Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, PC30
A cautionary tale regarding preparation of intestinal brush border membrane for detection of GLUT2 by Western blotting
X. Chen2, S. K. Srai2, E. S. Debnam1
1. Neuroscience, Physiology & Pharmacology, UCL, London, United Kingdom. 2. Structural & Molecular Biology, UCL, London, United Kingdom.
GLUT2 expression at the jejunal brush border membrane (BBM), particularly under conditions of increased luminal glucose concentration or in experimental diabetes mellitus (DM) has been previously demonstrated (Kellett & Helliwell et al 2000, Au et al 2002). However some studies have failed to detect expression of GLUT2 at the BBM, implying that glucose uptake across this membrane is exclusively SGLT1-mediated (Cui et al 2005, Batchelor et al 2011). Our present work provides a methodological explanation for the failure to detect GLUT2 at the BBM by western blotting. GK rats (a model of type 2 DM) and Wistar controls were used for the study. Jejunum was removed from animals anaesthetised with pentobarbitone sodium (60 mg. kg-1 I.P.). Glucose uptake across the BBM was measured using everted sleeves, an accepted method of BBM glucose uptake using intact tissue. Addition of the SGLT1 blocker phlorizin in the uptake buffer allowed quantitation of the SGLT1 and GLUT2 components of uptake. In parallel studies, purified BBM was prepared from either fresh intestine or using tissue that had been rapidly frozen in liquid N2 after its removal from the animal and stored at -80oC. In both cases BBM samples were stored at -20oC prior to their use for detection of GLUT2 and SGLT1 by western blotting. Blood glucose concentration in GK rats was higher than that in non-diabetic animals. Diabetes was without effect on SGLT1-mediated glucose uptake measured using 20 mM glucose, whereas the rate of phlorizin-insensitive (GLUT-mediated) uptake was 51.4% greater in GK jejunum (mean±SEM: 0.42±0.05 vs 0.63±0.04 nmoles/mg, p<0.005, Student’s unpaired t-test). GLUT2 was readily detectable in BBM prepared from unfrozen jejunum of GK rats, but the protein was present at only minimal levels in BBM that had been immediately prepared from jejunum of non-diabetic rats or from GK jejunum that had been frozen for 2-3 days prior to BBM preparation. SGLT1 expression levels were similar in BBM prepared from intestine of fresh and stored jejunum. Our study is the first demonstration of increased intestinal glucose uptake across the BBM in type 2 diabetes and it is likely that raised GLUT2 expression is a major contributor to upregulation of transport in this condition. The failure of previous studies to detect GLUT2 at the BBM by western blotting might be explained by the common practice of freezing whole intestine or mucosa for later preparation of BBM. The reason for the differential effect of freezing on BBM expression of GLUT2 and SGLT1 expression is unclear at the present time.
Where applicable, experiments conform with Society ethical requirements