The Regulation Mechanism Of Fibroblast Growth Factor 21 On Lipid Metabolism In Fully Differentiated Adipocyte In Goats

As an individual member of the FGF family, fibroblast growth factor 21 （FGF21） is an adipocytokine discovered in recent years. It is expresse predominantly in skeletal muscle, adipose, liver and pancreas. FGF21 have recently emerged in regulating energy homeostasis, glucose and lipid than the other FGFs. With the occurrence of obesity and diabetes, the effect of FGF21 on adipose metabolism is becoming a hot topic of research. For goat breeding, to research the the effect of FGF21 on adipose metabolism and energy balance in goat had a great theoretical significance.Here, cells were stained with Oil Red O to investigate the formation of lipids of goat adipocytes. Using Mito Tracker 7514 staining, we studied the effect of FGF21 on mitochondria mass. qPCR was employed to detect the relative expressions of lipolysis-related genes （adipose triglyceride lipase, hormone sensitive lipase, lipoprotein lipase, carnitine palmitoyltransferase 1, uncoupling protein 1） and lipogenesis-related genes （sterol regulatory element binding transcription factor 1, peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, acetyl-CoA carboxylase alpha） and expression of genes implicated in mitochondrial biogenesis, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha, nuclear respiratory factor 1, mitochondrial transcription factor A. The detailed results are as follows:1. Here, we obtained the full-length coding sequences of goat FGF21 （Accession number:KU375570）, containing an open reading frame （ORF） of 630 base pairs and encoding 209 amino acids. The putative goat FGF21 protein shared 99.04%,66.53%, 75.24%,91.39% and 85.71% identity with that of cattle （XP₀10813507.1）, sheep （XP₀11950505.2）, mouse （AAH49592.1）, pig （ACS34770.1）, and human （AAH 18404.1）, respectively. In addition, between goat and human the FGF domains （contained 126 residues from Gln43 to Pro168） were highly conserved （86.51% identity）. Especially, they had almost same receptor interaction binding sites （constructed by 17 residues） and similar heparin binding sites （glycine box）.2. Fully differentiated goat adipocytes were incubated with 75 nM FGF21 for 24 h,36 h and 48 h and stained with red O oil. Adipocytes displayed significantly smaller lipid droplets with FGF21 treatment. At 24 h after FGF21 treatment, the small lipid droplets （R<50 um） increased by 8% while middle （R:50-99.9 um） and large （R>100 um²） droplets were decreased by 3% and 5%（FGF21 treatment,34.63%, n=503; control,39.16%, n=453）, respectively. At 36 h the percentage of large lipid increased slightly, accompanied by slight decrease of small lipid droplets. Furthermore, the percentage of both middle and large size lipid droplets was increased whereas small droplets declined dramatically at 48 h after FGF21 treatment （P< 0.05）.3. qPCR analyses revealed that the expressed of fatty acid decomposition related genes. The experimental results show that the levels of ATGL, HSL, LPL, CPT-1 and UCP1 mRNAs were increased by FGF21 treatment at 24 h and at 36 h （Fig.3G） （P< 0.05）, but only ATGL mRNA levels maintained the significant higher level at 48 h, compared to control groups.4. FGF21 suppressed the mRNA levels ofSREBP1, PPARγ, CEBP/α and ACC at 24 h （P< 0.05）, and lowered the levels of the SREBP, PPARy, CEBP/a and ACC mRNA till 36 h and 48 h （P< 0.05）. Intriguingly, fatty acid synthase （FAS） expression did not affected by FGF21 treatment throughout the experiment （P> 0.05）.5. FGF21 treatment increased OPA1and Mfn1 levels at 24 h and 36 h but decreased the expression of OPA1, Mfn1 and Mfn2 at 48 h. Treatment with FGF21 decreased FIS1 level at 24 h,36 h and 48 h （P> 0.05）.6. We found out that mitochondrial content was significantly increased in cells treated with FGF21 for 24 h,36 h and 48 h （P< 0.05） compared with nontreated. Furthermore, the levels of PGC1a, NRF1, and TFAM mRNA were dramatically increased in adipocytes treated with FGF21 for 24 h （P<0.05） and 36 h （P<0.05）, while at 48 h only the expression of NRF1 and TFAM genes were slightly higher than control （P>0.05）.