| Early life exposures such as fetal malnutrition increase the incidence of obesity,type 2 diabetes and other metabolic diseases in the future.Low birth weight is an early manifestation of intrauterine malnutrition and intrauterine growth restriction.Epidemiological studies based on the Dutch famine population in 1944 and the offspring born in the famine years(1959-1961)in China show that the susceptibility of low birth weight individuals to obesity and type 2 diabetes increases significantly in adulthood.Studies believe that in the context of low birth weight,combined with long-term high-calorie diet,insulin resistance occurs,and the risk of obesity,diabetes and other metabolic diseases increases,namely the "health and disease developmental origin" hypothesis.However,the impact of low birth weight on adulthood insulin resistance and its molecular mechanisms remain unclear.Adipose tissue acts as the largest reservoir for energy storage,and dysfunctional adipose tissue plays a crucial role in the development of insulin resistance.When nutrition is in exceess,lipid storage capacity is reduced due to impaired nutrient buffering capacity of dysfunctional adipose tissue.Lipid is accumulated in the muscles and liver,leading to ectopic fat deposition.This lipotoxicity is an important cause of systemic insulin resistance.One Study had demonstrated that low birth weight mice completed catch-up growth at 3weeks of age and showed increased lipogenic gene expression and adipocyte size in adipose tissue.However,the role and molecular mechanisms of adipose tissue in low birth weight leading to insulin resistance in adulthood have not been explored.This study first observed the relationship between birth weight and overweight and obesity in children and adolescents aged 2-17 years.Then,a low birth weight mouse model was established by prenatal malnutrition method.At the same time,they were fed with high-fat diet to simulate the high-calorie diet pattern of modern society,further confirming the influence of early intrauterine nutrition on adult metabolism.Finally,proteomic techniques were used to study the changes of protein profiles in white adipose tissue of low birth weight mice exposed to high fat diet,and PRM and Western bolt techniques were used to further verify the specific changes of PPARγ-related signaling pathways in adipose tissue.It will provide a new intervention target and theoretical basis for the prevention and treatment of type 2 diabetes mellitus by elucidating the role and molecular mechanism of white adipose tissue in adult insulin resistance caused by low birth weight,Part one Association between birth weight and overweight and obesity in children and adolescentsObjective: To investigate the association between different levels of birth weight and the risk of overweight and obesity in children and adolescents aged 2-17 years.Methods: A total of 1011 children and adolescents aged 2-17 who were admitted to our hospital from October 2020 to October 2021 were included.The basic information was collected,including name,sex,age(accurate to month),height and weight,birth history,birth weight,and residential areas,etc.Overweight and obesity were defined for children and adolescents with body mass index above the 85 th and 95 th percentiles for specific sex and age,and analyzed stratified by age,birth weight and area of residence.The ages are divided into three categories: 2-6 years,7-13 years and 14-17 years.The odds ratio curve of birth weight to overweight and obesity in children and adolescents was obtained by using the restricted cubic spline(RCS)in the logistic regression program.According to this curve,birth weight was divided into five categories,namely Q1(<2500g),Q2(2500-2999g),Q3(3000-3499g),Q4(3500-3999g),Q5(≥4000 g).And Q3 was used as the reference group.Residential areas are divided into suburban and urban areas.Logistic regression models were used to evaluate the relationship between birth weight and the risk of overweight and obesity.Results:1.The prevalance of overweight and obesity in children and adolescentsThe prevalance of overweight and obesity in children and adolescents aged 2-17 years was 22.4%.The overweight rate was 8.7%,and obesity rate was 13.6%.By gender analysis,the incidence of overweight and obesity in girls(25.5%)was significantly higher than that in boys(19.4%).Stratified analysis according to age showed that the overweight and obesity rate of 7-13 years old and 14-17 years old were higher than that of 2-6years old,and there was no significant difference between the rates of 7-13 years old and 14-17 years old.According to the stratified analysis by residential area,the overweight and obesity rate of those living in suburban areas was 20.4%,while that of those living in urban areas was 24.3%,and the difference between the two group was not statistically significant.2.Overweight and obesity rates of different birth weight levels