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Functional glucokinase isoforms are expressed in rat brain
I Roncero1, E Alvarez, P Vázquez
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, Complutense University, Madrid, Spain.
Abstract:
Recently, the description of glucokinase mRNA in certain neuroendocrine cells has opened new ways to characterize this enzyme in the rat brain. In this study, we found glucokinase mRNA and a similar RNA splicing pattern of the glucokinase gene product in rat hypothalamus and pancreatic islets; the mRNA that codes for B1 isoform was the most abundant, with minor amounts of those coding for the B2, P1, P2, P1/B2, and P2/B2 isoforms. Glucokinase gene expression in rat brain gave rise to a protein of 52 kDa with a high apparent Km for glucose and no product inhibition by glucose 6-phosphate, with a contribution to the total glucose phosphorylating activity of between 40 and 14%; the hypothalamus and cerebral cortex were the regions of maximal activity. Low and high Km hexokinases were characterized by several criteria. Also, using RT-PCR analysis we found a glucokinase regulatory protein mRNA similar to that previously reported in liver. These findings indicate that the glucokinase present in rat brain should facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and the expression of glucokinase and GLUT-2 in the same hypothalamic neurons suggests a role in glucose sensing.
Insights
This study identified glucokinase mRNA and protein in the rat brain, particularly the hypothalamus, aiding adaptation to blood glucose changes. Its co-expression with GLUT-2 suggests a role in neural glucose sensing.
Area of Science:
- Neuroendocrinology
- Molecular Biology
- Biochemistry
Background:
- Glucokinase (GCK) mRNA has been identified in neuroendocrine cells, prompting further investigation into its role in the brain.
- Understanding GCK expression and function in the brain is crucial for comprehending glucose regulation and adaptation.
Purpose of the Study:
- To characterize glucokinase gene expression and protein activity in the rat brain, focusing on the hypothalamus.
- To investigate the presence and function of glucokinase regulatory protein (GCKRP) mRNA in the rat brain.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) to detect glucokinase mRNA isoforms and GCKRP mRNA.
- Enzyme activity assays to determine kinetic properties (Km, product inhibition) of brain glucokinase.
- Western blot analysis to identify the molecular weight of the brain glucokinase protein.
Main Results:
- Glucokinase mRNA, predominantly the B1 isoform, was detected in rat hypothalamus and pancreatic islets, with similar splicing patterns.
- Brain glucokinase exhibited high apparent Km for glucose, lacked glucose-6-phosphate product inhibition, and contributed significantly to glucose phosphorylating activity in the hypothalamus and cerebral cortex.
- RT-PCR confirmed the presence of glucokinase regulatory protein mRNA in the rat brain, similar to liver.
Conclusions:
- Glucokinase in the rat brain, especially the hypothalamus, plays a role in adapting to blood glucose fluctuations.
- Co-expression of glucokinase and GLUT-2 in hypothalamic neurons suggests a critical function in neural glucose sensing and regulation.