The Plasma Membrane-associated Protein RS1 Decreases Transcription of the Transporter SGLT1 in Confluent LLC-PK1 Cells

Thomas Korn,Thomas Kühlkamp,Christina Track,Irina Schatz,Katharina Baumgarten,Valentin Gorboulev,Hermann Koepsell

doi:10.1074/jbc.m105975200

Thomas Korn, Thomas Kühlkamp + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m105975200

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Abstract

Previously we cloned RS1, a 67-kDa polypeptide that is associated with the intracellular side of the plasma membrane. Upon co-expression in Xenopus laevis oocytes, human RS1 decreased the concentration of the Na(+)-D-glucose co-transporter hSGLT1 in the plasma membrane (Valentin, M., Kühlkamp, T., Wagner, K., Krohne, G., Arndt, P., Baumgarten, K., Weber, W.-M., Segal, A., Veyhl, M., and Koepsell, H. (2000) Biochim. Biophys. Acta 1468, 367-380). Here, the porcine renal epithelial cell line LLC-PK1 was used to investigate whether porcine RS1 (pRS1) plays a role in transcriptional up-regulation of SGLT1 after confluence and in down-regulation of SGLT1 by high extracellular D-glucose concentrations. Western blots indicated a dramatic decrease of endogenous pRS1 protein at the plasma membrane after confluence but no significant effect of D-glucose. In confluent LLC-PK1 cells overexpressing pRS1, SGLT1 mRNA, protein, and methyl-alpha-D-glucopyranoside uptakes were drastically decreased; however, the reduction of methyl-alpha-D-glucopyranoside uptake after cultivation with 25 mm D-glucose remained. In confluent pRS1 antisense cells, the expression of SGLT1 mRNA and protein was strongly increased, whereas the reduction of SGLT1 expression during cultivation with high D-glucose was not influenced. Nuclear run-on assays showed that the transcription of SGLT1 was 10-fold increased in the pRS1 antisense cells. The data suggest that RS1 participates in transcriptional up-regulation of SGLT1 after confluence but not in down-regulation by D-glucose.

Highlights

Because D-glucose has a key role in cellular metabolism, organisms dispose of highly efficient mechanisms to control transepithelial absorption of D-glucose in the small intestine and kidney and the D-glucose uptake into glucose-metabolizing cells
For example (i) one of the biological effects of insulin is the stimulation of D-glucose uptake into fat cells by GLUT4 [4, 5]. (ii) The D-glucose absorption in small intestine is adapted to diet by glucose-dependent regulation of the high affinity Naϩ-D-glucose co-transporter SGLT1 [6, 7]. (iii) The capacity of the D-glucose reabsorption in the proximal tubule is one of the factors that determined D-glucose plasma levels in diabetic patients [8]. (iv) The glomerulosclerosis observed in diabetes may be explained by the up-regulation of GLUT1 in mesangium cells, which triggers the overproduction of matrix proteins [9, 10]
As co-expression experiments of RS1 with SGLT1 in Xenopus oocytes suggested species-dependent up- or down-regulation of SGLT1 by RS1 [26, 29, 45], we investigated whether any alterations of porcine RS1 (pRS1) expression occur during the course of SGLT1 regulation by confluence and glucose concentrations

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The porcine renal epithelial cell line LLC-PK1 [15] was maintained in Dulbecco’s modified Eagle’s medium (DMEM) that contained 5 mM D-glucose and was supplemented with 10% (v/v) fetal bovine serum, 5 mM L-glutamine, 0.1 mg/ml streptomycin sulfate, and 100 units/ml penicillin G. Muristerone A binds to ecdysone/retinoid X receptor heterodimers that subsequently interact with a response element on the pIND vector, thereby activating the transcription of antisense pRS1 RNA from the minimal heat shock promoter [32]. As another control for RS1 antisense experiments, the porcine oxytocin receptor (nt 1–1184, GenBankTM accession number X71796) was cloned in reverse orientation into the pRcCMV vector (pRcCMV anti-oxytocin). LLC-PK1 cells were grown with 5 mM D-glucose in the medium until 5 days after confluence, washed with phosphate-buffered saline (0.01 M Na2HPO4, 0.15 M NaCl, pH 7.4), harvested, and lysed with lysis buffer that consisted of 0.01 M Tris-HCl, pH 7.4, 3 mM MgCl2, 0.5% (w/v) Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride (PMSF). All other chemicals were obtained as described earlier [26, 43]

RESULTS

The uptake measurements were performed with cells that were grown

DISCUSSION