The Regulatory Role Of PPARG Gene In Fatty Acid Metabolism Of Goat Mammary Gland

Goat milk is rich in short-and medium-chain fatty acids and unsaturated fatty acids,which has a good health care and can be effective in preventing certain metabolic diseases ofhumans. The unique characteristics for goat milk is due to its fatty acids content. The study onmechanism of fatty acid metabolism in goat mammary tissue will help to improve the qualityof goat milk. Peroxisome proliferator-activated receptor gamma (PPARG), a key transcriptionfactors related to fatty acid metabolism, influence the formation of lipid droplets anddeterminate fatty deposits in adipose tissue. However, the role of PPARG in regulating fattyacid metabolism in lactating mammary gland of goat is rarely reported.To investigate the role of PPARG in goat mammary gland, firstly, mammary tissue fromdifferent physiological periods of Saanen dairy goat was collected, and transcriptomesequencing was done to explore the mRNA profiling of goat mammary tissue. The PPARGgene was cloned and its expression in various tissue was analyzed. Its function was verifiedthrough pharmacological ligand treatment, RNAi and overexpression mediated by adenovirus.The results showed that PPARG can largely regulate genes related to fatty acid metabolism,and alter fatty acid composition in goat mammary epithelial cells (GMEC). PPARG plays animportant role in the goat milking process. The main results of this study are shown asfollows:1. We got effective and high-quality data about25Gb from the dairy goat mammary tissuetranscriptome sequencing. The total number of Unigenes is51,361, of which60.07%proteinsequence homology with NR, while there were55.65%,56.99%,53.11%and32.8%ofUnigenes could be annotated to SWISS-PROT, KEGG, KOG and Pfam database, respectively.Compared to NR database, the majority of annotated dairy goat mammary glandtranscirptome corresponded to sequences of Ovisaries with a match of31.2%, followed byBos taruus (15.7%), Bos grunniens (11.3%), Homo sapiens (2.9%), Orciuns orca (2.6%) andCeratotherium simum (2.4%). These Unigenes are involved to multiple metabolic pathways offatty acid metabolism, protein metabolism. In the lipid transport and metabolism category,FABP3, FASN, SCD are three top genes in mRNA expression, while the expression oftranscription factors such as PPARG, NR1H3and SREBP1is lower.2. The full cDNA of goat PPARG1is1906bp, containing:5’UTR114bp, CDS1428bp and3’UTR215bp, while complete CDS region of PPARG2is1528bp. Goat PPARG gene isconserved among species and the similarity is more than90%compared to other mammals.Tissue expression analysis showed that PPARG1widely expressed in many tissues, whilePPARG2have more tissue-specific expression. Both PPARG isoforms are highly expressed inadipose tissue, but PPARG1is primary form of PPARG in goat mammary gland.3. Luciferase assay showed that rosiglitazone (ROSI) could significantly activate PPARG,and its activity reached greatest at50μmol/L ROSI. After activated by ROSI in GMEC,PPARG’s expression is not significant changed, however, its downstream genes ACACA,FASN, SCD, FABP3, LPL, ADRP, PNPLA2SREBF1and NR1H3significantly increased,which are related to fatty acid metabolism. GMEC incubated with ROSI for24hours did notsignificantly change the composition of long-chain fatty acids including C16:0, C16:1, C18:0and C18:1and indices of unsaturated fatty acid desaturation. MTT assay showed that ROSIcan inhibit the growth of GMEC.4. Two effective shRNA sequence (sh614and sh1006) was screened. Two adenovirusvectors was constructed, and then Ad-sh614and Ad-sh1006adenovirus were packaged withthe titers of109and108U/mL, respectively. It was found that Ad-sh1006was more efficientto knockdown PPARG in GMEC. The decrease in expression observed after knockdown ofPPARG relative to the control group (Ad-NC) averaged65%,52%,67%,55%,65%,58%,85%,43%,50%, and24%for SCD, DGAT1, AGPAT6, SREBF1, ACACA, FASN, FABP3,SCAP, ATGL and PLIN3, respectively.5. Ad-PPARG1adenovirus was constructed and packed for overexpressing PPARG1.After overexpression of PPARG1in GMEC at the presence of ROSI, the mRNA expressionfor ACACA, CD36, DGAT1, INSIG1, FASN, LPL, NR1H3, PLIN2, PLIN3, PNPLA2, SCAP,SCD, SREBF1was significantly increased (P<0.05). The gene network regulated by PPARG1in GMEC were developed among different treatments. The ratio of both C16:1and C18:1increased significantly (P<0.05) at the expense of C16:0and C18:0after infection withAd-PPARG1adenovirus. Furthermore, the desaturation indices of both C16:1and C18:1wereincreased significantly (P<0.05) after overexpression of PPARG1. The increases fordesaturation indices of C16:1and C18:1were enhanced by the treatment of ROSI.6. We constructed and packed Ad-PPARG2adenovirus for overexpressing PPARG2. Atthe presence of ROSI, overexpression of PPARG2in GMEC significantly increased themRNA expression for CD36， DGAT1， INSIG1， FASN， NR1H3， PLIN2， PLIN3，PNPLA2，SCAP， SCD (P<0.05), meanwhile had no significant effect on SREBF1andACACA. However, overexpression PPARG2alone decreased the SREBF1and ACACA mRNAlevel(P<0.05) while had no significant effect on the expression PLIN3, PNPLA2, SCAP, and NR1H3. The ratio of both C16:1and C18:1increased significantly (P<0.05) at the expense ofC16:0and C18:0after infection with Ad-PPARG2adenovirus. The desaturation indices ofboth C16:1and C18:1were increased significantly (P<0.05) after overexpression of PPARG2.The increases for desaturation indices of C16:1and C18:1were enhanced by the treatment ofROSI.PPARG stimulates the synthesis of unsaturated fatty acids by promoting the activity ofSCD in GMEC. PPARG isoforms have different signaling pathways in terms of regulating FAbiosynthesis and TAG formation. The role of PPARG1in the regulation of mammary FAbiosynthesis appears strong. The regulation of PPARG1in fatty acid metabolism is partialmediated by SREBP1and NR1H3. PPARG2overexpression plus ROSI are more similar tothe negative regulation that PPARG elicits on FA biosynthesis in white adipose cells.