Expression and regulation of glucose transporters in the bovine mammary gland

Glucose is the primary precursor for the synthesis of lactose, which controls milk volume by maintaining the osmolarity of milk. Glucose uptake in the mammary gland plays a key role in milk production. Glucose transport across the plasma membranes of mammalian cells is carried out by 2 distinct processes: facilitative transport, mediated by a family of facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Transport kinetic studies indicate that glucose transport across the plasma membrane of the lactating bovine mammary epithelial cell has a K(m) value of 8.29 mM for 3-O-methyl-D-glucose and can be inhibited by both cytochalasin-B and phloretin, indicating a facilitative transport process. This is consistent with the observation that in the lactating bovine mammary gland, GLUT1 is the predominant glucose transporter. However, the bovine lactating mammary gland also expresses GLUT3, GLUT4, GLUT5, GLUT8, GLUT12, and sodium-dependent SGLT1 and SGLT2 at different levels. Studies of protein expression and cellular and subcellular localizations of these transporters are needed to address their physiological functions in the mammary gland. From late pregnancy to early lactation, expression of GLUT1, GLUT8, GLUT12, SGLT1, and SGLT2 mRNA increases from at least 5-fold to several hundred-fold, suggesting that these transporters may be regulated by lactogenic hormones and have roles in milk synthesis. The GLUT1 protein is detected in lactating mammary epithelial cells. Its expression level decreases from early to late lactation stages and becomes barely detectable in the nonlactating gland. Both GLUT1 mRNA and protein levels in the lactating mammary gland are not significantly affected by exogenous bovine growth hormone, and, in addition, GLUT1 mRNA does not appear to be affected by leptin.     

Glucose transporter and hypoxia-associated gene expression in the mammary gland of transition dairy cattle

Glucose is an important energy substrate, especially needed by dairy cows postpartum to support the onset of lactation. The prioritization and regulation of glucose uptake is accomplished, in part, by changes in expression of cellular glucose transport molecules (GLUT) within the mammary gland. The objectives of this study were to (1) evaluate the expression and cell-type specific localization of GLUT and hypoxia-associated genes that may regulate GLUT expression over the transition period and through lactation in bovine mammary tissue and (2) determine functionality of GLUT on primary bovine mammary endothelial cells (BMEC). Mammary tissue biopsies were taken from cows at 15 d before calving and again at 1, 15, 30, 60, 120, and 240 d post-parturition for quantitative real-time PCR analysis of GLUT and hypoxia-associated genes. Additional mammary tissue samples were used to localize GLUT within the cells of the lobulo-alveolar system via fluorescence microscopy. Cultures of primary bovine mammary endothelial cells were used to confirm the functionality of GLUT with a fluorescent glucose analog uptake assay. Significant increases in GLUT1 gene expression were observed during early lactation, whereas both GLUT3 and GLUT4 gene expression increased during late lactation. The gene expression for 2 receptors of vascular endothelial growth factor increased significantly during early lactation and remained increased throughout lactation when compared with gene expression during the transition period. All GLUT were detected on cultured BMEC and were capable of internalizing glucose through GLUT-mediated mechanisms. These data suggest mammary vascular tissues express GLUT during lactation and BMEC express functional glucose transporters. A better understanding of glucose uptake at the endothelial level may prove to be critical to improve glucose absorption from the blood for utilization by mammary epithelial cells.     

Regulation of mammary glucose uptake in goats: role of mammary gland supply, insulin, IGF-1 and synthetic capacity.

Variations in mammary glucose uptake were measured during the normal pregnancy-lactation cycle in dairy goats. In addition mammary glucose uptake was studied in response to somatotropin (ST) treatment in mid-lactation and acute increases in glucose concentration induced by sodium-propionate challenge in early lactation. Mammary glucose uptake was independent of arterial glucose, insulin and Insulin-like Growth Factor-1 (IGF-1) concentrations during lactation and during acute increases in arterial glucose concentration. Glucose uptake in the lactating mammary gland of the goat must therefore be carried out by an insulin-independent carrier, possible GLUT1, and glucose supply is not a limiting factor for uptake under in vivo conditions. Extraction of glucose uptake changed markedly during the normal course of lactation, following the overall changes in milk yield. Concentrations of glucose in skimmed milk, believed to reflect intracellular glucose concentration, changed in opposite directions, resulting in decreasing ratios of arterial: skimmed milk glucose concentration with progressing lactation. Thus, mammary synthetic capacity also involves a capacity for glucose uptake, which may be influenced by variations in glucose carrier numbers, as well as mammary metabolic activity (intracellular glucose concentration). In contrast to the situation during the normal course of lactation, ST stimulated milk yield, despite less efficient glucose extraction.     

