New optical tools for non-invasive imaging and quantification of glucose and nicotinamide riboside in living cells and animals

Glucose is one of the most important metabolites that plays a key role in living organisms, as it is the primary energy source. Glucose metabolism is also an integral part in the pathogenesis of diabetes and cancer. Despite this tremendous impact of glucose, there are no tools for imaging of glucose uptake non-invasively and in a real-time manner working in both living cells and animals without utilization of hazardous radiation and suitable for convenient preclinical use. The first part of this thesis is describing the development of a probe for imaging glucose uptake based on a highly sensitive and versatile bioluminescence technique. We set a goal to design a probe, which should be reliable, robust, easy to use and simply represent the glucose uptake in both living cells and animals. In addition the synthesis of the probe should be straightforward and high-yielding. This part of the thesis is composed of two projects utilizing different approaches to the probe design and probeâs mechanism of action. The first project was based on the Staudinger Ligation for imaging of glucose uptake by modifying the glucose molecule with an azide moiety and using luciferin caged with a reactive phosphine as a counter-partner for the assay. The second project describes the development of a cleavable disulfide glucose-conjugate probe. For the first project we aimed to synthesize a library of glucose pro bioluminescent probes, validate them in a cell-free and cell-based assay and identify the hit compound. Then we proved the GLUT-specificity of the uptake of our hit compound in living cells and animals. The robust and reproducible assay method for measurement of glucose uptake was established. The probe was applied for imaging of antidiabetic drugsâ influence on glucose uptake. We have also demonstrated its superior properties over 2-NBDG that is currently the most used probe in preclinical research. However, the probe utilized in the second project namely cleavable disulfide glucose conjugate probe was not so sensitive as glucose-azide. Nevertheless, we showed a successful application of this probe for imaging of glucose uptake in living cells and animals. The second part of this thesis describes the design and evaluation of the bioluminescent probe for imaging of nicotinamide riboside (NR) uptake. NR is a recently discovered NAD+ precursor that is naturally present in cow's milk. Despite its importance in modern research and a high potential as a new medicine for treatment of various types of diseases, including diabetes, there are no tools to visualize NR uptake in preclinical research. Also, there is a lack of knowledge on where and how NR can be absorbed in mammals. Here we established the Staudinger Ligation-based approach to develop an imaging tool for NR uptake. After the successful synthesis of azido-nicotinamide riboside molecule, we demonstrated its specificity and utility for imaging in living cells and animals. Findings described in this part cover not only the imaging of NR uptake, but also the possibility of existence of specific NR transporter in mammalian cells, which has not been discovered and described yet.

Development of novel tools for imaging of metabolic fluxes in vivo

Tamara Maric

Bioluminescent imaging is a powerful technique that enables imaging in living organisms with high sensitivity, low background signal, low cost and without the need for radioactivity. The emitted photons are produced in the oxidation reaction of luciferin catalyzed by luciferase. In the caged luciferin approach, the bioluminescent readout is used for measuring the activity of a specific biochemical process. Luciferin is caged in order to prevent catalysis by luciferase, and only when the cage is removed by a specific biochemical event of interest, luciferin is released and becomes a substrate of luciferase. Hence, emitted photons are proportional to the amount of free luciferin, thus allowing to monitor the dynamics of the investigated process. Recent progress in the development of bioorthogonal reactions enabled to answer several fundamental biological questions, improve clinical diagnosis, and to better evaluate therapy efficacy. For example, one of the most robust biorthogonal reactions is the Staudinger ligation, which occurs between a properly tuned azide and a phosphine. In this work, we have used the caged luciferin approach combined with the Staudinger ligation to monitor metabolite uptake in living animals. We have successfully applied this method for the monitoring uptake of two metabolites â glucose and nicotinamide riboside (NR). Glucose is a major source of energy for most living organisms and its aberrant uptake is linked to many pathological conditions. On the other hand, NR is a form of vitamin B3 and represents one of the salvageable NAD+ precursors. Low NAD+ level inside of cells is strongly linked to numerous metabolic and age-related disorders. Despite of their importance, in both cases our understanding of disease-associated glucose or NR flux is limited due to a lack of robust tools. To date, positron emission tomography (PET) imaging remains the gold standard for measuring glucose uptake and no optical tools exist for non-invasive longitudinal imaging of neither of the two metabolites in in vivo settings. Here we report the development of a novel bioluminescent glucose uptake probe (BiGluc) and bioluminescent NR uptake probe (BiNR) for real-time, non-invasive longitudinal imaging of glucose and NR absorption both in vitro and in vivo. The new imaging reagents enable a wide range of applications in the field of metabolism and drug development.

EPFL2020

Development of novel tools for imaging of metabolic fluxes in vivo

Tamara Maric

EPFL2020