We hypothesized that insulin-induced gene 1 (INSIG1) affects milk fat synthesis in buffalo. For this reason, the protein abundance of INSIG1 in the mammary tissue of buffalo during the peak period of lactation and dry-off period was evaluated. The results showed that the expression of INSIG1 at the peak of lactation was lower than that in the dry-off period. To explore the role of INSIG1 in milk fat synthesis, the buffalo mammary epithelial cells (BMECs) were isolated and purified from buffalo mammary tissue, and INSIG1 gene were overexpressed and knocked down by constructing the recombinant lentivirus vector of INSIG1 gene and transfecting into BMECs. Results revealed that INSIG1 overexpression decreased the expression of INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6, which led to reduction of triglycerides (TAG) content in the cell. In contrast, knockdown of INSIG1 had a positive effect on mRNA expression of the above genes. Overall, the data provide strong support for a key role of INSIG1 in the regulation of milk fat synthesis in BMECs.

Recent studies have shown elongase of very‐long‐chain fatty acids 6 (ELOVL6) is a vital protein for endogenous synthesis of saturated and monounsaturated long‐chain fatty acids in some mammals. Nevertheless, its role in lipid synthesis in buffalo mammary gland is still unclear. In this work, the full‐length coding sequence (CDS) of ELOVL6 was cloned and identified from buffalo mammary gland. As a result, the CDS of this gene is 795 bp, which encodes a polypeptide of 264 amino acid residues. The buffalo ELOVL6 contains an ELO domain which belongs to the ELO superfamily. Among the 10 tissues of buffalo in peak lactation detected by RT‐qPCR, the expression level of ELOVL6 was the highest in the brain, followed by the spleen, and then decreased in the mammary gland, muscle, kidney, heart, liver, rumen, intestine and lung. However, only the expression in the brain and spleen was statistically different from that in other tissues (p < 0.05). Compared with that of the dry‐off period, the mRNA abundance of ELOVL6 in the mammary gland was significantly increased in peak lactation. The experiments based on lentivirus transfection in buffalo mammary epithelial cells (BuMECs) displayed that the overexpression of ELOVL6 markedly promoted the expression of INSIG1, INSIG2, SREBP, PPARG, FASN, GPAM, DGAT2 and APGAT6 genes, and the knockdown of ELOVL6 significantly decreased the mRNA abundance of INSIG2, SREBP, FASN, SCD, GPAM, APGAT6 and TIP47 genes. In addition, the increase or decrease of ELOVL6 expression level also caused the corresponding change of total triglyceride content in the BuMECs. The results here suggest that the ELOVL6 can catalyse the synthesis of long‐chain fatty acids in the BuMECs, and it can indirectly affect the expression of genes related to milk fat synthesis through its catalytic products to promote the lipid biosynthesis of BuMECs.

No abstract

Abstract. The αS2-casein (αS2-CN) is a member of the casein family associated with milk traits in ruminants, but so far the buffalo CSN1S2 gene has not been well understood. In this work, the polymorphisms of CSN1S2 in river and swamp buffalo were detected using direct sequencing of polymerase chain reaction (PCR) products. As a result, 13 single nucleotide polymorphisms (SNPs) were identified in the coding sequence (CDS) of CSN1S2 in two types of buffalo, of which eight SNPs were non-synonymous. The amino acid changes caused by c.580T>C and c.642C>G may affect the function of buffalo αS2-CN. A total of 11 CSN1S2 CDS haplotypes were defined, and accordingly 11 variants of buffalo αS2-CN were inferred and named. The CSN1S2 CDSs of both types of buffalo were 669 nucleotides, which encoded a precursor of 222 amino acids (AAs), and the first 15 AAs constitute a signal peptide. The composition and physicochemical characteristics of two types of buffalo αS2-CNs were similar but slightly different from those of cattle αS2-CN. The αS2-CN mature peptides of buffalo and the species of Bos genus contained a casein domain, and their secondary structures were highly consistent, indicating that they are functionally similar. The results here provide initial insights into the variation, characteristics and biological function of buffalo CSN1S2.

Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase1 (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism. However, the functional role and regulatory mechanism of LPIN1 gene in milk fat synthesis of buffalo are still unknown. In this study, overexpression of buffalo LPIN1 gene transfected with recombinant fusion expression vector significantly increased the expression of AGPAT6, DGAT1, DGAT2, GPAM and BTN1A1 genes involved in triglyceride (TAG) synthesis and secretion, as well as PPARG and SREBF1 genes regulating fatty acid metabolism in the buffalo mammary epithelial cells (BMECs), while the lentivirus-mediated knockdown of buffalo LPIN1 dramatically decreased the relative mRNA abundance of these genes. Correspondingly, total cellular TAG content in the BMECs increased significantly after LPIN1 overexpression, but decreased significantly after LPIN1 knockdown. In addition, the overexpression or knockdown of PPARG also enhanced or reduced the expression of LPIN1 and the transcriptional activity of its promoter. The core region of buffalo LPIN1 promoter spans from − 666 bp to + 42 bp, and two PPAR response elements (PPREs: PPRE1 and PPRE2) were identified in this region. Site mutagenesis analysis showed that PPARG directly regulated the transcription of buffalo LPIN1 by binding to the PPRE1 and PPRE2 on its core promoter. The results here reveal that the LPIN1 gene is involved in the milk fat synthesis of BMECs, and one of the important pathways is to participate in this process through direct transcriptional regulation of PPARG, which in turn significantly affects the content of TAG in BMECs.