ASSAY and Drug Development Technologies Volume 4, Number 5, 2006 © Mary Ann Liebert, Inc. A Cellular Assay for Measuring the Modulation of Glucose Production in H4IIE Cells Anne-Marie de Raemy-Schenk, Solenne Troublé, Pascale Gaillard, Patrick Page, Jean-Pierre Gotteland, Alexander Scheer, Paul Lang, and Karen Yeow Abstract: Type II diabetes and its associated complications are a major health concern of the developed world. One of the hallmarks of diabetes is insulin resistance, where secreted insulin no longer has any effect on its target tissues, namely, liver, muscle, and fat. An important therapeutic strategy is to modulate blood glucose levels using pharmacological agents. Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that plays important roles in regulating glucose metabolism. It is a key negative regulator of insulin action and is an important contributing factor to insulin resistance in liver, muscle, and adipose tissue. We describe the development of a cell-based assay designed to measure glucose production in rat hepatoma cell line H4IIE liver cells in response to treatment with small molecule inhibitors, including GSK3 inhibitors. The assay is set up in a 96-well format, and glucose production is assessed using a convenient fluorescence-based readout. This disease-relevant cellular assay is a valuable tool for the progression of small molecules that modulate glucose production. 525 Introduction T YPE II INSULIN-RESISTANT diabetes mellitus and as- sociated conditions are among the primary human health concerns of the 21st Century. 1 Type II diabetes is characterized by insulin resistance, defined as a decrease in insulin action on muscle, fat, and liver, as well as the failure of pancreatic beta cells to secrete sufficient quan- tities of insulin. 2 In normal tissues, insulin stimulates glu- cose uptake in muscle and fat and inhibits hepatic glu- cose production. This response to insulin is defective in type II diabetics, resulting in long-term hyperglycemia, leading eventually to secondary complications and mor- tality. 1 Thus, a major focus of research and drug discov- ery efforts is on decreasing circulating blood glucose lev- els via pharmacological intervention. 3 Although there are multiple factors that contribute to the development of insulin resistance and type II diabetes, it is thought that one potential culprit is inadequate in- hibitory control of glycogen synthase kinase-3 (GSK3). 4 GSK3 is a serine-threonine protein kinase encoded by two highly homologous and ubiquitously expressed genes 5 and was originally identified through its ability to phosphory- late and inhibit glycogen synthase. 6 GSK3 activity opposes the actions of insulin, inhibiting glycogen synthesis and glucose uptake and altering the expression of insulin-reg- ulated genes. 7 GSK3 activity is higher in obesity-prone di- abetic rodents, 4 and GSK3 protein levels are increased in skeletal muscle from patients with type II diabetes. 8 In keeping with this, changes in the kinetic behavior of glyco- gen synthase in skeletal muscle cells from diabetic patients could be due to increased phosphorylation by GSK3. 9 GSK3 activity may also impair insulin signaling through inhibition of an insulin receptor-coupled protein, insulin receptor substrate-1. 10,11 Thus, in addition to direct mod- ulation of glycogen synthase, GSK3 may negatively mod- ulate insulin signaling via actions on other components of the insulin signaling pathway. The most well-established functional consequence of inhibition of GSK3 is the activation of glycogen syn- Serono Pharmaceutical Research Institute, Plan-les-Ouates, Switzerland. ABBREVIATIONS: DMSO, dimethyl sulfoxide; GSK3, glycogen synthase kinase-3; IC 50 , 50% inhibitory concentration; JNK, c-Jun N-ter- minal kinase; MTS, dimethyltetrazolium salt; OD, optical density; SD, standard deviation.