To explore the function of PPARγ in the goat mammary gland, we cloned the whole cDNA of the PPARγ gene. Homology alignments revealed that the goat PPARγ gene is conserved among goat, bovine, mouse, and human. Luciferase assays revealed that rosiglitazone enhanced the activity of the PPARγ response element (PPRE) in goat mammary epithelial cells (GMECs). After rosiglitazone (ROSI) treatment of GMECs, there was a significant (P < 0.05) increase in the expression of genes related to triacylglycerol synthesis and secretion: LPL, FASN, ACACA, PLIN3, FABP3, PLIN2, PNPLA2, NR1H3, SREBF1, and SCD. The decreases in expression observed after knockdown of PPARγ relative to the control group (Ad-NC) averaged 65%, 52%, 67%, 55%, 65%, 58%, 85%, 43%, 50%, and 24% for SCD, DGAT1, AGPAT6, SREBF1, ACACA, FASN, FABP3, SCAP, ATGL, and PLIN3, respectively. These results provide direct evidence that PPARγ plays a crucial role in regulating the triacylglycerol synthesis and secretion in goat mammary cells and underscore the functional importance of PPARγ in mammary gland tissue during lactation.

The scavenger receptor CD36 is involved in pathogen recognition, phagocytosis, and pathogen-induced signaling. This study investigated the relationship between CD36 and TLR4 in modifying lipopolysaccharide (LPS)-induced signaling pathways and mediating Escherichia coli (E. coli) endocytosis in primary goat mammary epithelial cells (pGMECs). The manipulation of CD36 expression significantly influenced TLR4 and nuclear factor kappa B (NF-κB) mRNA expression in pGMECs stimulated with LPS for 12 h. NF-κB and activator protein-1 (AP-1) activity was regulated by the manipulation of CD36 expression in LPS-induced pGMECs. However, CD36-mediated AP-1 activation occurred primarily through c-Jun N-terminal kinase (c-JNK). Adaptor proteins and proinflammatory cytokines were also involved in these signaling pathways and acted by regulating CD36 expression in LPS-stimulated cells. Moreover, CD36 cooperated with TLR4 in TLR4-mediated phagocytosis following E. coli simulation, but this complex was not induced by LPS treatment. Our study is the first to illuminate CD36 as a scavenger receptor in ruminants. Additionally, this study indicates that CD36 plays a vital role in the LPS-induced activation of downstream signaling cascades and mediates E. coli phagocytosis via TLR4 in pGMECs, which offers a novel treatment strategy for mastitis.

In nonruminants, the alternative splicing of peroxisome proliferator-activated receptor γ (PPARG) generates PPARG1 and PPARG2 isoforms. Although transcriptional control differences between isoforms have been reported in human adipose tissue, their roles in ruminant mammary cells are not well known. To assess which of these isoforms is more closely associated with the regulation of mammary lipogenic pathways, their tissue distribution was analyzed and the expression of key genes regulating lipogenic gene networks was measured after overexpression of the 2 isoforms in goat mammary epithelial cells (GMEC). The expression of PPARG2 was markedly greater in adipose tissue, whereas PPARG1 is the main isoform in goat mammary tissue (ratio of PPARG1:PPARG2 was close to 37:1). As was reported in previous work, PPARG1 upregulated the transcription regulators SREBF1 and PPARG and the lipogenic genes FASN, ACACA, and SCD. Along with a tendency for greater expression of AGPAT6, DGAT1, and PLIN2, these data suggest that PPARG1 is the isoform controlling lipogenesis in mammary cells. Addition of the PPARG ligand rosiglitazone (ROSI) to GMEC overexpressing both isoforms upregulated the expression of LPL and CD36, which help control uptake of long-chain fatty acids into mammary cells. Other responses to ROSI addition to GMEC overexpressing PPARG1 and PPARG2 included upregulation of AGPAT6, DGAT1, INSIG1, SREBF1, and NR1H3. Although the data suggest that both PPARG1 and PPARG2 could affect mammary lipogenesis via control of gene expression when stimulated (e.g., by ROSI), the fact that PPARG1 is more abundant in mammary tissue and that its overexpression alone upregulated key lipogenic gene networks suggest that it is the more important isoform in goat mammary cells.

Akt serine/threonine kinase acts as a central mediator in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, regulating a series of biological processes. In lipid metabolism, Akt activation regulates a series of gene expressions, including genes related to intracellular fatty acid synthesis. However, the regulatory mechanisms of Akt in dairy goat mammary lipid metabolism have not been elaborated. In this study, the coding sequences of goat Akt1 gene were cloned and analyzed. Gene expression of Akt1 in different lactation stages was also investigated. For in vitro studies, a eukaryotic expression vector of Akt1 was constructed and transfected to goat mammary epithelial cells (GMECs), and specific inhibitors of Akt/mammalian target of rapamycin (mTOR) signaling were applied to GMECs. Results showed that Akt1 protein was highly conserved, and its mRNA was highly expressed in midlactation. In vitro studies indicated that Akt1 phosphorylation activated mTOR and subsequently enhanced sterol regulatory element binding protein 1 (SREBP1), thus increasing intracellular triacylglycerol content. Inhibition of Akt/mTOR signaling down-regulated the gene expression of lipogenic genes. Overall, Akt1 plays an important role in regulating de novo fatty acid synthesis in goat mammary epithelial cells, and this process probably is through the mTOR/SREBP1 axis.

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