and GLP-1R mRNA in fluorescence quantitative analysis:According to the instructions to extract total RNA, pancreas total RNA extraction was using the Trizol Reagent, RNA was stored in aliquots at-80℃To determine mRNA expression in the OLETF rats, primers were designed to amplify the RKIP, GLP-1R, ChAT and M3R.(3) Islet cells HCNP-pp, GLP-1R protein expression:The protein of islet cells was extracted by lysate of protein. Protein quantity was measured by Bradford, expressed in mg/ml. The relative protein content of CT-HCNP-pp, NT-HCNP-pp and GLP-1R were measured by western blotting using and (3-actin as inner standards.Results:(1) OGTT experiment, Ins3O/AGlu3O and Ins-PCD:Althoμgh all rats FPG increased with age during the experimental period, placbo-OLETF rats exhibited significant increased compared with LETO and diffrent doses Liraglutide-OLETF rats(F=22.925, P<0.001), The same results were occurred in2hPG(F=40.805, P<0.001). However, the reverse results were occurred in Ins30/ΔGlu3O(F=8.895, P<0.001). Compared with the PBO group, Ins-PCDimproved significantly in L-200group and LETO group, however, the difference is not statistically significant in L-100group(F=15.968, P<0.001).(2) The rat pancreas HCNP-pp, ChAT, M3R and GLP-1R mRNA expression:At the transcriptional levels of HCNP-pp in PBO group rats were higher than different doses of Liraglutide-OLETF or LETO rats, however, at the transcriptional levels of ChAT, M3R and GLP-1R were occurred conversely results, the differences were statistically significant, HCNP-pp mRNA(F=8.563, P<0.001), ChAT mRNA(F=55.106, P<0.001), M3R mRNA(F=27.201, P<0.001), GLP-1R mRNA(F=6.012, P=0.003).(3) The rat pancreas HCNP-pp, GLP-1R protein expression:Compared with PBO group rats, L-100, L-200and Con group CT-HCNP-pp, NT-HCNP-pp protein content and the ratio of NT-HCNP-pp to CT-HCNP-pp were significantly reduced, In contrast, GLP-1R protein content of significantly increased, the differences were statistically significant,CT-HCNP-pp(F=12.973, P<0.001),NT-HCNP-pp (F=18.250, P<0.001), NT-HCNP-pp/CT-HCNP-pp(F=17.632,P<0.001), GLP-1R (F=20.467, P<0.001).(4) The correlation analysis between HCNP-pp and GLP-1R at protein expression level and gene expression level in rat islet cells:All different groups of rat islet cells, CT-HCNP-pp (0.711±0.061), NT-HCNP-pp (0.640±0.094) and NT-HCNP-pp/CT-HCNP-pp (0.897±0.071) were negatively correlated to GLP-1R (0.750±0.083) at protein levels, The same results were occurred at gene expression levels:the HCNP-pp mRNA (3.872±1.353), GLP-1R mRNA in (15.106±4.252). CT-HCNP-pp:y=-0.886X+1.380(R=0.649, t=-4.429, P<0.001); NT-HCNP-pp:y=-0.623X+1.148(R=0.701, t=-5.107, P<0.001); NT-HCNP-pp/CT-HCNP-pp:y=-0.685X+1.364(R=0.582, t=-3.717, P=0.001); HCNP-pp mRNA:y=-1.563X+2.11 (R=0.498, t=-2.982, P=0.006).Conclusions:(1) Liraglutide improved the high blood glucose, the index of early-phase insulin secretion and the islet β cells mass in IGT stage of OLETF rats, which to inhibit the progress of prediabetes.(2) After12weeks treatment, Placebo group rats progressed to type2diabetes status. In islet cells, the expression of HCNP-pp was increased, however, the expression of GLP-1R, HCNP were decreased, those indicate that type2diabetes is associated with GLP-IR, HCNP-pp and HCNP.(3) To analyse the correlation of HCNP-pp and GLP-1R in rat islet cells, The results showed that the gene and protein expression levels of HCNP-pp and GLP-1R were negatively correlated. Thus speculated that liraglutide prohibit the progress of pre-diabetes rats through activation of GLP-1R maybe correlated with the decrease of HCNP-pp expression. |
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