Unexpected severe consequences of Pikfyve deletion by aP2- or Aq-promoter-driven Cre expression for glucose homeostasis and mammary gland development.

Systemic deficiency of PIKfyve, the evolutionarily conserved phosphoinositide kinase synthesizing cellular PtdIns5P and PtdIns(3,5)P2 and implicated in insulin signaling, causes early embryonic death in mice. In contrast, mice with muscle-specific Pikfyve disruption have normal lifespan but exhibit early-age whole-body glucose intolerance and muscle insulin resistance, thus establishing the key role of muscle PIKfyve in glucose homeostasis. Fat and muscle tissues control postprandial glucose clearance through different mechanisms, raising questions as to whether adipose Pikfyve disruption will also trigger whole-body metabolic abnormalities, and if so, what the mechanism might be. To clarify these issues, here we have characterized two new mouse models with adipose tissue disruption of Pikfyve through Cre recombinase expression driven by adipose-specific aP2- or adiponectin (Aq) promoters. Whereas both mouse lines were ostensibly normal until adulthood, their glucose homeostasis and systemic insulin sensitivity were severely dysregulated. These abnormalities stemmed in part from accelerated fat-cell lipolysis and elevated serum FFA Intriguingly, aP2-Cre-PIKfyve(fl/fl) but not Aq-Cre-PIKfyve(fl/fl) females had severely impaired pregnancy-induced mammary gland differentiation and lactogenesis, consistent with aP2-Cre-mediated Pikfyve excision in nonadipogenic tissues underlying this defect. Intriguingly, whereas mammary glands from postpartum control and Aq-Cre-PIKfyve(fl/fl) mice or ex vivo mammary gland explants showed profound upregulation of PIKfyve protein levels subsequent to prolactin receptor activation, such increases were not apparent in aP2-Cre-PIKfyve(fl/fl) females. Collectively, our data identify for the first time that adipose tissue Pikfyve plays a key role in the mechanisms regulating glucose homeostasis and that the PIKfyve pathway is critical in mammary epithelial differentiation during pregnancy and lactogenesis downstream of prolactin receptor signaling.

Effect of prolactin on inositol uptake in mouse mammary gland explants.

Studies were carried out to assess the role of insulin (I), cortisol (H), and prolactin (P or PRL) in regulating myoinositol (inositol) uptake in the mammary gland. Using cultured mammary gland explants from pregnant mice (12-14 days into gestation), insulin and prolactin were found to stimulate inositol uptake, while cortisol impaired inositol uptake. Optimal inositol uptake was observed when tissues were treated with all three lactogenic hormones (I, H, and PRL). Further studies were designed primarily to characterize the PRL stimulation of inositol transport. Inositol uptake in the mammary explants increased linearly for 4 h, both in IH treated tissues and those treated with IHP; distribution ratios of greater than 14 were achieved at 4 h, suggesting an active inositol transport mechanism. The PRL effect on inositol uptake is sodium-dependent, temperature-dependent, and ouabain sensitive. DIDS and furosemide did not impair inositol uptake or the PRL effect on inositol uptake. PRL stimulated inositol uptake employing PRL concentrations of 10-1000 ng/ml. The PRL effect was manifested at all PRL-treatment times of 12 h or longer, but not at earlier times. PRL thus appears to be an important and essential hormone for the stimulation of inositol accumulation in milk during lactogenesis.

Hormone regulation of choline uptake and incorporation in mouse mammary gland explants.

Choline is a nutrient in milk that is essential for the nourishment and growth of newborns. In rat milk, choline is present in concentrations that are more than an order of magnitude higher than in maternal serum. Using cultured mammary tissues taken from 12-14-day pregnant mice, the effects of the three primary lactogenic hormones--prolactin (PRL), insulin (I), and cortisol (H)--on choline uptake and incorporation into lipids were determined. By itself or in the presence of H and/or PRL, I was the only hormone that increased the accumulation of choline in aqueous tissue fractions. In contrast, PRL, when present with I plus H, was the only hormone that stimulated the incorporation of choline into the lipid fraction of tissues. Choline uptake was found to be sodium and time dependent; maximum distribution ratios >18 were achieved after a 6-hr uptake time. In kinetic studies the apparent Km for choline uptake was calculated to be approximately 2.7 mM, whereas the Vmax was 7.4 mM intracellular water per 30 mins. These results suggest the existence of a sodium-dependent active transporter for choline in the mouse mammary gland that is specifically stimulated by I. PRL, in contrast, only stimulates the incorporation of choline into lipids.

Pendrin transporter carries out iodide uptake into MCF-7 human mammary cancer cells.

Previous studies have shown that iodide is actively taken up into mammary alveolar epithelial cells and secreted into milk. In the present studies we demonstrate that 125I also accumulates in MCF-7 cells against a concentration gradient; distribution ratios of greater than 30 were achieved. Iodide uptake into MCF-7 cells is transient, with peak accumulations occurring in about 5 min. The iodide is rapidly metabolized, probably to iodine, and it then exits the cells. The iodide transporter identified in MCF-7 cells is pendrin. DIDS, a nonspecific inhibitor of anion exchange, inhibits iodide uptake. Iodide uptake is impaired at reduced temperature, but is not dependent on sodium. Inhibitors of the sodium-iodide symporter (NIS) as well as ouabain did not affect the extent of iodide uptake. The pendrin transporter but not NIS was identified via western blotting techniques. Pendrin appears to be the primary iodide transporter in the MCF-7 cell line stocks that were employed for these studies.

