Abstract
BackgroundGlucokinase plays important tissue-specific roles in human physiology, where it acts as a sensor of blood glucose levels in the pancreas, and a few other cells of the gut and brain, and as the rate-limiting step in glucose metabolism in the liver. Liver-specific expression is driven by one of the two tissue-specific promoters, and has an absolute requirement for insulin. The sequences that mediate regulation by insulin are incompletely understood.Methodology/Principal FindingsTo better understand the liver-specific expression of the human glucokinase gene we compared the structures of this gene from diverse mammals. Much of the sequence located between the 5′ pancreatic beta-cell-specific and downstream liver-specific promoters of the glucokinase genes is composed of repetitive DNA elements that were inserted in parallel on different mammalian lineages. The transcriptional activity of the liver-specific promoter 5′ flanking sequences were tested with and without downstream intronic sequences in two human liver cells lines, HepG2 and L-02. While glucokinase liver-specific 5′ flanking sequences support expression in liver cell lines, a sequence located about 2000 bases 3′ to the liver-specific mRNA start site represses gene expression. Enhanced reporter gene expression was observed in both cell lines when cells were treated with fetal calf serum, but only in the L-02 cells was expression enhanced by insulin.Conclusions/SignificanceOur results suggest that the normal liver L-02 cell line may be a better model to understand the regulation of the liver-specific expression of the human glucokinase gene. Our results also suggest that sequences downstream of the liver-specific mRNA start site have important roles in the regulation of liver-specific glucokinase gene expression.
Highlights
Mutations in the human glucokinase (GCK) gene can lead to the MODY2 form of diabetes [1] and to persistent hyperinsulinemic hypoglycemia of infancy (PHHI) [2]
Our understanding of the sequences required for the regulation of liver-specific expression of the human glucokinase gene is incomplete [4]
In L-02, fragment I-3, yielded significantly lower reporter gene activity, but in contrast to HepG2, fragment I-4 had significantly lower reporter gene activity (Fig. 4A). These results suggest that sequences in intron 1 between bases 1951 and 2722 of the human glucokinase gene function to repress gene expression, with the possibility that a second sequence between bases 2720 and 3527 have some activity, at least in some types of liver cells (e.g., L-02, Fig. 4A)
Summary
Mutations in the human glucokinase (GCK) gene can lead to the MODY2 form of diabetes [1] and to persistent hyperinsulinemic hypoglycemia of infancy (PHHI) [2]. Phosphorylation of glucose is a key step in the metabolism of glucose and involved in the sensing of blood glucose levels, thereby regulating the release of insulin by pancreatic beta cells [3,4]. Glucokinase, has important roles both in regulating insulin release from pancreatic beta-cells and metabolism of glucose in liver cells, both of which have significant impact on blood glucose levels. Deficiency of glucokinase from either the pancreas or the liver has been shown to have important roles in the development of MODY2 [5]. Glucokinase plays important tissue-specific roles in human physiology, where it acts as a sensor of blood glucose levels in the pancreas, and a few other cells of the gut and brain, and as the rate-limiting step in glucose metabolism in the liver. The sequences that mediate regulation by insulin are incompletely understood
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