| Milk is an important nutrient source of human food,which can provide lipids,high-quality proteins,essential vitamins and minerals.Mammary epithelial cells are the minimum structure and functional unit of lactation of mammary gland.It is one of the important scientific questions in the field of life science to study the expression and metabolic regulation of genes involved in milk fat synthesis and reveal the related signal transduction pathways and molecular mechanisms.A lot of evidence have shown that amino acids and hormones participate in the regulation of milk fat synthesis,but the detailed of molecular mechanism is still unclear.Therefore,study of the molecular mechanism of milk fat synthesis,improve the regulatory mechanism of milk fat synthesis and its regulatory network is an important scientific question in the field of lactation biology.Whether FABP5 is an important regulator of milk fat synthesis and its molecular mechanism has yet to be revealed.Fatty acid binding protein?FABP?is a member of the intracellular lipid-binding protein?iLBP?family,which reversibly binds hydrophobic ligand and transports them between subcellular organelles.FABP5 is one of the most important member of the FABP family.Ongoing studies have shown that FABP5 plays an important role in signal transduction of lipid metabolism,which is widely involved in the uptake and transport of long-chain fatty acids,gene regulation,cell growth and differentiation.In this research,FABP5 has been proved to regulate milk fat synthesis in bovine mammary epithelial cells?BMECs?.FABP5 acts as a critical regulator of SREBP-1c gene expression induced by methionine?Met?and estrogen?E2?.In addition,we also found that fatty acids are necessary for FABP5-mediated SREBP-1c gene expression.This study enriched and improved the molecular regulatory network of milk fat synthesis,and also provided important experimental basis for the mechanism of nutrients and hormones regulating milk fat synthesis.Our study found that the expression of FABP5 in breast tissues of lactating period was significantly higher than that of puberty and dry period.Immunofluorescence microscopy detected that FABP5 was consistently enriched in the cytoplasm of alveolar epithelial cells Tissue culture method was used to isolate and purify BMECs in this study.The expression of Cytokeratin 18 and?-casein in BMECs was observed by immunofluorescence staining.These expression were positive showing that BMECs were successfully purified and had the ability of milk synthesis.We then established a cell model to stimulate milk synthesis by adding 0.6 mmol/L Met or 2.72×10-2 ug/L E2 to the culture medium of BMECs.We observed that both Met and E2 promoted lipid droplet formation by BODIPY staining.Western blotting analysis detected that the protein expression of FABP5 was also upregulated,and immunofluorescence analysis detected that Met and E2 could also promote the expression of FABP5 in cytoplasm.These data suggest that FABP5could response to extracellular stimuli such as Met and E2 and might play a regulatory role in milk fat synthesis.We next performed gene overexpression and knockdown experiments to detect the effects of FABP5 on milk fat synthesis.Western blotting analysis detected that the protein level of FABP5was significantly increased in cells transfected with the overexpression plasmid.FABP5overexpression led to increased lipid droplet accumulation in cells and TG secretion into the culture medium.Western blotting analysis also detected that the protein level of FABP5 was significantly decreased in cells transfected with FABP5 siRNA.FABP5 knockdown led to decreased lipid droplet accumulation in cells and TG secretion into the culture medium.These data reveal that FABP5 is a positive regulator of milk fat synthesis in BMECs.Western blotting analysis also detected that FABP5 overexpression markedly increased SREBP-1c expression but had little affect on PPAR?/?.qRT-PCR analysis also detected that the mRNA expression of SREBP-1c but not PPAR?/?was significantly increased.We further detected the mRNA levels of ACC and FAS which were two SREBP-1c-targeted genes,and found that FABP5 overexpression significantly increased expression of these two genes.As expected,FABP5knockdown had the opposed effects.FABP5 knockdown decreased SREBP-1c protein level but had little effect on PPAR?/?.qRT-PCR detected that FABP5 knockdown inhibited the mRNA expression of SREBP-1c but did not affect PPAR?/?.The mRNA levels of ACC and FAS were also significantly decreased.Together,these above data reveal that FABP5 positively regulates SREBP-1c gene expression for milk fat synthesis in BMECs.We found that FABP5 knockdown totally abrogated Met or E2-induced TG secretion of BMECs.Western blotting analysis detected that FABP5 knockdown also diminished the stimulation of SREBP-1c protein,but had little affects on PPAR?/?.qRT-PCR further detected that FABP5knockdown totally inhibited the mRNA expression of SREBP-1c stimulated by Met or E2,whereas the mRNA expression of PPAR?/?was little changed.We further investigated how Met and E2regulate FABP5 for SREBP-1c gene expression.We observed the effects of PI3K inhibitor wortmannin?Wm?on FABP5 expression.Wm significantly inhibited Met and E2-induced AKT phosphorylation.The protein levels of FABP5 increased by Met and E2 treatments were totally abolished by Wm treatment.These data reveal that FABP5 is required for Met and E2-induced SREBP-1c gene expression and suggest that Met and E2 regulate FABP5 via the PI3K signaling.We observed that FABP5 overexpression and knockdown had no effects on the AKT,mTOR,and NF?B1 phosphorylation,suggesting that the stimulation of FABP5 on SREBP-1c gene expression is independent on these signaling pathways.The fact that FABPs function as lipid chaperones prompted us to speculate that the stimulation of FABP5 on SREBP-1c gene expression is dependent on fatty acids.We observed that,when cells were cultured in fatty acid-free OPTI-MEM?medium,Met and E2 could not stimulate SREBP-1c gene expression.After fatty acids?the mixture of palmitic acid and oleic acid?were added into the culture medium,Met and E2 significantly stimulated SREBP-1c gene expression.We next observed that FABP5 overexpression could not stimulate SREBP-1c gene expression in cells cultured in fatty acid-free medium,while led to increased SREBP-1c gene expression after fatty acid stimulation.We further determined whether the regulatory role of FABP5 is dependent on its fatty acid-binding ability.For this purpose,we introduced point mutations in fatty acid-binding sites of FABP5 into C-terminal Flag-tagged FABP5 vectors and transfected these vectors into cells.Western blotting analysis detected that the stimulation on SREBP-1c gene expression by the mutants R129A,Y131A,or R129A/Y131A were significantly reduced,compared with the wild-type?WT?vector group.Together,these data reveal that FABP5-mediated Met and E2-induced SREBP-1c gene expression depends on its ability to effectively transport fatty acids.In summary,FABP5 could regulate SREBP-1c gene expression and milk fat synthesis induced by methionine?Met?and estrogen?E2?in BMECs.Methionine and estrogen activate FABP5 via PI3K signaling pathway,and FABP5 affects SREBP-1c gene expression dependent on its fatty acid binding function. |
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