Prolactin, cortisol, and insulin regulation of nucleoside uptake into mouse mammary gland explants.

Nucleosides are essential components of milk that are used for the nourishment of newborns. Effects of the three primary lactogenic hormones, including prolactin (PRL), insulin (I), and cortisol (H), on nucleoside uptake and incorporation into cultured mammary tissues taken from 12- to 14-day pregnant mice were determined; most experiments focused on the regulation of uridine uptake. Insulin alone, as well as PRL in the presence of insulin and cortisol, was shown to stimulate uridine uptake and incorporation into RNA in mammary explants taken from 12- to 14-day pregnant mice. The PRL effects were expressed at concentrations of 25 ng/ml and above, which are physiological plasma concentrations. In the absence of sodium, uridine uptake and incorporation were diminished, suggesting the presence of a sodium-dependent uridine transporter. In kinetic studies the apparent Km for uridine uptake was calculated to be 312 microM, and the Vmax 2.90 micromol/hr/L cell water; PRL had no effect on the Km but increased the Vmax to 5.88 micromol/hr/L cell water. When assessing uridine uptake in the presence of the other nucleosides at 0.1 mM, only cytidine competed with uridine uptake. The fact that distribution ratios of greater than 15:1 were achieved with uridine indicates that uridine uptake may be via an active transporter. These studies show that PRL enhances uridine update in mammary tissues by stimulating the activity, and probably synthesis, of a sodium-dependent, active uridine and cytosine transporter.

Prolactin regulation of the pendrin-iodide transporter in the mammary gland.

Iodide is an essential constituent of milk that is present in concentrations more than an order of magnitude higher than in the maternal plasma. Earlier, a sodium-iodide symporter was identified in the mammary gland; this transporter is presumed to take iodide from the maternal plasma into the alveolar epithelial cells of the mammary gland. We now report the existence of a second iodide transporter, pendrin, which is also essential for iodide accumulation in milk. Via Western blotting methods, high levels of the transporter were detected in lactating tissues; lesser amounts were found in tissues from midpregnant and virgin mice. Prolactin, at physiological concentrations, stimulated the expression of the pendrin transporter in cultured mammary tissues taken from 12- to 14-day-pregnant mice. The prolactin effect on iodide uptake into cultured mammary tissues was abolished by pendrin transport inhibitors, including DIDS, furosemide, and probenecid. These studies suggest that the prolactin stimulation of pendrin activity is an essential element in the prolactin stimulation of iodide uptake into milk.

Effect of prolactin on phosphate transport and incorporation in mouse mammary gland explants.

Inorganic phosphate is present in milk at a concentration that is severalfold higher than in maternal plasma. In cultured mammary tissues from 12- to 14-day-pregnant mice, the intracellular concentration of (32)PO(4) was six times higher than in the culture medium after a 4-h treatment with (32)PO(4). Of the principal lactogenic hormones [insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in the presence of H and I) stimulated (32)PO(4) uptake into cultured mammary tissues; H, by itself or in the presence of I or PRL, inhibited (32)PO(4) uptake. All three lactogenic hormones together effected the greatest stimulation of (32)PO(4) uptake. Similar hormone effects were observed with regard to (32)PO(4) incorporation into lipids and trichloroacetic acid-insoluble molecules. In a time course study, the onset of the PRL stimulation of (32)PO(4) uptake and incorporation occurred 8-12 h after PRL addition; in dose-response studies, the PRL effect was manifested with PRL concentrations of 50 ng/ml and above. From kinetic studies, the apparent maximal velocity of PO(4) uptake was determined to be approximately 7.7 mM x h(-1) x l cell water(-1); the apparent Michaelis-Menten constant was approximately 3-5 mM. The PRL effect on (32)PO(4) uptake was abolished when sodium was absent from the uptake medium. These studies thus demonstrate a complex interaction of three hormones (I, H, and PRL) in the regulation of (32)PO(4) uptake and incorporation into macromolecules in cultured mouse mammary tissues.

Effect of insulin on iodide uptake in mouse mammary gland explants.

Studies were carried out primarily to assess the role of insulin in regulating iodide uptake in the mammary gland. Using cultured mammary gland explants from virgin and pregnant mice (12-14 days into gestation), insulin (1 microg/ml) was shown to stimulate iodide uptake after a 2-day exposure period. The effect of insulin was manifested by itself, as well as in the presence of cortisol and prolactin. Optimal iodide uptake was observed when tissues were treated with all three lactogenic hormones (insulin, cortisol, and prolactin). In a time-course experiment, the effect of insulin alone was initially observed after a 10-hr treatment; the effect was maintained for 30 hr. In dose-response studies, 1 ng/ml insulin elicited a significant effect after 24 hr in culture; a maximal effect was achieved with 50-100 ng/ml insulin. The optimal cortisol concentration for a maximum stimulation of iodide uptake was 10(-7)M. In a quantitative Western blot analysis employing an antibody to the sodium-iodide symporter, insulin stimulated an upregulation of the transporter protein after a 4-, 8-, or 20-hr treatment with insulin. Perchlorate and thiocyanate abolished the insulin effect on iodide uptake, further suggesting that the insulin response occurs via a stimulation of the sodium-iodide symporter. Clearly, insulin is an important and essential hormone in the lactogenic hormone complex for regulating iodide uptake in the mammary gland, but maximal expression of iodide uptake is only expressed when all three lactogenic hormones are present.