Developing and utilizing a robust mass spectrometry-based approach to study glucose metabolism and transport in the lens

Abstract

The lens is an essential component of the ocular system and has a high metabolic demand. However, the delivery of energy sources such as glucose and waste removal cannot occur via the vasculature as this would obstruct the path of light through the lens. Thus, the lens requires a highly developed glucose delivery system to maintain its transparency. This thesis not only examines glucose uptake in the lens and the microcirculation that can transport glucose within the lens, but also develops a highly spatially resolved methodology to be able to study this. Firstly, I optimised a paradigm that would allow Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) analysis of glucose uptake and metabolism in ocular lenses. Specifically, the matrix choice, tissue preparation steps and normalisation strategies were carefully optimised to glucose and its metabolites using MALDI-IMS applied to organ cultured bovine lens tissue. Next, this optimised method was applied in combination with MALDI-IMS and gas chromatography mass spectrometry (GC-MS) to understand the mechanisms of glucose uptake, transport and metabolism of glucose in the bovine lens under physiological normoglycemic conditions. The results revealed that glucose uptake first occurs in the equatorial region, followed by the anterior and posterior poles. Additionally, I spatially correlated the pattern of glucose uptake to the expression of glucose transporters (GLUTs) in the bovine lens. Using spatial proteomics, I discovered that GLUT1 is the predominant isoform expressed in the lens fibres, and GLUT3 is predominantly expressed in the lens epithelium. The location of GLUT1 was further confirmed using immunohistochemistry. Subsequently, MALDI-IMS demonstrated that, once glucose is taken up, it can be metabolised in the epithelium, outer cortex and even the lens nucleus. In addition, I attempted to investigate whether glucose delivery to deeper regions of the lens occurs via an intracellular or extracellular pathway. Unfortunately, MALDI-IMS and GC-MS methods did not have the required resolution to visualize extracellular delivery using current stable isotopically-labelled tracers. However, GC-MS showed that glucose delivery to the nucleus could be reduced by inhibiting the microcirculation using the Na+/K+ ATPase inhibitor ouabain, a result that provided indirect support of the hypothesis that an extracellular pathway is used to deliver glucose to the lens nucleus. Further research will be needed to confirm this hypothesis. Overall, this thesis provides high-resolution information about the pattern of glucose uptake, its correlations with the expression of glucose transporters, and the distribution of the glucose metabolites, thereby enhancing our understanding of lens metabolism. The tools developed in this thesis to study lens tissue in physiological conditions can be applied to advance our understanding of lens pathologies such as diabetic and age-related cataracts in which glucose metabolism in the lens is perturbed. Therefore, this thesis establishes a consolidated research pipeline to study metabolomics (the dynamics of nutrient uptake, transport and metabolic flux) and proteomics in the lens to enable future spatially-resolved screening of metabolic changes in pathological models.