| Peroxisome proliferator-activated receptor gamma(PPARG), one central transcription factor associated with lipid metabolism, affect triglyceride(TAG) synthesis and the key factor of fatty deposits in fat tissue. However, the mechanism of PPARG in regulating lipid metabolism in goat lactation is rare. Five different long chain fatty acid(LCFA)(C16, C18, CLA, DHA, and EPA), agonist(ROSI) and antagonist(GW) of PPARG and the combination of LCFA with its antagonist were treated in goat mammary epithelial cells(GMEC) together with PPARG over-expression, we analyzed the regulation of PPARG on genes related to fatty acid metabolism and also the function of Adipo R1 and LPL, PPARG target genes in GMEC. The main results of this study are shown as follows:1. Treatments with 50 μM GW(PPARG antagonist) in GMEC found that m RNA expression of Adipo R1, LPIN1, PPARG, LXRA, LPL, ACSL1, FABP3 and FABP4 were down-regulated; Treatments of 50 μM ROSI(PPARG agonist) in GMEC found that m RNA expression of Adipo R1, FADS1, AGPAT6, LPIN1, SCD, FABP3, LPL, FABP4 and SREBF2 gene were all down-regulated. The effects of saturated fatty acids(C16 and C18) are stronger than ROSI and most of the detected genes can be significantly increased. However, the expression of FASN, FADS1, LPIN1, SREBF1, SREBF2, INSIG1, RXRA, NCOR1 and FABP3 was greatly down-regulated by CLA(P<0.05). The combination of GW and saturated fatty acids in GMEC did not completely eliminate the effects of saturated fatty acids on genes related to lipid metabolism, while GW and unsaturated long chain fatty acids(CLA, DHA and EPA) in GMEC had a complex effect on genes related to lipid metabolism.2. After GMEC being infected with adenovirus mediated the PPARG over-expression vector, we found over-expression of PPARG up-regulated(P < 0.05) the expression of some lipogenic enzymes(ACSS2, ACSL1, LPL, SCD, DGAT1, FADS1, GPAM, and LPIN1), transcription regulators(Adipo R1, LXRA, MLXIPL, PPARG, PPARGC1B), and components of the circadian CLOCK network(CRY1 and CRY2). After GMEC treated with ROSI together with over-expression of PPARG, there was a further and marked increase(P < 0.05) in the m RNA expression of lipogenic enzymes(LPL, FASN, ACSS2, GPAM, LPIN1, ACSL1, SCD), transcription regulators(Adipo R1, LXRA, PPARG, SREBF1, and PPARGC1B), and components of the circadian CLOCK network(CLOCK, CRY1 and CRY2) compared to the over-expression of PPARG without adding ROSI in GMEC. Taken together, Adipo R1 and LPL were two important target genes of PPARG in GMEC.3. Cloning of Adipo R1 gene in goat mammary gland showed that the full length of Adipo R1 gene was 2032 bp which encode 375 amino acid(AA) and 7 transmembrane domains and no signal peptide were included in the entire sequence. Furthermore, Adipo R1 gene had the most abundant expression in lung, followed by the small intestine and mammary gland with the minimal expression was observed in heart. Meanwhile, the expression of Adipo R1 gene in peak lactation is significantly higher than dry period(P<0.05). Adipo R1 can be affected by insulin and prolactin in dose dependent manner. GMEC treated with different concentrations of insulin(1 nmol/L, 50 nmol/L and 100 nmol/L) showed that the m RNA level of Adipo R1 was decreased and there was the most obvious effect at insulin concentration of 50 nmol/L(P<0.05); GMEC treated with different concentrations of prolactin(10 ng/m L, 100 ng/m L and 1000 ng/m L) increased the m RNA level of Adipo R1 gene and there was the most obvious effect at prolactin concentration of 100 ng/m L(P<0.05). After Adipo R1 interference in GMEC, m RNA expression of Adipo R1 genes was obviously down-regulated(P<0.05) and expression of genes related to de novo fatty acid synthesis including SREBF1, ACACA, FASN and SCD were significantly down-regulated(P<0.05), and also m RNA expression of genes related to TAG hydrolysis including ATGL and HSL both are significantly lower after Adipo R1 interference in GMEC(P<0.05).4. Cloning of LPL gene in goat mammary gland showed that the full length of LPL gene was 3555 bp which encode 478 AA and no transmembrane domains were included in the entire sequence and signal peptide was existing between 23-24 AA. Furthermore, LPL gene had the most abundant expression in adipose tissue, followed by the heart and mammary gland with the minimal expression was observed in liver and muscle. Meanwhile, the expression of LPL was significantly increased from dry period to early lactation, followed by the decrease at peak and middle lactation and increased again at late lactation. The expression of LPL throughout the entire lactation presented "N" type curve. After LPL RNA interference mediated by sh RNA, LPL decrease was associated with reduced m RNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3(P<0.05). LPL antagonist Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression(P<0.05).This study suggests that PPARG can control fatty acid de novo synthesis, desaturation, lipid droplet formation and TAG synthesis in goat mammary epithelial cells via its target genes. Furthermore, PPARG can play an important role in regulation of fatty acid metabolism... |
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