Vitamin C Promotes Adipogensis Of 3T3-L1 Preadipocytes By Attenuating ERK Signaling To Up-regulate The Collagen Ⅵ

White adipocytes are embedded in an extracellular matrix(ECM)network composed of collagens and proteoglycans.Collagens are the most abundant proteins that make up interstitial fibrin and cellular basement membranes.Studies have shown that collagen VI(Col VI)gradually increased in adipogenic differentiation of white preadipocytes and highest in mature adipocytes.Extracellular regulated protein kinases(ERKs)are members of the mitogen-activated protein kinase family and regulate cell proliferation and differentiation.Vitamin C(VC)has the dual role of promoting triglyceride accumulation and lipolysis,and is involved in the hydroxylated modification of collagen after translation.VC has the role of promoting adipogenic differentiation of white preadipocytes,while its regulatory mechanism is still not fully understood.I made assumptions based on the previous research results and our preliminary experimental results,which is that VC up-regulated the expression of Col VI and lipid-related molecules by attenuating the phosphorylation of ERK1/2,eventually promoting the adipogenic differentiation of animal white preadipocytes.This article explores the molecular mechanism by which VC promotes adipogenic differentiation of 3T3-L1 mouse preadipocytes and validates this hypothesis.1.Experimental grouping3T3-L1 preadipocytes were divided into 6 groups.The cells were induced to mature adipocytes at D0 to D14 simultaneously treated with AA2 P,ERK1/2 phosphorylation inhibitor(U0126),total collagen synthesis inhibitor(EDHB),Lenti-Col VI-GFP and EDHB + U0126,respectively.Experimental groups are as follows.Group 1 were non-induced group of complete medium and complete medium + AA2 P group,respectively(N.I.and N.I.+ AA2P);Group 2 were adipogenic induction group and adipogenic induction + AA2 P group,respectively(I and I + AA2P);Group 3 were adipogenic induction + U0126 group and adipogenic induction + U0126 + AA2 P group,respectively(I + U0126 and I + U0126 + AA2P);Group 4 were adipogenic induction + EDHB group and adipogenic induction + EDHB + AA2 P group,respectively(I + EDHB and I + EDHB + AA2P);Group 5 were adipogenic induction + lentiviral-Col VI group and adipogenic induction + lentivirus-Col VI + AA2 P group,respectively(I + Lenti-Col VI and I + Lenti-Col VI + AA2P);Group 6 were adipogenic induction + U0126 + EDHB group and adipogenic induction + U1026 + EDHB + AA2 P group,respectively(I + EDHB + U0126 and I + EDHB + U0126 + AA2P).2.Results(1)AA2P promotes adipogenic differentiation of 3T3-L1 mouse preadipocytes(I)The characteristic of morphological changes: the adipogenic differentiation of 3T3-L1 preadipocytes could be initiated by AA2 P treatment alone,but its adipogenic rate was much lower than that of the classical inducer(the adipogenic differentiation group,I group).The combination of AA2 P on the basis of the addition of classical inducers could significantly promote the adipogenic differentiation of 3T3-L1 preadipocytes,and the adipogenic rate was higher than that of the purely induced group,indicating that the effect of AA2 P and classic inducers on the adipogenic differentiation of preadipocyte is positive superimposed.(II)The characteristics of target molecule transcription and translation level changes: AA2 P significantly up-regulated mRNA and protein expression of Col VI,SREBP-1c,PPARγ2 and C/EBPα throughout adipogenic differentiation of preadipocytes,and delayed the down-regulation of C/EBPβ in early and middle stages of adipogenic differentiation(D0-D7);The phosphorylation level of ERK1/2(p-ERK1/2)was significantly decreased in early and middle stages of adipogenic differentiation by AA2 P,but had no effect on total ERK1/2.(2)Inhibition of ERK1/2 phosphorylation could block the adipogenic differentiation of preadipocytes(I)The characteristic of morphological changes: U0126(100 μM),which is a specific inhibitor of ERK1/2 phosphorylation and was added at the early stage of adipogenic differentiation(D0-D3),could completely block the process of adipogenic differentiation of preadipocytes,while AA2 P partially(15.2 ± 5.4%)reversed this inhibitory effect.(II)The characteristics of target molecule transcription and translation level changes: U0126 significantly reduced the phosphorylation of ERK1/2 at the early stage of adipogenic differentiation of preadipocytes;The expressions of C/EBPβ,PPARγ2 and C/EBPα mRNA and protein were significantly down-regulated,while the expression of Col VI mRNA and protein were significantly up-regulated throughout the process of adipogenic differentiation.The expression of SREBP-1c mRNA and protein were significantly down-regulated at the late stage of adipogenic differentiation.The expression of SREBP-1c,C/EBPβ,PPARγ2 and C/EBPα mRNA and protein were up-regulated at the late stage of adipogenic differentiation(D8-D14)after treated with AA2P;the phosphorylation of ERK1/2 was completely blocked by AA2 P in the early stage of adipogenic differentiation.