Extra-hepatic tissues contain G6PC3, a different isoform of the glucose-6-phosphatase catalytic subunit than that found in liver (Guionie et al, 2003). The regulation of G6PC3 is poorly understood. This protein is thought to be involved in local glucose supply. Inability to upregulate its synthesis appropriately may be associated with intra uterine growth retardation, delayed or impaired neural development, sudden infant death, diabetes, or other problems caused by disordered glucose metabolism. A luciferase reporter gene system was used to investigate the regulation of the G6PC3 promoter by physiological concentrations of various metabolites. HeLa cells were transiently transfected with a DNA construct containing one of 4 common polymorphisms of the G6PC3 promoter coupled to the luciferase gene. Tranfected cells were exposed to glucose concentrations representing extreme hypoglycaemia (1mM), cord blood/ fasting blood glucose (3.5mM), postprandial blood glucose (5.5mM) and moderate (15mM) to high (25mM) diabetic levels (n=5). The effects on G6PC3 promoter activity of 18 hours exposure to 3.5-10 mM lactate, pyruvate, acetate and β-hydroxybutyrate (β-HBA) were also investigated. The cells were harvested and luciferase activity and protein concentration were measured. There was no significant difference between the four promoter polymorphisms in their response to glucose (2-way ANOVA: F(3, 99) = 1.09; p =0.359). At 1mM glucose luciferase activity was 17.87± 2.13 times higher than that of the empty vector. Promoter activity at all other glucose concentrations was significantly higher than at 1mM glucose (2-way ANOVA: F(4, 99); p<0.001). Activity increased 4.8 ± 0.75 fold from 1mM to 5.5mM glucose, then plateaued. This differs markedly from the response of the catalytic subunit isoform found in liver, G6PC1, which is only stimulated by fasting conditions or high (20mM+) glucose concentrations (Massillon et al, 1996; Massillon, 2001). The equivalent number of carbon units of pyruvate to that found in 5.5mM glucose media was added to the two lowest glucose concentrations. This restored G6PC3 promoter activity to levels similar to that seen at 5.5mM glucose (ANOVA: p>0.05), suggesting that other metabolic fuels can regulate activity of the gene. However, lactate, β-HBA and acetate were without effect (ANOVA: p>0.05). The G6PC3 promoter does not contain any known consensus glucose responsive or carbohydrate responsive sequences and the mode of regulation by glucose and pyruvate is unknown. Studies to further characterise the mechanism of carbohydrate regulation of G6PC3 are underway.