Genetic Screen For Controlling Milk Fat Synthesis In Lactating Sows And Studies On The Effects Of Long-chain Fatty Acids On Milk Fat Synthesis In Porcine Mammary Epithelial Cells

The aim of this study was to establish the developmental pattern of key factors that participate in milk TAG synthesis and secretion in porcine mammary tissue from late pregnancy to peak lactation, and to select the some key factors or proteins that may play key role in controlling the milk TAG synthesis and secretion in sows. Additionally, the effect of four different long-chain fatty acid(LCFA)(palmitate, oleate, linoleate, stearate) on the lipid synthesis in porcine mammary epithelial cells(p MEC) was also evaluated and the potential mechanism involved in lipid synthesis and secretion was also studied here. This study consisted of two parts of research.1. The objective of this experiment was to determine the degree of change in porcine mammary gene and protein expression that is associated with milk triacylglycerol(TAG) synthesis and secretion from late pregnancy to lactation. Percutaneous biopsies were excised from the right or left anterior mammary gland of each sow at d-17(± 2)(late pregnancy), d 1(early lactation), and d 17(± 2)(peak lactation) relative to parturition. Colostrum samples were collected on the day of parturition, within 4 h of the birth of the first piglet and milk samples were collected on d 17 post-farrowing. TAG content and fatty acid compostion of colostrum and milk were determined. The mammary transcriptome for seventy genes and thirteen proteins involved in TAG synthesis and secretion from six sows each at d-17(late pregnancy), d 1(early lactation), and d 17(peak lactation) relative to parturition was analyzed using quantitative real-time PCR and western-blot analysis respectively. The results were shown as follows:(1) The TAG content in mammary gland in early-(1 d) and peak-lactation(17 d) are two folds or two and half folds of that from late pregnancy(-17 d)(P < 0.05).(2) The TAG content of milk was significantly increased(P < 0.05) relative to colostrum. The concentrations of de novo synthesized FAs(mainly composed of C4-C14), saturated FAs and monounsaturated FAs were higher in milk than in colostrum(P < 0.05).(3) SLC27A3, ACSS2, ACSL3, FABP3, FADS1, ELOVL1, DGAT1, AGPAT1, LPIN1, PLIN2, SREBP1, INSIG1, INSIG2 and PPARγ are the most abundant genes in their respective family members in porcine mammary gland, and their m RNA expression, except PPARγ, in mammary gland were increased with lactation(P < 0.05).(4) Marked up-regulation with high relative m RNA abundance was evident during lactation for genes associated with FA uptake(VLDLR, LPL, CD36), FA activation(ACSS2, ACSL3, ACSL6) and intra-cellar transport(FABP3), de novo FA synthesis(ACACA, FASN), FA elongation(ELOVL1), FA desaturation(SCD, FADS1), TAG synthesis(GPAM, AGPAT1, LPIN1, DGAT1), lipid droplet formation(BTN2A1, XDH, PLIN2) and transcription factors and nuclear receptors(SREBP1, SCAP, INSIG1/2, PPARα).(5) Western blot analysis of CD36, FABP3, ACACA, SCD, FASN, GPAM, AGPAT1, LPIN1, DGAT1, PLIN2, SREBP1 and PPARα proteins confirmed this increase in porcine mammary tissue metabolic activity during lactation as compared to lipogenic activity during late pregnancy(-17 d); the protein expression of PPARγ wasincreased at early lactation(1 d), but not increased at peak lactation(17 d).2. To evaluate the effect of LCFA(palmitate, oleate, linoleate, stearate) on the TAG synthesis and potential mechanism involved, The p MECs were treated with varied concentrations(0, 25, 50, 100, 200, 400 and 600 μM) of palmitate, oleate, linoleate or stearate alone for 24 h, respectively. The m RNA expression of genes and key proteins related to lipid synthesis and secretion were determined. The results were shown as follows:(1) Effects of different LCFAs on the cellular TAG contentAll LCFAs except oleate caused a significant increase in cellular TAG accumulation at 25 ~ 600 μM(P < 0.05). Oleate caused a significant increase in cellular TAG accumulation at 50 ~ 600 μM(P < 0.05).(2) Effects of different LCFAs on cellular expression of genes related to lipid synthesis(1) The cellular m RNA expression of genes related to lipid synthesis, whose m RNA expression were increased linearly(P < 0.05) with increasing concentration of all fatty acids, were involved in: FA uptake(LPL, CD36), TAG synthesis(GPAM, AGPAT6, LPIN2, DGAT1), lipid droplet formation(PLIN2) and transcription factors and nuclear receptors(PPARγ).(2)The m RNA expression of genes related to lipid synthesis, whose m RNA expression were decreased linearly(P < 0.05) with increasing concentration of all fatty acids, were involved in: de novo FA synthesis(ACACA, FASN), transcription factors and nuclear receptors(SREBP1, INSIG1).(3)ACSL3(FA activation) and FABP3(intra-cellar transport) m RNA expression in p MECs were significantly(P < 0.05) up-regulated linearly or quadratically with the increasing concentration of palmitate and stearate, while their expression were significantly(P < 0.05) down-regulated linearly or quadratically with the increasing concentration of oleate and linoleate.(3) Effects of different LCFAs on the expression of proteins related to lipid synthesis(1) The cellular protein expression of CD36(fatty acid uptake) and DGAT1(TAG synthesis) were increased(P < 0.05) with increasing concentrations of all LCFAs.(2)Expression of ACACA(de novo synthesis of fatty acid) was decreased with increasing concentrations of palamitate(P < 0.05) when increased with the addition of oleate and stearate, but the cellular protein expression of ACACA was not affected by linoleate.(3)The protein expression of PPARγ(nuclear receptors) was increased(P < 0.05) with all fatty acids except palmitate, and its expression was not affected by palmitate.(4) The protein expression of SREBP1 decreased(P < 0.05) with increasing unsaturated fatty acids(oleate and linoleate), but not effected by saturated fatty acids(palmitate and stearate).Based on the results of this study, it is concluded that: during the transition from pregnancy to lactation of sows, the metabolic pathway related to lipid synthesis capacity are increased, involved in FA uptake, FA activation and intra-cellar transport, de novo FA synthesis, FA elongation, FA desaturation, TAG synthesis, lipid droplet formation and transcription factors and nuclear receptors. And 24 genes and 13 proteins were selected as key genes or proteins that control the lipid synthesis in porcine mammary gland. Treatment with palmitate, oleate, linoleate or stearate alone increased lipid synthesis, which is involved in fatty acid uptake, TAG synthesis and lipid droplet synthesis capacity in p MECs, but at the same time, inhibited fatty acid de novo synthesis and its upstream regulator.