2292-PUB: Luminescent Assays Quantitate the Biology of Peptide Hormones

The interest in metabolic diseases like diabetes, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), continues to increase due to their growing impact on cell health. To facilitate studies and uncover novel drug targets, there is a significant need to develop disease relevant in vitro cellular models and robust, easy to use detection technologies. Here we demonstrate that luminescent assays coupling specific dehydrogenases to the production of NAD(P)H and generation of light can be used to study insulin biology and its effects on key metabolic pathways. Insulin stimulated glucose uptake was measured in 3T3-L1 MBX adipocytes with 10-fold assay window and the expected pharmacology using a glucose uptake assay. Using a glycerol detection assay, we showed 4-fold increase in lipolysis upon isoproterenol stimulation that was suppressed by insulin. Effects of insulin on gluconeogenesis was studied by measuring glucose production in liver microtissues, which are also a useful model for NAFLD studies and measurement of steatosis using a triglyceride assay. This assay does not require organic extraction and has the sensitivity required for quantitative triglyceride detection using low cell numbers (<1000 cells/per well). Pancreatic islets are critical for regulated glucose homeostasis and measuring hormone production and secretion is the most direct method for assessing the quality and function of islets. To facilitate those studies, we developed bioluminescent, in solution, no wash insulin and glucagon immunoassays that are significantly more rapid and cost-effective than current commonly used approaches. The assays show high sensitivity and wide dynamic range (10 pM to 8 nM for insulin and 0.5 pM to 1 nM for glucagon) and are amenable to miniaturization making them well suited for rapid measurements of large numbers of samples that are generated, for example, in perifusion or high throughput screening experiments.