Metabolism of arginine and ornithine in the cow and rabbit mammary tissue.

Amino acid uptake by the bovine mammary gland was determined by arteriovenous difference. Extraction of arginine from the plasma by the lactating bovine mammary gland was in excess of requirements for milk protein synthesis. Ornithine and citrulline also were extracted by the gland but are not in milk protein. Incubations of slices of lactating mammary tissue from cows and rabbits indicate that nonessential amino acids, especially proline and glutamate, are the major end products of arginine and ornithine metabolism in the lactating mammary gland.     

Effects of branched-chain amino acids on glucose uptake and lactose synthesis rates in bovine mammary epithelial cells and lactating mammary tissue slices

Even though supplementations of essential AA (EAA) are often related to increased lactose yields in dairy cows, underlying mechanisms connecting EAA availability to the mammary glands and lactose synthesis are poorly understood. The objective of this study was to examine the effects of branched-chain AA (BCAA) including Leu, Ile, and Val on (1) glucose transporter (GLUT1) abundance and glucose uptake, (2) the abundance of proteins regulating lactose synthesis pathway, and (3) fractional synthesis rates of lactose (FSR) using bovine mammary epithelial cells (BMEC) and mammary tissues slices (MTS). The BMEC (n = 4) were allocated randomly to regular Dulbecco's Modified Eagle Medium with Ham's F12 (DMEM/F12) medium (+EAA) or +EAA deficient (by 90%) in all EAA (?EAA), all BCAA (?BCAA), only Leu (?Leu), only Ile (?Ile) or only Val (?Val). Western immunoblotting analyses, depletion of glucose in media, and a proteomic analysis were performed to determine the abundance of GLUT1 in the cell membrane, net glucose uptake, and the abundance of enzymes involved in lactose synthesis pathway in BMEC, respectively. The MTS (n = 6) were allocated randomly to DMEM/F12 medium having all EAA and ¹³C-glucose at concentrations similar to plasma concentrations of cows (+EAA_p), and +EAA_p deprived of all BCAA (?BCAA_p) or only Leu (?Leu_p) for 3 h. The ¹³C enrichments of free glucose pool in MTS (E_Glu-free) and the enrichments of glucose incorporated into lactose in MTS and media [E_{Lactose-bound (T&M)}] were determined and used in calculating FSR. In BMEC, ?BCAA increased the fraction of total GLUT1 translocated to the cell membrane and the fraction that was potentially glycosylated compared with +EAA. Among individual BCAA, only ?Leu was associated with a 63% increase in GLUT1 translocated to the cell membrane and a 40% increase in glucose uptake of BMEC. The ?BCAA tended to be related to a 75% increase in the abundance of hexokinase in BMEC. Deprivation of Leu tended to increase glucose uptake of MTS but did not affect E_Glu-free, E_{Lactose-bound (T&M)}, or FSR relative to +EAA_p. On the other hand, ?BCAAp did not affect glucose uptake of MTS but was related to lower E_{Lactose-bound (T&M)}, or FSR relative to +EAA_p. Considering together, decreasing Leu supply to mammary tissues enhances GLUT1 and thus glucose uptake, which, however, does not affect lactose synthesis rates. Moreover, the deficiency of other BCAA, Ile, and Val alone or together with the deficiency of Leu seemed to decrease lactose synthesis rates without affecting glucose uptake. The data also emphasize the importance of addressing the effect of the supply of other nutrients to the mammary glands than the precursor supply in describing the synthesis of a milk component.     

