INTERACTION OF GLUCOSE AND LIPID WITH CELL SURVIVAL IN LACTATION

乳腺上皮细胞分化为分泌细胞用于哺乳，对新生哺乳动物的存活具有重要意义。催乳素在这一过程中起着关键作用，目前的研究重点是催乳素对乳蛋白基因转录的调控。仅根据乳汁蛋白质合成的调节来表征乳房分化提供了对这一复杂过程的一维观察。除了刺激牛奶蛋白基因的转录外，催乳素还提供生存信号，并可能刺激其他生理过程，包括葡萄糖运输和脂肪生物合成。我们用转基因小鼠进行的研究表明，乳腺上皮细胞中Akt和葡萄糖水平的调节可能对于1)保持平衡是必要的 2)促进乳腺上皮细胞的存活。葡萄糖转运蛋白GLUT1是乳汁中表达的主要转运蛋白。我们推测，乳源激素激活Akt可以刺激GLUT1转位到质膜，并增加己糖激酶活性。这些变化导致胞浆中GLUT1浓度的增加。 葡萄糖和葡萄糖-6-磷酸。因此，葡萄糖和葡萄糖-6-磷酸的增加通过糖酵解增加丙酮酸和NADPH的产生，刺激脂肪酸的生物合成，并保持线粒体膜电位，从而促进细胞存活。我们建议使用磁共振波谱来确定 MMTV-MYR-Akt1转基因小鼠脂肪生物合成增加的代谢基础。我们推测，激活Akt的过表达应该以细胞内葡萄糖和葡萄糖-6-磷酸为代价增加乳腺细胞中的丙酮酸浓度，增加柠檬酸水平，并减少相对于脂肪合成的乳糖合成。在第二个目标中，我们将确定Akt1基因缺失的小鼠是否存在哺乳缺陷。在第三个目标中，我们将建立转基因小鼠，在其中使用Tet-On系统有条件地调节己糖激酶-I在乳腺中的表达，以验证这样的假设，即在哺乳期过度表达己糖激酶会由于脂类和乳糖的合成失衡而导致哺乳失败。在第四个目的中，我们将过表达GLUT1，以确定这是否会抑制乳腺的退化。这些目标解决了以下基本问题 利用体内模型研究调节脂类和乳糖生物合成的代谢途径，以及葡萄糖代谢在调节细胞凋亡中的作用。

The differentiation of mammary epithelial cells into secretory cells for lactation is of great importance to survival of newborn mammals. Prolactin is critical in this process and the focus has been upon the regulation of milk protein gene transcription by prolactin. Characterizing mammary differentiation solely on the basis of the regulation of milk protein synthesis provides a one-dimensional view of this complex process. In addition to stimulating transcription of milk protein genes, prolactin provides a survival signal and may stimulate other physiologic processes including glucose transport and lipid biosynthesis. Our research with transgenic mice that express constitutively activated Akt1 in the mammary gland suggests that regulation of Akt and glucose levels in mammary epithelial cells may be necessary to 1) maintain "balanced aerobic glycolysis" which leads to the proper ratio of lipid and lactose in the milk, and 2) promote survival of mammary epithelial cells. The glucose transporter GLUT1 is the predominant transporter expressed in the lactating mammary gland. We hypothesize that activation of Akt by lactogenic hormones stimulates translocation of GLUT1 to the plasma membrane and increases hexokinase activity. These changes result in an increase in the cytosolic concentrations of glucose and glucose-6-phosphate. As a consequence the increase in glucose and glucose-6-phosphate stimulates fatty acid biosynthesis due to the increased production of pyruvate and NADPH via glycolysis; and maintains mitochondrial membrane potential thus promoting cell survival. We propose to use magnetic resonance spectroscopy to determine the metabolic basis for the increased lipid biosynthesis in MMTV-myr-Akt1 transgenic mice. We hypothesize that overexpression of activated Akt should increase the pyruvate concentration in mammary cells at the expense of intracellular glucose and glucose-6-phosphate, increase citrate levels, and decrease lactose synthesis relative to lipid synthesis. In the second aim we will determine whether there is a lactation defect in Akt1 null mice. In the third aim, we will generate transgenic mice in which the expression of hexokinase-I in the mammary gland is conditionally regulated using the Tet-On system, to test the hypothesis that overexpression of hexokinase during lactation will result in a lactation failure due to an imbalance in the synthesis of lipids versus lactose. In the fourth aim we will overexpress GLUT1 to determine whether this will suppress involution of the mammary gland. These aims address fundamental questions about the metabolic pathways regulating biosynthesis of lipids and lactose, and the role of glucose metabolism in regulating apoptosis using in vivo models.