| The intramuscular fat (IMF) content is a key factor that influences the pork qualities,such as the pork juiciness, tenderness and flavor. Increasing the IMF content is an important way to improve pork quality. However, IMF content and back fat thickness (BFT) are positively related to each other. Confirming the differences of physiological characteristics and regulation pathways between intramuscular adipocytes (IMA) and subcutaneous adipocytes (SA) could contribute to regulate IMA specifically. Glucocorticoids (GCs) play vital roles in promoting fat deposition, were able to induce the proliferation and differentiation of pre-adipocytes. Recent studies showed that the GCs sensitivity decided the ability of fat deposition. In present study, we analyze the differences of GCs sensitivity between SA and IMA at first. Due to the special location of IMA, we examine the effects ofmuscle on the adipocytes by muscle tissue culture fluid and myocytes-adipocytes co-culture methods. Besides, we continue to seek the possible myokines which can regulate the GCs sensitivity in order to explore the mechanisms of the GCs sensitivity differences between SA and IMA.The main results achieved are as follows:1. The differences of GCs sensitivity between SA and IMABoth subcutaneous and intramuscular pre-adipocytes were treated with dexameson(DEX) in different concentration to induce their proliferation. Cell viability were detected by CCK8. The results showed that DEX promotes the proliferation of these two types of cells. 100nM DEX have a more significant effect on subcutaneous pre-adipocytes than on intramuscular pre-adipocytes after treated for 24, 48, 72 hours (P<0.5). 2.5?M DEX were used to induce pre-adipocytes differentiation and then TG level in cells were detected,we found that TG accumulation level in subcutaneous pre-adipocytes was 29% higher than intramuscular pre-adipocytes after treated with DEX (P<0.5).Both subcutaneous and intramuscular mature adipocytes were treated with DEX, the results indicated that both cells had no significant TG increase but a significant adipolysis levels (P<0.5), whereas no significant difference between the adipolysis levels of two cells.Insulin (INS) has a synergistic effect with DEX to promote fat deposition, DEX combined with the INS can increase the TG accumulation of mature adipocytes and inhibit the adipolysis, and their function of promoting TG accumulation and inhibition of adipolysis in subcutaneous mature adipocytes were significantly higher than that in intramuscular mature adipocytes (P<0.5).14 sows were treated with adrenocorticotropic hormone (ACTH) (which can promote the release of endogenous glucocorticoids) to study the subcutaneous adipose tissue (SAT)and intramuscular adipose tissue (IMAT) glucocorticoid sensitivity in vivo. The results indicated that ACTH treatment promotes adipogenesis maker gene such as FASN, STAT5,SRC2 and SRC3 expression in these two types of cells,and its function in SAT significantly higher than that in IMAT(p<0.05). This treatment also promotes adipolysis maker gene such ATGL and HSL expression, furthermore its function in SAT significantly higher than that in IMAT (P<0.05). The result showed that endogenous glucocorticoid sensitivity of subcutaneous fat is stronger than that in intramuscular fat.2. The differences of glucocorticoid receptor levels between SAT and IMATIn order to reveal the mechanism of different GCs responsiveness, we detected the expression patterns of Glucocorticoid receptor (GR) in SAT and IMAT. The results showed that GR total RNA expression levels in SAT is 1.9 times of that in IMAT (P <0.05). The function subtype GRa mRNA in SAT mRNA levels is 2.2 times of that in IMAT (P<0.05).It is suggested that because of its higher GRa level SAT owns a higher glucocorticoid responsiveness than IMAT.To analyze the difference of GRa level between SAT and IMAT, nine kinds of GR gene Exon1 variants were detected. The results showed that expression level of E1-C was the highest, it is 65% to 76% of the total GR mRNA, thus E1-C expression had a maximum effect on the total GR expression. Expression levels of variants E1-F, E1-D, E1B, E1A,E1-H were medium (1.6%-10.2%), and expression levels of variants E1-E, E1-J, E1-G were lower (0.4%-2.8%). Moreover, variants E1-A, E1-C and E1-H in SAT were significantly higher than that in IMA (P<0.05).Exon 1 variants expression patterns in GRa and GR? shows that variants E1-A and E1-B expression in GR? were significantly higher than that in GRa (P<0.05). Thus E1-A and E1-B tend to have more effect on GR? expression. Whereas E1-H expressed only in GRa indicating that variant E1-H transcripts encoding only GRa subtypes, moreover E1-H level in SAT was 1.9 times of that in IMAT(P<0.05). Therefore, GRa level in SAT was higher than that in IMAT mainly because that SAT had a higher main variant E1-C level and a higher GRa specific variant E1-H level.3. Muscle tissue culture fluid influences the proliferation, differentiation and apoptosis of adipocytes by inhibiting GRa expressionBecause of its special location, we assume that IMF glucocorticoids sensitivity was influenced by muscles via inhibition of GRa level. Pre-adipocytes were treated with muscle tissue culture fluid (TCF), the results showed that GR variants E1-C and E1-H levels werereduced by 32% and 73% respectively and GRa level reduced by 46% (P<0.05).Cell counting results showed that after treated with TCF the number of cells decreased by 41% (P<0.05) compared