| [Background]Diabetes mellitus(DM)is a chronic metabolic disorder characterized by hyperglycemia resulting from the interaction between genetic and environmental factors,and its main pathological features include metabolic disturbance of glucose,fat and protein caused by insulin resistance and defects in insulin secretion.The report from the World Health Organization demonstrated that diabetes mellitus has become the third most common disease following cardiac-cerebral vascular disease and cancer.Insulin resistance(IR)is a pathophysiological state in which a given concentration of insulin produces a less biological effect in target tissues such as liver,skeletal muscle and fat due to their reduced sensitivity to insulin,which is manifested by reduction in glucose uptake and glycogen synthesis as well as lower ability to maintain normal blood glucose levels.Insulin resistance is one of important causes of type 2 diabetes and its accompanying pathophysiological state as well.With the rapid development of global industry,chemicals are widely used since the 1950s,with higher increase in both category and amount,accompanying this increase is growing incidence of diabetes mellitus,therefore,the relationship between chemicals in environment and diabetes,insulin resistance attracts widespread attention of researchers worldwide.Lee et al.report that persistent organic pollutants in serum,including organochlorine pesticides,polychlorinated biphenyls(PCBs)and so on,are strongly associated with diabetes epidemic in the US general population.Epidemiological studies carried out by Fierens et al.found that persistent organic pollutants included PCB77,PCB81,PCB126,PCB169 and other PCBs congeners that were highly accumulated in the body of type 2 diabetic patients from the US,Europe and Asia.The National Toxicology Program(NTP)organized a workshop in January 2011 titled "Role of Environmental Chemicals in the Development of Diabetes and Obesity".The objective of this workshop was to examine the literature for evidence of associations between certain chemicals and obesity or diabetes.Research recommendations were given by the workshop.A few research groups working on this issues at the early stage of the study.PCBs,also known as chlorinated biphenyl,are synthetic organic chemical compound in which chlorine atoms replace the hydrogen atoms on benzene ring,including 209 congeners.PCBs which are one of important endocrine disruptors(EDs)are considered a potential chemical causative factor contributing to type 2 diabetes and insulin resistance because of its lipid solubility,strong endocrine disrupting nature and other properties.However,there is still lack of experimental evidence that may prove the casual correlation between type 2 diabetes and PCBs exposure,although a close link between them has been observed in epidemiological studies.Intake of high fat diet is the main route of exposure to PCBs in human,which tends to co-occur with PCB exposure.High-fat diet-induced metabolic abnormalities and insulin resistance have been widely reported,however,experimental studies on metabolic disturbulance caused by persistent exposure to low dose of PCBs are still rare,specially the exploration of metabolic abnormalities resulting from combined exposure to high-fat diet and PCBs is still in underway.In this study,an attempt has been made to initially identify whether exposure to PCBs or high fat diet alone or in combination led to insulin resistance in SD rats and its possible mechanisms by simulation of long-term combined exposure.Aroclor 1254,a PCB congener with 54%chlorine,is one of the most widely used commercial polychlorinated biphenyl(PCB)mixture.On the basis of experimental data about Aroclor 1254 reported in the literatures and pilot experiment results of this study,male SD rats in two groups were given daily Aroclor1254 1.0mg/kg or 0.2mg/kg by oral gavage,respectively,while another two groups were given daily Aroclor1254 1.0mg/kg or 0.2mg/kg,respectively,in combination with high-fat diet,and compared with the control group and the high-fat diet group.In this study,Part1 was designed to initially assess the effect of exposure to PCBs and high fat diet alone or in combination on glucose and fat metabolism in SD rats by measuring changes in body weight,body composition,glucose tolerance test,insulin tolerance test,blood biochemistry,pathology and other indicators;Part 2 was designed to explore the mechanisms responsible for the effect of exposure to PCBs and high fat diet alone in combination on metabolism,to identify its effect on IRS2,PI3K,AKT,FOXol and other protein which were essential to PI3K/Akt,the insulin signaling pathway in liver of rats;Part 3 was designed to determine changes in amino acid,carbohydrates,inorganic salt and other major small molecules which were capable of directly reflecting in vivo biochemical changes in urinary metabolome of rats after exposure to PCBs and high fat diet alone in combination using Nuclear Magnetic Resonance(NMR)based metabolomics.Part 1 Insulin Resistance Induced by Combined Exposure to PCBs and High Fat Diet in SD Rats[Objective]After a 10-week exposure to PCBs and high fat diet alone or in combination,changes in body weight,body composition,glucose tolerance,insulin tolerance,hematology,blood biochemistry,pathology were measured in SD rats for a preliminary assessment of the effect of exposure to assess the alterations of glucose and fat metabolism,discuss the possible mechanisms and causes of the changes.