LEP Gene Promotes Milk Fat Synthesis via the JAK2-STAT3 and mTOR Signaling Pathways in Buffalo Mammary Epithelial Cells.

Leptin (LEP), a protein hormone well-known for its role in metabolic regulation, has recently been linked to lipid metabolism in cattle. However, its function in buffalo mammary glands remains unclear. To address this issue, we isolated and identified the LEP gene and conducted experiments to investigate its function in buffalo mammary epithelial cells (BuMECs). In this study, two transcript variants of LEP, designated as LEP_X1 and LEP_X2, were identified. The coding sequences (CDS) of LEP_X1 and LEP_X2 are 504 bp and 579 bp in length, encoding 167 and 192 amino acid residues, respectively. Bioinformatics analysis revealed that LEP_X2 is a hydrophobic protein with an isoelectric point below 7 and contains a signal peptide, while LEP_X1 is hydrophilic and lacks a signal peptide. Our study found that LEP gene expression in lactating BuMECs was significantly higher than in non-lactating cells, with LEP_X2 expression remarkably higher than LEP_X1 in lactating BuMECs. Overexpression of both LEP_X1 and LEP_X2 significantly promoted the expression of genes related to milk fat synthesis in lactating BuMECs, including STAT3, PI3K, mTOR, SCD, and SREBF1, accompanied by an increase in cellular triglycerides (TG). Interestingly, LEP_X2 overexpression significantly suppressed LEP_X1 expression while increasing intracellular TG concentration by 12.10-fold compared to LEP_X1 overexpression, suggesting an antagonistic relationship between the two variants and supposing LEP_X2 plays a dominant role in milk fat synthesis in lactating BuMECs. Additionally, four nucleotide substitutions were identified in the buffalo LEP CDS, including a nonsynonymous substitution c.148C>T (p.Arg50Cys), which was predicted to decrease the stability of the LEP protein without affecting its function. These results collectively underscore the significant role of LEP in milk fat synthesis and can provide a basis for molecular breeding strategies of buffalo.

Effect of INSIG1 on the milk fat synthesis of buffalo mammary epithelial cells.

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.

Molecular characterization, tissue expression and polymorphisms of buffalo PPARGC1A gene.

PPARGC1A exerts important functions in activating many nuclear receptors and transcription factors that are related to energy balance. Previous studies have shown that PPARGC1A gene is associated with lactation traits of dairy cattle. However, the functional role of the buffalo PPARGC1A gene is still unknown. In this work, the complete coding sequence (CDS) of buffalo PPARGC1A was isolated and characterized for swamp and river buffalo. The CDS length of PPARGC1A for both types of buffalo was the same, which was composed of 2394 nucleotides and encoded a peptide composed of 797 amino acid residues. This protein belonged to a hydrophilic protein and contained one RRM_PPARGC1A domain (AA 674-764) without a signal peptide or a transmembrane domain. The differential expressions of this gene in 10 buffalo tissues in lactation and non-lactation displayed that the PPARGC1A was highly expressed in the muscle, heart, liver, brain and kidney of both non-lactating and lactating periods, but its expression was significantly different in the muscle, heart, liver, small intestine, mammary gland, rumen, spleen and lung between the two periods. Eight single nucleotide polymorphisms (SNPs) were found in buffalo, in which the c.778C > T, c.1257G > A and c.1311G > A were shared by two types of buffalo with similar allele frequencies, while the c.419C > T, c.759A > G, c.920C > A, c.926G > A and c.1509A > T were only observed in river buffalo. The SNP419, SNP920 and SNP926 were non-synonymous, which led to the amino acid changes of p.Ser140Phe, p.Pro307His and p.Arg309Lys. Seven nucleotide differential sites were identified in the PPARGC1A gene between buffalo and other Bovidae species. Phylogenetic analysis indicated that buffaloes were independently clustered into one branch, but they were closely related to the species of the Bos genus. The results indicate that buffalo PPARGC1A is an inducible transcriptional coactivator involved in regulating carbohydrate and fat metabolism. It can exert a functional role in a variety of buffalo tissues and may participate in milk fat synthesis and development in the mammary gland.

