Bioluminescence imaging of hepatic nutrient fluxes

Bioluminescence imaging (BLI) is a powerful technology for studying molecular and cellular features in living animals. Impressive advances in luciferase engineering and ease of targeting by genetic encoding permit the broad use of bioluminescence for in vivo imaging for diverse purposes. First, we generated transgenic mice with liver-specific expression of luciferase and used BLI with a FFA probe to enable non-invasive real-time imaging of FFA flux in the liver. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. We found that our novel imaging system is a useful and reliable tool to study the dynamic changes in hepatic FFA flux in preclinical model systems. We also utilize this imaging approach to study the role of fatty acid transport proteins in hepatic carcinogenesis. We generated a genetically engineered liver cancer mouse model via hydrodynamic transfection and determined the changes of fatty acid uptake during tumor formation. Our results demonstrated that the rate of FFA uptake is significantly decreased during the development of hepatocellular carcinoma (HCC), but not that of intrahepatic cholangiocarcinoma (ICC). We additionally showed that FATP5 knockdown suppresses ICC tumor formation and that the down regulation of FATP5 expression delays tumor growth even after the onset of ICC. These findings uncovered the critical role of FATP5 in ICC development and demonstrated the potential utility of these proteins as therapeutic targets for the prevention and treatment of ICC. We expanded our bioluminescent imaging approaches to other nutrients and copper in particular. We demonstrated Copper-Caged Luciferin-1 (CCL-1), a bioluminescent copper-responsive probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of non-alcoholic fatty liver disease (NAFLD) reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. Lastly, we also utilized bioluminescent approaches to detect liver damage and apoptosis. We shown that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. We applied this "split luciferin" ligation reaction to the imaging of apoptosis associated with caspase 3/7. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. These imaging approaches for nutrient fluxes should open up our understandings of complex metabolic networks in various metabolic processes and diseases states