Live imaging of GLUT2 glucose-dependent trafficking and its inhibition in polarized epithelial cysts

Abstract

GLUT2 is a facilitative glucose transporter, expressed in polarized epithelial cells of the liver, intestine, kidney and pancreas, where it plays a critical role in glucose homeostasis. Together with SGLT1/2, it mediates glucose absorption in metabolic epithelial tissues, where it can be translocated apically upon high glucose exposure. To track the subcellular localization and dynamics of GLUT2, we created an mCherry–hGLUT2 fusion protein and expressed it in multicellular kidney cysts, a major site of glucose reabsorption. Live imaging of GLUT2 enabled us to avoid the artefactual localization of GLUT2 in fixed cells and to confirm the apical GLUT2 model. Live cell imaging showed a rapid 15 ± 3 min PKC-dependent basal-to-apical translocation of GLUT2 in response to glucose stimulation and a fourfold slower basolateral translocation under starvation. These results mark the physiological importance of responding quickly to rising glucose levels. Importantly, we show that phloretin, an apple polyphenol, inhibits GLUT2 translocation in both directions, suggesting that it exerts its effect by PKC inhibition. Subcellular localization studies demonstrated that GLUT2 is endocytosed through a caveolae-dependent mechanism, and that it is at least partly recovered in Rab11A-positive recycling endosome. Our work illuminates GLUT2 dynamics, providing a platform for drug development for diabetes and hyperglycaemia.

Activation of nuclear receptor PXR impairs glucose tolerance and dysregulates GLUT2 expression and subcellular localization in liver

“…In polarized enterocytes, the GLUT2 concentration in apical membrane is regulated by intracellular trafficking [33]. Furthermore, dynamic basal to apical translocation has been observed in cultured, polarized, kidney epithelial cell line in response to glucose [34]. There are also some reports describing internalization of GLUT2 in liver in response to feeding mediated by interaction with insulin receptor [35,36].…”

Modulation of Renal GLUT2 by the Cannabinoid-1 Receptor: Implications for the Treatment of Diabetic Nephropathy

“…This was achieved by the dynamic tracking of its localization in an MDCK II cell line expressing a C-terminal hGLUT2-mCherry fusion protein. 13 When the cells in a monolayer were cultured in media containing PBS, GLUT2 was localized to the plasma membrane, corresponding to the BLM. 13 Hyperglycemia resulted in rapid GLUT2 internalization to the perinuclear region (Supplemental Figure 4, A and B), corresponding to the BBM of three-dimensional RPTCs.…”

“…6,7,12 Additionally, a shift in its localization from the RPTC's basolateral membrane (BLM) to the apical/brush border membrane (BBM), contributing to increased glucose reabsorption, was also reported. 6,13 Plasma or luminal glucose concentrations have been shown to regulate GLUT2 expression and/or translocation, 10 accounting for the deleterious effects of hyperglycemia on the proximal tubule. Although reports regarding the transcriptional regulation of GLUT2 have revealed a few transcriptional factors that may directly control the expression of GLUT2 under diabetic conditions, [14][15][16] the upstream molecular mechanism underlying these processes has yet to be determined.…”

Structure of, and functional insight into the GLUT family of membrane transporters

“…[158][159][160][161][162][163] In contrast, stress, a high-fat diet, and glucocorticoids inhibit GLUT2 trafficking to the brush border membrane. [164][165][166] Most recently, subcellular localization studies using live cell imaging demonstrated that GLUT2 is endocytosed through a caveolae-dependent mechanism, which is partially recovered in Rab11A-positive recycling endosomes. 99 However, what is still not entirely clear is the role that this transporter plays while it is transiently expressed at the cell surface.…”