Effect And Mechanism Of AMPK On Apoptosis And Energy Metabolism Of Gastric Smooth Muscle Cells During The Development Of Diabetic Gastroparesis

Diabetic gastroparesis(DGP)is a common gastrointestinal complication in diabetes.Although the motility of gastric smooth muscle is regulated by neural and humoral factors,but the contribution of the functional status of gastric smooth muscle cells in regulating smooth muscle motility cannot be neglected.The contractile movement of gastric smooth muscle is the source of power for gastric emptying,and the effective number of smooth muscle cells and sufficient cellular energy supply are the guarantee of this power.The level of apoptosis can change the number of effective cells,and changes in cellular energy metabolism can affect the energy supply of cells.So determining the mechanism of apoptosis and energy metabolism in gastric smooth muscle cells under high glucose state will play a positive role in the treatment of DGP and diabetes.Study has found that AMP-activated protein kinase(AMPK)is closely associated with apoptosis and energy metabolism.AMPK is a heterotrimeric protein composed of a catalytic a subunit and two regulatory subunits,? and y.AMPK activity is regulated by phosphorylation on threonine-172 located on the a catalytic subunit.The activation of AMPK is affected by increases in adenosine monophosphate AMP/ATP ratio and ADP/ATP ratio.Increased AMP can bind to the AMPK y-subunit,and cause conformational change that promote activation of AMPK.However,the AMP allosteric stimulation is only a 3-5 fold increase in AMPK activity,complete activation of AMPK by upstream AMPK kinase such as LKB1,CaMKK?,TAK1 is required.AMPK is a key molecule in the regulation of energy metabolism,exists in almost all eukaryotic species and is also known as "Energy receptor".AMPK activity can change with the different energy status which regulate energy metabolism.Many studies have indicated that AMPK may be involved in apoptosis by affecting reactive oxygen species(ROS),mammalian target of rapamycin(mTOR),Bim,and p53 pathways.However,the specific mechanism underlying the involvement of AMPK in apoptosis and cell energy metabolism under high glucose condition is still unclear.Objective(1)The study aimed to investigate the changes in apoptosis and energy metabolism of gastric smooth muscle cells during the DGP development,and determine the activation of AMPK and its activation modes.(2)To investigate the effect of AMPK activation on apoptosis and energy metabolism of rat gastric smooth muscle cells under high glucose conditions(3)To explore the changes in phosphorylation of AMPK signal pathway,and elucidate the mechanism of action of AMPK activation on apoptosis and energy metabolism.The aim of this study was to further elucidate the pathogenesis of GDP,and provide an experimental and theoretical basis for developing new treatments for diabetic gastroparesis.Method(1)Diabetic rat model was established,and the model group was randomly divided into DM4w group,DM6w and DM8w group,and normal rats were used as the control group.Spectrophotometer was used to detect the changes of gastric residual pigment ratios,intestinal conduction velocity and gastrointestinal propulsion rate in each group,and the establishment of DGP model was determined.Isolated muscle strip experiments were performed to observe the changes in spontaneous contraction of gastric smooth muscle in DGP rats.Immunohistochemical staining and flow cytometry were used to detect cells apoptosis in each group.The ATP,ADP,and AMP contents and energy charge in rat gastric smooth muscle tissues of each group was detected by enzyme-linked immunosorbent assay(ELISA).The phospho-AMPK,AMPK,LKB1,TAK1,CaMKK? protein expression in rat gastric smooth muscle tissues of each group was detected by western blot,and analysis to determine the activation of AMPK and its activation modes.(2)Rat gastric smooth muscle cells were cultured in vitro,and treated with AMPK inhibitors and agonists.The experiment was divided into the normal glucose group(glucose 5 mmol/L),the high glucose group(glucose 35 mmol/L),the normal glucose+2-DG group,the high glucose+2-DG group,the normal glucose+Compound C group,the high glucose+Compound C group.The detection time was at 24h and 48h.The effect of AMPK on gastric smooth muscle cell apoptosis and energy metabolism at different time points was observed.(3)We established a DGP rat model,and cultured rat gastric smooth muscle cells in vitro,phosphorylated protein kinase antibody array was used to observe the phosphorylation changes of AMPK signal pathway and identify the differential proteins.After silencing AMPK by RNA interference,changes in the above identified differential proteins were detected in high glucose 24h group and high glucose 48h group and high glucose+siRNA 48h group by western blot assay and ELISA.The mechanism of action of AMPK activation on apoptosis and energy metabolism were elucidated.Results(1)After diabetic model establishment,the gastric emptying ability of diabetic rats gradually declined with prolonged disease duration.The gastric residual pigment ratio was increased(P<0.05),the intestinal transit rate(P<0.01)and intestinal propulsion rate(P<0.05)were decreased in rats after 6 week of model establishment,indicating that DGP formed at 6 weeks after diabetic rat model establishment.The frequency and amplitude of spontaneous contraction