Cationic amino acid transport by bovine mammary tissue

Cationic amino acid transport (arginine and lysine) into bovine mammary tissue occurs by a sodium independent and saturable mediated system. Concentrative uptake ratios (cell concentration/media concentration) for both arginine and lysine varied between 6 and 22. High concentrations of specific inhibitors of the neutral amino acid transport systems had no effect upon arginine or lysine uptake. Both arginine and lysine were strong inhibitors of each others uptake, whereas ornithine showed less specificity for inhibition of arginine and lysine uptake. In the presence of all amino acids, cationic amino acid uptake occurred at a rate equivalent to that with cationic substrates alone. The presence of the independent cationic amino acid transport system in bovine mammary tissue accounts for the high cationic amino acid uptake by measuring arteriovenous differences of plasma across the bovine mammary gland. This transport system is responsible for excess uptake (beyond milk protein synthesis requirements) of cationic amino acids for catabolism to other amino acids or potential oxidation.     

Relationship between glucose transport and metabolism in isolated bovine mammary epithelial cells

Glucose transport by isolated bovine mammary epithelial cells involves translocation across the cell membrane into a compartment that exchanges slowly with the bulk cytosol. The significance to glucose metabolism of this compartmentalization was examined by generation, modeling, and analysis of transport and metabolism data. Net uptake of 5 mM 3-O-methyl-d-glucose by isolated bovine mammary epithelial cells was measured at 37°C. Time-course curves were better fitted by a double exponential equation than a single exponential equation and were subjected to compartmental analysis to obtain glucose transport model parameters. Lactose synthesis and glucose oxidation rates and cellular concentrations of intermediary metabolites, glucose-6-phosphate and glucose-1-phosphate, were measured at varied media glucose concentrations. A model that integrates both glucose transport and metabolism under-predicted the rates of lactose synthesis and glucose oxidation by a factor of 3. To account for the observed glucose use rates, glucose must be available for phosphorylation once translocated across the cell membrane (intermediate compartmentalization of translocated glucose does not exclude access to hexokinase). Metabolic control analysis indicated that, at physiological glucose concentrations, phosphorylation by hexokinase exerts 80% of the control of glucose metabolism to lactose and CO₂, and transport exerts the remaining 20%.     

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.     

Duodenal infusion of glucose decreases milk fat production in grass silage-fed dairy cows

Four lactating dairy cows were arranged in a 4 x 4 Latin square design to study the effect of intestinal glucose supply on milk fat synthesis. Glucose (0, 443, 963, and 2398 g/d) was continuously infused in the duodenum over 14-d periods. Grass silage-based diets were formulated to be isoenergetic and isonitrogenous and met 100 and 110% of energy and protein requirements according to INRA (1989). Mammary uptake of nutrients was estimated through assay of arteriovenous differences and blood flow measurements. Glucose infusions decreased arterial concentrations of acetate, beta-hydroxybutyrate, and nonesterified fatty acids linearly and total glycerides curvilinearly. Milk fat yield was slightly decreased (- 52 g/d) between 0 and 963 g/d of glucose and milk fatty acid composition was modified by a marked decrease in long-chain fatty acids and an increase in de novo synthesis. The decrease in long-chain fatty acids, related to the decreased mammary uptake of plasma total glycerides, was likely due to a decrease in lipoprotein lipase and esterification activities. In regards to the evolution of metabolite concentrations in milk, the enhanced de novo synthesis and chain elongation was probably allowed by a greater availability of NADPH synthesized through pentose phosphate pathway. The greatest dose of glucose clearly decreased milk fat yield (-234 g/d). A mammary cell mediated intracellular reaction likely caused a homothetic decrease in milk fatty acids. However, reduced synthesis was not due to a shortage of glycerol-3-phosphate because its milk concentration remained unchanged. In conclusion, changes in exogenous glucose supply, in cows fed a grass silage-based diet, decreased milk fat production and modified milk fatty acid composition.     

