Mechanism Of Cell Death-inducing DNA Fragmentation Factor-α-like Effector C In Regulating Inflammatory Response And Insulin Resistance Of Adipocyte Of Dairy Cows

Fat mobilization is a physiological adaptive response to energy deficiency in mammals.It is a phenomenon of the imbalance of lipogenesis and lipolysis in adipose tissue.Perinatal dairy cows require energy to support fetal growth and lactation,however,dry matter intake(DMI)decrease which trigger negative energy balance(NEB),and further adipose tissue is mobilized and decomposed to non-esterified fatty acids(NEFA)as energy.Nevertheless,severe and long lipolysis cause high level of NEFA in the blood and energy metabolism disorders such as ketosis and fatty liver in perinatal cows.Adipose tissue not only store energy,but also secrete a variety of inflammatory factors such as tumor necrosis factor-α(TNF-α),which affects the insulin sensitivity and inflammatory response.Studies have shown that the metabolism of adipose tissue is disordered and exhibits systemic inflammatory response and insulin resistance in ketotic cows.However,mechanisms controlling mobilization of adipose tissue is still unclear.Lipid droplets(LDs),as the main energy storage site of adipose tissue,is inlaid with various lipid droplet coating proteins in surface.Among,including the cell death-inducing DNA fragmentation factor-α-like effector c(CIDEC),recent studies have found CIDEC plays a key role in regulating lipid metabolism and metabolic-related diseases.However,the mechanism of CIDEC in regulation of lipolysis in adipocytes of dairy cows is still unclear.Whether CIDEC affects inflammatory response and insulin sensitivity of adipocytes is still unclear.Therefore,the purpose of the current study was to investigate the change of CIDEC,lipolysis,and inflammation signaling in adipose tissue of ketotic cows;the role of CIDEC in regulation of lipolysis,inflammatory response and insulin sensitivity in isolated adipocytes.In this experiment,adipose tissues of ketotic cows and healthy cows were collected to detect the abundance of CIDEC and the status of lipolysis,inflammatory response,and insulin signaling pathway.The results showed that protein and m RNA abundance of CIDEC in adipose tissue of ketotic cows were significantly downregulated,and protein abundance of adipose triglyceride lipase(ATGL)and phosphorylated hormone-sensitive lipase(p-HSL),phosphorylated p65(p-p65)and phosphorylated nuclear factor kappa B inhibitory kinase(p-IκB),and the m RNA abundance of pro-inflammatory factors TNF-α and IL-1β were increased.In contrast,m RNA abundance of anti-inflammatory factor IL-10 and protein abundance of phosphorylated kinase B(p-AKT)and phosphorylated insulin receptor(p-IR)were decreased.It shows that enhanced lipolysis,activated nuclear factor kappa B(NF-κB)inflammatory signal pathway,and impaired insulin sensitivity in adipose tissue of dairy cows with clinical ketosis.In vitro,transfection of adipocytes of dairy cows with si-CIDEC found that protein abundance of CIDEC was decreased,and protein abundance of lipolytic enzymes ATGL and p-HSL and the glycerol content of the medium supernatant,protein abundance of p-p65 and p-IκB,and m RNA abundance and secretion of pro-inflammatory factors TNF-α and IL-1β were increased,while m RNA abundance and secretion of anti-inflammatory factor IL-10 and protein abundance of p-AKT and p-IR in the insulin signaling pathway were decreased.These data indicate that silencing CIDEC induces lipolysis,activates the inflammatory response,and impairs the insulin signaling pathway.Inflammatory factors are important factors to induce lipolysis in adipose tissue.Our work found treating with 0.1,1,and 10 ng/m L TNF-α treatment for 3 h reduced protein and m RNA abundance of CIDEC,and increased protein abundance of ATGL and p-HSL and the medium supernatant glycerol content,protein abundance of p-p65 and p-IκB,and the secretion of pro-inflammatory factor IL-1β,while the secretion of anti-inflammatory factor IL-10 and protein abundance of insulin signaling pathway p-AKT and p-IR were decreased.However,overexpression of CIDEC by adenovirus alleviated the downregulation of CIDEC expression,reduced protein abundance of ATGL and p-HSL and the glycerol content of the medium supernatant,inhibited the activation of NF-κB inflammatory signaling pathway,and relieved the damage of insulin signaling pathway induced by TNF-α.These data indicate that the high expression of CIDEC alleviate lipolysis,inflammatory response and insulin resistance.Therefore,our results revealed that decreased expression of CIDEC accompanied with enhanced lipolysis,over-activated NF-κB signaling pathway,and impaired insulin signaling pathway in adipose tissue of ketotic cows.In vitro results showed that silencing CIDEC enhanced the lipolysis,activated NF-κB signaling pathway and impaired insulin sensitivity in calf adipocytes.Besides,overexpression of CIDEC in calf adipocytes attenuated the excessive lipolysis,over-activated NF-κB signaling pathway and impaired insulin sensitivity induced by TNF-α.These results further clarify the mechanism of excessive lipolysis in dairy cows with ketotic and provide a target for the prevention and treatment of ketosis from the perspective of limiting lipolysis.