The Effect Of GPR120 On Placental Development And Its Role In Adipogenesis In Sows With High Backfat Thickness

Adipose tissue is known as both an energy storage organ and an important endocrine organ.Excessive deposition of body fat not only affects the carcass quality of pigs,but also affects the reproductive performance of pigs.Therefore,to elucidate the mechanism of adipose tissue formation from the perspectives of adipocyte production and lipid metabolism,and to explore the regulation of body fat distribution in nutritional ways has become one of the hot issues in current research.We have reported that sows with higher backfat thickness will get more low birth weight piglets,which has not been well clarified the mechanism as well as the fat accumulation of sows.Over the past two decades,it has been found that fat cell production is regulated by highly complex and precise transcriptional cascade networks.The core regulator is the peroxisome proliferator activated receptor γ(PPARγ).Nuclear receptor PPARγ is a key transcription factor to regulate adipogenic differentiation.It can respond to Long Chain Polyunsaturated Fatty Acid(LCPUFA)and play an important role in promoting adipogenic differentiation,placental formation,and angiogenesis.Recent studies have found that G protein-coupled receptor 120(GPR120)on the cell membrane also responds to LCPUFA-induced adipogenic differentiation,but its mechanism of regulation is unknown.Notably,what signals transduced by GPR120 are activated in the cell nucleus? How do these signals activate PPARγ to promote adipogenic differentiation? In addition,under conditions of maternal obesity during pregnancy,whether the expression of GPR120 and PPARγ is affected and cause placental dysfunction? These important issues are worth further studying.Therefore,this study aims to explore the mechanism how the maternal body condition during pregnancy affect the production of low birth weight(LBW);on the other hand,to elucidate the mechanism that GPR120 regulates adipogenesis through PPARγ.The mechanism of production of LBW piglets reveals the effect of normal expression of GPR120 and PPARγ on placental development and function.This study may help to clarify the mechanism of LBW production and adipogenic differentiation and lipid metabolism during maternal obesity.1,Maternal obesity aggravates the abnormality of porcine placenta by increasing N6-methyladenosine(1)1090 multiparous sows were divided into three groups(Group 1: backfat thickness ≤17mm,Group 2: backfat thickness 18-20 mm,Group 3: backfat thickness ≥21mm)to analyze the percentage of IUGR piglets.56,38,and 56 placentas were collected from the sows in each group respectively for the further research.Analysis of triglyceride levels in placental tissue revealed that group 3 was significantly higher than group 2(P<0.05),and there was an increased trend compared with group 1.(2)Compared with the other two groups,the sows with high backfat thickness have increased percentage of the piglets with birth weight ≤0.9kg and ≤1.0kg significantly.We focus on the LBW placental tissue from these three groups.The expression of key genes related to placental angiogenesis,PPARγ and vascular endothelial growth factor A(VEGFA)showed remarkable decrease in the LBW placentas from group 3.Furthermore,the placentas of LBW from group 3 also showed low vascularity density.Placental epigenetic modifications regulate the key genes related to placental development.Intriguingly,we for the first time found that the novel RNA modification m6 A was significantly increased in the LBW placentas from group 3.In line with the m6 A level,the expressions of the demethylase the fat mass and obesity-associated protein(FTO)were decreased at mRNA level as well as the protein level.Using the Me RIP-QPCR technology,the specific m6 A methylations modified in the PPARγ and VEGFA were found higher in the LBW placentas from group 3.This may help explain the low expression of the two genes.