The effects of L-type amino acid transporter 1 on milk protein synthesis in mammary glands of dairy cows

The mammary gland requires the uptake of AA for milk protein synthesis during lactation. The L-type amino acid transporter 1 (LAT1, encoded by SLC7A5), found in many different types of mammalian cells, is indispensable as a transporter of essential AA to maintain cell growth and protein synthesis. However, the function of LAT1 in regulating milk protein synthesis in the mammary gland of the dairy cow remains largely unknown. For the current study, we characterized the relationship between LAT1 expression and milk protein synthesis in lactating dairy cows and investigated whether the mammalian target of rapamycin complex 1 (mTORC1) signaling controls the expression of LAT1 in their mammary glands. We found that LAT1 and the heavy chain of its chaperone, 4F2, were expressed in mammary tissues of lactating cows, with the expression levels of LAT1 and the 4F2 heavy chain being significantly greater in lactating mammary tissues with high-milk protein content (milk yield, 33.8 ± 2.1 kg/d; milk protein concentration >3%, wt/vol,; n = 3) than in tissues from cows with low-milk protein content (milk yield, 33.7 ± 0.5 kg/d; milk protein concentration <3%, wt/vol; n = 3). Immunofluorescence staining of sectioned mammary tissues from cows with high and low milk protein content showed that LAT1 was located on the whole plasma membrane of alveolar epithelial cells, suggesting that LAT1 provides essential AA to the mammary gland. In cultured mammary epithelial cells from the dairy cows with high-milk protein content, knockdown of LAT1 expression decreased cell viability and β-casein expression; in contrast, overexpression of LAT1 had the opposite effect. Inhibition of mTORC1 by rapamycin attenuated the phosphorylation of molecules related to mTORC1 signaling and caused a marked decrease in LAT1 expression in the cultured cells; expression of β-casein also decreased significantly. These results suggest that LAT1 is involved in milk protein synthesis in the mammary glands of lactating dairy cows and that the mTORC1 signaling pathway might be a control point for regulation of LAT1 expression, which could ultimately be used to alter milk protein synthesis.     

Increasing milking intervals decreases the mammary blood flow and mammary uptake of nutrients in dairy cows

Increasing the milking intervals reduces milk yield. The aims of this study were to determine whether the reduction in milk yield could be explained by a decrease in mammary uptake of the nutrients or a decrease in the efficiency of the mammary gland in using the milk precursors to synthesize milk components, or both. In a Latin square design with 5 periods, 4 multiparous lactating dairy cows in midlactation were milked at 8-, 12-, 16-, or 24-h intervals over a period of 7 d. The cows were surgically prepared to estimate the net mammary balance of nutrient precursors of milk components (glucose, α-amino nitrogen, acetate, β-hydroxybutyrate, and total glycerol). The efficiency of the mammary gland in synthesizing milk components was estimated by the mammary uptake:milk output ratio. After 7 d of treatment, the decrease in milk yield of 6.1 kg/d between 8- and 24-h milking intervals was associated with a reduction in the uptake of nutrients by the mammary gland, whereas the efficiency of the mammary gland in synthesizing milk components remained relatively unchanged. The mammary uptake decreased by 26% for glucose, 32% for α-amino nitrogen, 18% for acetate, 24% for total glycerol, and 24% for β-hydroxybutyrate, respectively. These reductions in nutrient uptake were due to a decrease in the mammary blood flow (1.23 ± 0.24 L/min). For milk fat precursors (acetate, β-hydroxybutyrate, and total glycerol), the decrease in mammary blood flow explained the entire reduction in the mammary uptake. For glucose and the milk protein precursors, the reduction in the mammary blood flow explained 60% of the decrease in the mammary uptake, with the other 40% being accounted for by a reduction in the mammary extraction of nutrients. The nutrient uptake was altered as milk yield decreased. These decreases began with the 16-h milking interval and were higher at the 24-h milking interval.     

Coordination of mammary metabolism and blood flow after refeeding in rats

The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 μmol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed.     