in children and adolescentsFor the general population,the incidence of overweight and obesity in group Q5 was higher than those in group Q1,Q2 and Q3,and the incidence of overweight and obesity in Q4 group was higher than those in group Q2 and Q3.Compared with Q2 and Q3 groups,the incidence of overweight and obesity in group Q1 showed an increasing trend,but the difference was not statistically significant.Among girls,the overweight and obesity rates in Q5 group and Q4 group were higher than those in Q1 group,Q2 group and Q3 group.Among boys,the overweight and obesity rates of Q5 group and Q4 group were higher than those of Q2 group and Q3 group.3.Association between birth weight and overweight and obesity rates among children and adolescentsIn this study,birth weight,as a continuous variable,was non-linear correlated with the risk of overweight and obesity in children and adolescents aged 2-17 years.The birth weight of total population and boys showed a U-shaped correlation with the risk of overweight and obesity,while the birth weight of girls showed a positive correlation with the risk of overweight and obesity in children and adolescents aged 2-17.After adjusting for confounding factors,Q3 group was used as the reference group,the OR values of overweight and obesity in the Q1group(OR 1.595;95%CI: 1.005-2.531),Q4 group(OR 1.879;95%CI: 1.237-2.855)and Q5 group(OR 2.778;95% CI:1.629-4.738)were significantly higher.According to gender analysis,the OR values of overweight and obesity in Q4 group(OR 2.450;95% CI: 1.326-4.527)and Q5 group(OR 2.806;95%CI:1.318-5.973)were significantly higher among girls;In boys,the OR values of overweight and obesity in Q1 group(OR 2.934;95% CI:1.495-5.757)and Q5 group(OR 2.765;95% CI: 1.285-5.952)were significantly higher.Summary: In summary,low birth weight(<2500g)and higher levels of birth weight(birth weight ≥3500g)were associated with an increased risk of overweight and obesity in children and adolescents.The birth weight of boys showed a U-shaped correlation with the risk of overweight and obesity,while the birth weight of girls showed a positive correlation with the risk of overweight and obesity in children and adolescents.Part Two Effects of low birth weight mice exposed to high fat diet on adulthood insulin sensitivityObjective:The changes in food intake,physical activity and body weight of low-birth weight mice fed with high fat were observed,and the metabolic indexes such as blood glucose,blood lipid and insulin were measured.The lipid deposition in each tissue was observed.The influence of early intrauterine nutrition on the the adult metabolism of mice was confirmed and the effects of low-birth weight mice on inducing adulthood insulin sensitivity was preliminarily investigated under the background of high fat diet.Methods: ICR mice aged 6-8 weeks was used to establish a low birth weight model by caloric restriction during pregnancy.Pregnant female mice were randomly divided into restricted diet group and normal diet group.The restricted diet group was subjected to 50% dietary restriction(referring to the food intake of the normal diet group)on the 12.5-18.5 days of pregnancy.The body weight of all newborn mice was measured within 24 hours after delivery.Nine male pups were randomly selected from the offspring of restricted diet mice and normal diet mice and labeled as LBW group and NBW group.They were weted for 3 weeks,and then all given a high-fat diet.The body weight of the offspring mice were measured weekly,and the food intake,physical activity of the offspring mice were measured through the metabolic cage.Intraperitoneal glucose tolerance test(IPGTT)was conducted at 11 weeks of high fat intervention(14 weeks of age).Blood glucose was measured at 17 weeks of high fat intervention(20 weeks of age)before death.Blood and tissue specimens were tested.And serum insulin,total cholesterol(TC)and triglyceride(TG)levels were measured.Weight ratios of liver,skeletal muscle and adiposity index(%,(epididymal fat+ perirenal fat)/body weight ×100)were calculated.Hematoxylin and eosin staining(HE staining)were used to visualize adipose tissue morphology.Oil red O staining was used to observe the lipid content in liver,and skeletal muscle triglyceride(TG)content was determined to understand the lipid deposition in skeletal muscle.Results:1.Establishment of a low birth weight mouse modelThe low birth weight mouse model was established by 50% foodrestriction during pregnancy.The birth weight of the LBW group was significantly lower than that of the NBW group,indicating that the LBW mouse model was successfully established.There was no significant difference in body weight between the two groups after 3 weeks of weaning,suggesting that mice in LBW group achieved catch-up growth.2.Comparison of body weight,food intake and and physical activity of mice in two groupsAt the 17 th weeks of high fat intervention(20 weeks of age),there