| Milk fat is a main nutritional component of milk. Milk fat can provide nutrient and energy for humans. The production of high quality milk with enough milk fat is the developmental tendency of health food. However, milk fat content in milk is generally low in our country, which weaken competitiveness of dairy products in domestic and foreign markets. Therefore, the improvement of milk yield and milk fat content become a problem that we need to solve immediately. Milk fat precursor of dairy cows mainly are acetic acid, β-hydroxy butyrate and long-chain fatty acids which mammary gland take in from blood. The previous studies mainly focus on the effect of milk fat precursor on production performance of dairy cows. But, little is reported about the interaction between milk fat precursors and functional genes in milk fat synthesis of mammary gland.This study firstly screen the differentially expressed genes and proteins of milk fat synthesis in mammary glands between dry milk cows, low milk quality cows and high milk quality cows by qRT-PCR and Western blotting. These differentially expressed genes and proteins serve as the evaluation indexs of the in vitro experiment. A dairy cow mammary epithelial cell (DCMECs) line was established through culture method of tissue block in the study. DCMECs were supplemented with sodium acetate, β-hydroxy sodium butyrate, the mixture of sodium acetate and (3-hydroxy sodium butyrate, palmitic acid and stearic acid. The mRNA expression levels of genes about fatty acid uptake, transport, activation related proteins (CD36, FABP3, ACSS2, ACSL1), fatty acid synthases and desaturase (ACC, FAS, SCD), triglyceride synthetases (GPAT, AGPAT6, DGAT1), and transcriptional regulation factors (PPARy, PPARGC1A, SREBP1, INSIG1and SCAP) in milk fat synthesis of DCMECs were determined by qRT-PCR. The protein expression levels of transcriptional regulation related factors in milk fat synthesis were determined by Western blotting. The activities of triglyceride synthetases were determined by enzyme activity kits.The location of PPARy in DCMECs was detected by immunofluorescence cell staining. Gene silencing and overexpression of PPARy in DCMECs were conducted in this study. The change of concentration demand to fatty acids of DCMECs with PPARy mRNA interference or PPARy overexpression were determined by detecting triglyceride contents in culture medium. Simultaneously, the effect of fatty acids on the mRNA expression levels of related gene, the protein expression levels of transcriptional regulation related factors, and the activities of triglyceride synthetases in milk fat synthesis of DCMECs with PPARy mRNA interference or PPARy overexpression were determined by qRT-PCR, Western blotting and enzyme activity kits. The results showed as follows:1. The mRNA expression levels of CD36, FABP3, ACSS2, ACSL1, ACC, FAS, SCD, GPAT, AGPAT6, DGAT1, PPARy, PPARGC1A, SREBP1, INSIGland SCAP were significantly increased in mammary glands of the low milk quality cows and the high milk quality cows compared to dry milk cows (P<0.05). Moreover, the mRNA expression levels of these genes in mammary glands of the high milk quality cows were obviously higher than the low milk quality cows (T<0.05). The protein expression levels of CD36, FABP3, PPARγ, PPARGC1A, SREBP1, INSIG1and SCAP were significantly elevated in mammary glands of the low milk quality cows and the high milk quality cows compared to dry milk cows (P<0.05). Simultaneously, the protein expression levels of these proteins in mammary glands of the high milk quality cows were markedly higher than the low milk quality cows (P<0.05).2. The results of the effect of different fatty acids on milk fat synthesis in DCMECs showed as follows:(1) Sodium acetate at12mmol/L significantly increased the mRNA expression levels of FABP3, ACSS2, ACC, FAS, SCD (P<0.05) and markedly decreased CD36mRNA level (P<0.05). Sodium acetate obviously upregulated the mRNA and protein expression levels of PPARy, PPARGC1A, SREBP1, INSIGland SCAP (P<0.05). Sodium acetate significantly enhanced the mRNA expression levels and the activities of GPAT、AGPAT6、DGAT1(P<0.05).(2) β-hydroxy sodium butyrate at1mmol/L obviously upregulated the mRNA expression levels of FABP3, ACSS2, ACC, FAS, SCD (P<0.05) and significantly reduced CD36mRNA level (P<0.05).(3-hydroxy sodium butyrate markedly enhanced the mRNA and protein expression levels of PPARγ, PPARGC1A, SREBP1, INSIG1, SCAP (P<0.05). β-hydroxy sodium butyrate significantly increased the mRNA expression levels and the activities of GPAT、AGPAT6、 DGAT1(P<0.05).(3) The concurrent treatment of sodium acetate at12mmol/L and P-hydroxy sodium butyrate at1mmol/L significantly increased the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, ACC, FAS, SCD (P<0.05). The synergistic action of sodium acetate and β-hydroxy sodium butyrate obviously promoted the mRNA and protein expression of PPARy, PPARGC1A, SREBP1, INSIG1and SCAP (P<0.05). The mRNA expression levels and the activities of GPAT、AGPAT6、DGAT1were enhanced by the synergistic action of sodium acetate and p-hydroxy sodium butyrate (P<0.05).(4) Palmitic acid at150μmol/L obviously enhanced the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, SCD (P<0.05). Palmitic acid significantly upregulated the mRNA expression levels and the activities of GPAT, AGPAT6and DGAT1(P<0.05). Palmitic acid markedly increased the mRNA and protein expression levels of PPARy, PPARGC1A, SREBP1, INSIG1and SCAP (P<0.05).(5) Stearic acid at125μmol/L markedly upregulated the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, SCD (P<0.05). Stearic acid significantly enhanced the mRNA expression levels and the activities of GPAT, AGPAT6and DGAT1(P<0.05). Stearic acid obviously increased the mRNA and protein expression levels of PPARy, PPARGC1A, SREBP1, INSIG1and SCAP (P<0.05). 