Applying Dynamic Nuclear Polarization to Measure Pancreatic Metabolism using Hyperpolarized [1-¹³C]Pyruvate

Anna Rushin, Dalton Graham, Matthew Merritt

University of Florida

Dynamic Nuclear Polarization (DNP) transfers polarization from a free radical electron to a coupled nuclear spin bath, significantly enhancing the signal in the resulting nuclear magnetic resonance (NMR) spectra. Dissolution DNP can be used in combination with ex vivo organ perfusions to measure central carbon metabolism in real time. This research used a novel pancreas perfusion technique that primarily targets the exocrine pancreas, which produces digestive enzymes through the ductal system. The exocrine pancreas is known to be impacted in Type 1 Diabetes (T1D), but the underlying mechanisms of this dysfunction are severely understudied. This work used the NOD.Rag1^-/-.AI4^α/β mouse model of T1D and the NOD.Rag1^-/- control to examine pancreatic metabolic flux in T1D in the fed and fasted states. Pancreata from these mice were cannulated through the common bile duct, excised, attached to a perfusion rig, and allowed to perfuse for 30 minutes. Oxygen consumption was measured throughout the perfusion to confirm tissue viability. Dissolution DNP was performed with 15 mM trityl radical using an Oxford HyperSense 3,35 T polarizer and a final concentration of 4 mM hyperpolarized [1-¹³C]pyruvate. Pseudo-2D NMR spectra were collected on a 600 MHz instrument with a 10mm [¹³C]-optimized cryoprobe and analyzed with Bruker TopSpin and Mestrenova. The kinetics of lactate-C1, alanine-C1, malate-C1, and HCO₃^- were similar between experimental groups. Approximately 50 individual spectra were summed based on the appearance of lactate for quantification of individual metabolites. Peak assignments were confirmed using 2D NMR experiments on pancreas extracts. Fumarate-C1 and aspartate-C1 were significantly increased in the diabetic mice, suggesting increased flux through pyruvate carboxylase. Alanine to lactate and bicarbonate to lactate ratios were significantly decreased in the diabetic mice in both fed and fasted states compared to the control, indicating a shift in the tissue redox balance and in oxidative flux respectively. These measurements of real time metabolic turnover provide unique insights into alterations in the exocrine pancreas in T1D.