was no significant difference in body weight between NBW and LBW mice.The food intake and physical activity of the two groups were not markedly different.3.Comparison of glucose tolerance and insulin sensitivity between two groups1)At the 11 th week of high fat intervention(14 weeks of age),the fasting blood glucose level of the LBW group was significantly higher than that of the NBW group,and the blood glucose levels of the LBW group were higher than those of the NBW group at 1h and 2h after intraperitoneal injection of glucose.2)At the end of 17 weeks(20 weeks of age)after high fat intervention,fasting blood glucose and serum insulin levels in LBW group were higher than those in NBW group,and HOMA-IR values in LBW group were significantly higher than those in NBW group.4.Comparison of lipid metabolism between the two groupsCompared with NBW group,serum total cholesterol(TC)in LBW group was increased,and TG showed an increasing trend.The weight ratio of liver and skeletal muscle in LBW group was not significantly increased,but the adiposity index was significantly increased,suggesting the accumulation of visceral adipose tissue..5.Morphological observation of adipose tissue and ectopic lipid deposition in liver and skeletal muscle of mice in two groupsHE staining of adipose tissue showed that adipose cells in LBW group were significantly enlarged compared with those in NBW group.A large number of red lipid droplets in both LBW and NBW groups were observed according to Oil red O staining of liver,suggesting lipid deposition in the liver.And the liver lipid deposition in the LBW group is more pronounced.Compared with NBW group,TG content in muscle of LBW group was significantly increased,suggesting lipid deposition in skeletal muscle.Summary: A low birth weight mouse model was successfully established by caloric restriction during pregnancy.Low birth weight mice fed with high fat could lead to decreased adulthood glucose tolerance,abnormal lipid metabolism and insulin resistance.Part Three The potential molecular mechanism of adipose tissue invovled in adulthood insulin resistance induced by low birth weightObjective:To reveal the potential molecular mechanism of adipose tissue invovled in adulthood insulin resistance induced by low birth weight by investigating changes in the protein expression profile of white adipose tissue in low-birth weight(LBW)mice with high-fat diets.Methods: The proteins was extracted from the epididymal adipose tissue of ICR mice,and they were degraded into peptides by pancreatic enzymes.The peptides from the LBW and NBW groups were identified by TMT labeling and LC-MS/MS analysis.The identified DEPs were analyzed by GO analysis,KEGG pathway enrichment analysis and protein domain enrichment analysis.Six samples from LBW and NBW groups were selected for PRM quantization of target proteins,and two or more unique peptide segments were used for each protein.Meanwhile,Western blotting(Western blot)was used to verify the expression of target proteins.A LBW mouse model was established by caloric restriction during pregnancy,and normal birth weight(NBW)mice were used as controls.After 17 weeks of high fat intervention(20 weeks of age),total protein was extracted from epididymal adipose tissue of mice in both groups.After the tissue lysate was digested by pancreatic enzymes,the obtained peptide segments were quantitatively analyzed by tandem mass labeling(TMT)and liquid chromatography-mass spectrometry(LC-MS/MS).The difference ratio FC >1.2 or <0.83 and P<0.05(Student’s t test)were used as thresholds.The differentially expressed proteins(DEPs)of LBW and NBW groups were screened.In order to further screen the key biological processes that change in LBW adipose tissue,the functions of these proteins were analyzed using Fisher exact test(P<0.05)based on the GO(Gene Ontology)analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)PATHWAY database respectively.Finally,Parallel Reaction Monitoring(PRM)technology and Western Blotting(Western Blot)were used to verify the expression levels of the screened differential proteins,further identifying the key pathway of adulthood insulin resistance induced by LBW.Results:1.Expression of differential proteins in adipose tissueA total of 5,854 proteins were identified in adipose tissue of high-fat fed mice by using TMT tag-based proteomics techniques.And 4,940 proteins containing quantitative information were included.According to FC>1.2 or<0.83 and P<0.05(t test),a total of 996 differential proteins with significant changes in the low birth weight group were identified,of which 325 proteins were significantly up-regulated and 671 proteins were significantly down-regulated.2.GO analysis of adipose tissue DEPs in two groups of mice1)Functional classification of DEPs in GO analysisThe GO database contains three types of functional annotations:biological process(BP),molecular function(MF)and cellular