The Regulation And Mechanism Of Fatty Acid Binding Protein 4 In Ageing-mediated Metabolic Disorders

Objective This work is aimed to screen the biomarkers of ageing and FABP4 is one such biomarker identified through plasma proteomics.The impact of FABP4 on aging-related cognitive ability,cardiac and metabolic function were then examined in mice.The potential mechanism for FABP4-mediated action on ageing was explored in LO2,a hepatocyte cell line.MethodsPartⅠ.A cross-sectional study was conducted,in which 102 healthy young people（22-25 years old）and 170 healthy elderly people（62-65 years old）were enrolled.All participants were divided into four groups based on gender and age.Their clinical characteristics and biochemical parameters were collected and analyzed accordingly.To screen the different expression proteins（DEPs）during ageing,three mixed samples from each group were subjected to TMT protein profile identification.Cluster analysis,GO and KEGG pathway enrichment analysis of DEPs were performed by Blast2GO software.Multiple linear regression analysis was conducted to investigate covariates that were associated with plasma FABP4.PartⅡ.To study the impact of loss-of-function of Fabp4,sh RNA-mediated gene knockdown was performed in mice.A Fabp4-targeting and a non-targeting control sh RNA were cloned into Adeno-Associated Viral Vector 2/9（AAV2/9-GFP）respectively,and the efficacy was verified in vivo.A total of thirty 14-month-old male wild type（WT）mice were randomly divided into two groups as the following:（1）old mice injected with AAV9-sh Fabp4 by tail vein and fat in situ injection,n=15;（2）old mice injected with AAV9-sh Neg with the same way,n=15.All the animals were kept until 20 month-old after AAV injection.A group of 8-week-old WT mice were used as young control group（n=15）.All three groups were subjected to ultrasonic cardiogram,water maze and metabolic cage to evaluate their cardiac function,cognitive ability and basal energy metabolism.A glucose tolerance test（IPGTT）was performed to determine the change in glucose tolerance.After completing the above observations,all the mice were anesthetized,and blood was collected from the eyeballs.Plasma lipid profiles,glucose,fasting insulin and FABP4 levels were examined.A portion of the tissues were fixed for staining,while the rest were frozen and kept until needed.HE staining was utilized to observe the pathological changes in adipose tissue and liver,while oil red staining was used to assess the lipid deposition in liver.RT q PCR was used to examine the m RNA levels of genes related to ageing,inflammation,and glucose and lipid metabolism in liver.Five liver samples were selected randomly from each group for RNA-seq to understand the possible mechanism of FABP4’s action in ageing.PartⅢ.To dissect the mechanism of Fabp4’s action,overexpression of Fabp4 was conducted in human hepatocytes line LO2.The cells were cultured in two different mediums,which contains 0.5%BSA and palmitic acid combined with oleic acid（50m M+100m M）,respectively.Evaluation of the effect of FABP4 on cell senescence and fat accumulation was performed byβ-gal staining and oil red staining,respectively.Finally,the differential expression of MAPK/JAK-STAT pathway identified by RNA-seq was validated by Western blot.The potential interacting proteins for FABP4 were predicted through bioinformatic analysis.Co-immunoprecipitation（Co-IP）was conducted to verify the speculated interaction between FABP4 and GDF5.PartⅣ.Crtc1^-/-mouse line was generated using the CRISPR/Cas9 system,and littermate wild-type(Crtc1^+/+)mice were as normal control.The tissue harvet and measurement at the end point was the same as the PartⅡ.ResultsPartⅠ.The body mass index（BMI）,blood pressure,plasma glucose and lipids,in the elderly were significantly higher than the young people.Plasma proteomics identified nine proteins that were DEPs in male and female.Among the nine proteins,ARMH4,CILP2,IGFBP3,IGF-1and IGFBP5 were down-regulated in the elderly,while FGG,IGHV3-72,FABP4,FLG2 were up-regulated.The changes of plasma IGF-1 and FABP4 levels by ageing were confirmed further by ELISA.Correlation and regression analysis indicated that FABP4 was positively correlated with age,however,age was only an independent risk factor for male but not for female.Compared to male,BMI had more profound impact on plasma FABP4 in female.PartⅡThe plasma FABP4 levels and FABP4 expression in WAT were increased significantly in 24-month-old mice compared to 2-month-old mice.As expected,the old mice had decreased memory function and spatial exploration ability.Knockdown of FABP4 in the old mice alleviated the negative impact of ageing on cognition.Metabolic profiling（O2 consumption,CO2 production,calorie consumption and activities within 24 hours）revealed that the overall energy metabolism of old mice was significantly decreased,whereas knockdown of FABP4 restored the metabolism rate,and promoted the locomotor activity in the old mice.Knockdown of FABP4 protected ageing-associated weight gain and glucose intolerance,which was accompanied by decreased serum low-density lipoprotein（LDL）,free fatty acids（FFA）and pyruvate.HE staining and Oil red staining revealed that FABP4 knockdown lowered ectopic lipid deposition in liver.Consistently,knockdown of FABP4 reduced gene expression of IL-1β,IL-6 and G-6-pase,indicating a protective effect on ageing-associated increase of inflammation and gluconeogenesis in liver.Meanwhile,FABP4 knockdown up-regulated fatty acid oxidation presumably by promoting the expression of PPARγ,the key regulator of fatty acidβ-oxidation.PartⅢ.In the absence of fatty acid supply,overespression of FABP4 promoted cell senescence as shown by increasedβ-galactosidase staining and increased expression of ageing-related genes（P16,P21,IL-1β,IL-6）.Oil red O staining indicated that overexpression of FABP4 promoted lipid deposition in hepatocytes presumably by up-regulation of SREBP1c,the key regulator of fatty acid synthesis.In contrast,FABP4overexpression did not accelerate cell senescence in the presence of fatty acid supply.Western-blot showed that ERK1/2 and p-38 MAPK activation is increased by FABP4overexpression,suggesting a MAPK pathway mediated mechanism for FABP4’s action in ageing.The results of Co-IP revealed that FABP4 interacted with GDF5.PartⅣ.Under normal feeding conditions,Crtc1^-/-mice exhibited an obese phenotype resultant from the abnormal expansion of the white adipocytes.The development of obesity in Crtc1^-/-mice is independent of alterations in food intake or energy expenditure.Moreover,Crtc1^-/-mice were more prone to insulin resistance and dyslipidemia,as evidenced by higher levels of plasma glucose,insulin and FABP4 than wildtype mice.Transcriptome analysis in liver and epididymal white adipose tissue（e WAT）showed that the fat accumulation caused by Crtc1 deletion was mainly related to lipid metabolism in adipose tissue,but not in liver.GSEA and KEGG analysis identified PPAR pathway to be of the highest impact on lipid metabolism in e WAT.This regulation was independent of a direct interaction between CRTC1 and PPARγ.Conclusion Plasma FABP4 levels is an ageing-related biomarker for male.Knockdown of FABP4 partially reverses ageing-associated deterioration of metabolic health.FABP4 might promote cell senescence and fat deposition in hepatocytes through a GDF5-mediated activation of MAPK signaling pathway.Meanwhile,CRTC1 deletion can increase the level of FABP4 in vivo and induce obesity.