Study On The Mechanism Of Calmodulin In Lipolysis And Inflammation Of Dairy Cows With Clinical Ketosis

Clinical ketosis of dairy cows is one of the most important nutritional and metabolic diseases that harm the healthy development of dairy farming in the world.Due to the decrease of dry matter intake and lack of energy before and after parturition,the lipolysis of cows is enhanced,and the large amount of NEFA produced by fat lipolysis exceeds the oxidative capacity of liver,leading to ketosis.At present,the pathogenesis and pathological changes of cow ketosis involve in negative energy balance,fat mobilization,abnormal liver function and inflammation.In addition,our previous proteomics study of adipocytes of ketosis cows found that the fold difference of calmodulin(Ca M)was 2.401.Although Ca M has many biological functions,little is known about the relationship between Ca M and inflammation,lipolysis in adipose tissue of dairy cows.Therefore,this study proposed a hypothesis that Ca M might participate in lipid metabolism and inflammation of ketosis cows.Two experiments in vivo and in vitro are used to understand the Ca M regulation on inflammation and lipolysis in adipose tissue,to clarify the potential effect mechanism of Ca M on clinical ketosis in dairy cows,and to enrich and develop the ketosis mechanism and provide a foundation for new strategy for prevention of ketosis in dairy cows.Experiment Ⅰ: Analysis of hemopathology and Ca M,lipolysis and inflammation in adipose tissue of dairy cows with clinical ketosis.In this experiment,twelve multiparous Holstein cows with similar parities and milk yield(> 9 tons/yrs)were selected as experimental animals.According to the serum levels of three biochemical indexes and clinical manifestations of dairy cows on 14～21 d postpartum,the experimental cows were divided into healthy control group(CON,n=6,BHBA < 1.20 mmol/L,Glu > 3.5 mmol/L,NEFA < 0.7 mmol/L)and clinical ketosis group(CK,n=6,BHBA > 3.00 mmol/L,Glu < 2.8 mmol/L,NEFA > 0.7 mmol/L).In addition,serum of the two groups were collected on fasting in the morning for detecting energy metabolism,liver function,inflammation and other indicators,and adipose tissue from tails were collected for Western blot of inflammation-related proteins and lipolysis-related proteins,and immunohistochemical analysis.The results showed that(1)serum levels of BHBA,NEFA,AST,ALT,IL-6,IL-1β and TNF-α in CK cows were significantly higher than those in CON cows(P <0.01),and levels of Glu and liver function index(LFI)were significantly lower than those in CON cows(P < 0.01).(2)The expression level of TLR4,p-NF-κB/NF-κB,Ca M,ATGL,p-HSL/HSL in adipose tissue of CK cows were significantly higher than those of CON cows(P < 0.01),and the expression level of PLIN1 was significantly lower than that of CON cows(P < 0.01).(3)The expression level of Ca M in adipose tissue of CK cows was significantly higher than that of CON cows(P < 0.01),and fat droplets were fatter and more irregular than those of CON cows.These results suggest that cows with clinical ketosis not only had clear clinical signs,but also had the increased lipolysis(low blood glucose,high BHBA and high NEFA),liver damage(the increased levels of AST and ALT,and low LFI)and inflammation(the increased levels of IL-1β,IL-6 and TNF-α).Meanwhile,the enhanced lipolysis of adipose tissue was closely associated with increased Ca M expression,activation of inflammatory pathway(TLR4/IKK/NF-κB),and the increased lipolysis-related proteins expression(ATGL,p-HSL/HSL and PLIN1).Experiment Ⅱ: Effects of Ca M silencing / overexpression on lipolysis and inflammation in primary cultured adipocytes of calves.In this experiment,adenovirus overexpression / si-RNA were respectively used in Ca M overexpression / silencing tests and LPS was used in adipocyte inflammatory model.Western blot was used to detect Ca M inflammation-related proteins(TLR4/IKK/NF-κB)and lipolysis-related proteins(ATGL,p-HSL /HSL,PLIN1)in adipocytes.The results showed that(1)LPS significantly upregulated the expression of Ca M,TLR4,IKK,pNF-κB/NF-κB,ATGL,p-HSL/HSL(P < 0.01),and down-regulated the expression of PLIN1(P <0.01).(2)The results of Ca M overexpression were consistent with those of LPS except that there was no significant effect on ATGL,while the results of silent Ca M were opposite to those of Ca M overexpression.(3)Ca M overexpression and LPS stimulation could further up-regulate the expression of HSL(P < 0.05),down-regulated the expression of PLIN1(P < 0.05)and upregulated the expression of ATGL(P < 0.05).These results suggest that Ca M can promote adipocytes lipolysis by HSL and PINL1,enhance TLR4/IKK/NF-κB inflammatory pathway,and then enhance lipolysis.LPS can enhance adipocytes Ca M expression,promote lipolysis by ATGL,HSL and PINL1.Conversely,the increased lipolysis also promotes inflammatory responses by the TLR4/IKK/NF-κB pathway,suggesting that Ca M plays a positive regulating role in inflammation and lipolysis in adipocytes,and there is a positive interaction between Ca M,inflammation and lipolysis.Conclusion: Clinical ketosis cows were in a condition of negative energy balance,enhanced lipolysis,liver damage,and inflammation,which was associated with the increased expression of Ca M,the enhanced inflammatory pathway of TLR4/IKK/NF-κB and the increased lipolysis of ATGL P-HSL /HSL and PINL1 in adipose tissue.The positive regulation of Ca M on inflammation and lipolysis in adipocytes suggests that Ca M plays an important role in the development of clinical ketosis in dairy cows,which provides a new direction for further revealing the mechanism and prevention and control strategies of clinical ketosis in dairy cows.