Isolation, identification, expression and subcellular localization of PPARG gene in buffalo mammary gland.

Peroxisome proliferator-activated receptor gamma (PPARG), as a member of the nuclear receptor superfamily, plays an important role in adipocyte differentiation and regulation of lipid and glucose metabolism. In this study, the transcripts of PPARG gene were isolated and identified in buffalo mammary gland. The results showed that two types of transcripts (PPARG1 and PPARG2) of PPARG gene produced by alternative 5' end use were expressed in buffalo mammary gland, and each of them had four different alternative splicing variants. The PPARG1 includes PPARG1a, PPARG1b, PPARG1c and PPARG1d, while the PPARG2 contains PPARG2a, PPARG2b, PPARG2c and PPARG2d. Among them, only PPARG1a, PPARG2a and PPARG2d can encode complete functional proteins with three complete functional domains, and the rest encode truncated proteins with incomplete functional domains. All the eight variants of PPARG protein do not contain transmembrane regions and signal peptides, but their conserved domain, secondary and tertiary structure and subcellular localization were different. Subcellular localization confirmed that the main transcripts PPARG1a and PPARG2a played a functional role in the nucleus, which was consistent with the results by in silico prediction. RT-qPCR analysis of buffalo mammary tissue showed that the mRNA expression levels of PPARG1 and PPARG2 in lactation were higher than those in non-lactation, and the expression levels of transcripts PPARG2d and PPARG1b + PPARG2b in lactating stage were also higher than those in non-lactating stage, but the mRNA abundance of transcripts PPARG1c, PPARG1d and PPARG2c in non-lactating period was higher than that in lactating period. The results of this study suggest that PPARG1 and PPARG2 may play important role in buffalo milk fat synthesis, and the eight alternative splicing variants found here are likely to be related to the post-transcriptional regulation of lactation.

Identification, molecular characteristics, and tissue differential expression of DGAT2 full-CDS cDNA sequence in Binglangjiang buffalo (Bubalus bubalis).

It has been found that diacylglycerol acyltransferase-2 (DGAT2) plays a crucial role in the synthesis of triglycerides (TGs) in some mammals, but its role in buffalo lactation is unclear. In the present study, the DGAT2 full-CDS cDNA sequence of Binglangjiang buffalo was isolated, and the physicochemical characteristics and structure of its encoding protein were characterized. Furthermore, the differential expressions of this gene in 10 tissues of lactating and non-lactating buffalo were analyzed by real-time quantitative PCR (RT-qPCR). The results showed that the coding region (CDS) of this gene was 1086 bp in length, encoding a peptide composed of 361 amino acid residues. The deduced amino acid sequence shared more than 98.6 % identity with that of cattle, zebu, yak, and bison in the Bovidae family. Buffalo DGAT2 protein is a slightly hydrophobic protein with a transmembrane region, which functions in membrane of endoplasmic reticulum. Besides, this protein belongs to the LPLAT_MGAT-like family and contains a conserved domain of DAGAT that has a function in the synthesis of TGs. The multi-tissue differential expression analysis demonstrated that DGAT2 was expressed in the heart, liver, mammary gland, and muscle in both non-lactating and lactating buffalo. And its expression level in the heart, liver, and mammary gland during lactation was significantly higher than that during non-lactation. The results indicate that buffalo DGAT2 may be involved in milk fat synthesis. This study can establish a foundation for further elucidating mechanisms of the buffalo DGAT2 gene in milk fat synthesis.

[Relationship between aberrant FHIT transcripts and hepatocellular carcinoma].

OBJECTIVE: To study the relationship between aberrant FHIT transcripts and hepatocellular carcinoma (HCC). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and single strand conformational polymorphism (SSCP) assays were used to analyze the transcripts and mutations of FHIT gene in 24 matched tumorous tissues and para-tumorous tissues from patients with HCC and in 4 normal liver tissues. RESULTS: Aberrant FHIT transcripts were observed in 11 out of 24 (46%) tumorous tissues and in 2 (8%) of the matched para-tumorous tissues. CONCLUSION: FHIT aberrant transcripts may play an important role in the pathogenesis of hepatocellular carcinoma.