Bile acids are a diverse class of amphipathic molecules originally formed in liver and modified by microorganisms in the intestine. In addition to their conventional role in dietary lipid absorption, bile acids have been recognized as signaling molecules that regulate the synthesis and metabolism of bile acids, glucose homeostasis, and energy expenditure. The G protein-coupled membrane receptor for bile acid (TGR5 or GP-BAR1) is the most extensively studied receptor for bile acids. Activation of TGR5 by agonists leads to increase in intracellular cAMP, which signals the reduction of inflammatory cytokine expression in macrophage, increase of glucagon-like peptide (GLP-1) secretion in intestine, and energy expenditure in brown adipose tissue and muscle. Therefore, it has been an interesting drug target in metabolism and inflammation. Cayman''s TGR5 (GP-BAR1) Reporter Assay Kit consists of a 96-well plate coated with a transfection complex containing DNA constructs for expressing TGR5 and a cAMP response element regulated secreted alkaline phosphatase (SEAP) reporter (TGR5 Reverse Transfection Strip Plate). Cells grown on the transfection complex will express TGR5 at the cell surface within 24 hours. Binding of agonists to TGR5 initiates a signal transduction cascade through the GαS and adenylate cyclase pathway resulting in the expression of SEAP which is secreted into the cell culture medium. Aliquots of culture medium are collected at 6-8 hours after stimulation, and SEAP activity is measured following the addition of a luminescence-based alkaline phosphatase substrate provided in the kit. The kit is easy to use and can be readily applied to high-throughput screening for therapeutic compounds regulating activation of TGR5. A selective synthetic agonist, the TGR5 Receptor Agonist (3-[2-chlorophenyl]-N-[4-chlorophenyl]-N,5-dimethyl-4-isoxazolecarboxamide), is included in the kit for use as a positive control. The kit provides sufficient reagent to measure SEAP activity at three time points using the three included white assay plates.
