Integrated Analysis Of Metabolic Adaptation On Adipose Tissue In Ketotic Dairy Cows Based On Multi-omics Data

Ketosis is a major metablic disorder in perinatal high-yield dairy cows.In order to meet the the energy requirements of body and lactation,cows have a natural genetic drive to mobilize adipose tissue during transiton period.Although lipolysis ensures adequate energy supply around calving,massive and prolonged lipolysis can limit the energy buffering capacity of adipose tissue and increase the secretion of pro-inflammatory cytokines for a long time,making perinatal dairy cows more susceptible to other metabolic and infections disease.Therefore,metabolic adaptation of adipose tissue is critical for successful perinatal period,but other changes in adipose tissue during the onset of ketosis are unclear after the lipolysis process.With the development of high-throughput sequencing technology,it provides a new way for the screening and identification of key functional genes and proteins in the metabolic adaptation process of adipose tissue in transition dairy cows.Based on multi-omics data,this study explored important functional genes,proteins and regulatory elements in the metabolic adaptation process of adipose tissue from the transcriptome and proteome levels,and we further performed functional verification of important differentially expressed gene in vitro.It is expected that the metabolic adaptation mechanisms of adipose tissue in ketosis cows will be well understood from the level of transcriptome and proteome.In order to excavate and verify the key genes and regulatory elements of metabolic adaptation of adipose tissue in ketosis cows from the transcriptome level,RNA-seq technology was used to identify mRNA and lncRNA changes in adipose tissue of 5 healthy and 5 ketosis Holstein cows.The results showed that a total of 241 differentially expressed mRNAs and 191 differentially expressed lncRNAs were found.Combined with GO,KEGG and CNC analysis,we initially identified six differentially expressed genes encoded by five differentially expressed lncRNAs as key differentially expressed genes and lncRNAs,which mainly involved in the regulation of lipolysis,inflammatory response,oxidative stress and fatty acid biosynthesis affecting perinatal metabolic adaptation of adipose tissue in dairy cows.This proteomics analysis revealed alterations in protein abundance in the adipose tissue of transitioning cows under ketotic or healthy conditions during early lactation.Ketotic cows have impaired immune function and altered carbohydrate,lipid and amino acid metabolism in the adipose tissue,as indicated by the top canonical pathways and biological functions identified by KEGG and GO.Based on differential abundance of proteins in these pathways,it seems that ketotic cows have increased ATP synthesis,shifts in precursor supply for gluconeogenesis,massive lipolysis and impaired inflammatory immune response in their adipose tissue that may contribute to infectious diseases.Thus,these changes in adipose tissue function may be associated with transition-related diseases and poor lactation performance.These findings can facilitate further studies to better understand the molecular mechanisms through which altered proteins may promote inflammation and hence ketosis.By integrating the transcriptome and proteomic results,we showed that transforming growth factor ?1(TGF?1)was significantly differentially expressed in both mRNA and protein levels,implying that TGF?1 may play a key role in the regulation of adipose tissue inflammation in ketotic cows.Using gene overexpression and RNA interference(RNAi)technology,we overexpressed and silenced the TGF?1 gene in inflammatory model of primary cultured bovine adipocytes.The results showed that TGF?1 overexpression significantly reduced the mRNA and protein expressions of interleukin-1?(IL-1?),interleukin-6(IL-6),interleukin-17(IL-17)and tumor necrosis factor alpha(TNF?)in LPS-induced bovine adipocyte inflammatory responses.TGF?1 gene silencing increased the mRNA expression and secretion of IL-1?,IL-6,IL-17 and TNF?,which further aggravated the inflammatory responses of adipocytes compared with the control group.TGF?1 overexpression inhibited the expression of Toll-like receptor 4(TLR4),nuclear factor-kappa B(NF?B)and the degradation of inhibitor of nuclear factor kappa-B kinase(I?B)compared with the control group.TGF?1 gene silencing further increased the activation of TLR4,NF?B and the degradation of I?B induced by LPS.The results showed that TGF?1 plays an anti-inflammatory role in the adipose tissue inflammatory responses of dairy cows,and its mechanism may be through inhibition of TLR4/NF-?B inflammatory signaling pathway.In summary,this study investgated the metabolic adaptation of adipose tissue to ketosis by transcriptome and proteome sequencing of ketotic or healthy cow in early lactation from long-chain non-coding RNA,protein-encoding RNA and protein levels.The results showed that the metabolic mechanism of adipose tissue in dairy cows is complicated.Due to the influence of many non-coding genes,coding genes and regulatory pathways related in transcriptome,a variety of functional lncRNAs may be produced in adipose tissue of ketotic cows,which are regulated by the interaction with mRNA and affected the protein expression,and ultimately affect lipid metabolism,carbohydrate metabolism,inflammatory response,oxidative stress and fatty acid biosynthesis in adipose tissue.The multi-omics systems was used to analyze the metabolic adaptation of adipose tissue in ketotic cows.The related research results provide new insights for understanding the energy metabolism and immune response mechanism in ketotic cows.