Glucose transport in proliferating and post-confluent cultured renal epithelial cells

Author

Pamela Jean Galloway

Date of Award

12-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

John S. Cook

Committee Members

Frank W. Larimer, Raymond A. Popp, Margaret Terzaghi-Howe

Abstract

The kinetics of facilitated diffusion (Na+-independent, basolateral) hexose transport were measured in the cultured renal epithelial cell lines, LLC-PK₁ and OK, at two time points during their growth in culture. These cells are proximal tubule in origin. The two growth states, proliferating and post-confluent, were chosen because the capacity for Na+-dependent (apical) hexose transport, as measured by the concentrative uptake of α-meGlc, is nearly undetectable in proliferating cells, and reaches maximal values in post-confluent cell populations. This work tested the hypothesis that a shift from low to high K_m for facilitated diffusion glucose transport would occur in these cells as they developed in culture and acquired the capacity for apical transport. The K_{m, app} values for 2-dGlc uptake measured in 3-O-meGlc preloaded LLC-PK₁ cells were 14 mM and 37 mM for proliferating and post-confluent cells, respectively. A similar result was obtained in nonpreloaded LLC-PK₁ cells. The K_{m, app} values for 2-dGlc uptake in proliferating and post-confluent OK cells were 7 mM and 63 mM, respectively. The presence of high K_m transport in post-confluent OK cells was also detected by 3-O-meGlc efflux under equilibrium exchange. Thus both cell lines show a growth state-dependent shift in K_m for facilitated diffusion hexose transport. Chromatographic analysis of intracellular 2-dGlc after uptakes revealed that concentrations of nonphosphorylated 2-dGlc remain low relative to both extracellular 2-dGlc and the K_m for transport in post-confluent cells, so that uptake measurements are not significantly affected by efflux. Antisera to GLUT1 (low K_m) and GLUT2 (high K_m) recognized proteins on Western blots of cell lysates and membrane preparations from proliferating and post-confluent cell populations of both lines. GLUT1 and GLUT2 riboprobes detected homologous transcripts in Northern blots of LLC-PK₁ cells. Western blotting taken together with kinetics results indicated that GLUT2 may be synthesized but not functional in proliferating cells. Immunofluorescence staining revealed that GLUT2 protein was present in the perinuclear region of proliferating OK cells. In contrast, lateral membrane staining of GLUT2 was clearly visible in post-confluent OK cells. Western blotting of biotinylated, cell-surface associated proteins indicated that GLUT2 is present at the plasma membrane of post-confluent, but not proliferating, OK cells. Immunogold labeling of OK cell sections showed basal membrane staining by GLUT2 antiserum. The immunofluorescence staining pattern of GLUT1 in OK cells was distinctly fibrillar, appearing to encircle most nuclei and radiate towards cell peripheries. The GLUT1 fibrillar arrays colocalized with vimentin filaments in all interphase cells. In mitotic cells, GLUT1 signal was localized in spheroidal aggregates, while vimentin fibers were visible in the area of condensed chromosomes. Western blots show that two distinct proteins were recognized. Colchicine treatment resulted in the collapse of both GLUT1 and vimentin arrays, and both systems continued to be colocalized.

Recommended Citation

Galloway, Pamela Jean, "Glucose transport in proliferating and post-confluent cultured renal epithelial cells. " PhD diss., University of Tennessee, 1994.
https://trace.tennessee.edu/utk_graddiss/10352