dDNP as a method to assess altered cellular metabolism in vitro

Abstract

Lifestyle diseases are expanding global health problems that are contributing to the global burden of chronic diseases. To link diet to metabolic outcome, it is necessary to understand the metabolic fate and interaction of the nutritional components in living organisms. The link between metabolic perturbations and human diseases has led to growing interest in metabolic research. The objective of this project is to study perturbed metabolism using dissolution Dynamic Nuclear Polarization (dDNP) in several disease models in vitro. By overcoming the sensitivity issues related to traditional magnetic resonance, dDNP offers the advantage of non-invasive metabolic visualization in vitro and in vivo. In the first part of the thesis, dDNP is used to probe slow biochemical reactions in combination with Stable Isotope-Resolved Metabolomics (SIRM). By application of this method, the timeframe of the experiment can be extended from minutes to hours or longer. The dDNP-SIRM approach is applied to investigate early handling of excess fuel in insulin producing β-cells before they reach a glucotoxic state which is a pathogenic factor in type 2 diabetes. Glucose-derived pyruvate is found to correlate with a high fuel burden for the cells and is hypothesized to be a potential biomarker in the development of insulin impairment. In conclusion, this study shows that -cells actively use different metabolic pathways to reduce excess metabolites formed due to uncontrolled glycolysis. Glycerol- and fatty acid metabolism is the most likely candidate for this deviation pathway. Further studies are needed to elucidate this fundamentally important and relatively overlooked defense mechanism important for protecting the -cell against glucotoxicity. In the second part of the thesis, dDNP is applied to study real time kinetics using hyperpolarized [113C] pyruvate to visualize metabolism in cancer cells. The biological model represents pancreatic cancer, demonstrated by different cell lines representing various stages of the cancer. For this purpose, a bioreactor with a home-built flow cell was constructed and tested. It was demonstrated that the cells grown on microcarriers showed pyruvate to lactate conversion in the flow cell. Furthermore, the bioreactor was found suitable for longitudinal cell studies over several hours, but also revealed that flow stress is an important limitation for many cell systems on microcarriers. The third part of the thesis concerns three different bioprobes for novel applications, in vivo and in vitro. The sample formulation and solid-state DNP polarization were optimized for each bioprobe. Biological applications are discussed for each probe, and initial studies were performed to assess potential for hyperpolarization studies. In summary, this thesis shows the versatility of dDNP for metabolic research and potential diagnostic applications demonstrated by the polarization of 13C labeled substrates in vitro.