Influencing Factors And Mechanisms Of The Changes In Islet Function During Pregnancy

Part 1 Effect of artemisinin on improving pancreatic islet function in pregnant rats fed with high-fat dietPurpose:The current high incidence of gestational diabetes mellitus is related to the lifestyle of high-fat diet,maternal obesity and older pregnancy.It is the result of genetic changes and acquired environmental factors.However,the specific impact mode and molecular mechanism need to be further clarified.Whether artemisinin can improve the function of overloaded islets and its mechanism are still unclear.The purpose of this study was to investigate the effects of high-fat diet,pregnancy and high-fat diet pregnancy on the function of pancreatic islet beta cells in female rats and to clarify the molecular basis of the changes.To further explore the mechanism of artemisinin in improving the function of pancreatic islet beta cells in high-fat diet and pregnant rats,and to provide some clues and basis for the future study of the proliferation and transdifferentiation of gestational diabetes mellitus and beta cells.Method:Female SD rats were fed with common diet,high-fat diet,high-fat diet and artemisinin water for 21 days for 12 weeks,and then were fed with 1:1 female and male cages to establish pregnancy models under different dietary intervention.They were divided into six groups:ODC,HFC,ARTHFC,ODP,HFP and ARTHFP.In vivo glucose tolerance test was performed by intragastric administration in the second and third trimesters of pregnancy.Serum insulin level was measured by ELISA.Islets were extracted by common bile duct puncture.Islets were stimulated by high and low glucose in vitro.The expression of islet-related transcription factors was measured by QT-PCR.The morphological changes of islets were observed by histopathology(immunohistochemistry or immunofluorescence).Changes in proinsulin and insulin levels,expression of transcription factor proteins related to islet proliferation and processed shear proteins were detected by western-blot test.To further analyze the mechanism of high-fat diet,pregnancy and artemisinin in the changes of islet morphology and function.Results:Female 21-day weaned SD rats were treated with different diets and artemisinin.(1)During the feeding process,the body weight of the high-fat group grew fastest,while that of the high-fat+artemisinin group grew slowly,similar to that of the general diet group.Fasting blood glucose in the high-fat+artemisinin group increased first and then decreased,peaking at the fourth week.The blood sugar in hyperlipidemia group increased gradually and stabilized for 8-12 weeks.There was no significant change in blood sugar in the general diet group.At 12 weeks of feeding,the body weight of high fat diet group(265.90+9.92g)was 20%higher than that of normal diet group(215.11+19.25g)and high fat+artemisinin group(207.25+15.99g)(23.7%and 28.3%respectively).There was no significant difference in fasting blood glucose among the three groups.(2)After cage placement,before execution in the second and third trimesters of pregnancy:(1)Weight:Compared with ODC and HFP,HFC gained weight(P<0.01).ODP was 58.28%heavier than ODC,HFP 54.14%heavier than HFC,and ARTHFP 10.41%heavier than ARTHFC.(2)glucose:Compared with ODC and ARTHFC,HFC increased fasting blood glucose(P<0.01).The fasting blood sugar in each pregnancy group was lower than that in the control group(P<0.01).(3)OGTT test in vivo:(1)Blood glucose:Glucose tolerance in high fat group was lower than that in common diet group and artemisinin group.(2)Area under blood sugar curve:HFC is higher than ODC and ARTHFC(HFC:44.03±1.85 vs ODC:35.26±0.85?ARTHFC:32.36±0.92)(P<0.05);HFP is higher than ODP and ARTHFP(HFP:34.70±1.43 vs ODP:23.63±0.71?ARTHFP:22.69±0.81)(P<0.01).There was no difference between general diet and artemisinin group.The area under the glucose tolerance curve in the control group was higher than that in the pregnant group,regardless of diet and drug intervention.(ODC:35.26±0.85 vs ODP:23.63±0.71;HFC:44.03±1.85 vs HFP:34.70±1.43;ARTHFC:32.36±0.92 vs ARTHFP:22.69±0.81)(P<0.01).