of gastric smooth muscle tissues were both reduced(P<0.001).Apoptotic gastric smooth muscle cells were present during the development of DGP,and the apoptosis was obvious after 6 weeks(P<0.05)and after 8 weeks of model establishment(P<0.01).The presence of energy metabolism disorder in rat gastric smooth muscle tissue was observed during DGP,energy metabolism disorder was more obvious after 4 weeks(P<0.05)and after 6 weeks(P<0.05)and after 8 weeks of model establishment(P<0.001).During DGP,AMPK activity in rat gastric smooth muscle tissue was inhibited at 6 weeks of model establishment(P<0.05),which was activated at 8 weeks(P<0.05).The activity of upstream kinase of AMPK,LKB1 was gradually increased at 4 weeks(P<0.05)and at 6 weeks(P<0.01)and at 8 weeks(P<0.01)after model establishment,and the expression of CaMKK? was gradually increased at 6 weeks(P<0.05)and at 8 weeks(P<0.01),there was no change in TAK1 activity.(2)AMPK activity in rat gastric smooth muscle cells increased with time prolonged under high glucose condition(P<0.01),but the increase was not obvious compared with under normal glucose condition(P<0.05).Under high glucose condition,the intracellular free Ca2+was increased(P<0.05);the apoptosis of rat gastric smooth muscle cells was increased after the cells were treated with AMPK agonist(P<0.05),whereas the apoptosis was significantly decrease after treatment with AMPK inhibitor(P<0.01).(3)During the early stage with high glucose,AMPK was the main promotion factor of mitochondrial metabolism pathway,but did not increase the ATP production,AMPK also promoted glycolysis pathway,but it is only one of the factors that promote glycolytic pathway.During the late stage with high glucose,AMPK is a major inhibitor of mitochondrial pathway,and still played a role in promoting glycolytic pathway which acted as the main regulator.(4)Results from AMPK Signaling Phospho-Antibody Array found that a total of 73 differentially expressed proteins were identified,of which,30 phosphorylated antibody were identified.After removing non-specific expression changes,there were 18 phosphorylated antibody(such as p53,AKT,4E-BP1,p70S6K,CaMK?,CaMK ?,PLC-?3,PKA,ACC1,and eEF2)were identified as differentially expressed.(5)The expression of p53 was increased in high glucose 48h group(P<0.01),after silencing AMPK it was decreased(P<0.001).The microarray found that activated AMPK could increased p53Ser315 phosphorylation and decreased p53Ser392 phosphorylation.The activity of Akt was decreased in high glucose 48h group(P<0.001),after silencing AMPK it was increased(P<0.001).The microarray found that activated AMPK could change Aktl Ser124?Akt1S1Thr246?AktTyr326?Akt Ser473 phosphorylation.The activity of p70S6K was decreased in high glucose 48h group(P<0.001),after silencing AMPK it was increased(P<0.001).The microarray found that activated AMPK could inhibit p70S6KThr389 phosphorylation and p70S6KSer411 phosphorylation.The activity of PKA was increased in high glucose 48h group(P<0.01),after silencing AMPK it had an decreasing trend but it was not obvious.The microarray found that activated AMPK could increase PKA-R2BSer113 phosphorylation.The expression of BAX and Caspase-3 was increased in the high glucose 48h group(P<0.05),after silencing AMPK it was unchanged.The expression of Bcl-2 was decreased in high glucose 48h group(P<0.001),after silencing AMPK it was increased(P<0.001).(6)The expression of 4E-BP1 was unchanged.The microarray found that activated AMPK could increase 4E-BP1 Thr70 and 4E-BP1 Ser65 phosphorylation.(7)The expression of PLC-?3 was decreased in high glucose 48h group(P<0.001),after silencing AMPK it had an increasing trend but it was not obvious.The microarray found that activated AMPK could increase PLC? Ser1105 phosphorylation.The activity of CaMK? was increased in high glucose 48h group(P<0.001),after silencing AMPK it was decreased(P<0.001).The microarray found that activated AMPK could increase CaMK II Thr305 phosphorylation.The activity of CaMK? was increased in high glucose 48h group(P<0.001),after silencing AMPK it was unchanged.The microarray found that activated AMPK could increase CaMK?Thr196/200 phosphorylation.(8)The expression of eEF2 was increased in high glucose 48h group(P<0.01),after silencing AMPK it was unchanged.The microarray found that activated AMPK could decrease eEF2Thr56 phosphorylation and increase eEF2KSer366 phosphorylation.The expression of ACC1 was increased in high glucose 48h group(P<0.001),after silencing AMPK it was decreased(P<0.01).The microarray found that activated AMPK could decrease ACCSer79 and ACCSer80 phosphorylation.Conclusion1.There were apoptosis and energy metabolism in rat gastric smooth muscle tissue during the development of DGP,and AMPK was activated.2.Activated AMPK promoted apoptosis of rat gastric smooth muscle cells in high glucose state,and the effect was more obvious with time,and the mechanism was that activated AMPK regulated the expression and activity of p53,Akt,p70S6K,PKA,PLC-?3,CaMKII,CaMKIV and 4E-BP1 in rat gastric smooth muscle cells.3.Activated AMPK first promoted the mitochondrial metabolic pathway then inhibited this pathway,and always promoted the glycolysis pathway in the rat gastric smooth muscle cells in high glucose state and the mechanism was that activated AMPK regulates the expression and activity of ACC1,p53,PKA,PLC-?3,CaMKII,CaMKIV and eEF2 in rat gastric smooth muscle cells.