Effects Of Specifical Inactivation Of PP2Ac? On Glucose Metabolism In Mouse

Background and Objective:Protein phosphatases 2A(PP2A)is a major kind of serine/threonine protein phosphatase in eukaryotic cells.Via dephosphorylation of serine or threonine residues on various protein substrates,PP2 A play important roles in regulation of cell metabolism,differentiation,apoptosis,signal transduction,cytoskeleton and pathophysiological mechanism of tumorigenesis.Previous research has shown that depletion of the catalytic subunit of protein phosphatase 2A(PP2Ac)markedly attenuates glucose-stimulated insulin secretion in pancreatic ?-cells in vitro.Insulin secretory defect is one of the pathogenesis of diabetes mellitus,Which show that PP2A is closely related to incidence of diabetes mellitus.So far,depletion of the catalytic subunit of PP2Ac in vivo remains unexplored.The research intends to establish PP2Ac? inactivated mouse model in pancreatic(3-cells and investigate the role of PP2Aca in glucose metabolism.Methods:The Ins-2 transgenic mice in which Cre enzyme was exclusively detected in pancreatic ?-cells bred with PP2Acaflox/flox mice to obtain PP2Acaflox+:Ins-2 mice,then bred with the PP2Acaflox/flox mice again to obtain PP2Acaflox/flox:Ins-2 mice(KO mice).Identify the 2nd exon of PP2Aca knockdown by PCR and Western Blot.Interperitoneal glucose tolerance test(IPGTT)was performed in transgenic mice at 2,3,4 months,and PP2Acaflox/flox mice were employed with control(n=8).0,30,60,120 min blood glucose were tested,respectively.Used glucose oxidase test method to determine fasting blood glucose,fasting insulin was tested by radioimmunoassay.Insulin resistance index(HOMA-IR,FINS × FPG/22.5)and insulin sensitivity index(HOMA-?,20 × FINS/FPG-3.5)were calculated.Pancreas were picked with paraffin embedding from the both groups(glucose tolerance impaired was defected in KO mice)on 4 months old mice,which used to line 5 microns serial section.Effects of inactivation of PP2Aca on pancreatic islet morphology,quantity,the expression of insulin/glucagon and the islet fibrosis were observed by HE staining,immunohistochemical staining,immunofluorescence technique and Masson staining.The insulin secretory function of pancreatic islets was evaluated by glucose stimulated insulin secretion(GSIS).The expression levels of gene with respect to insulin synthesis,cells proliferation or apoptosis were detected with real-time PCR,such as Ins-1,Bcl-2,Bax,Akt,Leptin,Caspase-3.Results:The PCR shows that PP2Aca transcripts were shorter in KO mice an in control mice.Western blot shows the protein level of PP2Ac? expression in KO mice was significantly lower than that of control mice(2.25 ± 0.17 vs.2.97 ± 0.16,P<0.05).The results of IPGTT indicated that,among 2 months old mice the blood glucose levels of KO mice at 0 min,30 min,60 min and 120 min were 79 ± 10 mg/dL,235 ± 36 mg/dL,152 ± 17 mg/dL and 105 ± 11 mg/dL,of PP2Acaflflox/flox mice at four points were 82 ± 11 mg/d,219 ± 45 mg/dL,152 ± 36 mg/dL and 95 ± 11 mg/dL,of PP2Aca flox/+:Ins2-Cre mice at four points were 97 ± 23 mg/dL,232 ± 43 mg/dL,151 ± 18 mg/dL and 107 ± 15 mg/dL.Among 3 months old mice the blood glucose levels of KO mice at 0 min,30 min,60 min and 120 min were 87 ± 21 mg/dL,244 ± 42 mg/dL,154 ± 19 mg/dL and 105 ± 20 mg/dL,of PP2Aca flox/flox mice at at four points were 87 ± 15 mg/dL,219 ± 45 mg/dL,156 ± 38 mg/dL and 98 ± 16 mg/dL,of PP2Aca flox/+:Ins2-Cre at four points were 122 ± 14 mg/dL,243 ± 42 mg/dL,155 ± 19 mg/dL and 108 ± 19 mg/dL.Among 4 months old mice the blood glucose levels of KO mice at 0 min,30 min,60 min and 120 min were 92 ± 21 mg/dL,449 ± 84 mg/dL,366 ± 78 mg/dL and 185 ± 41 mg/dL,of PP2Aca flox/flox mice at at four points were 86 ± 22 mg/dL,202 ± 66 mg/dL,153 ± 42 mg/dL and 98 ± 23 mg/dL,of PP2Aca flox/+:Ins2-Cre mice at at four points were 122 ± 14 mg/dL,243±42 mg/dL,155 ± 19 mg/dL and 108 ± 19 mg/dL?There were no significant difference(P>0.05)at 2 and 3 months old mice,but significantly higher than those of controls(P<0.05)at 4 months old mice.No significant difference(4.6 ± 0.8 vs.4.4 ± 0.6,P>0.05)of HOMA-IR and HOMA-p(172 ± 16 vs.164 ± 14)were found between KO mice and control mice at 4 months.Besides that,the number(266 ± 123 vs.164 ± 14)and size(1922 ± 731 vs.1958 ± 1236)of pancreatic islets were no significant difference(P>0.05)between two groups,and there were no apparent changes in expression of insulin/glucagon in KO mice.Fibrosis was not found in pancreatic islets.Fluorescence intensity was no significant difference between two groups(160 ± 24 vs.81.3 ± 29.3).The results of GSIS indicated that the insulin from PP2Aca inactived islets fed with high concentration glucose was significantly inhibited(0.50 ± 0.16 vs.0.82 ± 0.15,P<0.05)in KO group,and no significant difference(0.21 ± 0.08 vs.0.23 ± 0.1,P>0.05)in low concentration glucose.Expression of mRNA was no statistical difference between group KO and control,results showed as bellow:Caspase-3(1.55 ± 0.78 vs.1.01 ± 0.19)?Bax(2.19 ± 0.57 vs.1.04 ± 0.40)?Leptin(1.06 ± 0.57 vs.1.02 ± 0.26)?Bcl-2(2.99 ± 0.68 vs.1.07 ± 0.22)?Akt(2.52 ± 1.16 vs.1.03 ± 0.33)?INS-1(2.59 ±1.42 vs.1.14±0.64).Conclusions:Specifical inactivation of PP2Aca in pancreatic P-cells mouse model is successfully established,and glucose tolerance impairs at 4 months in PP2Ac?flox/flox:Ins2-Cre mice.Insulin secretion in PP2Acaflox/flox:Ins2-Cre mice significant fewer than control mice.