The Effect Of Long-term Exposure Under Low-level Nonylphenol On Glucose Metabolism In Rats

It was reported that exposed Environmental endocrine disruptors could induced glucose and lipid metabolism disorders,which increased the risk of metabolic syndrome such as type 2 diabetes and obesity.Nonylphenol,one of the typical EEDs,can be absorbed by organism through the gastrointestinal tract and skin contact,and it accumulates with blood to various tissues and organs.NP could be detected in Chinese,American and Germany’s urine.In vitro tests showed that:NP could lead to secretory dysfunction of beta cells.Animal experiments proved that exposed to NP during perinatal period could lead to the typical symptoms of metabolic syndrome in F1 and F2 progeny.Although the relevant studies have confirmed that NP would have a harmful effect on the organism,and the doses were far higher than the actual exposure doses of NP in the environment population.In order to approach the environmental exposure of NP in general population,and further explore whether NP has low dose effect,the same concentration of environmental concentration is designed for NP dosage.It is set to three,0.02,0.2,2 g/kg/day,respectively.In this study,the main pathway of NP exposure was simulated by gavage,and a long-term low concentration of NP exposure was performed to establish animal models.We investigated preliminarily whether long-term exposure to low concentration of NP effects the function of pancreatic sugar metabolism and its possible mechanism.High-fat diet is one of the critical factors that can lead to insulin resistance.This experiment also explored whether different doses of NP exposure could promote the effects of high-fat diet on glucose metabolism disorders.Part 1 The effect of long-term exposure under low-nonylphenol on glucose metabolism in ratsObjective:Constructing an animal model of Sprague-Dawley rats and exploring the effects of low concentration NP long-trem exposure on the glucose metabolism of rat pancreas.Methods:Eighty Sprague-Dawley rats(150±10)g with half males and half females were assigned into 4 groups(n=20 per group),and each group was treated with a specific testing sample for 180 days.The first group was fed a normal diet and received gavage with groundnut oil alone,and served as the normal-diet(ND)control(C);the second,third and fourth groups were fed a normal diet and were gavaged with NP,which was dissolved in groundnut oil,at a dose level of 0.02 μg/kg/day(L),0.2 μg/kg/day(M)or 2 μg/kg/day(H),respectively.The rats fasted before gavaging with 5 ml/kg in every morning.The rats were selected for oral glucose tolerance test(OGTT)at the 90 th day,and rats were killed after 180 days.Body weight and fasting blood glucose levels(FBG)was detected regularly.Serum insulin and serum leptin were analyzed byenzyme-linked immune sorbent assay(ELISA)kit.The histopathologic change of rat pancreatic tissue were observed.The NP concentrations of serum and pancreas in rats were tested by high performance liquid chromatography(HPLC).The expressions of key genes and proteins,Glucokinase,uncoupling protein uncoupling protein-2,Glucose transporter-2,were detected by reverse transcription-quantitative polymerase chain reaction(RT-q PCR)and Western Blot.Results: 1.Compared with the control group,NP exposure could lead to a decrease in pancreatic mass(F=109.905,P<0.001)and a decrease in pancreatic coefficient(F=136.200,P<0.001).There was a dose-effect relationship between NP exposure and pancreas weight and coefficient.2.Compared with the control group,NP exposure could lead to increase fasting blood glucose levels of rats(F90d=189.311,P90d<0.001;F180d=160.785,P180d<0.001),There was a dose-effect relationship between NP exposure and the fasting blood glucose level.NP exposure decreased fasting insulin levels(F=100.89,P<0.001).There was a dose-effect relationship between NP exposure and the fasting insulin levels.Compared with the control group,the levels of leptin in the rats exposed to NP increased(F=76.628,P<0.001).3.Compared with the control group,exposure to NP for 90 days caused oral glucose intolerance abnormal in rats and the area under the OGTT curve increased(F=361.423,P<0.001).There was a dose-effect relationship between NP exposure and the area under curve of OGTT(OGTT-AUC).4.Compared with the control group,exposure to NP caused an increase in insulin resistance index(F=196.857,P<0.001)and insulin sensitivity index(F=199.032,P<0.001)at 90 day.5.Compared with the control group,the NP concentration in serum and pancreas increased(Fserum90d=322.289,P<0.001,Fserum180d=396.446,P<0.001,Fpancreas90d=501.091,Ppancreas180d<0.001).There was a dose-effect relationship between the NP concentration in serum and pancreatic tissue and the NP exposure.6.The pathological findings of pancreas showed:compared with the control group,there were pathological changes of edema,necrosis and inflammatory cell infiltration in the pancreas tissue of NP exposed group.In the same field of vision,the number of insulin cells decreased and the volume of islets decreased in the middle and high dose groups.7.RT-q PCR’s results showed that: compared with the control group,NP exposure induced