3. The protein of PPARy was mainly located in the nucleus of DCMECs by the laser confocal microscope.4. The mRNA expression levels of CD36, FABP3, ACSL1, ACC, FAS and SCD (P<0.05), the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05), the mRNA and protein expression of PPARy, PPARGC1A, SREBP1, INSIG1and SCAP (P<0.05) were significantly decreased in DCMECs with PPARy mRNA interference compared with nontransfected group and negative control group. The mRNA expression levels of CD36, FABP3, ACSL1, ACC, FAS and SCD (P<0.05), the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05), the mRNA and protein expression of PPARy, PPARGC1A, SREBP1, INSIG1and SCAP (P<0.05) were obviously increased in DCMECs with PPARy overexpression compared with nontransfected group and pGCMV-IRES-EGFP group.5. Compared with the normal DCMECs, the required quantity of different fatty acids to DCMECs with PPARy mRNA interference were increased. But, the required quantity of different fatty acids to DCMECs with PPARy overexpression were decreased.6. The results of the effect of different fatty acids on milk fat synthesis in DCMECs with PPARy mRNA interference showed as follows:(1) Compared to DCMECs with PPARy mRNA interference, the mRNA expression levels of FABP3, ACSL1, ACSS2, ACC, FAS, SCD, PPARGC1A, INSIG1and SCAP (P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05), and the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05) all were significantly improved in DCMECs with PPARy mRNA interference and16mmol/L sodium acetate.(2) Compared to DCMECs with PPARy mRNA interference, the mRNA expression levels of FABP3, ACSL1, ACSS2, ACC, FAS, SCD, PPARGC1A, INSIG1and SCAP (P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05), and the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05) all were obviously increased in DCMECs with PPARy mRNA interference and1mmol/L β-hydroxy sodium butyrate.(3) Compared to DCMECs with PPARy mRNA interference, the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, ACC, FAS, SCD, PPARGC1A, INSIGI and SCAP (P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05), and the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05) all were markedly improved in DCMECs with PPARy mRNA interference,8mmol/L sodium acetate and1.25mmol/L β-hydroxy sodium butyrate.(4) Compared to DCMECs with PPARy mRNA interference, the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, SCD, PPARGC1A, INSIGI and SCAP (P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05), and the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05) all were obviously enhanced in DCMECs with PPARy mRNA interference and150μmol/L palmitic acid.(5) Compared to DCMECs with PPARy mRNA interference, the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, SCD, PPARGC1A, INSIGI and SCAP (P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05), and the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P< 0.05) all were significantly increased in DCMECs with PPARy mRNA interference and125μmol/L stearic acid.7. The results of the effect of different fatty acids on milk fat synthesis in DCMECs with PPARy overexpression showed as follows:(1) Compared to DCMECs with PPARy overexpression, the mRNA expression levels of FABP3, ACSL1, ACSS2, ACC, FAS, SCD, PPARGC1A, INSIG1and SCAP (P<0.05), the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05) were all markedly improved in DCMECs with PPARγ overexpression and8mmol/L sodium acetate.(2) Compared to DCMECs with PPARy overexpression, the mRNA expression levels of FABP3, ACSL1, ACSS2, ACC, FAS, SCD, PPARGC1A, INSIG1and SCAP (P<0.05), the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05) were all significantly increased in DCMECs with PPARy overexpression and0.75mmol/L β-hydroxy sodium butyrate.(3) Compared to DCMECs with PPARy overexpression, the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, ACC, FAS, SCD, PPARGC1A, INSIG1and SCAP (P<0.05), the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05) were all obviously increased in DCMECs with PPARy overexpression,12mmol/L sodium acetate and0.75mmol/L P-hydroxy sodium butyrate.(4) Compared to DCMECs with PPARy overexpression, the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, SCD, PPARGC1A, INSIG1and SCAP (P<0.05), the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05) were all significantly enhanced in DCMECs with PPARy overexpression and125μmol/L palmitic acid.(5) Compared to DCMECs with PPARy overexpression, the mRNA expression levels of CD36, FABP3, ACSL1, ACSS2, SCD, PPARGC1A, INSIG1and SCAP (P<0.05), the mRNA expression and the activities of GPAT, AGPAT6, DGAT1(P<0.05), the mRNA and protein expression of PPARy and SREBP1(P<0.05) were all markedly improved in DCMECs with PPARy overexpression and100umol/L stearic acid.This study indicated that fatty acids such as sodium acetate, β-hydroxy sodium butyrate, the mixture of sodium acetate and β-hydroxy sodium butyrate, palmitic acid and stearic acid were invovled in regulating the mRNA and protein expression of fatty acid uptake, transport, activation related proteins, fatty acid synthases and desaturase, triglyceride synthetases, transcriptional regulation factors in milk fat synthesis of DCMECs and promoted milk fat synthesis by increasing the mRNA and protein expression of PPARy. The study on gene silencing and overexpression of PPARy in DCMECs indicated that PPARy was a key transcriptional regulatory factor of milk fat synthesis in DCMECs and PPARy played a pivotal role to the regulating of milk fat synthesis of dairy cows. The concentration demand of DCMECs with PPARy gene silencing and overexpression on different fatty acids had changed. Different fatty acids can regulate the expression of genes and proteins related to milk fat synthesis in DCMECs with PPARy gene silencing and overexpression, which further indicated that fatty acids regulated milk fat synthesis through PPARy. This study provided the references for nutrition regulation of milk fat synthesis in ruminants and had an important guiding significance to improving milk fat and milk quality by using nutrient elements. |
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