Cardiovascular responses and mammary substrate uptake in Jersey cows treated with pituitary-derived growth hormone during late lactation

Pituitary-derived bovine growth hormone (bGH) was administered to Jersey cows during late lactation for 7 d. Milk yield increased significantly during treatment and by a maximum of 49.6% on d 7. The magnitude of the increase was similar to that of mammary plasma flow (47.8 +/- 18.3%) over the same period. By 15-21 d after treatment, both variables had returned to pretreatment values. With respect to milk composition, bGH had negligible effect on lactose and fat concentrations but there were significant decreases in protein, sodium and chloride. Arterial plasma concentrations of bGH increased substantially during treatment, but the associated rise in insulin was not statistically significant. Haematocrit decreased significantly, the lowest value being recorded 3 d after bGH treatment ceased. Mammary respiratory quotient fell progressively after the start of bGH treatment and reached the lowest recorded value 3 d after treatment ceased (62.2 +/- 7.3% of pretreatment value). Glucose and acetate uptake by the mammary gland increased significantly during treatment, increase in glucose uptake being due both to a greater arterio-venous difference and to mammary plasma flow. There was strong evidence that the acute response in increased milk yield was associated with multiple effects in terms of mammary plasma flow and metabolism, as well as haematocrit changes indicative of increased plasma volume.     

Evaluation of whole blood and plasma in the interorgan supply of free amino acids for the mammary gland of lactating dairy cows

We investigated the contribution of plasma and red blood cells to amino acid (AA) supply for milk protein synthesis during a combination of treatments that included abomasal infusion of casein and AA and utilization of a hyperinsulinemic-euglycemic clamp. Treatments resulted in substantial differences in circulating concentrations of AA, mammary uptake of AA, and rates of milk protein synthesis. Arterial concentrations of all AA in plasma were highly correlated with that of whole blood. Concentrations of AA in red blood cells were either higher (Asn+Asp, Gly, His, Leu, Met, Orn, Ser, Tau, Thr, and Tyr), lower (Ala, Arg, Cit, Cys, Ile, and Val), or similar (Gln+Glu, Phe, and Pro) to that of plasma. Arteriovenous difference measurements demonstrated that interorgan transfer of AA to the mammary gland was primarily by plasma. There was little involvement of red blood cells except for small quantities of Leu, Met, and Thr to the mammary gland; this contribution was greatest for Met and accounted for 14% of the total mammary uptake. Countercurrent transport of Gln + Glu, Asn + Asp, and Pro was also evident where these AA were extracted from plasma, but were released into red blood cells as blood passed through the mammary gland. This net influx of Gln+Glu, Asn+Asp, and Pro into red blood cells was equivalent to 26, 17, and 30% of their mammary uptake from plasma. Overall, the interorgan transport of free AA for the mammary gland was predominantly by plasma, and red blood cells were limited to minor contributions in mammary uptake for a few AA. Furthermore, arteriovenous differences of essential AA across the mammary gland were highly correlated between plasma and whole blood.     

Short communication: Protein kinase C regulates glucose uptake and mRNA expression of glucose transporter (GLUT) 1 and GLUT8 in lactating bovine mammary epithelial cells

The aim of this study was to determine the role of protein kinase C (PKC) in regulating glucose uptake in lactating bovine mammary epithelial cells (BMEC). The BMEC were cultured and treated with different concentrations of phorbol 12-myristate 13-acetate (PMA;0, 10, 25, 50, 100, and 200 ng/mL), the classic activator of PKC, for 48 h. Compared with the cells with no PMA treatment, 50 and 100 ng of PMA/mL significantly stimulated the glucose uptake of the BMEC, whereas the glucose uptake by the cells treated with the lowest and the highest amounts of PMA (25 and 200 ng/mL, respectively) did not show a significant difference. Consistently, the mRNA expression of glucose transporter (GLUT) 1 and 8 showed a similar pattern of increase under the treatments of PMA. Furthermore, when the cells were pretreated with GF1090203X (0, 0.25, 0.5, 1, and 2 μM), an inhibitor of PKC, for 30 min before exposed to PMA (50 ng/mL), the PMA-induced glucose uptake and GLUT1 and GLUT8 expression were decreased by GF1090203X in a dose-dependent manner. These results demonstrate that PKC is involved in the regulation of glucose uptake by BMEC, and this function may work, at least partly, through upregulating the expression of GLUT1 and GLUT8.