[Methods]1.In this study,male SD rats were enrolled and exposed to Aroclor 1254 containing 54%chlorine that was PCB congeners mixture at low dose for 10 weeks.The rats were randomly divided into six groups(n=5 in each group):the control group,high fat diet(HFD)group,Aroclor1254 1.0mg group,Aroclor1254 0.2mg group,Aroclor1254 1.0mg + HFD group and Aroclor1254 0.2mg + HFD group.High fat diet was composed of 70%basal diet,20%lard,5%cholesterol and 5%sucrose.2.Body composition was measured with Echo MRI 700 body composition analysis device before rats were sacrificed..3.Glucose tolerance test was performed a week prior to sacrifice.Rats were fasted for 12 hours before the experiment,and then given 2g/kg glucose solution by oral gavage.Glucose levels in rats were determined before(fasting)and 30,60,90,120 and 180 min after gavage of glucose solution using Roche blood glucose meter.3 days later,insulin tolerance test was performed on SD rats that were given intraperitoneal injection of 1 IU/kg insulin,measure blood glucose level before(fasting)and 15,30,45,60,120 and180 min after injection of insulin.4.Blood cell count,leukocyte differential count and major serum biochemical indicators were measured when rats were sacrificed.Major organs were stained with HE and liver tissues were stained with oil red O for pathological examination.[Results]1.Aroclor1254 1.0mg + HFD group and Aroclor1254 0.2mg + HFD group showed a continuous weight gain throughout the experiment,while Aroclor12541.0mg group exhibited a slow weight gain from Week 7 to 10 after treatment and a downward trend in weight by the end of the experiment.2.Fat content in Aroclorl254 1.0mg + HFD group and Aroclor1254 0.2mg +HFD group was significantly increased when compared with the control group(p<0.01),especially a significantly higher level in fat content was observed in Aroclor1254 0.2mg + HFD group than in HFD group(p<0.05).As compared with control group,HFD group showed a slower growth rate of fat than two combined treatment group(p<0.05).In contrast,Aroclor1254 1.0mg group showed significantly lower fat content than HFD group(p<0.01).3.In Aroclor12541.0mg + HFD group,Aroclorl254 0.2mg + HFD group and Aroclor1254 0.2mg group,white blood cell count was significantly increased(p<0.05),when compared with control group.The number of lymphocytes was significantly increased(p<0.05),but the percentage of monocytes was significantly decreased(p<0.05)than in the control group in that three groups.Aroclor 12541.0mg group and Aroclor1254 1.0mg + HFD group showed elevated levels of direct bilirubin(p<0.01)when compared with the control group.Aroclor12541.0mg + HFD group,Aroclor1254 0.2mg + HFD group,HFD group and Aroclor1254 0.2mg group showed significantly lower urea content(p<0.01).ALT and AST levels in the five treatment groups were lower than those in the control group,but no regular trend was observed.Total cholesterol levels in Aroclor1254 1.0mg group were significantly higher than those in the control group(p<0.05).4.In glucose tolerance test,60 min after the gavage with glucose,in Aroclor1254 1.0mg+HFD group(p<0.01),Aroclor1254 0.2mg +HFD group(p<0.05),the level of blood glucose was significantly higher than that of the control group.Afterwards,at the three time points of 90,120 and180min,the blood glucose of the two combined treatment groups was always at a higher level(p<0.01),compared with control group.In Aroclor1254 1.0mg group and HFD group,the blood glucose level was at a relatively high level only at the time point of 90 min.In Aroclor12541.0mg+HFD group and Aroclor1254 0.2mg +HFD group,the AUC value was significantly higher than that of the control group(p<0.01).It is more significant that the AUC value of Aroclor1254 1.0mg+HFD group was significantly higher than that of HFD group(p<0.05).5.In insulin tolerance test,15 min after injection of insulin,the level of blood glucose in Aroclor1254 1.0mg+HFD group was significantly higher than that of the control group(p<0.05).Afterwards,at the five time points of 30,45,60,120 and180min,the blood glucose of the Aroclor12541.0mg+HFD group and Aroclor1254 0.2mg +HFD group were at a significantly higher level(p<0.01)when compared with the control group and the difference was statistically significant.In Aroclor1254 1.0mg group,Aroclor1254 0.2mg and HFD group,the blood glucose levels remained higher at the time points of 30,45,60 min than those in the control group(p<0.05),and the difference was not statistically significant at the time points of 120 and 180 min when compared with control group.In five treatment groups,the AUC value was significantly higher than that of the