(3)insulin:The peak points of ODC and ODP were all 30 minutes.Compared with ODC,insulin levels of ODP at 30minutes and 60 minutes increased(P<0.05).The peak release delay of HFC was 60min,the peak of HFP was 30 min,and the insulin level of HFP at 0 min was significantly higher than that of HFC(P<0.05).The peaks of OGTT insulin levels in ARTHFC and ARTHFP were all at 30 minutes.Compared with ARTHFC,the insulin levels in ARTHFP at 0,30,60 and 120 minutes increased(30min P<0.01;Surplus P<0.05).(4)Isolated islet GSIS test:(1)Supernatant insulin results:Insulin secretion was stimulated by 2.8 mM glucose:HFP and ARTHFP were higher than ODC(P<0.05);HFP was higher than HFC.16.7mM glucose stimulated insulin secretion:ARTHFC and ARTHFP were higher than HFC(P<0.05,P<0.01);ARTHFP was higher than HFP(P<0.05).Insulin secretion before and after 2.8 mM,16.7 mM glucose stimulation:The insulin secretion level after 16.7 mM stimulation in ODC,ODP,ARTHFC and ARTHFP groups was significantly higher than that after 2.8 mM stimulation(P<0.05,P<0.01;P<0.05);there was no significant difference in the high-fat group before and after stimulation.(2)Insulin in islets:ARTHFC,ARTHFP were higher than ODC and HFC(P<0.05,P<0.01;P<0.05);compared with other five groups,the insulin level of HFP was significantly higher(P<0.01).(5)Western blot detected the changes of insulin and proinsulin in islets:ODP was higher than ODC,and the ratio of proinsulin to proinsulin+insulin in HFP was higher than that in HFC(P<0.05).However,ARTHFP did not change significantly compared with ARTHFC(ODC:11.8±0.9%vs ODP:26.7±0.8%,P<0.05;HFC:32.9±1.5%vs HFP:47.1±0.6%,P<0.05;ARTHFC:10.1±0.5%vs ARTHFP:16.4%±0.7%,P>0.05)?Compared with ODP,ARTHFP,the proportions of proinsulin in islets of HFP were higher(P<0.05),compared with ODC?ARTHFC,the proportions of proinsulin in islets of HFC were higher(P<0.05).(6)The expression of ALDH1a3,PAX4,ARX and PC1/3 protein in islets:(1)ALDH1a3 protein level:ODP was higher than ODC and HFP was higher than HFC(P<0.01).ARTHFP had no significant change compared with ARTHFC.Compared with ODP and ARTHFP,the level of ALDH1a3protein in HFP was higher(P<0.05).(2)PAX4 protein level:ODP was higher than ODC(P<0.01);HFP was higher than HFC,and ARTHFP had no significant change compared with ARTHFC.The level of PAX4 protein in ODP was higher than that in HFP and ARTHFP(P<0.05).(3)ARX protein level:There was no difference between groups and within groups(P>0.05).(4)PC1/3 protein level:Pregnancy and high fat diet could decrease PC1/3 protein level compared with normal control group(P<0.01).The expression of PC1/3 increased after high-fat diet combined with artemisinin(ARTHFC),which was similar to that in ODC group(P>0.05).The expression level of PC1/3 in hyperlipidemic pregnancy increased after artemisinin(ARTHFP)was administered at the same time,which was significantly different from that of ODP and HFP(P<0.01).(7)Histology of pancreas:(1)Compared with the control group,the area of pancreas islets in pregnancy and the average cross-sectional area of pancreas islets increased in different degrees.The area of pancreas islets in high-fat diet was larger than that in common diet,and the area of islets decreased after artemisinin intervention in high-fat diet model.Pancreatic islets account for the size of the pancreas(%):ODC:1.20±0.45%vs ODP:2.26±0.51%;HFC:1.72±0.57%vs HFP:2.56±0.19%;ARTHFC:1.60±0.48%vs ARTHFP:2.26±0.32%;Average cross-sectional area of islets in different groups(×10~6?m~2):ODC(0.35±0.24)vs ODP(3.52±1.52);HFC(3.01±2.35)vs HFP(4.18±1.87);ARTHFC(0.91±0.74)vs ARTHFP(3.04±1.57).(2)HE staining showed that intra-islet vasodilation could be seen in high-fat diet,pregnancy and high-fat pregnancy group,and the intra-islet and peripheral vasodilation could be further increased after artemisinin intervention.(3)Immunofluorescence staining showed that pregnancy and high fat diet promoted islet enlargement mainly through the compensatory increase in the number of beta cells.Beta cells account for the area of islets(%):ODC:80.22±5.11%vs ODP:91.85±6.94%;HFC90.23±4.06%vs HFP:96.03±1.11%;ARTHFC:85.34±5.02%vs ARTHFP:93.77±1.40%.Beta cells account for the area of islets was increased in HFC,compared with ODC and ARTHFC(P<0.05).ODP increased significantly compared with ODC,ARTHFP increased significantly compared with ARTHFC(P<0.05).