Abstract
Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes.
Highlights
The liver plays a vital role in blood glucose homeostasis by production of glucose in the fasted state and efficient removal of glucose in the post-prandial state in response to portal hyperglycaemia for storage of glucose as glycogen or conversion to triacylglycerol [1]
Fractional changes in response to 25 mM glucose or glucagon were greater for the N/C ratio than for nuclear or cytoplasmic intensities (Table 1) and the power calculations showed that a larger sample size is required to detect significant differences in translocation from nuclear or cytoplasmic intensities as opposed to N/C ratio (N/C ratio: 60) (Table 1)
Two methods have been used for semi-quantitative analysis of glucokinase translocation: either based on the determination of the ratio of cellular nuclear-tocytoplasmic (N/C) mean pixel intensity [23,24,25] or from the automated nuclear or cytoplasmic intensity multiplied by the total cytoplasmic or nuclear area estimated with an independent stain [13,26,37]
Summary
The liver plays a vital role in blood glucose homeostasis by production of glucose in the fasted state and efficient removal of glucose in the post-prandial state in response to portal hyperglycaemia for storage of glucose as glycogen or conversion to triacylglycerol [1]. Central to this process is the responsiveness of the liver to the hormone glucagon [1,2,3]. After a carbohydrate-containing meal, the elevation in insulin suppresses glucagon secretion and thereby hepatic glucose production. Whilst the mechanisms involved in the regulation of glycogenolysis and gluconeogenesis by glucagon have been well characterised, the effects of glucagon excess on glucose utilisation have not been fully elucidated
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