| Background and Objective:Protein pHospHatases 2A(PP2A)is a number of serine/threonine protein phosphatase family in eukaryotic cells.PP2A play an important role in regulation of cell metabolism,differentiation,apoptosis,and modulation of singnal transduction,cytoskeleton and pathopHysiological mechanism of tumorigenesis via dephosphorylation of serine or threonine residues on various protein substrates.In vitro studies have shown that the depletion of the catalytic subunit of protein pHospHatase 2A(PP2Ac)lead to significant attenuation of glucose-stimulated insulin secretion in pancreatic β-cells.We previously found that specific knockout(KO)PP2Aca in islet β cells resulted in glucose tolerance impaired in mice.This study was aimed to determine mechanism of the impaired blood glucose tolerance in KO mice with conditional knockout protein pHospHatases 2Aca(PP2Aca)in pancreatic β cells.Methods:Selected mice which special knockout PP2Aca of islet β cells as KO group,genetype was PP2Acα flox/flox:Ins2-Cre,the genetype of control(CON)mice was PP2Ac a flox/flox and PP2Aca flox/+:Ins2-Cre.Interperitoneal glucose tolerance test(IPGTT)was performed in both group mice(sex,age matched)at 4 months old.Fasting blood glucose levels and 30,60,120 min blood glucose concentrations after glucose loading were monitored,respectively.Enzyme-linked immuno sorbent assay was performed in testing fasting insulin,2,10,30,60,120 min serum insulin after glucose loading.Fasting blood glucose and glucose concentrations at 15 min,30 min,45 min,60 min and 120 min after insulin injected of two groups of mice were tested.Serial paraffin embedding pancreas sections of both groups of mice(5 μm)were performed to Hematoxylin-Eosin staining,immunohistochemical staining,immunofluorescence technique and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL)in order to observe the islet quantity,morpHology,apoptosis,and the expression of insulin/glucagon.Glucose stimulated insulin secretion(GSIS)was performed to evaluate β cells founction.The expression levels of Ins-1,Ins-2,TRPA1,Akt,Bcl-2,Bax and Caspase-3 were detected by quantitative-real-time PCR(qRT-PCR).Results:The data of IPGTT indicated that the fasting blood glucose levels were similar between KO and CON mice groups(88±23.7 vs.84 ± 27.1 mg/dL,P>0.05),while after glucose loading at 30 min(424 ± 71.8 vs.272±62.3 mg/dL,P<0.05),60 min(341 ± 98.9 vs.177 ± 51.7 mg/dL,P<0.05)and 120 min(179±57.2 vs.101±29.2 mg/dL,P<0.05)glucose levels in KO mice were significant higher than those of control mice.Fasting serum insulin(0.45±0.17 vs.0.48 ± 0.20 μg/L,P>0.05)and serum insulin concentrations after glucose loading at 2 min(0.69 ± 0.20 vs.1.10±0.29 μg/L,P>0.05),10 min(0.85±0.20 vs.1.36 ± 0.38 μg/L,P>0.05),60 min(1.24 ± 0.29 vs.1.84±0.51 μg/L,P>0.05)and 120 min(0.66±0.28 vs.0.88±0.18μg/L,P>0.05)between KO and CON mice were similar,but insulin level at 30 min after glucose loading in KO mice was obviously lower than that of CON mice(1.03 ±0.18 vs.2.23 ± 0.44 μg/L,P<0.05).Fasting blood glucose(188±29.1 vs.164±26.6 mg/dL,P>0.05)and glucose levels after insulin injected on 15 min(157 ± 36.0 vs.117± 61.8 mg/dL,P>0.05),30 min(127 ± 47.9 vs.91±39.6 mg/dL,P>0.05),45 min(100 ± 48.4 vs.102 ± 36.9 mg/dL,P>0.05),60 min(112±31.9 vs.106 ± 49.9 mg/dL,P>0.05)and 120 min(105±39.8 vs.111±27.1 mg/dL,P>0.05)were no obviously difference.Besides,there were no significant differences in pancreatic islet number(263 ± 50 vs.240±66,P>0.05)and area(2275±457 vs.2266 ± 400 μm2,P>0.05)between the two groups.There was no change in the expression of insulin/glucagon between KO and control mice.Fluorescence intensity was also no difference between the two groups(120.5±21 vs.90±24.3 IOD,P>0.05).The results of GSIS indicated that insulin secretion of KO mice was significantly lowered stimulated by high glucose(16.7 mmol/L,0.42±0.1 vs.0.81 ± 0.06 μIU/L,P<0.05),although insulin levels remained almost the same(0.22±0.05 vs.0.28±0.02 μIU/L,P>0.05)in the two groups exposed to low glucose concentration(5.5 mmol/L).The relative of expression of Caspase-3(0.922±0.917 vs.1.00±0.075,P>0.05),Bax(0.51 ± 0.37 vs.1.00 ± 0.795,P>0.05),Bcl-2(0.865 ± 0.218 vs.1.00±0.445,P>0.05),Akt(1.389±0.603 vs.1.00 ± 0.11,P>0.05),Ins-1(0.979 ± 0.982 vs.1.00 ±0.578,P>0.05),Ins-2(2.66±2.486 vs.1.00 ± 0.862,P>0.05),Epac-2(0.747 ±0.559 vs.1.00±0.348,P>0.05)in KO group did not differ with CON group mice.We observed signifcantly lower expression of TRPA1 in KO islets compared with mice in CON group(0.579 ± 0.099 vs.1.00 ± 0.236,P<0.05).Conclusions:The injured insulin secretion of pancreatic islet β cell in conditional knockout PP2Aca mice might contribute to the impaired glucose tolerance in mice in vivo. |
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