(4)Number of Ki67+beta cells/islets:HFC is higher than ODC and ARTHFC(P<0.05).ODP is higher than ODC(P<0.01).ARTHFP?HFP is higher than ARTHFC?HFC(P<0.05).ODC:1.06±0.57 vs ODP:2.31±0.36;HFC1.99±1.18 vs HFP:2.34±0.74;ARTHFC:1.23±0.60 vs ARTHFP:2.21±0.57.Furthermore,high fat and pregnancy promote the proliferation of pancreatic beta cells.Artemisinin has a certain effect on alleviating the compensation of beta cell overload.(8)Changes of PAX6,Foxo1 and PDX1 genes in islets:(1)PAX6:The expression of PAX6 gene in ODP was lower than that in ODC,and the expression of HFP was higher than that in HFC(P<0.01).HFC and ARTHFC were lower than ODC,there were significant statistical differences in the increase of HFP compared with ARTHFP and ODP(P<0.01).(2)Foxo1:The expression of Foxo1 gene in HFP was higher than that in HFC,while that in HFC was lower than that in ODC(P<0.01).(3)PDX1:The expression of PDX1 in HFP was higher than that of ODC,ODP and HFC(P<0.01).Conclusion:(1)The maternal islet load increased,islet proliferation,mainly islet beta cells,compensatory physiological insulin secretion increased,so that fasting blood glucose and blood sugar AUC decreased.But its insulin synthesis reserve and secretion capacity are good.The expression of ALDH1a3 and PAX4 protein in normal pregnant islets increased under normal diet.(2)High-fat diet aggravated islet load in female SD rats,proliferation of islet beta cells,increase of proinsulin,obstruction of insulin processing and shearing process,insulin secretion response to high-glucose stimulation was weakened in vitro,and insulin release was delayed.The fasting blood glucose and AUC in high-fat diet group were higher than those in general diet group.High fat diet inhibited the expression of Foxo1 and PAX6 in female rats.(3)Under the effect of high-fat diet and pregnancy,the load of islets and proinsulin increased further,and the fasting insulin level and fasting blood sugar were higher than those of pregnant women.ALDH1a3 transdifferentiation and expression of PAX6,Foxo1 and PDX1 in pancreatic islets of high-fat pregnant rats increased.(4)High-fat diet in female rats with artesunate intervention reduced islets,decreased the number of beta cells,and improved islet microcirculation;reduced proinsulin/insulin ratio,through PC1/3 increase to improve insulin processing shear process.The function of islet was restored.Part II:Study on the role of gastrointestinal hormones in islet function of gestational diabetes mellitus womenObjective:As a polygenic hereditary disease,type 2 diabetes mellitus has certain family and hereditary tendencies.Gestational diabetes mellitus accounts for about90%of gestational hyperglycemia,which is becoming more and more common in clinic.In this study,blood samples were collected from mild gestational diabetes mellitus?normal pregnancy?new type 2 diabetes mellitus and normal women,to further clarify the changes of proinsulin in gestational and gestational diabetes mellitus,and the role and correlation of gastrointestinal hormones in them.It will provide a certain basis for the study of the pathogenesis of gestational diabetes mellitus in the future.Methods:According to the diagnostic and exclusive criteria of gestational diabetes mellitus and type 2 diabetes mellitus,blood samples of glucose tolerance(gestational0 min,60 min,120 min or non-gestational 0 min,30 min,60 min,120 min,180 min)were collected,and blood samples of normal pregnancy and normal control women(conditionally matched)were collected.Glucose was measured by hexokinase method,insulin and C-peptide by chemiluminescence method,and pro-insulin concentration by chemiluminescence immunosandwich method.The levels of glucagon,pancreatic polypeptide,growth hormone-releasing peptide,glucose-dependent insulin-releasing peptide and glucagon-like peptide 1 in blood samples of each group were measured by milliplex MAP Luminose magnetic bead panel technique for 0 min and 120 min.Statistical analysis was conducted to compare the differences of indicators in each group.Result:(1)Glucose:Compared with NP group,the blood sugar of DMG group increased at 0 min,60 min and 120 min(P<0.01).The blood sugar in DM2 group was higher than that in NC group(P<0.01).