Expression Profiling of Glucose Transporter 1 (GLUT1) and Apoptotic Genes (BAX and BCL2) in Milk Enriched Mammary Epithelial Cells (MEC) in Riverine Buffalo during Lactation

Abstract

Lactation is an important physiological process in dairy animals. During lactation, up to 85% of the body glucose is directed toward the mammary glands for milk synthesis. Studies related to lactation physiology are generally carried out on mammary biopsies, which may adversely affect animal health. In the present study, milk enriched MEC were used to study the expression pattern of GLUT1 and apoptotic genes (BAX and BCL2) across different stages of lactation in riverine buffalo in relation to milk yield. MEC were enriched from milk using cytokeratin-8 antibodies coated magnetic beads. Total RNA isolated from enriched MEC showed significant correlation (r(2) = 0.92 ± 0.02) with the milk yield at different stages of lactation. GLUT1 expression pattern correlated with the milk yield as highest GLUT1 expression (4.68 ± 0.79) was observed during peak-lactation (90 days post-parturition), whereas low GLUT1 expression (1.01 ± 0.1, 15 d; 0.71 ± 0.03, 30 d) was observed during early lactation. The BAX/BCL2 ratio was high (1.02 ± 0.2, 15 d; 0.94 ± 0.06, 30 d) during the early phase of lactation, indicating high rate of apoptosis, whereas low BAX/BCL2 ratio (0.25 ± 0.03, 60 d; 0.42 ± 0.04, 90 d) was observed during mid-lactation coinciding with the increase in RNA concentration and milk yield. Highest BAX/BCL2 ratio (1.41 ± 0.3, 120 d; 4.02 ± 0.6, 240 d) was observed during late lactation i.e., 240 days, which was also reflected as decline in milk yield and RNA concentration. Also, BAX/BCL2 ratio in milk enriched MEC was in accordance with RNA concentration in MEC and milk yield at different phases of lactation. Our study showed that expression pattern of genes under study (GLUT1, BAX, and BCL2) in milk enriched MEC correlated well with important physiological properties such as milk yield in buffalo.

Comparative transcriptome analysis of mammary epithelial cells at different stages of lactation reveals wide differences in gene expression and pathways regulating milk synthesis between Jersey and Kashmiri cattle

“…[24] could explain the relatively low levels of CSN2 , CSN3 , CSN1S1 and CSN2S2 when compared with our data, suggesting that the antibody-captured milk MEC technique used by these authors was probably not optimal. The validity of gene expression results obtained by using purified MEC has been demonstrated in cows and buffalo [31, 33–36] and supported by our data. The advantages of purified MEC as a non-invasive source of RNA for mammary gland transcriptome analysis include but not limited to possibility for repeat sampling over a period of time on the same animals without causing damage to mammary tissue and ability to specifically study milk secreting cells.…”

Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts

“…Furthermore, the effect of Quinagolide, a prolactin-release inhibitor, was similar regardless of the anti-cytokeratin antibody used: anti-cytokeratin 8 ( Boutinaud et al, 2012 ) or anti-cytokeratin 1, 5, 10, and 14 ( Lollivier et al, 2015 ). In addition, the effect of the lactation stage was concordant in two bovine studies ( Sigl et al, 2012 ; Boutinaud et al, 2013b ) as well as one bovine study and one buffalo study ( Boutinaud et al, 2013b ; Yadav et al, 2014 ) where different antibodies were used ( Table 1 ). Finally, the agreement of these different studies suggests that the immunomagnetic method of MEC purification is reliable.…”

“…Goat milk contains a higher concentration of MEC than bovine milk ( Boutinaud and Jammes, 2002 ). Thus, 1.8–3.6 kg of bovine milk ( Boutinaud et al, 2008 , 2012 , 2014 ; Krappmann et al, 2012 ; Sigl et al, 2014 ), 0.9–1.6 kg of caprine milk ( Ben Chedly et al, 2011 , 2013 ) and 1 kg of buffalo milk ( Yadav et al, 2014 ) were used to extract purified MEC from milk in order to analyze mammary transcripts. During the second step of MEC isolation, an immunomagnetic separation technique is used to isolate the MEC from the total milk somatic cells and to remove the leukocytes.…”

“…In contrast to milk protein transcripts, BAX and BCL2 transcripts were shown to rise as lactation progressed in milk-purified MEC of Holstein dairy cows ( Boutinaud et al, 2013b ). Accordingly in buffalo, an abrupt rise in BAX and BCL2 transcripts was observed in milk-purified MEC during late lactation ( Yadav et al, 2014 ). The effect of lactation stage has also been studied on the expression profiles of the lipogenic genes using milk-purified MEC in buffalo, which is one of the species among dairy animals with the richest fat content ( Yadav et al, 2015 ).…”

Cell adhesion downregulates the expression of Homer1b/c and contributes to drug resistance in multiple myeloma cells

“…So the Bcl-2/Bax ratio is important to MM cell fate after chemotherapy. However, it has been reported that downregulation of Homer1b/c using specific siRNA protects neurons against neurotoxicity-induced apoptosis through an increase in Bcl-2 with a decrease in Bax (14,35,36). Thus, we suspected that, after chemotherapy, increased expression of Homer1b/c might promote cell apoptosis through the classical mitochondrial apoptotic pathway.…”