Expression of the Human Glucokinase Gene: Important Roles of the 5′ Flanking and Intron 1 Sequences

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.

Evolution of Hepatic Glucose Metabolism: Liver-Specific Glucokinase Deficiency Explained by Parallel Loss of the Gene for Glucokinase Regulatory Protein (GCKR)

“…Since genome assemblies for some species are still at a draft stage it might be expected that the failure to identify a GCKR -like sequence may simply be due to gaps in the available genomes. In contrast, our searches of these same genomes for GCK gene-like sequences resulted in the identification of complete or partial genes in all of these mammalian [32] and non-mammalian vertebrate species (Table S1), suggesting that the absence of sequences similar to GCKR in at least some (but possibly not all) of the genomes is due to the loss of this gene. Gene loss can be due to either deletion of a sequence from the genome, or failure to find using the blast similarity search algorithm as the sequence became non-functional and degenerated into a pseudogene.…”

“…As these species do not demonstrate symptoms of diabetes or poor glucose metabolism, they most likely possess an intact glucose sensing mechanisms; thus, mutations that inactivate GCK function seem unlikely. This conclusion is supported by our recent characterization of GCK genes from diverse species, where intact GCK genes were found in the genomes of most vertebrates examined [32]. An exception was the genome sequences of two bat species (flying fox and little brown bat), where the GCK genes were found to be missing their liver-specific first exon, which might prevent expression of GCK in the liver but not other sites [32].…”

Evolution of glucose utilization: Glucokinase and glucokinase regulator protein

“…GCKR is also absent from the livers of the cat, a species who are deficient in liver glucokinase activity (Hiskett et al, 2009). These observations prompted searches for glucokinase and GCKR genes from the genomes of diverse vertebrate species, including those that do and do not have reported liver glucokinase activity (Wang et al, 2012(Wang et al, , 2013. Potentially intact glucokinase genes could be found in every genome searched, however, mutated GCKR genes were found in the genomes of multiple species (Wang et al, 2012(Wang et al, , 2013.…”

“…These observations prompted searches for glucokinase and GCKR genes from the genomes of diverse vertebrate species, including those that do and do not have reported liver glucokinase activity (Wang et al, 2012(Wang et al, , 2013. Potentially intact glucokinase genes could be found in every genome searched, however, mutated GCKR genes were found in the genomes of multiple species (Wang et al, 2012(Wang et al, , 2013. GCKR genes that contain mutations that should prevent function (i.e., frame-shift or splicing mutations) were found in species that were previously reported to lack (or have low) glucokinase activity in the liver; while potentially intact (i.e., have complete open reading frames and splice consensus sequences) GCKR genes were found in species that were reported to have activity (Wang et al, 2013).…”

“…The cat provides an example of a carnivore that has lost GCKR and thus glucokinase activity (Hiskett et al, 2009;Verbrugghe et al, 2012). However, there are many other carnivores, such as the dog that have glucokinase activity (Tanaka et al, 2005) and have retained both GCKR and glucokinase genes (Wang et al, 2012(Wang et al, , 2013. The retention of glucokinase activity in the liver may allow dogs (Batchelor et al, 2011) or carnivorous fish (Panserat et al, 2000b;Polakof et al, 2011) to eat more varied diets or to regulate blood glucose levels when glucose is synthesized from other substrates.…”