High glucose causes a decrease in expression of thiamine transporters in human proximal tubule epithelial cells in vitro
Larkin JR, Thornalley PJ
Talk at EASD Annual Meeting, Rome, Italy (2008)
Diabetologia 51 Supp 1 S97. DOI: 10.1007/s00125-008-1117-6
Abstract
Background and aims: It has been shown recently that both type 1 and type 2 diabetic patients have markedly decreased plasma concentrations of thiamine whilst maintaining nutritional sufficiency of the vitamin. Low plasma thiamine concentration was linked inversely to renal clearance of thiamine - attributed to decreased re-uptake of thiamine in the renal tubule epithelium. Similar decreases in plasma thiamine concentration and increases in thiamine clearance were found in streptozotocin-induced diabetic rats, and were also associated with decreased expression and activity of the thiamine pyrophosphate-dependent enzyme transketolase. Transketolase is a critical rate-controlling enzyme of the pentose phosphate pathway which helps resist adverse effects of high glucose concentration and the development of nephropathy and other microvascular complications in diabetes. In diabetic patients, low plasma thiamine concentration was also linked inversely with abnormally high plasma levels of soluble vascular cell adhesion molecule-1—a marker of microvascular and macrovascular disease. The cause of increased thiamine clearance in diabetes remains unknown. Thiamine clearance is normally minimised by efficient re-uptake of thiamine from the glomerular filtrate by the thiamine transporters of renal proximal tubule epithelial cells. The aim of this study is to investigate if hyperglycaemia changes the expression of the thiamine transporter THTR-1 in human renal tubule epithelial cells in vitro.
Materials and methods: The HK-2 cell line was used as a model source of human proximal tubule epithelium cells. HK-2 cells were cultured in D-MEM/F-12 medium supplemented with 10% foetal bovine serum and 23 nM thiamine - a typical physiological level - for five days before harvesting. Cultures contained either a low glucose concentration (5 mM) or high glucose concentration (26 mM). After culture, cells were lysed and the membrane fraction separated by centrifugation (20,000xg, 30 min, 4 °C). Proteins of the membrane fraction were separated by 12% SDS-PAGE, transferred to nitrocellulose membrane and blotted with anti-THTR-1 antibodies (Alpha Diagnostics), stripped of antibody and re-blotted for the housekeeping protein β-actin with a second anti-β-actin antibody (AbCam, UK). Western blotting results are expressed as a THTR-1/ β-actin band intensity ratio.
Results: Thiamine transporter antigen was present in HK-2 cell membrane extracts as a protein of ca. 50 kDa in SDS-PAGE analysis. The THTR-1/ β-actin band intensity ratio was decreased 58% in cultures of HK-2 cells with high glucose concentration, relative to low glucose concentration controls. Intensity ratio: low glucose 0.255; high glucose 0.107 (p<0.01, n = 4).
Conclusion: High glucose concentration decreased the expression of the thiamine transporter in human proximal tubule HK-2 epithelial cells in vitro. This is expected to decrease uptake of thiamine across the tubular epithelium. A similar effect of hyperglycaemia in vivo may contribute to the increased clearance and plasma deficiency of thiamine in experimental and clinical diabetes.
Supported by: University of Warwick