to the control group, it indicates that TCF treatment inhibits the proliferation of pre-adipocytes. Also we found that GR antagonist RU486 has a similar effect on the inhibition of proliferation of pre-adipocytes, it leads to the number of adipocytes decreased by 38% (P<0.05); DEX can rescue the effects of TCF on inhibition of adipocytes proliferation, the number of cells in TCF+DEX group was significantly higher than TCF group (P<0.05). Determination of TG content and oil red O staining cells showed that TCF significantly inhibited the differentiation of pre-adipocytes and TG deposition decreased by 45% (P<0.05).Flow cytometry was used to measure pre-adipocytes cycle,the results showed that G2-M phase cells increased by 60%, while the G0-G1 phase cells decreased by 23% which indicates that pre-adipocytes were arrested in the G2-M phase by TCF. RU486 has a similar effect on cell cycle which led G2-M phase cells increased by 38%, while G0-G1 phase cells decreased by 14%. Adding DEX to TCF can rescue the inhibition of TCF on cell cycle.Flow cytometry was used to detected the apoptosis of pre-adipocytes, the results showed that late apoptosis rate increased 3.3-fold, cell necrosis rate increased 8-fold which indicates that TCF promotes the apoptosis of pre-adipocytes. RU486 has a similar effect on cell apoptosis, late apoptosis rate increased 2.4-fold, cell necrosis rate increased 6.8-fold.Adding DEX to TCF can rescue the inhibition of TCF to cell apoptosis.4. C2C12 myotubes influence proliferation, differentiation and apoptosis of 3T3-L1 adipocytes by reducing GRa expressionTo study the effects of mature myotubes on GRa level and glucocorticoid response in adipocytes, we established a myoblasts C2C12 and pre-adipocytes 3T3-L1 co-culture model by using trans-well chamber. The co-culture results showed that the total number of cells decreased by 34% and EdU negative cells increased by 89% which indicates that mature myotubes significantly inhibited the proliferation of 3T3-L1 pre-adipocytes. Results from Oil Red O staining and determination of TG content experiment showed that TG deposition decreased by 39% which indicates that C2C12 significantly inhibited adipocyte differentiation. Meanwhile C2C12 myotubes leads to a significant caspase 3 activity increase in 3T3-L1 pre-adipocytes, the proportion of early apoptotic cells increased from 3.8% to 14.1%, the proportion of late apoptotic cells increased from 6.8% to 9.1% (P<0.5).The mRNA and protein of GRa were detected, we found that C2C12 significantly inhibited 3T3-L1 cells GRa level (P<0.5). The expression of Cyclin D (cell proliferation marker gene), PPARy (adipocytes differentiation marker gene) and FAS (apoptotic marker gene) in 3T3-L1 pre-adipocytes were significantly altered after co-cultured with C2C12,and DEX can rescue the C2C12 effects, however expression of these gene cannot return to the same level as control group. All these results indicated that C2C12 myotubes can reduce glucocorticoid response of 3T3-L1 pre-adipocytes, inhibit the proliferation, differentiation of 3T3-L1 pre-adipocytes and promote their apoptosis. Thus, the results of TCF function in previous chapter were verified.5. Myostatin influence proliferation and apoptosis of adipocytes by reducing the GRa expressionIn our previous study we found that a special factor secreted by muscle called myostatin (MSTN) can inhibit the differentiation of adipocytes. Then we treated adipocytes with MSTN, the results showed that compared to the control group GRa and variants E1-C,El-H mRNA levels was reduced by 36%, 35% and 33% respectively, GRa protein levels decreased by 24% (P<0.5). CCK8 and EdU staining method were used to detected cell proliferation,the results showed that MSTN inhibits the proliferation of adipocytes,cell viability reduced significantly,EdU negative cells ratio increased 1.83 times (P<0.5). Flow cytometry results showed that cells in G0-G1 phase increased by 8.3%, cells in S phase decreased by 5.1% (P<0.5). Apoptosis assay results showed that early and late apoptosis rate were increased by 220% and 90% respectively (P<0.5).6. Myostatin increased El-C and E1-H promoter methylation levels, inhibit the expression of GRaGR gene promoter DNA methylation can suppress the expression of GR, moreover it is reported that MSTN can regulate DNA methylation of adipocytes. Bisulfite sequencing results showed that within all the methylation sites detected, the methylation level of variant E1-C promoter in IMAT tissues was 66%, which is significant higher than the 46%in SAT tissues (P<0.5). The whole methylation level of El-H promoter was 15%,which is significant higher than the 9% in SAT tissues(P<0.5). Different methylation status wereexhibited between SAT and IMAT especially in the sites CpG8, CpG14, CpG30, CpG37 and CpG38 (P<0.5). The influences of MSTN to E1-C and E1-H promoter methylation levels in adipocytes were detected by DNA methylation immunization (MeDIP), the results showed that the methylation levels of E1-C promoter increased from 15% to 29%, andlevels of E1-H promoter increased from 15% to 26% (P<0.5).In summary, muscle secretes MSTN, increases GR variants E1-C and E1-H promoter methylation levels in IMAT, leading to the inhibition of GRa expression, thus glucocorticoid sensitivity in IMAT were reduced which blocked the Glucocorticoid-induced proliferation, differentiation and anti-apoptosis effects, resulting in inhibiting of intramuscular fat deposition. |
PDF Full Text Request