control group,and the difference was statistically significant(p<0.01).The AUC values of Aroclor1254 1.0mg+HFD group and Aroclor1254 0.2mg +HFD group were significantly higher than the other three treatment groups.6.After exposure to PCBs and high fat diet alone or in combination,SD rats in Aroclorl2541.0mg+HFD group and Aroclor1254 0.2mg+HFD group developed widespread vacuolations structure in hepatocyte,with visible scattered inflammatory infiltration.With oil red O staining,lesions of liver tissues were identified as vacuolar fatty degeneration of the hepatocyte.Aroclor12541.Omg + HFD and Aroclor1254 0.2mg + HFD developed more severe lesion in terms of severity and extent than Aroclor1254 group or high fat diet alone group.It showed no significant pathological changes in the remaining organs.[Conclusion]1.Exposure to PCBs in combination with high fat diet is capable of causing body gain and significant increase in fatty content in SD rats.Fat content was higher in Aroclorl254 0.2mg +HFD group than that in HFD group,indicating that combined exposure to PCBs and high fat diet on fatty metabolism exerted more significant effect than exposure to HFD alone.Significantly elevated blood leukocytes and lymphocytes were observed in rats exposed to PCBs in combination with high fat diet,thus suggesting the occurrence of chronic inflammation in rats.2.Exposure to PCBs in combination with high fat diet is capable of causing hepatic steatosis which is more severe than exposure to PCBs or high fat diet alone in term of lesion degree and extent.Blood biochemical results demonstrate that exposure to PCBs in combination with high fat diet leads to liver dysfunction in SD rats.3.Rats that are exposed to PCBs in combination with high fat diet develop more severe insulin resistance as compared with those treated with PCBs or high fat diet alone.Abnormal lipid metabolism in hepatocyte caused by exposure to PCBs in combination with high fat diet may be associated with insulin resistance,which leads to hepatic steatosis as a result of accumulation of fat in liver cells.Hepatic insulin resistance may further aggravate liver steatosis which contributes to glucose and fat metabolic imbalance,thus ultimately resulting in type 2 diabetes.Part 2 Effect of Exposure to PCBs in Combination with High Fat Diet on Hepatic PI3K/AKT Signaling Pathway in SD Rats[Objective]Liver plays an important role in glucose metabolism.Significant insulin resistance occurred in SD rats after 10-week exposure of PCBs in combination with high-fat diet,as described in Part1.The aim of this section was to initially explore the molecular mechanisms of hepatic IR resulting from exposure to PCBs in combination with high-fat diet by measuring changes in proteins that were essential to PI3K/Akt signaling pathway in hepatocytes.[Methods]Male SD rats were randomly grouped,treated,dissected and biopsized with the same procedures as described in Part1.Western blot was used to detect relative changes in levels of major proteins such as p-IRS-2,PI3K p110?,AKT,p-AktSer473 and p-FoxO1 Ser256 in PI3K/Akt signaling pathway in liver of SD rats.[Results]Compared with control group,p-IRS-2 of Aroclor1254 1.0mg+HFD group(P<0.01),Aroclor1254 0.2mg+HFD group(P<0.01)decreased significantly.Compared with the HFD group,p-IRS-2 of Aroclor1254 1.0mg+HFD group(P<0.01),Aroclorl254 0.2mg+HFD group(P<0.01)in the liver of rats were significantly reduced.Compared with control group,PI3K p110? relative content of HFD group(P>0.05),Aroclor1254 1.0mg group(P>0.05),Aroclor1254 0.2mg group(P>0.05)decreased but has not statistically significant difference.Aroclor12541.0mg+HFD group(P<0.01),Aroclor1254 0.2mg+HFD group(P<0.05)compared with control,the relative contents of PI3K p110? reduced and statistically significant difference.Akt content between different treatment groups had not changed significantly,but the relative contents of p-AktSer473 changes significantly.Compared with the control group,Aroclor1254 1.0mg+HFD group(P<0.05),Aroclor1254 0.2mg+HFD group(P<0.05)in the liver of the rats were significantly reduced,and had statistically significant difference.Compared with the HFD group,in Aroclor1254 1.0mg+HFD group(P<0.05),Aroclor1254 0.2mg+HFD group(P<0.05)the relative contents of p-AktSer473 level decreased significantly,and has a statistically significant difference.The relative contents of p-AktSer473 level in HFD group,Aroclor1254 1.0mg group,Aroclor1254 0.2mg group slightly lower compared with controls,but not statistically significant.Compared with control group,in the HFD group(P<0.05),Aroclor12541.0mg group(P<0.05),Aroclor1254 0.2 mg group(P<0.05),Aroclor12541.Omg+HFD group(P<0.05),p-FoxO1Ser256 levels decreased,Aroclor1254 0.2mg+HFD group (P<0.01)in the liver of rats decreased significantly,and has a statistically significant difference.Compared with the HFD group,Aroclor1254 1.Omg+HFD group(P<0.01),Aroclor1254 0.2mg+HFD group(P<0.05)p-FoxO1Ser256 levels decreased significantly,and has a statistically significant difference.