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Journal of Biomolecular Screening
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Activation and Translocation of Glucokinase in Rat Primary Hepatocytes Monitored by High Content Image Analysis

Michael Wolff

Department of Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

Stefan G. Kauschke

Department of Metabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

Susanne Schmidt

Department of Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

Ralf Heilker

Department of Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany, Ralf.Heilker{at}bc.boehringer-ingelheim.com

In the liver, glucokinase (GK) regulatory protein (GKRP) negatively modulates the metabolic enzyme GK by locking it in an inactive state in the nucleus. Here, the authors established a high content screening assay in the 384-well microplate format to measure the nucleus-to-cytoplasm translocation of GK by reagents that destabilize the interaction between GK and GKRP. As a cellular model system, primary rat hepatocytes endogenously expressing both GK and GKRP at physiological levels were used. The GK translocation assay was robust, displayed limited day-to-day variability, and delivered good Z' statistics. The increase of the glucose concentration in the extracellular medium from a low glucose situation (2.8 mM) to beyond its physiological set point value of 5 mM was found to drive GK from the nucleus into the cytoplasm. Likewise, both fructose (converted intracellularly into fructose-1-phosphate) and a known allosteric GK activator were found to induce the export of GK from the nucleus and to synergistically enhance the effects of medium or high glucose concentrations with respect to GK translocation. Transfer of the high content screening format to a semiautomated medium throughput screening platform enabled the profiling of large compound numbers with respect to allosteric activation of GK. (Journal of Biomolecular Screening 2008:837-846)

Key Words: high content screening • glucokinase • glucokinase regulatory protein • rat primary hepatocytes • allosteric activation

This version was published on October 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 9, 837-846 (2008)
DOI: 10.1177/1087057108323910
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