(3)Blocking the synthesis of total collagens inhibited the adipogenic differentiation of preadipocytes(I)The characteristic of morphological changes: ethyl-3,4-dihydroxybenzoate(EDHB,100 μM),an inhibitor of collagen synthesis,could completely inhibit adipogenic differentiation of 3T3-L1 preadipocyte.AA2 P could partially(50.6 ± 4.8%)reversed the inhibitory effect of EDHB on adipogenic differentiation of preadipocytes.(II)The characteristics of target molecule transcription and translation level changes: EDHB significantly up-regulated the expression of Col VI mRNA during the process of adipogenic differentiation of preadipocytes,whereas the expression of Col VI protein decreased in the early stage,increased in the middle stage and then decreased again in the late stage of adipogenic differentiation,the phosphorylation of ERK1/2 was opposite;The expression of SREBP-1c,C/EBPβ,PPARγ2 and C/EBPα mRNA and protein were significantly down-regulated in adipocytes.the expression of mRNA and protein of Col VI,SREBP-1c,C/EBPβ,PPARγ2 and C/EBPα mRNA and protein were up-regulated at the late stage of adipogenic differentiation by AA2P;the phosphorylation of ERK1/2 was reduced at the early stage of adipogenic differentiation.(4)Knockdown of Col VI inhibited the adipogenic differentiation of preadipocyte(I)The characteristic of morphological changes: knockdown of Col VI significantly inhibited preadipocyte adipogenic differentiation.AA2 P could partially(20.4 ± 4.2%)reverse the inhibition.(II)The characteristics of target molecule transcription and translation level changes: The Col VI mRNA knockdown rate was 65.3 ± 0.5%;the expressions of SREBP-1c,C/EBPβ,PPARγ2 and C/EBPα mRNA and protein were significantly down-regulated throughout the adipogenic differentiation of preadipocytes,while phosphorylation of ERK1/2 was significantly increased at the early stage of adipogenic differentiation(D3).The expression of SREBP-1c,C/EBPβ,PPARγ2 and C/EBPα were upregulated by AA2 P,while the expression level was still lower than that of Col VI non-knockdown group.In addition,ERK1/2 phosphorylation was significantly X reduced at the early stage of adipogenic differentiation.(5)Blocking ERK1/2 phosphorylation and total collagen synthesis at the same time could inhibit the preadipocyte inhibit adipogenic differentiation(I)The characteristic of morphological changes: combined use of EDHB and U0126 completely inhibits adipogenic differentiation of preadipocytes.AA2 P could partially(10.5 ± 3.2%)reversed the inhibitory effect of U0126 + EDHB on adipogenic differentiation of preadipocytes.(II)The characteristics of target molecule transcription and translation level changes: the combined use of EDHB and U0126 could significantly reduce the phosphorylation level of ERK1/2.The expression of Col VI mRNA in adipocytes was significantly up-regulated while the expression of Col VI protein was significantly down-regulated throughout the process of adipogenic differentiation;the expression of SREBP-1c,C/EBPβ,PPARγ2 and C/EBPα mRNA and protein were significantly down-regulated.Although the expression of Col VI mRNA,SREBP-1c and C / EBPα mRNA and protein were up-regulated by AA2 P,there was no significant difference compared with the U0126 + EDHB group;Col VI protein was increased by AA2 P in the late adipogenic differentiation;the expression of C/EBPβ mRNA and protein were significantly up-regulated by AA2 P at the early stage of adipogenic differentiation;the expression of PPARγ2 mRNA and protein were significantly up-regulated at the late stage of adipogenic differentiation;the phosphorylation of ERK1/2 was further decreased by AA2 P at the early stage of adipogenic differentiation and disappeared to the late stage.3.Conclusions(I)AA2P alone could activate adipogenic differentiation of 3T3-L1 preadipocytes without addition of adipogenic inducers;the effect of AA2 P and adipogenic inducers was positive superposition;the simultaneous addition of AA2 P and adipogenic inducers could maximize the adipogenic differentiation of preadipocytes,suggesting that the effect of AA2 P and adipogenic inducers is positively additive;(II)U0126,a specific inhibitor of ERK1/2 phosphorylation could significantly up-regulate the expression of Col VI,indicating that ERK1/2 phosphorylation could inhibit the expression of Col VI;(III)EDHB,protein an inhibitor of total collagens synthesis,and knockdown of Col VI could increase ERK1/2 phosphorylation,indicating that Col VI could inhibit ERK1/2 phosphorylation;(IV)AA2P could up-regulate the expression of Col VI by attenuating ERK1/2 phosphorylation at the early stage of adipogenic differentiation.The increased Col VI may further attenuate the phosphorylation of ERK1/2,thereby releasing the phosphorylation of PPARγ by phosph-ERK1/2,indirectly enhances its transcriptional activity and promotes the adipogenic differentiation of 3T3-L1 preadipocytes.