(3)Compared with the comparative groups,the expression levels of PPARγ,VEGFA,ABHD5 and GPR120 in both mRNA and protein decreased noticeably in the LBW group.It was also observed that the density of the H&E stained vessels became attenuated in LBW group.Importantly,for the first time,the increased m6 A levels were found in LBW placentas.Lower protein level of FTO(the key demethylase of m6A)was observed in LBW placentas,whereas no difference was found among the four groups in the expression levels of METTL3,the main methyltransferase of m6 A.By using Me RIP-QPCR technology,the m6 A modification in PPARγ,VEGFA,ABHD5 and GPR120,as well as FTO,was considerably enhanced in the placentas from LBW group.We infer that in maternity obesity,the higher m6 A modification displayed in the genes related to placental development,lipid metabolism and angiogenesis may result in the down regulation of these genes,which could be associated with m6 A demethylase FTO.Hence,it is important to uncover the mechanism of adipogenesis in sows.We focus on the role of GPR120 in adipose differentiation.2,Identification of porcine GPR120 transcripts and signals propertyIn the current study,we found that p GPR120 had 3 spliced variants.Transcript 1 encoded 362-amino acids(aa)wild type p GPR120-WT,which shared 88% homology with human short form GPR120.Transcript 1 was the mainly expressed transcript of p GPR120.It was expressed predominantly in ileum,jejunum,duodenum,spleen,and adipose.Transcript 3(coding 320-aa isoform)was detected in spleen,while the transcript 2(coding 310-aa isoform)was only slightly expressed in spleen.A selective agonist for human GPR120(TUG-891)and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293 T cells transfected with construct for p GPR120-WT but not p GPR120-V2.However,320-aa isoform was not a dominant negative isoform.Extracellular signal-regulated kinase 1/2(ERK1/2)phosphorylation levels in cells transfected with construct for p GPR120-WT were well activated by PUFAs,especially n-3 PUFA.These results showed that although p GPR120 had 3 transcripts,transcript 1 which encoded p GPR120-WT was the mainly expressed transcript.TUG-891 and PUFAs,especially n-3 PUFA,well activated p GPR120-WT.3,The function and mechanism of GPR120 in regulating adipocyte differentiation.This study was aimed to interpret the relevant function mechanism of GPR120 in the differentiation of 3T3-L1 adipocytes.(1)Collected 0,0.5,1,2,4,6,8 days of MDI-induced mouse 3T3L1 cells,the results showed that GPR120 expression was dramatically increased along with the adipogenic differentiation of 3T3-L1 adipocytes.Porcine adipose tissue vascular stroma cells also showed similar results when induced by MDI.The adipogenic ability was significantly inhibited in sh GPR120-transfected cells by lentivirus.(2)ALA and TUG-891,a selective agonist of GPR120,promoted the intracellular triglyceride accumulation in a dose-dependent manner and did not enhance adipogenesis in sh GPR120-transfected cells.Markedly,ALA and TUG-891 increased the activation of PPARg in a GPR120-dependent pathway as assessed by luciferase reporter assay.(3)Another endogenous agonist of GPR120,DHA can also activate the activity of PPRE but inhibit the expression of PPARγ,thereby inhibiting the adipogenic differentiation.(4)Furthermore,in the adipogenic differentiation process of 3T3-L1 adipocytes,TUG-891 increased the [Ca2+]i and phosphorylation level of ERK1/2.Pretreatment with inhibitors of either ERK1/2(U0126)or [Ca2+]I(BAPTA-AM)notably attenuated the GPR120-mediated adipogenesis.In summary:(1)Maternal obesity(backfat thickness ≥21mm)could influence normal fetal growth,causing LBW.In maternal obesity,considerable differences were found between LBW fetuses and normal ones in the expressions of the genes related to lipid metabolism and angiogenesis.We deduce that the higher m6 A modification in the key genes in LBW placenta may participate in the regulation of their expressions;FTO,the demethylase,could be the vital regulator in placental development and fetal growth.(2)The mechanism of p GPR120 promoting adipogenic differentiation is that with the activation of ALA and specific agonists,the expression of nuclear receptor PPARγ is upregulated by the intracellular calcium-ERK1/2 signaling pathway,accounting for promoting adipogenesis.Only ALA,but not DHA,activates PPARγ expression and promotes adipogenic differentiation in the p GPR120-dependent.