| The enzymatic hydrolysis of the triglyceride in adipocytes plays the key function in process of energy metabolism of animal. The triglyceride may be hydrolyzed into diacylglycerol, monoglyceride, glycerol and free fatty acids (FFAs) by turns under the regulation of lipase. Recent studies showed that adipose triacylglyceride lipase (ATGL) functioned as a key enzyme for adipose lipid mobilization.ATGL specifically hydrolyzes the first ester bond of triglycerides and is a rate-limiting enzyme in triacylglycerol hydrolysis.The expression and activity of ATGL are regulated at both transcriptional and posttranslational levels by hormone, cytokine and lipolysis pathway etc.Isoprenaline (ISO) and TNF-αmay affect the expression of ATGL and significantly promote the process of lipolysis in adipocytes from rodent and human being. As we know, porcine has the highest deposition of body fat among all livestock, and is more similar as a model of the lipidosis to human being than rodent which are common experimental animal in researching of medical science. So, porcine is regarded as an potential animal model for application to human obesity, diabetes, hepatic steatosis, and other related metabolic disorders that associated with ATGL.At present, there are no reports about regulatoion of ISO and TNFαon the expression of ATGL in porcine adipocytes.In this study, porcine primary-dipocytes were incubated with ISO, TNFαand inhibitors of lipolysis pathway (PKA、p38MAPK and p42/44 MAPK) respectively. Expression of ATGL mRNA and protein were detected by RT-PCR and Western blot. The recombinant lentivirus was processed with siRNA of ATGL. Then, porcine mature adipocytes were infected by lentivirus. After that, the centration of the glycerol and FFAs in the media was measured as indicators of the lipolysis and FFA consumption. Further, Distribution of ATGL and coactivator CGI-58 was located in poricine dipocytes by applying immuno-fluorescence and staining method, and the interactions were detected between ATGL, CGI-58 and perilipin by co-immunoprecipitation method. The results were as follows:1. ISO and TNFαsignificantly promoted lipolysis in porcine adipocytes with dose and time dependent way respectively, along with the down regulation of expression of ATG. Both PKA inhibitor and p42/44 MAPK inhibitor may significantly reduce the down regulation of expression of ATG which was stimulated by ISO and TNFα, while p38 MAPK inhibitor failed to repress that.2. It showed the production recombinant lentivirus successfully, and 1×108 TU/mL in virus titre after dectecting DNA sequence, recombinant plasmid, packaging plasmid, 293Tcells infected by lentivirus and expression of green fluorescin. Supplement of RNAi of ATGL may reduce the centration of the glycerol and FFAs from the porcine adipocytes under the quiescent condition, and the porcine adipocytes stimulated by ISO and TNFαrespectively. Meanwhile, the centration rate of the glycerol and FFAs did not accord with the relation of 1:3 which was rational rate in the process of normal lipolysis. Although RNAi of ATGL existed in the porcine adipocytes, the centration of the glycerol and FFAs was significantly upgraded for stimulating of ISO and TNFαcompared with the Control.3. Both ATGL and CGI-58 located in the surface of lipid droplet in the porcine adipocytes, while they partly extended to the cytoplasm at the same time with the stimuli ISO. Under the conventional culture of porcine adipocytes, CGI-58 was directly bonded to ATGL and perilipin respectively. Supplement of ISO may promote the combination of ATGL and CGI-58, but weeken the combination of CGI-58 and perilipin.In summary, ISO and TNFαsignificantly step down expression of key lipase—ATGL in porcine adipocytes. Both PKA inhibitor and p42/44 MAPK pathway affected the expression regulation of ATGL stimulated by ISO and TNFα. ISO and TNFαaffected the lipolysis in porcine adipocytes with assisting of ATGL. ISO implemented the regulation of lipolysis by promoting the combination of ATGL and CGI-58. |
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