Date of Award
Doctor of Philosophy (PhD)
Stable isotope tracers are widely used to study the in vivo kinetics of central carbon metabolism in diseases such as obesity, type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). However, despite their common use, 13C-labeled acetate, lactate, and propionate have led to large incongruities and inconsistencies in their in vivo measurement of hepatic metabolism. To resolve major discrepancies and recent controversies of hepatic metabolism we developed and validated [1,2-13C2]-L-glutamine as a novel tracer strategy for measuring hepatic central carbon metabolism. In our rodent studies [1,2-13C2]-L-glutamine generated a straightforward labeling pattern of key metabolites suitable for the interpretation of LC-MS/MS spectra using Mass Isotopomer Multi Ordinate Spectral Analysis (MIMOSA) principles and thereby enabled an unencumbered independent assessment of Krebs cycle fluxes. In a head-to-head comparison, we found that [1,2-13C2]-L-glutamine had many favorable qualities and few liabilities compared to the other tracer strategies. Additionally, given that in vivo NMR studies of mitochondrial metabolism often depend on the signal for glutamate and other abundant metabolites that act as label-trapping pools for Krebs Cycle intermediates, we were able to directly measure α-Ketoglutarate-Glutamate exchange rate (Vx) and show evidence for the assumption that the intramitochondrial metabolite pool is in rapid exchange with NMR observable cytosolic metabolites. Here we report the development and in vivo kinetic and steady state validation of [1,2-13C2]-L-glutamine as an independent tracer of hepatic metabolism.
SIEBEL, STEPHAN, "Minimizing Tracer Interference to Assess in vivo Hepatic Metabolism with Glutamine-generated Mass Isotopomers" (2021). Yale Graduate School of Arts and Sciences Dissertations. 412.