Polyamine regulation of Na⁺/glucose symporter expression in LLC‐PK₁ cells

Abstract

Addition of polyamines or their analogs to newly confluent LLC-PK1 cells resulted in down-regulation of Na(+)-dependent glucose transport (symport) activity. Polyamines prevented the induction of this symporter by the differentiation inducer hexamethylene bisacetamide (HMBA) but did not influence induction by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Partial depletion of endogenous polyamines after addition of alpha-difluoromethylornithine (DFMO) resulted in a 4 to 5-fold increase in symporter expression. Symporter induction by either HMBA or DFMO was inhibited by the protein kinase inhibitor H-7 but H-7 did not affect symporter induction by IBMX. Changes in symporter activity were accompanied by changes in levels of the 75 kD symporter subunit detected by Western blot. Cultures exposed to HMBA exhibited reduced levels of ornithine decarboxylase activity. Our results suggest that induction of symporter expression by HMBA may be mediated in part by its effects on polyamine metabolism, and point to parallel roles of polyamines and cyclic AMP in regulating the expression of this physiologically important renal transport system.

Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC‐PK1 cells

“…Levels of both transcripts of SGLTl mRNA reached a plateau value after 3 weeks exposure to 5 mM HMBA (Fig. 4A) and exhibited temporal correlation with the previously described increase in SGLTl transport activity (Peng and Lever, 1993).…”

Regulation of Na+-coupled Glucose Transport in LLC-PK1 Cells

“…Cell Culture-The porcine renal cell line LLC-PK 1 clone G8 was maintained in a 50:50 mixture of Dulbecco's modified Eagle's medium: Ham's F-12 (ICN-Flow Laboratories) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 5 mM glutamine, 0.12% sodium bicarbonate, and 15 mM HEPES, pH 7.0, in 5% CO 2 , as described previously (Peng and Lever, 1993). Cells were plated at a density of 10 4 cells/cm 2 .…”

“…Much of what is currently known about regulation of the expression of the SGLT type of glucose transporter has been derived from studies using the cell line LLC-PK 1 , derived from porcine renal proximal tubule (Amsler and Cook, 1982;Peng and Lever, 1993). This cell line permits the reconstitution of a fully functional polarized epithelium in vitro.…”

“…The development of a confluent monolayer is accompanied by the appearance of a number of differentiated functions characteristic of this epithelium including the formation of tight junctions and microvilli, vasopressin responsiveness, transepithelial salt and water transport, several brush-border marker enzyme activities, and apical membrane Na ϩ -coupled glucose transport activity (reviewed in Lever (1989)). Elevation of intracellular levels of cyclic AMP or treatment with the differentiation inducer hexamethylene bisacetamide (HMBA), 1 greatly increased both the levels of expression and rate of appearance of several of these differentiated functions including Na ϩ -coupled glucose transport (Amsler and Cook, 1982;Yoneyama and Lever, 1984;Peng and Lever, 1993;Yet et al, 1994). These agents appear to act by independent and synergistic mechanisms (Peng and Lever, 1993).…”

“…Elevation of intracellular levels of cyclic AMP or treatment with the differentiation inducer hexamethylene bisacetamide (HMBA), 1 greatly increased both the levels of expression and rate of appearance of several of these differentiated functions including Na ϩ -coupled glucose transport (Amsler and Cook, 1982;Yoneyama and Lever, 1984;Peng and Lever, 1993;Yet et al, 1994). These agents appear to act by independent and synergistic mechanisms (Peng and Lever, 1993). SGLT1 transcripts of 2.2 and 3.9 kb, differing in the length of the 3Ј-untranslated region (3Ј-UTR) are observed in LLC-PK 1 cells as a result of alternative cleavage and polyadenylation (Ohta et al, 1990).…”