Ranslation elongation [18]. The EEF1A1 and EEF2 expression were both up-regulated significantly in GMGE cells transfected with pcDNA3.1-GLUT1 and pcDNA3.1-GLUT12 simultaneously. However, 10781694 transfectionwith pcDNA3.1-GLUT1 and pcDNA3.1-GLUT12 respectively did not affect the EEF1A1 and EEF2 expression in GMGE cells. The phosphoinositide-3-kinase class 3 (PIK3C3) expression was significantly up-regulated to activate protein synthesis in GMGE cells transfected with pcDNA3.1-GLUT1 and pcDNA3.1GLUT12 simultaneously. However, Ras homolog enriched in brain (RHEB) expression was significantly down-regulated. Furthermore, the STAT5B expression was not changed, while PRLR expression was significantly decreased in GMGE cells transfected with pcDNA3.1-GLUT1 and pcDNA3.1-GLUT12 respectively or simultaneously.Functional Analysis of GLUT1 and GLUTFigure 3. Glucose uptake (A and C) and lactose secretion (B and D) in GT1-GMGE and GT12-GMGE respectively. Glucose uptake was detected in 24 h and 48 h in GT1-GMGE and GT12-GMGE, while lactose secretion was detected in 48 h. Vertical coordinate means glucose uptake or lactose concentration and total protein radio. Horizontal coordinate means different groups. Data are expressed as means 6 SE (n = 3). *P,0.01, compared with GMGE. doi:10.1371/journal.pone.0065013.gDiscussionGLUTs are expressed in every cell of the body and provide the metabolic energy and building blocks for the synthesis of 50-14-6 biomolecules and control glucose utilization, glucose production and glucose sensing [19]. GLUT1, responsible for basal glucose uptake, is considered to be the primary monosaccharide transporter. In contrast, GLUT12 is mainly expressed in skeletal muscle, adipose tissue, the small intestine and placenta [14]. Rogers et al. speculated that human GLUT12 is expressed in prostate cancer and breast cancer [20], whereas it is absent in normal prostate and expressed at very low levels in normal breast tissue [21]. However, the biological function of GLUT12 is not clear. Moreover, no data regarding goat GLUTs are currently available. In this study, we cloned goat GLUT1 and GLUT12 from goat mammary gland tissue. The prediction of the transmembrane helices demonstrated that both goat GLUT1 and GLUT12 have 12 transmembrane structures and belong to the class I and III proteins of the GLUT family, respectively. Goat GLUT1 and GLUT12 are highly homologous to other mammalian GLUTs and exhibited all of the motifs that are presumably required for sugar transport activity [22?4]. We inserted goat GLUT1 and GLUT12 into the pcDNA3.1 (+) plasmid and transfected these constructs into GMGE cells to assess the functions of goat GLUT1 and GLUT12 in mammary gland cells. In the GT1-GMGE cells, the mRNA expression of GLUTwas significantly increased, whereas the expression of GLUT12 mRNA was unchanged. In the GT12-GMGE cells, the GLUT12 expression increased significantly, and the GLUT1 expression decreased significantly. These results demonstrated that the transcription of goat GLUT1 and GLUT12 was driven by the cytomegalovirus (CMV) promoter. Because GLUT12 expression is restricted mainly to insulin-sensitive tissues, it is postulated to be a second insulin-responsive glucose transporter, along with GLUT4 [14]. The GLUT4 protein also acts in a cooperative manner with GLUT1, which is evident in insulin-sensitive tissues (fat and muscle) where the GLUT1 protein is localized to the plasma membrane and the BI-78D3 web tissue-specific GLUT 4 is distributed in an intracell.Ranslation elongation [18]. The EEF1A1 and EEF2 expression were both up-regulated significantly in GMGE cells transfected with pcDNA3.1-GLUT1 and pcDNA3.1-GLUT12 simultaneously. However, 10781694 transfectionwith pcDNA3.1-GLUT1 and pcDNA3.1-GLUT12 respectively did not affect the EEF1A1 and EEF2 expression in GMGE cells. The phosphoinositide-3-kinase class 3 (PIK3C3) expression was significantly up-regulated to activate protein synthesis in GMGE cells transfected with pcDNA3.1-GLUT1 and pcDNA3.1GLUT12 simultaneously. However, Ras homolog enriched in brain (RHEB) expression was significantly down-regulated. Furthermore, the STAT5B expression was not changed, while PRLR expression was significantly decreased in GMGE cells transfected with pcDNA3.1-GLUT1 and pcDNA3.1-GLUT12 respectively or simultaneously.Functional Analysis of GLUT1 and GLUTFigure 3. Glucose uptake (A and C) and lactose secretion (B and D) in GT1-GMGE and GT12-GMGE respectively. Glucose uptake was detected in 24 h and 48 h in GT1-GMGE and GT12-GMGE, while lactose secretion was detected in 48 h. Vertical coordinate means glucose uptake or lactose concentration and total protein radio. Horizontal coordinate means different groups. Data are expressed as means 6 SE (n = 3). *P,0.01, compared with GMGE. doi:10.1371/journal.pone.0065013.gDiscussionGLUTs are expressed in every cell of the body and provide the metabolic energy and building blocks for the synthesis of biomolecules and control glucose utilization, glucose production and glucose sensing [19]. GLUT1, responsible for basal glucose uptake, is considered to be the primary monosaccharide transporter. In contrast, GLUT12 is mainly expressed in skeletal muscle, adipose tissue, the small intestine and placenta [14]. Rogers et al. speculated that human GLUT12 is expressed in prostate cancer and breast cancer [20], whereas it is absent in normal prostate and expressed at very low levels in normal breast tissue [21]. However, the biological function of GLUT12 is not clear. Moreover, no data regarding goat GLUTs are currently available. In this study, we cloned goat GLUT1 and GLUT12 from goat mammary gland tissue. The prediction of the transmembrane helices demonstrated that both goat GLUT1 and GLUT12 have 12 transmembrane structures and belong to the class I and III proteins of the GLUT family, respectively. Goat GLUT1 and GLUT12 are highly homologous to other mammalian GLUTs and exhibited all of the motifs that are presumably required for sugar transport activity [22?4]. We inserted goat GLUT1 and GLUT12 into the pcDNA3.1 (+) plasmid and transfected these constructs into GMGE cells to assess the functions of goat GLUT1 and GLUT12 in mammary gland cells. In the GT1-GMGE cells, the mRNA expression of GLUTwas significantly increased, whereas the expression of GLUT12 mRNA was unchanged. In the GT12-GMGE cells, the GLUT12 expression increased significantly, and the GLUT1 expression decreased significantly. These results demonstrated that the transcription of goat GLUT1 and GLUT12 was driven by the cytomegalovirus (CMV) promoter. Because GLUT12 expression is restricted mainly to insulin-sensitive tissues, it is postulated to be a second insulin-responsive glucose transporter, along with GLUT4 [14]. The GLUT4 protein also acts in a cooperative manner with GLUT1, which is evident in insulin-sensitive tissues (fat and muscle) where the GLUT1 protein is localized to the plasma membrane and the tissue-specific GLUT 4 is distributed in an intracell.
