Rat glucokinase gene: structure and regulation by insulin.

Abstract

The glucokinase gene is 15.5-kilobases long, appears to be present as a single copy, and contains 10 exons that range in size from 96 to 977 base pairs. The transcription start site was located 127 nucleotides upstream from the translation initiation codon. The 5' flanking DNA contains several regions similar to dermed promoter elements. These include a probable "TATA box," an Spl binding site, and several elements related to liver-specific gene expression. In addition, we determined that transcription of the glucokinase gene increased at least 20-fold when diabetic rats were treated with insulin for 2 hr.Glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) plays a key role in the regulation of glucose homeostasis by catalyzing the first step in glycolysis (1). Expression of the enzyme is limited to hepatocytes and pancreatic ,B cells (2, 3), and it is regulated differently in these two tissues. The hepatic enzyme is induced by insulin and repressed by cAMP (4) whereas in the p cell glucokinase activity is increased by glucose (5). The glucokinase gene is, therefore, of interest both because of its tissue-specific expression and because of the several regulatory processes that can be analyzed. Before the cis-acting DNA elements responsible for the tissuespecific expression and hormonal regulation of this enzyme can be identified and studied, the structure of the glucokinase gene, the transcription unit in each tissue, and the sequence of its 5' flanking DNA must be determined.Glucokinase is thought to be a member of a family of hexokinases that have a common evolutionary origin (6). This concept was based on indirect evidence because none of the complete structures of the mammalian hexokinases were available. We recently deduced the structure of rat liver glucokinase and found that it shares 33% and 53% amino acid sequence identity with yeast hexokinase and the carboxylterminal portion of rat brain hexokinase I, respectively (7). We now have determined the structure of the glucokinase genet as an initial step toward answering how the various hexokinase isozymes are related to each other. Primer-Extension Analysis. A 36-base oligonucleotide (5'-ATGTTCCTGACTCCTGAGGCCACCTGTTGCAGGTGA-3') complementary to sequences near the 5' end of the glucokinase mRNA was synthesized and 5'-end-labeled with [_y-32P]ATP (>5000 Ci/mmol; 1 Ci = 37 GBq) and T4 polynucleotide kinase. The primer (3 x 10s cpm) was annealed in a total volume of 20 gl to 20 pug to poly(A)+ RNA; the annealing buffer contained 20 mM Tris-HCl (pH 7.5), 250 mM NaCl, and 1 mM EDTA. After hybridization for 1 hr at 60TC the reaction mixtures, containing the annealed primer and RNA, were diluted with 130 1A ofa solution containing 50 mM Tris-HCl (pH 7.5), 40 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, actinomycin D (75 ,ug/ml), deoxyribonucleotides at 0.5 mM each, and 2 units of avian myeloma virus reverse transcriptase (Promega Biotec) and then incubated at 37TC for 1 hr. The products of the reactions were size-fractionated on a 5% polyacrylamide/7 ...

Hexokinase as a sugar sensor in higher plants.

“…AtHXKI and AIHXK2 share 82% nucleotide identity, and AtHXKI and AtHXK2 share 85% amino acid identity. Results of data base searches and sequence comparisons showed that AtHXK proteins share significant sequence identity with human (Nishi et al, 1992) and rat (Magnuson et al, 1989) GLK (34 to 35%) and yeast (Stachelek et al, 1986;Prior et al, 1993) HXKs (36 to 38%) ( Figure 2). Conserved ATP and sugar binding domains (Bork et al, 1993) were found in the deduced protein sequences of both AtHXK genes (Figure 2).…”

“…A t h x k Z H w a n R a t Y e a m t l Y e a m t l Y e a r t 3 A t h x k l A t h x k Z H w a n R a t Y e a r t l Y e a m t Z Y e a m t 3 A comparison of predicted amino acid sequences of Arabidopsis AtHXK1 and AtHXK2, human GLK (Nishi et al, 1992), rat GLK (Magnuson et al, 1989), S. cerevisiae HXKl (Yeastl) and HXK2 (Yeast2) (Stachelek et al, 1986), and Kluyveromyces lactis RAG5 (Yeast3) (Prior et al, 1993) is shown. The ATP binding site (Bork et al, 1993) includes underlined regions 1, 2, and A, indicating the conserved phosphate 1 and 2 and adenosine interaction regions, respectively.…”

Liver glucokinase gene expression is controlled by the onecut transcription factor hepatocyte nuclear factor-6

“…The phenotype of the hnf6-/-mice is not fully penetrant (our unpublished observations) and 20 to 25% of the mice do not show glucose intolerance. As expression of the glucokinase gene is stimulated by insulin [21,22], we separated non-diabetic from diabetic hnf6-/-mice based on glucose tolerance tests. We found that, both in the 3.…”

Transcriptional control of genes that regulate glycolysis and gluconeogenesis in adult liver

“…The liver and ,-cell mRNAs are identical with the exception of their 5' end that codes for a different 15-amino-acid stretch. In the rat [8,9] and in the human [10,11], there is a single glucokinase gene containing 11 exons Abbreviations used: AF, accessory factor; b-HLH, basic region/helix-loop-helix; C/EBP, CCAAT/enhancer-binding protein; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; CRE, cyclic AMP response element; CREB, cyclic AMP response element-binding protein; DBP, Dbinding protein; FBPase-1, fructose-1,6-bisphosphatase; FBPase-2, fructose-2,6-bisphosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GIRE, glucose/insulin response element; GRU, glucocorticoid-responsive unit; HNF, hepatocyte nuclear factor; IRE, insulin response element; IRS, insulin response sequence; NF-I, nuclear factor-I; NFY, nuclear factor Y; Oct-1, octamer factor-1; PEPCK, phosphoenolpyruvate carboxykinase; PFK-1, 6-phosphofructo-i-kinase; PFK-2, 6-phosphofructo-2-kinase; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; RAR, retinoic acid receptor; RARE, retinoic acid response element; STAT, signal transducer and activator of transcription; T3, tri-iodothyronine; T3R, T3 receptor; USF, upstream stimulating factor.…”