[Conclusion]1.The SD rats in HFD group,Aroclor1254 1.Omg group and Aroclor1254 0.2mg group showed a decrease in relative levels of proteins in p-IRS-2,PI3K p110?,P-AktSer473 and p-FoxO1Ser256 which were major molecules in PI3K/Akt signaling pathway in liver,the level of these proteins in Aroclor1254 1.Omg+HFD group and Aroclor1254 0.2mg+HFD group decreased further compared with PCBs and HFD exposure alone.This implies that exposure to PCBs combined with high fat diet had a more significant impact on the protein in hepatic PI3K/Akt pathway which played a major role in glucose and fat metabolism than exposure to PCBs or high fat diet alone,thus leading to exacerbation of metabolic abnormalities in liver.The effects induced by the exposure could result in protein phosphorylation level and it is non-specific.2.The above findings are consistent with major conclusion as described in sectionl of this study,namely exposure to PCBs combined with high fat diet may exacerbate insulin resistance and fatty degeneration of liver caused by exposure alone.A preliminary interpretation is also given to molecular mechanisms responsible for these pathological changes from the view of PI3K/Akt pathway.Part 3 Metabonomics analysis of effects induced by PCBs and high fat diet combined exposure in SD rats[Objective]NMR(Nuclear Magnetic Resonance(NMR)-based metabonomic analysis was employed to determine the urinary metabolic changes in SD rats after exposure to PCBs and high fat diet alone or in combination,to explore the pathophysiological status in the body combining with changes in animal body composition,blood biochemical test,pathological test,OGTT,IPITT,PI3K/AKT signaling pathway in liver tissue and other indicators.[Methods]1.Male SD rats were randomly grouped and treated with the same procedures as described in Part1.Urine samples were collected from SD rats 24 hour before sacrifice,and samples were stored in-80? refrigerator.2.Urine samples were pre-treated and transferred into NMR tubes,1H NMR spectra of all the urine samples were obtained at 599.69 MHz 1H frequency by using a Varian INOVA 600 NMR spectrometer.After data acquisition,FID signals were converted to NMR spectra via Fourier transformation.The peak of the chemical shift of TSP was used as a reference position and set to 0.3.All NMR spectra were phased and baseline corrected and then data-reduced to 225 integrated regions of equal width(0.04 ppm)corresponding to the region of 80.52 to89.48 using the VNMR 6.1C software package(Varian,Inc).SIMCA-P + software was used for principal component analysis(PCA).Analytical results were expressed as scores plot and loadings plot.[Results]1.A multivariate statistical analysis was performed on the 1H NMR peak integrated value of urine of SD rats of 6 groups,and the PCA score plot indicated that,in the control group,HFD group,Aroclor 1254 1.0mg group,Aroclor12540.2mg group and Aroclor1254 0.2mg+HFD group,there was the phenomena of partial intersection and overlapping,demonstrating that the difference of metabolites in urine after corresponding treatment given to 5 groups of rats was relatively small.Aroclor1254 1.Omg+HFD group was far from other 5 groups with a few overlapped parts,indicating that the difference between Aroclor1254 1.Omg+HFD and other 5 groups in terms of metabolic characteristics of animals urine was relatively high.The PCA score plot was made between each 5 treatment groups and the control group respectively indicated that changes occurred in the metabolic features of rats urine in 5 treatment groups.2.Aroclor1254 1.0mg + HFD group and Aroclor1254 0.2mg + HFD group showed an increase in lactate,glucose,succinate,2-hydroxy-isovaleric acid and hippuric acid but a decrease in citrate,taurine,?-ketoglutarate and creatinine in urine when compared with the control group.[Conclusion]1.After a 10-week exposure to PCBs and high fat diet alone or in combination,metabolic changes were observed in SD rats.Especially Aroclor1254 1.0mg + HFD group showed significant changes in metabolic feature,which were manifested by abnormalities in the content of tricarboxylic acid cycle intermediates such as succinate and citrate,etc.in urine,or elevated level caused by accumulation of energy-related metabolites.2.Considering the role of differential metabolites in cells,these changes may result from injury of mitochondrial function and dysregulation of energy metabolism.Tricarboxylic acid cycle is the most effective way by which the body gets its energy via oxidization of glucose and other substances as well as the common pathway for complete oxidative decomposition of glucose,of protein and fat.Hence,abnormalities in tricarboxylic acid cycle and energy-related metabolite accumulation induced by exposure to PCBs in Combination with high fat diet may exacerbate metabolic syndrome of glucose,fat and other nutrients,thus contributing to development of type 2 diabetes,metabolic syndrome,insulin resistance.The altered metabolites may be considered biomarker candidates of the toxicity induced by exposure of PCBs and high fat diet. |
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