relative gene expression of pancreatic glucokinase(GK)and glucose transporter 2(GLUT-2)downregulation(FGLUT-2=245.555,P GLUT-2<0.001;FGK = 131.434,PGK<0.001),and the relative gene expression of GK and GLUT-2 were decreased with the increase of exposure dose of NP.Compared with the control group,NP exposure induced pancreatic uncoupling protein 2(UCP-2)gene relative expression upregulation(F=109.761,P < 0.001).8.WB’s result showed that: compared with the control group,NP exposure could induce the peotein relative expression of GK,GLUT-2 and UCP-2 down regulation(FGK=240.362,PGK<0.001;FGLUT-2=360.413,PGLUT-2<0.001;FUCP-2=103.312,PUCP-2<0.001).Conclusion:1.Low concentration of NP(0.2,2 g/kg/day)can accumulate in serum and pancreatic tissue after long-term exposure,resulting in pathological damage of pancreas tissue,inducing glucose tolerance in rats,causing insulin resistance and inducing glucose metabolism disorder in rats.2.The expression levels of key genes and proteins such as UCP-2,GK and GLUT-2 in pancreas were abnormal after exposure to NP(0.2,2 g/kg/day)for a long time,which might be one mechanism of glucose metabolism disorder.Part 2 Nonylphenol promotes the effect on glucose metabolism disorder exposure to high-fat diet in ratsObjective:This study investigated whether there was interaction effect of different doses of NP combined with high-fat diet exposure on glucose metabolism in rats.Methods: One hundred and sixty Sprague-Dawley rats(150±10)g with half males and half females were assigned into 8 groups(n=20 per group),and each group was treated with a specific testing sample for 180 days.The groups were as follows: normal diet control group(group C,corn oil),normal diet with low-dose(group L,0.02 g/kg/day NP),common feed dose group(group M,0.20 g/kg/day NP),normal diet high-dose group(H group,2 g/kg/day NP),high-fat diet control group(group CH,corn oil + high-fat diet),high-fat diet and low-dose group(LH group,0.02 g/kg/day NP+ high-fat diet),high-fat diet group(group MH,0.20 g/kg/day NP+ high-fat diet),high-fat diet and high-dose group(HH group,2 g/kg/day NP+ high-fat diet).The rats fasted for 10 hrs before gavaging of 5 ml/kg testing sample in every morning.Methods of biochemical indexes related to glucose metabolism are the same as the first part.Results: 1.Compared to normal diet control group,body weights showed an increasing trend at 90 day and 180 day(P90 d=0.556,P180 d=0.970),and fast blood glucose levels showed an increas(P90 d<0.001,P180 d<0.001),and oral glucose intolerance was abnormal,and OGTT area under the curve(OGTT-AUC)increased(P<0.001),and IRI was enlarged(P90d<0.001,P180d<0.001),ISI was minished(P90d=0.034,P180d<0.001).2.Compared with the normal feed control group,pancreatic tissue was represented mild edema in high fat diet control.RT-q PCR results showed,compared with the normal feed control group,the relative expression levels of GK and GLUT-2 genes in the pancreas of the high-fat diet control group decreased(PGK<0.001,PGLUT-2<0.001),UCP-2 gene relative expression increased(PUCP-2<0.001).WB results showed,the high-fat diet control group pancreatic GK,GLUT-2 and UCP-2 protein relative expression levels decreased(PGK<0.001,PGLUT-2<0.001,PUCP-2=0.001).3.There was interaction effect on fasting blood glucose levels at day 180 by co-exposure NP and high-fat diet(Flow=14.582,Plow=0.001;Fmedium=4.981,Pmedium=0.032;Fhigh=12.359,Phigh=0.001).There was interaction effect on OGTT-AUC by co-exposure NP and high-fat diet(Flow=34.865,Plow0.001;Fmedium=4.853,Pmedium=0.034;Fhigh=65.007,Phigh<0.001).There was interaction effect on IRI by co-exposure NP and high-fat diet(Flow=31.488,Plow<0.001;Fmedium=49.972,Pmedium<0.001;Fhigh=15.139,Phigh<0.001).There was interaction effect on pancreatic UCP-2 gene expreesion by co-exposure NP and high-fat diet(Flow=54.624,Plow<0.001;Fmedium=45.16,Pmedium<0.001;Fhigh=246.982,Phigh<0.001).There was interaction effect on pancreatic GLUT-2 protein expreesion by co-exposure NP and high-fat diet(Flow=17.137,Plow < 0.001;Fmedium=38.198,Pmedium < 0.001;Fhigh=4.847,Phigh=0.048).4.There was interaction effect on fasting blood glucose levels by co-exposure low-dose NP and high-fat diet(F=8.785,P=0.005).There was interaction effect on insulin resistance index at 90 day by co-exposure low-dose NP and high-fat diet(F=6.429,P= 0.016).5.There was interaction effect on fasting insulin levels by co-exposure medium-dose NP and high-fat diet(F90d=5.521,P90d=0.024;F180d=15.884,P180d<0.001).There was interaction effect on GK gene expreesion and protein expreesion by co-exposure medium-dose NP and high-fat diet(Fgene=10.307,Pgene=0.004;Fpro=14.191,Ppro=0.003).6.There was interaction effect on the gene expressions of pancreatic GK and UCP-2by co-exposure high-dose NP and high-fat diet(FGK=6.629,PGK=0.018;F GLUT-2=7.45,P GLUT-2=0.013).There was interaction effect on the protein expressions of pancreatic GK and UCP-2 by co-exposure high-dose NP and high-fat diet(FGK=4.847,PGK=0.048;FUCP-2=103.780,PUCP-2<0.001).Conclusion:Co-exposure to NP and high-fat diet could increase the risk of glucose metabolism disorder,and the interaction effects of the dose at 0.2 and 2.0 ug/kg/day NP were remarkable among the groups.