ABSTRACT
Alterations in hepatic free fatty acid (FFA) uptake and metabolism contribute to the development of prevalent liver disorders such as hepatosteatosis. However, detecting dynamic changes in FFA uptake by the liver in live model organisms has proven difficult. To enable noninvasive real-time imaging of FFA flux in the liver, we generated transgenic mice with liver-specific expression of luciferase and performed bioluminescence imaging with an FFA probe. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. By using this method, we detected a decrease in FFA accumulation in the liver after mice were given injections of deoxycholic acid and an increase after they were fed fenofibrate. In addition, we observed diurnal regulation of FFA hepatic uptake in living mice. Our imaging system appears to be a useful and reliable tool for studying the dynamic changes in hepatic FFA flux in models of liver disease.
Subject(s)
Fatty Acids/metabolism , Liver/diagnostic imaging , Liver/metabolism , Luminescent Measurements , Animals , Biological Transport/drug effects , Cholagogues and Choleretics/pharmacology , Circadian Rhythm , Deoxycholic Acid/pharmacology , Fenofibrate/pharmacology , Hypolipidemic Agents/pharmacology , Luciferases/genetics , Male , Mice , Mice, Transgenic , PhotographyABSTRACT
Novel, clinically relevant, approaches to shift energy balance are urgently needed to combat metabolic disorders such as obesity and diabetes. One promising approach has been the expansion of brown adipose tissues that express uncoupling protein (UCP) 1 and thus can uncouple mitochondrial respiration from ATP synthesis. While expansion of UCP1-expressing adipose depots may be achieved in rodents via genetic and pharmacological manipulations or the transplantation of brown fat depots, these methods are difficult to use for human clinical intervention. We present a novel cell scaffold technology optimized to establish functional brown fat-like depots in vivo. We adapted the biophysical properties of hyaluronic acid-based hydrogels to support the differentiation of white adipose tissue-derived multipotent stem cells (ADMSCs) into lipid-accumulating, UCP1-expressing beige adipose tissue. Subcutaneous implantation of ADMSCs within optimized hydrogels resulted in the establishment of distinct UCP1-expressing implants that successfully attracted host vasculature and persisted for several weeks. Importantly, implant recipients demonstrated elevated core body temperature during cold challenges, enhanced respiration rates, improved glucose homeostasis, and reduced weight gain, demonstrating the therapeutic merit of this highly translatable approach. This novel approach is the first truly clinically translatable system to unlock the therapeutic potential of brown fat-like tissue expansion.
Subject(s)
Adipocytes/metabolism , Adipose Tissue, Brown/transplantation , Ion Channels/metabolism , Mitochondrial Proteins/metabolism , Stem Cells/metabolism , Thermogenesis/physiology , Tissue Scaffolds , Adipose Tissue, Brown/metabolism , Animals , Body Temperature/physiology , Cell Adhesion/physiology , Cell Differentiation/physiology , Cold Temperature , Energy Metabolism/physiology , Mice , Uncoupling Protein 1ABSTRACT
Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.
Subject(s)
Diagnostic Imaging/methods , Luminescent Measurements/methods , Neoplasms/diagnosis , Nitroreductases/metabolism , Animals , Cell Line , Female , Genetic Therapy/methods , Humans , Mice , Mice, Inbred BALB C , Prodrugs/pharmacologyABSTRACT
The ability of white and brown adipose tissue to efficiently take up long-chain fatty acids is key to their physiological functions in energy storage and thermogenesis, respectively. Several approaches have been taken to determine uptake rates by cultured cells and primary adipocytes including radio- and fluorescently labeled fatty acids. In addition, the recent description of activatable bioluminescent fatty acids has opened the possibility for expanding these in vitro approaches to real-time monitoring of fatty acid uptake kinetics by adipose depots in vivo. Here, we will describe some of the most useful experimental paradigms to quantitatively determine long-chain fatty acid uptake by adipocytes in vitro and provide the reader with detailed instruction on how bioluminescent probes for in vivo imaging can be synthesized and used in living mice.