Studies On The Regulation Of Glucose Transport And Utilization In Bovine Mammary Epithelial Cells

This study were conducted to investigate the regulation of glucose uptake and milk synthesis in bovine mammary epithelial cells (BMEC). Firstly, a lactating BMEC model was established; then the effects of lactogenic hormones and monosaccharide substrates on the gene expression of glucose transporters (GLUTs) and glucose uptake were determined; finally, the effects of glucose availability on milk synthesis and the possible mechanism was studied.1.Establishment and characterization of lactating BMEC modelThe objective of this study was to establish an in vitro lactating BMEC model. Mammary tissues were dispersed and cultured in DMEM/F12 medium containing insulin, prolactin, hydrocortisone, transferrin, epidermal growth factor and fetal calf serum. After the cells migrating from the tissue reach-80% of confluency, the tissues were removed and secretory epithelial cells were enriched by digesting with 0.25% trypsin repeatly to remove fibroblasts. The BMEC displayed a monolayer, cobblestone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates. The isolated cells were identified as epithelial origin by staining with antibody against cytokeratine 18. A one-half logarithmically growth curve, abundant microvilli, cytoplasmic lipid droplets, transcription of asl casein gene and synthesis of as caseins were observed. Thus, the lactating BMEC model can be an effective model in vitro for studies of milk synthesis of bovine mammary gland.2. The study on the regulation of glucose transporters gene expression and signaling pathwayThe study was conducted to determine the effects of lactogenic hormones (prolactin, insulin and hydrocortisone) on expression of GLUTs gene and the possible signaling pathway involved in the process in BMEC. The results showed that inclusion of prolactin and insulin did not affect abundance of GLUT1 mRNA (P> 0.05); prolactin at high down-regulated, whereas insulin at all concentrations up-regulated the expression of GLUT8 mRNA (P< 0.05), indicating that GLUT8 may be responsive to insulin in BMEC; the abundance of GLUT1 and GLUT8 mRNA increased by low concentration of hydrocortisone, but decreased when treated at higher level. Interaction of hydrocortisone and insulin on GLUT8 gene expression was observed (P< 0.01), and hydrocortisone counteract the insulin-stimulated expression of GLUT8 mRNA. When three hormones were added together at their physiological concentrations during lactation (100 ng/mL prolactin,5 ng/mL insulin and 100 ng/mL hydrocortisone), the expression of GLUT1 and GLUT8 mRNA was depressed (P< 0.05), similar to the result with 100 ng/ml of hydrocortisone. It is inferred that lactogenic hormones may not be involved in the regulation of abruptly increased expression of GLUT1 and GLUT8 mRNA in bovine mammary gland during the early lactation. Pretreatment with SB203580, an inhibitor of p-38 MAPK, did not influence the insulin-induced glucose uptake (P> 0.05). In contrast, LY294002, a specific inhibitor of PI3-K, for 30 min, significantly reduced the insulin-stimulated glucose uptake (P< 0.05). PKC is the downstream effector of PI3K, and addition of PMA (50 and 100 ng/mL), the agonist of PKC, stimulated the gene expression of GLUT1/GLUT8 and glucose uptake (P< 0.05). Furthermore, addition of PKC inhibitor (GF1090203X) (0.5,1 or 2μM), decreased the GLUT1/ GLUT8 gene expression and glucose uptake stimulated by PMA (P< 0.05). These results indicate that insulin may stimulate glucose uptake primarily via PI3K/PKC linked signaling pathways.3. The study on mechanism of glucose transport regulated by monosaccharide substrate and glucose availability in BMECGlucose and fructose were used to investigate the effect and mechanism of monosaccharide on cell viability and glucose transport by BMEC. Compared with the group without any monosaccharide, addition of glucose or fructose (5 or 10 mmol/L) increased the cell viability (P < 0.05), and the cell viability was higher with fructose than that with the same concentration of glucose (P< 0.05); viability of cells increased firstly and then decreased with increasing concentration of glucose/fructose, with the highest value at 10 mmol/L (P< 0.05); the uptake of each monosaccharide increased with increasing concentration of glucose/fructose, with the largest uptake at 20 mmol/L (P< 0.05). The plasma of mammalians has only few of fructose, and then we focus on the glucose transport in the following experiments. When incubated for 12 h, compared with control (2.5 mmol/L),5 and 10 mmol/L glucose did not influence abundance of GLUT1 mRNA (P< 0.05), while the expression of GLUT8 mRNA was not affected by any concentration of glucose (P> 0.05). For 24 h, GLUT1 and GLUT8 gene expression decreased with increasing concentration of glucose (P< 0.05), and the GLUT1 and GLUT8 gene expression at all concentrations of glucose was higher than their counterparts for 12 h (P< 0.05), except for at 20 mmol/L glucose. The HKs activity at high concentration of glucose (5,10 or 20 mmol/L) was higher than that at 2.5 mmol/L (P< 0.05). The expression of HK2 other than HK1 mRNA was detected in the BMEC, and abundance of HK2 mRNA was not affected by any concentration of glucose compared with 2.5 mmol/L glucose for 12 h (P< 0.05); the expression of HK2 gene decreased with increasing concentration of glucose for 24 h and had no difference with that of their counterpart for 12 h (P> 0.05). Furthermore, addition of 3-bromopyruvate (3-BrPA), the inhibitor of HK2, resulted in the decrease of glucose uptake and cell viability at both 2.5 and 10 mM glucose (P< 0.05), respectively. Therefore, the glucose concentrations might affect glucose uptake partly by altering activity of HKs, and HK2 might play important role in the process.4. Effect of glucose availability on milk synthesis and glucose metabolism in BMECThe BMEC were used to investigate the effect of glucose availability on milk synthesis and glucose metabolism. When treated for 12 h, compared with 5 mmol/L glucose, the increasing concentration of glucose (10 or 20 mmol/L) did not affect the mRNA abundance of acety-CoA carboxylase (ACC), fatty acid synthase (FAS), diacyl glycerol acyl transferase (DGAT) and glycerol-3 phosphate acyl transferase (GPAT) gene (P< 0.05); for 24 h, the mRNA abundance of ACC, FAS, DGAT and GPAT gene decreased with increasing concentration of glucose (P< 0.05). The expression profile of sterol regulatory element binding protein-1 (SREBP-1) showed the same pattern with ACC, FAS, DGAT and GPAT cultured with different concentrations of glucose. In addition, the content of triglyceride in the BMEC was higher with the high glucose availability group than that with lower one for 12 h (P< 0.05).The effects of glucose availability (2.5,5,10 or 20 mmol/L) on lactose synthesis and glucose metabolism for 12 h were also investigated. The gene expression of beta-1,4-galactosyl transferase (B4GALT) increased firstly and decreased lately with increasing concentration of glucose, and the gene expression ofα-lactalbumin (LA) was not affected by any concentrations. While the content of lactose synthase increased with increasing concentration of glucose, with the highest value at 20 mmol/L. Besides, the increased glucose concentration stimulated the activity of pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PH), and elevated the energy status of the BMEC (P< 0.05).In summary, a lactating BMEC model was established, and it is found that the glucose metabolism may be regulated by Iactogenic hormones. Insulin plays important role in regulation of glucose uptake in BMEC and it may work by influencing the GLUT8 expression through PI3K/PKC signaling pathway. Glucose avalibility may affect the glucose transport mainly by altering activity of HKs and increasing glucose availaility may stimulate the milk fat and lactsoe synthesis by affecting the glucose metabolism in the BMEC.