| Milk fat, primary components of milk, have great influence on milk nutritional and technological properties. Besides adipose tissue, mammary gland is another major fat-synthesizing tissue in the whole body during lactation. Recent research has suggested that short-chain fatty acids(SCFAs) are ligands for G protein-coupled receptors 41(GPR41) which belongs to one member of G protein-coupled receptors superfamily in cell surface membrane. Activation of GPR41 by SCFAs has a role of regulating lipid metabolism in adipose tissue. However, the potential role of GPR41 on regulating milk fat metabolism is still unknown.In this sudy, to explore the role of GPR41 on milk fat metabolism regulation, mammary tissue from different physiological periods of Saanen dairy goat was collected and we analyzed the m RNA expression of GPR41 in mammary gland at different physiological periods. Primary cultured mammary gland epithelial cells were treated with GPR41 ligands SCFAs. Adenovirus-mediated gene overexpression, si RNA-mediated gene silencing and GPR41–coupled Gi/o protein inhibitor was used to investigate the role of GPR41 in milk fat metabolism. Futhermore, we exprlore the underlying mechanisms by which GPR41 mediate milk fat metabolism regulation in mammary gland. The main results of this study are as follows:(1) Real-Time quantitative PCR(RT-PCR) results revealed that the content of TAG and lipid droplet formation were significantly stimulated by propionate and butyrate. The expression of FABP3, SCD1, PPARG, SREBP1, DGAT1, AGPAT6 and ADRP were upregulated by propionate(P<0.05) and butyrate(P<0.05) treatment. In contrast, the m RNA expression of FASN and LXR was not affected by propionate, but reduced by butyrate(P<0.05). Acetate had no obvious effect on the content of TAG and lipid droplet formation but increased the m RNA expression of SCD1(P<0.05) and FABP3(P<0.05) in GMECs.(2) Twenty Xinong Saanen dairy goats were used for analysis expression of GPR41 at diffirent lactation stages. RT-q PCR results showed that GPR41 was most adequately expressed at the peak lactation stage, which had a remarkable higher expression than any other detected stages(P<0.05). There were no differences in GPR41 expression level among the early-lactation, mid-lactation, and late-lactation stages. The lowest m RNA expression of GPR41 was found at dry period.(3) Recombinant adenovirus vector(Ad-GPR41) containing GPR41 c DNA sequence was constructed, and then was packaged and propagated in 293 A cells. GMECs were infected with control adenovirus(Ad-GFP) and Ad-GPR41 adenovirus respectively in the presence of GPR41 ligand propoioate. Results showed that compared with control groups(Ad-GFP), overexpression of GPR41 enhanced the expression of SCD1, LPL, FABP3, SREBP1, PPARG and INSIG-1(P<0.05) but had no effect on the expresson of CD36, FASN and ACACA(P>0.05). Moreover, overexpression of GPR41 in the presence of GPR41 ligand propoioate did not enhanced the TAG content and lipid droplet formation significantly.(4) According to the goat GPR41 sequence, we synthesized two pairs of complementary single-strand DNA oligonucleotides(si GPR41-39 and si GPR41-948) which targeting two different sites of GPR41 m RNA. Si GPR41-948 sequences caused an obvious interference effect. After transfection with si GPR41-948 for 48 h, the expression of PPARG, SREBP1, INSIG-1, SCD1, FABP3, LPL and CD36 was decreased significantly(P<0.05) but had no effect on the expresson of FASN and ACACA(P>0.05). Meanwhile, decreased expression of GPR41 did not reduce the TAG content and lipid droplets formation.(5) GMECs were pretreated with PTX(200 ng/ ml) for 16 h to block the Gi/o pathway, and then the cells were treated with or without propionate(1.5 or 3 m M) for 24 h. The results showed that PTX pre-treatment significantly blocked the expression of SCD1, FABP3, PPARG, SREBP1 and INSIG-1 induced by propionate(P<0.05), but had no effect on the expression of CD36 and LPL induced by propionate in GMECs(P>0.05).(6) SREBP1 involves in GPR41-mediated fatty acids related to genes expression. Propionate-stimulated FABP3, SCD1 and INSIG-1 expression was further enhanced by increased SREBP1(P<0.05) and lower expression of SREBP1 completely abolished the m RNA level of FABP3, SCD1 and INSIG-1 induced by propionate(P<0.05).(7) PPARG involves in GPR41-mediated fatty acids metabolism associated with genes expression. After treatment with 50 μmol/L PPARG activator rosiglitazone, propionate-induced FABP3 expression was enhanced in GMECs significantly(P<0.05). Meanwhile, GMECs were pre-incubated with PPARG inhibtor for 24 hours blocked the proponate-induced FABP3 expression(P<0.05).In conlusion, GPR41 has a potential role of regulating lipogenic gene expression in GMECs, especially, has a great effect on the expression of PPARG, SREBP1, SCD1, FABP3 and INSIG-1, which suggested that GPR41 may participate in fatty acids desaturation and transport. Furthermore, we found that GPR41 regulated the expression of SCD1, FABP3 and INSIG-1 through controlling the expression of SREBP1. PPARG also involves in GPR41-regulated FABP3 expression, indicating that GPR41 regulated lipogenic gene expression via the concerted action of those transcription regulators. |
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