component(CC).In biological processes(BP),DEPs in LBW/NBW group are mainly involved in cellular process,single-organism processes,metabolic process and biological regulation.It was worth mentioned that 586 proteins were enriched into the metabolic process,accounting for 58.9% of the annotated proteins,suggesting that DEPs may play an important role in regulating the metabolic process.In terms of cell composition(CC),DEPs of LBW and NBW groups were mainly distributed in cell,organelle,membrane,extracellular region and other subcellular location.In terms of molecular function(MF),the above differential proteins are significantly enriched in binding and catalytic activity,which may be related to their participation in metabolic process.2)GO enrichment analysisWe conducted GO enrichment analysis of differentially expressed proteins,and a total of 14 GO items were enriched in biological process(BP),among which 8 pieces of GO items(57.1%)were related to metabolism referring to organic acid metabolic process,oxoacid metabolic process,carboxylic acid metabolic process,carbohydrate metabolic process,neutral lipid metabolic process,triglyceride metabolic process,acyglycerol metabolic process,regulation of fatty acid regulation metabolic process.Among the items related to metabolic process,4 pieces of GO items(50%)were related to lipid metabolism.It was suggested that DEPs played a crucial role in the regulation of lipid metabolism.3.KEGG Enrichment analysis of adipose tissue DEPs in two groups of miceDifferential proteins in white adipose tissue of LBW and NBW mice were mainly enriched in 34 pathways,among which 13 pathways(38.2%)were related to metabolism.It was worth mentioned that among the 9metabolism-related pathways,the P value of PPAR pathway enrichment test was 0.000209,which was the most significant enrichment among all metabolic pathways.It was the only signaling regulation pathway and associated with the regulation of lipid metabolism.These results suggest that the PPAR pathway may be a key regulatory pathway for lipid metabolism in low birth weight mice.Compared with the NBW mice,the LBW mice showed decreased glucose tolerance and abnormal lipid metabolism,so LBW may regulate lipid metabolism through PPAR pathway and affect glucose metabolism.4.Validation of PPARγ signaling pathway of adipose tissueConsidering the important role of PPAR signaling pathway in lipid metabolism regulation of differential proteins,we further screened differential proteins of this pathway.It included 33 differential proteins,and the PPARγsignaling pathway was closely related to lipid metabolism in adipose tissue.CD36,SCD1 and PCK1 were significantly up-regulated in the LBW group.This result was verified by PRM quantitative proteomics(n=6/group,P<0.05,Student’s t test).Because PPARγ information was not quantified by proteomics,so we also detected the expression of PPARγ in white adipose tissue of LBW and NBW mice by Western blot analysis,suggesting that PPARγ was significantly up-regulated in the LBW group.Summary:1.GO analysis showed that the differentially expressed proteins in the LBW/NBW group played a very important role in regulating metabolic processes,especially lipid metabolism.2.KEGG pathway enrichment analysis indicated that the abnormal expression of PPAR signaling pathway was related to lipid metabolism and played an important role in insulin resistance of low birth weight mice.3.When exposed to high-fat diets,LBW mice exhibited insulin resistance and disorder of lipid metabolism compared with NBW mice.The expression of PPARγwas elevated,as well as upstream CD36,downstream SCD1 and PCK1 of the PPARγ in the adipose tissue of LBW mice,further confirming the activation of PPARγ signaling pathway.It was suggested that the activation in CD36/PPARγ/SCD1 and CD36/PPARγ/PCK1 pathways may induce adipose dysfunction,thereby increasing susceptibility to insulin resistance.Conclusions:1.Low birth weightand higher levels of birth weight were associated with an increased risk of overweight and obesity in children and adolescents.The birth weight of boys showed a U-shaped correlation with the risk of overweight and obesity,while the birth weight of girls showed a linear positive correlation with the risk of overweight and obesity in children and adolescents.2.A low birth weight mouse model was successfully established by caloric restriction during pregnancy.Low birth weight mice fed with high fat could lead to decreased adulthood glucose tolerance,abnormal lipid metabolism and insulin resistance.3.It was suggested that the abnormal expression of PPAR signaling pathway was related to lipid metabolism and played an important role in insulin resistance of low birth weight mice.4.The activation of the CD36/PPARγ/ SCD1 and CD36/PPARγ/PCK1 pathways may induce adipose dysfunction,thereby increasing susceptibility to insulin resistance. |
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