A majority of studies reported a percentage of methylated tumors and were thus easily comparable

at room temperature. To estimate percent conversion of ATP to ADP, mixtures of ATP and ADP were tested in duplicate, containing 0, 3, 5, 10, 30, 50, 70, and 100% ADP. The luminescence value at 10% ADP corresponded to the luminescence generated by 4 nM GCK. The ADP-GloTM kit is linear throughout the range tested and Nigericin (sodium salt) chemical information provides an excellent range of detection. The effects of GKRP inhibition were then assessed using the same reaction conditions at pH 7.1. ADP generation was measured using a matrix of five concentrations each of GCK and GKRP in quadruplicate. Reactions were incubated for 75 minutes. Luminescence of 15 nM GCK was reduced by approximately 60% by 15 nM GKRP, and the mean S/B at 15 nM GCK +15 nM GKRP was 3.7. Accordingly, the bioluminescence reaction was capable of measuring GCK enzymatic activity and its inhibition by GKRP. We therefore pursued miniaturization of the assay to 1536-well plate format and tested the effects of S6P and F1P in three different assay formats: 0.04 mM ADP in the absence of GCK and GKRP, 4 nM GCK, and 15 nM GCK with 15 nM GKRP. All assays provided robust luminescence signal in the absence of F1P or S6P. As expected, response to F1P and S6P were dependent on the presence of GKRP, with F1P activating the GCK enzymatic reaction and resulting in an increase in luminescence signal, and S6P decreasing luminescence. Following these validations, reaction conditions of 15 nM GCK, 15 nM GKRP, and 2 mM S6P were selected for screening of the LOPAC1280 library at 0.4 mM ATP and 5 mM glucose. Compounds were added to assay mix containing all components except ATP, and the reaction was then initiated by ATP. Controls included GKA-EMD, all assay components without GCK but with 15 nM GKRP, and all assay components with 15 nM GCK only to represent the maximum uninhibited signal. The screening assay performed well. Cellular Analysis of GCK Translocation A number of previous studies have demonstrated high-content methodologies for analyzing GCK nuclear-to-cytoplasmic translocation in the presence of glucose, GKAs, and F1P precursors such as fructose and sorbitol in freshly isolated rat hepatocytes. We investigated whether PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639073 these protocols could be extended to cryopreserved pooled Sprague-Dawley rat hepatocytes. Twenty-four hours after plating in 96-well plates, hepatocytes were glucose-starved for 12 hours, and then treated with glucose or GKA-EMD for 2 hours before immunostaining for both GCK and GKRP localization. While GKRP predominantly localized to the nucleus in all conditions tested, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639073 we observed strong glucose-dependent GCK translocation, as well as GKAdependent GCK translocation, suggesting cryopreserved hepatocytes can be utilized to model GCK translocation. Previous attempts to develop human cellular models of GCK translocation, either using endogenous proteins in Hep G2 or HepaRG cells or generating overexpression systems by transfection of human GCK and/or GCKR in HeLa cells or primary mouse hepatocytes, have failed to reproduce results observed in rodent models. We therefore investigated whether cryopreserved GCK/GKRP Assays human hepatocytes from two different donors, without known T2D, dyslipidemia, or drug use, could be utilized as models for GCK translocation. In all conditions tested, there was a significant sub-population of GCK-positive cells with complete cytoplasmic localization that were negative for GKRP. Accordingly, to detect translocation, we developed an analysis methodology to restrict quantitati