Department of Physiology, David Geffen School of Medicine, Univeristy of California at Los Angeles, Los Angeles, California 90095-1751, USA.
Am J Physiol Cell Physiol. 2012 Aug 1;303(3):C348-54. doi: 10.1152/ajpcell.00115.2012. Epub 2012 Jun 6.
The human Na(+)-glucose cotransporter SGLT2 is expressed mainly in the kidney proximal convoluted tubule where it is considered to be responsible for the bulk of glucose reabsorption. Phosphorylation profiling has revealed that SGLT2 exists in a phosphorylated state in the rat renal proximal tubule cortex, so we decided to investigate the regulation of human SGLT2 (hSGLT2) by protein kinases. hSGLT2 was expressed in human embryonic kidney (HEK) 293T cells, and the activity of the protein was measured using radiotracer and whole cell patch-clamp electrophysiology assays before and after activation of protein kinases. 8-Bromo-adenosine cAMP (8-Br-cAMP) was used to activate protein kinase A, and sn-1,2-dioctanoylglycerol (DOG) was used to activate protein kinase C (PKC). 8-Br-cAMP stimulated D-[α-methyl-(14)C]glucopyranoside ([(14)C]α-MDG) uptake and Na(+)-glucose currents by 200% and DOG increased [(14)C]α-MDG uptake and Na(+)-glucose currents by 50%. In both cases the increase in SGLT2 activity was marked by an increase in the maximum rate of transport with no change in glucose affinity. These effects were completely negated by mutation of serine 624 to alanine. Insulin induced a 250% increase in Na(+)-glucose transport by wild-type but not S624A SGLT2. Parallel studies confirmed that the activity of hSGLT1 was regulated by PKA and PKC due to changes in the number of transporters in the cell membrane. hSGLT1 was relatively insensitive to insulin. We conclude that hSGLT1 and hSGLT2 are regulated by different mechanisms and suggest that insulin is an SGLT2 agonist in vivo.
人源 Na(+)-葡萄糖协同转运蛋白 SGLT2 主要在肾脏近曲小管表达,被认为负责大部分葡萄糖重吸收。磷酸化谱分析表明,SGLT2 在大鼠肾近曲小管皮质中处于磷酸化状态,因此我们决定研究蛋白激酶对人源 SGLT2(hSGLT2)的调节。在人胚肾 293T 细胞中表达 hSGLT2,并用放射性示踪剂和全细胞膜片钳电生理学检测方法在蛋白激酶激活前后测量蛋白的活性。8-溴-腺苷环磷酸(8-Br-cAMP)用于激活蛋白激酶 A,sn-1,2-二油酰基甘油(DOG)用于激活蛋白激酶 C(PKC)。8-Br-cAMP 刺激 D-[α-甲基-(14)C]吡喃葡萄糖苷([(14)C]α-MDG)摄取和 Na(+)-葡萄糖电流增加 200%,而 DOG 增加[(14)C]α-MDG 摄取和 Na(+)-葡萄糖电流增加 50%。在这两种情况下,SGLT2 活性的增加都伴随着转运最大速率的增加,而葡萄糖亲和力没有变化。丝氨酸 624 突变为丙氨酸完全消除了这些作用。胰岛素诱导野生型 SGLT2 的 Na(+)-葡萄糖转运增加 250%,但 S624A SGLT2 则没有。平行研究证实,由于细胞膜上转运体数量的变化,hSGLT1 的活性受 PKA 和 PKC 调节。hSGLT1 对胰岛素相对不敏感。我们得出结论,hSGLT1 和 hSGLT2 的调节机制不同,并提示胰岛素在体内是 SGLT2 的激动剂。