Effect Of Overexpression Of Leptin And Adiponectin On Muscle Fiber Type And Key Functional Genes Related To Lipolysis In Muscle

Differences in meat quality traits are closely related to gene expression. Therefore, in a previous study, we used microarray analysis to compare longissimus dorsi muscle gene expression profiles between Jinhua pigs and Landrace. We got a number of high or low expression of genes in longissimus dorsi muscle of Jinhua pigs from the result of microarray experiments. Leptin and Adiponectin were selected to test the functions. The effect of Leptin and Adiponectin on muscle fiber typing, lipid metabolism and functional genes related to lipolysis were studied by hydrodynamics-based gene delivery, ELISA, real-time PCR and western blotting techniques. The study reveals the possible mechanisms of the two genes regulatetd different meat quality between varities and systematically studied the gene regulation status in vivo from the overall level of biological to the molecular level. Results are as following:1 Comparison of gene expression of Leptin and Adiponectin in longissimus muscle tissue between Jinhua pigs and LandraceGene expression of Leptin and Adiponectin in longissimus muscle tissue were compared between Jinhua pigs and Landrace by microarray. The results showed that: 90 days, the mRNA levevl of Leptin and Adiponectin were 2.20,2.58 times than that of Landrace respectively. The results of real-time PCR shows that the result in microarray were positive (p< 0.01); The mRNA level of Leptin, Adiponectin of Jinhua pigs were higher than Landrace. Significant differences were observed in 30, 90,120 days (p< 0.01).2 Construction of mouse pVRmob and pTarget-Adiponectin and establishment of mouse Leptin and Adiponectin overexpression modelIn order to study the function of Leptin and Adiponectin, mice overexpression plasmid and model were established by hydrodynamics-based gene delivery, and verified by ELISA, RT-PCR, Western blot techniques. The results of construction of mouse pVRmob and pTarget-Adiponectin showed that:Mice pVRmob plasmid inserts and NM₀08493.3 GI:34328437 sequence provided the same; Mice pTarget-Adiponectin plasmid inserts and NM₀09605.4 GI:87252710 sequence provided the same. The result of establishment of mouse pVRmob and pTarget-Adiponectin overexpression model showed that:Leptin mRNA and protein were produced adequately in the liver after hydrodynamic gene delivery in vivo. The Leptin concentration in the plasma after the gene transfer increased. The same result was in the mouse pTarget-Adiponectin overexpression model. The results suggest that we have successfully established overexpression model of mouse Leptin and Adiponectin.3 Study on the effect of Leptin on the functional genes related to meat qualityIn order to study the function of Leptin, we establish a living mouse model for gene overexpression by constructing of pVRmob plasmid and injecting the plasmid into the mice by hydrodynamics-based gene delivery. The result showed that the mRNA abundance of myosin heavy chain type I (MyHC-I) mRNA in gastrocnemius muscle and extensor digitorum longus after administration of Leptin for 1 week were significantly increased 118.75%(p<0.05) and 140.05%(p<0.05) compared with the control group. Gene expression level of proliferator-activated receptor-y coactivator-la (PGC-la) were increased 104.67%(p<0.01) in gastrocnemius muscle, corresponding increase in protein level. Gene expression level of PGC-la was increased 61.73%(p<0.05) in extensor digitorum longus, corresponding increase in protein level. The mRNA abundance of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL) genes in gastrocnemius muscle were significantly increased 144.79%(p<0.05),102.43%(p<0.01),80.09% (p<0.05) respectively. The mRNA abundance of ATGL, HSL and LPL genes in extensor digitorum longus muscle were significantly increased 162.25%(p<0.05), 124.69%(p<0.05),110.13%(p< 0.05) respectively. Gene expression of FOXO3a mRNA was increased 43.73%(p<0.05) in gastrocnemius muscle. Gene expression of SIRT1 mRNA was increased 61.45%(p<0.05) in extensor digitorum longus, corresponding increase in protein level. These results suggest that a high dose Leptin may affect muscle fiber type of wild type mice via PGC-1 a, and affect lipolysis in the skeletal muscles of wild type mice via SIRT1, FOXO 3 a.4 Study on the effect of Adiponectin on the functional genes related to meat qualityIn order to study the function of Adiponectin, we establish a living mouse model for gene overexpression by constructing of pTarget-Adiponectin plasmid and injecting the plasmid into the mice by hydrodynamics-based gene delivery. The result showed that the mRNA abundance of MyHC-I m RNA in gastrocnemius muscle and extensor digitorum longus after administration of Adiponectin for 1 week were significantly increased 61.35%(p<0.05) and 104.81%(p<0.05), compared with the control group. Gene expression and protein levels of PGC-1αwere increased 271.31%(p< 0.01) in gastrocnemius muscle. Gene expression of PGC-la was increased 246.26% (p<0.01) in extensor digitorum longus, corresponding increase in protein level. The amounts of ATGL, HSL and LPLmRNA in gastrocnemius muscle were significantly increased 64.84%(p<0.01),23.62%(p<0.05),83.81%(p<0.05) respectively. The amounts of ATGL, HSL and LPLmRNA in extensor digitorum longus muscle were significantly increased 20.12%(p<0.05),67.87%(p<0.05),44.45%(p<0.05) respectively. Gene expression of SIRT1 and FOXO3a mRNA was increased 29.30% (p<0.05),76.25%(p<0.01) in gastrocnemius muscle respectively. Gene expression of SIRT1 and FOXO3a mRNA was increased 29.30%(p<0.05),76.25%(p<0.01) in gastrocnemius muscle respectively. Gene expression of SIRT1 and FOXO 3a mRNA were increased 27.78%(p<0.05),38.97%(p<0.05) in extensor digitorum longus, corresponding increase in SIRT1 protein level. These results suggest that a high dose Adiponectin may affect muscle fiber type of wild type mice via PGC-1 a, and affect lipolysis in the skeletal muscles of wild type mice via SIRT1, FOXO 3a.This research revealed that Leptin and Adiponectin play a similar function in regulation of meat quality. They may affect the content of MyHC I by regulating the expression of PGC-la, affect the expression of gene related to lipoclasis by regulating the expression of SIRT1, FOXO3a, thus affect meat quality.