(2)Insulin and C-peptide:The peak of insulin release in DMG,NP,DM2 and NC groups was 60 min,60 min,120 min and30 min,respectively.The insulin level of DMG was higher than that of NP group at 0min,60 min and 120 min(P<0.05).The insulin level in DMG group was significantly higher than that in DM2 group at 60 minutes(P<0.05).The fasting insulin levels of NP were lower than those of NC,DM2 and DMG(P<0.05).There was significant difference in fasting C-peptide between the four groups(P<0.01).(3)Proinsulin:The peak of proinsulin release was 30 minutes in NC group,120 minutes in DMG group,NP group and DM2 group.Compared with NC,NP and DMG group,the peak of proinsulin release was 0 minutes,60 minutes and 120 minutes in DM2 group(P<0.05,P<0.05,P<0.01),and the release was delayed.There was no significant difference in proinsulin level between DMG group and NP group(P>0.05),but fasting insulin had an increasing trend.(4)Gastrointestinal related hormones:(1)Glucagon:Glucagon in DM2 group was higher than that in NP,DMG group at 0 min and 120 min(P<0.05);Glucagon in NP group was lower than that in NC group at 0 min and 120min(P<0.05).(2)Pancreatic polypeptide:P P levels in fasting pregnancy and 120minutes were lower than those in normal non-pregnant women and type 2 diabetes mellitus(P<0.01).The level of fasting PP in type 2 diabetes mellitus was lower than that in normal group(P<0.05),but the secretion rhythm of PP in type 2 diabetes mellitus still existed after glucose stimulation.There was no significant difference in PP level between gestational diabetes mellitus and normal pregnancy(P>0.05).(3)Ghrelin:DMG decreased at 0 min and 120 min compared with NP and NC groups(P<0.01).The Ghrelin of DM2 group decreased at 0 min and 120 min compared with NC group(P<0.01).The Ghrelin of normal subjects increased at 120 min after glucose stimulation,and the rhythm of gestation and type 2 diabetes mellitus lost.(4)GIP:The level of fasting GIP in pregnancy was lower than that in normal women and type 2 diabetes mellitus(P<0.05).There was no significant difference in fasting GIP between DMG and NP group,or DM2 and NC group(P>0.05).After glucose stimulation,GIP levels in each group were higher than those in fasting group for 120minutes.Compared with other groups,GIP level decreased only in 120 min of normal pregnancy(P<0.01).(5)GLP-1:There was no significant difference between 4groups at 0 min and 120 min(P>0.05).(5)Correlation analysis of gastrointestinal hormones and proinsulin in pregnant women:Proinsulin 0 min was positively correlated with GLP-1 0 min in gestational diabetes mellitus group and Proinsulin120 min was positively correlated with GLP-1 120 min,r=0.909(P=0.012),r=0.874(P=0.023),respectively.Conclusion:(1)Pregnancy aggravates islet load and delays the release of insulin and proinsulin.Insulin levels in mild GDM were higher than those in normal pregnancy after fasting and glucose stimulation,but glucagon did not play a key role in mild GDM.(2)Ghrelin plays a certain role in pregnancy(especially gestational diabetes mellitus)and newly developed type 2 diabetes mellitus.GLP-1 is associated with proinsulin in mild gestational diabetes mellitus.GIP may play a role in glycometabolism during pregnancy.Full text conclusion:(1)Pregnancy and high-fat diet both aggravate islet load and delay the release of insulin and proinsulin,but the mechanisms are different.(2)The expression of ALDH1a3 and PAX4 protein in normal pregnant islets increased under normal diet;the high-fat diet inhibits the expression of Foxo1 and PAX6 in female rats;ALDH1a3 transdifferentiation and expression of PAX6,Foxo1 and PDX1 in pancreatic islets of high-fat pregnant rats increased.(3)Artemisinin can reduce the ratio of proinsulin to insulin by PC1/3 and improve islet microcirculation to restore islet function.(4)There was no significant change in proinsulin level in mild gestational diabetes mellitus women compared with normal pregnancy.Insulin level increased after fasting and glucose stimulation.GIP and Ghrelin play a role in the functional adaptation of islets during pregnancy,while Ghrelin and GLP-1 play a role in mild gestational diabetes mellitus.