Abstract
Purpose :
Glucose is the major nutrient in the lens, which is taken up by the lens from the aqueous humor. This study aims to map glucose uptake and metabolism in cultured lenses, and correlate the pattern of glucose uptake to glucose transporter distributions and abundance.
Methods :
Ex vivo bovine lenses were incubated in AAH containing normoglycaemic stable isotopically-labelled (SIL) glucose (5mM) for 5min-20h. Lenses were then fixed in 2%PFA for 72h for immunofluorescence microscopy analysis, frozen for MALDI imaging mass spectrometry (IMS) analysis, or manually dissected into epithelium flat mount and cortical fibre cells for either MALDI IMS or proteomic analysis. For MALDI-IMS studies, 20um thick axial lens tissue sections were analysed by a MALDI-MS. For proteomics studies, membrane protein preparations generated by centrifugation were separated by SDS-PAGE, gel bands digested by trypsin, and peptides analysed by LC-MS/MS. For immunohistochemistry studies, 16um thick axial lens sections were labelled with primary antibodies specific for GLUT 1 or GLUT 3. Fluorescent secondary antibodies, and cell membrane marker were added, and images collected by confocal microscopy.
Results :
MALDI-IMS maps of SIL glucose showed uptake at 5min was concentrated in the peripheral epithelium. At later timepoints, glucose distributed throughout the epithelium and the cortical lens fibres. Proteomic analysis of central and peripheral regions of the lens epithelium detected GLUT1 and 3, with GLUT 3 in higher abundance than GLUT1 throughout the epithelium, but with GLUT1:GLUT3 ratio increased in the peripheral epithelium relative to central. Microscopy localized GLUT1 and GLUT3 to epithelial cell membranes and young fibre cells. SIL glucose metabolites found in glycolysis, the sorbitol pathway, and UDP-glucose formation were mapped to cortical lens fibres, with distinct signal changes relative to endogenous glucose metabolite signal up to 20h incubation.
Conclusions :
Mass spectrometry and immunohistochemistry have spatially mapped the major uptake site of glucose in bovine lens to the peripheral epithelium and cortical fibres. SIL glucose is rapidly metabolized in epithelial and fibre cells to many metabolites, which are most abundant in the metabolically more active cortical fibre cells in comparison to central fibres.
This is a 2021 ARVO Annual Meeting abstract.