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Function of SREBP1 in the milk fat synthesis of dairy cow mammary epithelial cells.

Li N, Zhao F, Wei C, Liang M, Zhang N, Wang C, Li QZ, Gao XJ - Int J Mol Sci (2014)

Bottom Line: Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis.SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum.These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China. linan0829@163.com.

ABSTRACT
Sterol regulatory element-binding proteins (SREBPs) belong to a family of nuclear transcription factors. The question of which is the most important positive regulator in milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) between SREBPs or other nuclear transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), remains a controversial one. Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis. Thus far, however, the interaction between the SREBP1 and mTOR (the mammalian target of rapamycin) pathways in the regulation of milk fat synthesis remains poorly understood. This study aimed to identify the function of SREBP1 in milk fat synthesis and to characterize the relationship between SREBP1 and mTOR in DCMECs. The effects of SREBP1 overexpression and gene silencing on milk fat synthesis and the effects of stearic acid and serum on SREBP1 expression in the upregulation of milk fat synthesis were investigated in DCMECs using immunostaining, Western blotting, real-time quantitative PCR, lipid droplet staining, and detection kits for triglyceride content. SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum. These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.

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Effect of sterol regulatory element-binding protein 1 (SREBP1) overexpression on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs). Three groups of DCMECs were assessed: nontransfected group, pGCMV–IRES–EGFP empty vector group (control group) and pGCMV–IRES–EGFP–SREBP1 group. (A) Relative mRNA levels (gene of interest/β-actin) of indicated genes were determined by qRT-PCR after gene overexpression of SREBP1; (B) Expression levels of indicated proteins were assessed by Western blotting. P and N denote the precursor and cleaved nuclear forms of SREBP1, respectively; (C) Quantification of protein of interest/β-actin relative fold by gray scale scan; and (D) Triglyceride (TG) content in the culture supernatant of DCMECs. Values represent means ± SE (standard error) (n = 3). * p < 0.05, ** p < 0.01 compared with the pGCMV–IRES–EGFP empty vector group.
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ijms-15-16998-f001: Effect of sterol regulatory element-binding protein 1 (SREBP1) overexpression on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs). Three groups of DCMECs were assessed: nontransfected group, pGCMV–IRES–EGFP empty vector group (control group) and pGCMV–IRES–EGFP–SREBP1 group. (A) Relative mRNA levels (gene of interest/β-actin) of indicated genes were determined by qRT-PCR after gene overexpression of SREBP1; (B) Expression levels of indicated proteins were assessed by Western blotting. P and N denote the precursor and cleaved nuclear forms of SREBP1, respectively; (C) Quantification of protein of interest/β-actin relative fold by gray scale scan; and (D) Triglyceride (TG) content in the culture supernatant of DCMECs. Values represent means ± SE (standard error) (n = 3). * p < 0.05, ** p < 0.01 compared with the pGCMV–IRES–EGFP empty vector group.

Mentions: The mRNA expression of SREBP1, ACC, FAS, SCD, m-TOR, and FABP3 (fatty acid-binding protein) was significantly increased in the pGCMV–IRES–EGFP–SREBP1 group compared with the empty vector group (Figure 1A), whereas PPARγ was found to be downregulated. The protein expression levels of SREBP1, p-SREBP1, mTOR, and p-mTOR were notably increased in cells transfected with SREBP1 compared with cells in the empty vector group (Figure 1B,C). Overexpression of SREBP1 in DCMECs was found to significantly increase triglyceride secretion (Figure 1D). These findings reveal that the overexpression of SREBP1 increases milk fat synthesis in DCMECs.


Function of SREBP1 in the milk fat synthesis of dairy cow mammary epithelial cells.

Li N, Zhao F, Wei C, Liang M, Zhang N, Wang C, Li QZ, Gao XJ - Int J Mol Sci (2014)

Effect of sterol regulatory element-binding protein 1 (SREBP1) overexpression on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs). Three groups of DCMECs were assessed: nontransfected group, pGCMV–IRES–EGFP empty vector group (control group) and pGCMV–IRES–EGFP–SREBP1 group. (A) Relative mRNA levels (gene of interest/β-actin) of indicated genes were determined by qRT-PCR after gene overexpression of SREBP1; (B) Expression levels of indicated proteins were assessed by Western blotting. P and N denote the precursor and cleaved nuclear forms of SREBP1, respectively; (C) Quantification of protein of interest/β-actin relative fold by gray scale scan; and (D) Triglyceride (TG) content in the culture supernatant of DCMECs. Values represent means ± SE (standard error) (n = 3). * p < 0.05, ** p < 0.01 compared with the pGCMV–IRES–EGFP empty vector group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4200870&req=5

ijms-15-16998-f001: Effect of sterol regulatory element-binding protein 1 (SREBP1) overexpression on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs). Three groups of DCMECs were assessed: nontransfected group, pGCMV–IRES–EGFP empty vector group (control group) and pGCMV–IRES–EGFP–SREBP1 group. (A) Relative mRNA levels (gene of interest/β-actin) of indicated genes were determined by qRT-PCR after gene overexpression of SREBP1; (B) Expression levels of indicated proteins were assessed by Western blotting. P and N denote the precursor and cleaved nuclear forms of SREBP1, respectively; (C) Quantification of protein of interest/β-actin relative fold by gray scale scan; and (D) Triglyceride (TG) content in the culture supernatant of DCMECs. Values represent means ± SE (standard error) (n = 3). * p < 0.05, ** p < 0.01 compared with the pGCMV–IRES–EGFP empty vector group.
Mentions: The mRNA expression of SREBP1, ACC, FAS, SCD, m-TOR, and FABP3 (fatty acid-binding protein) was significantly increased in the pGCMV–IRES–EGFP–SREBP1 group compared with the empty vector group (Figure 1A), whereas PPARγ was found to be downregulated. The protein expression levels of SREBP1, p-SREBP1, mTOR, and p-mTOR were notably increased in cells transfected with SREBP1 compared with cells in the empty vector group (Figure 1B,C). Overexpression of SREBP1 in DCMECs was found to significantly increase triglyceride secretion (Figure 1D). These findings reveal that the overexpression of SREBP1 increases milk fat synthesis in DCMECs.

Bottom Line: Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis.SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum.These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China. linan0829@163.com.

ABSTRACT
Sterol regulatory element-binding proteins (SREBPs) belong to a family of nuclear transcription factors. The question of which is the most important positive regulator in milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) between SREBPs or other nuclear transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), remains a controversial one. Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis. Thus far, however, the interaction between the SREBP1 and mTOR (the mammalian target of rapamycin) pathways in the regulation of milk fat synthesis remains poorly understood. This study aimed to identify the function of SREBP1 in milk fat synthesis and to characterize the relationship between SREBP1 and mTOR in DCMECs. The effects of SREBP1 overexpression and gene silencing on milk fat synthesis and the effects of stearic acid and serum on SREBP1 expression in the upregulation of milk fat synthesis were investigated in DCMECs using immunostaining, Western blotting, real-time quantitative PCR, lipid droplet staining, and detection kits for triglyceride content. SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum. These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.

Show MeSH