Development and Application of Metabolomic Techniques for Identification and Quantification of Intercellular Metabolites Relevant to Glucose Stimulated Insulin Secretion in

Abstract

β-cells found in the islets of Langerhans secrete insulin in response to elevated blood glucose levels. Disruption of this process causes diabetes which is characterized by poor control of blood glucose and results in serious long-term complications. Despite years of extensive β-cell research the precise metabolic pathways that facilitate glucose-stimulated insulin secretion (GSIS) are not fully understood. We report novel insights into GSIS gained through development of improved cell quenching, metabolite extraction, and chromatographic separation techniques and their application to the time-resolved metabolomic study of GSIS in the clonal β-cell line INS-1 832/13. We report a hydrophilic interaction liquid chromatography - mass spectrometry method using a propyl-amine stationary phase and high pH mobile phase that affords excellent sensitivity and chromatographic performance for a wide range of metabolites including the sugar phosphates, amino acids, organic acids, and nucleotides. We report a rapid sampling technique for adherent mammalian cells that improves sensitivity, extract stability, and throughput yielding quantitative recovery of a wide range of metabolites in a single 1 minute extraction step. We demonstrate that a rapid water rinse step with novel LN2 quenching conveniently improves sensitivity for metabolites up to 20-fold and removes interferences without altering the metabolome. To gain insight into the dynamic metabolic changes associated with biphasic insulin release and assess dysreguation in disease states, we quantified an unprecedented number of β-cell metabolites (~90) impacted by glucose stimulation in healthy cells and in lipotoxic and glucotoxic models of disease. We quantified these metabolites in both time-resolved and glucose dose-response experiments and generated results that allowed us to test and extend several hypotheses of the biochemical mechanisms of GSIS in parallel. These metabolites included those studied previously as well as several metabolites identified by undirected analysis that are novel to GSIS research including long-chain acyl-CoAs, GDP-mannose, and ZMP. Dramatic alterations in glucose metabolism were measured in both lipotoxic and glucotoxic models indicative of an increase in energy metabolism at basal glucose levels. Stimulation experiments with [U13C]-glucose demonstrated increased glycolytic flux resulting in elevated concentrations of pentose phosphate pathway metabolites as high as 200-fold in the glucotoxic model.