Glucose is an essential nutrient for the human body. It is the major energy source for many cells, which depend on the bloodstream for a steady supply. Blood glucose levels, therefore, are carefully maintained. The liver plays a central role in this process by balancing the uptake and storage of glucose via glycogenesis and the release of glucose via glycogenolysis and gluconeogenesis. The several substrate cycles in the major metabolic pathways of the liver play key roles in the regulation of glucose production. In this review, we focus on the short- and long-term regulation glucose-6-phosphatase and its substrate cycle counter-part, glucokinase. The substrate cycle enzyme glucose-6-phosphatase catalyzes the terminal step in both the gluconeogenic and glycogenolytic pathways and is opposed by the glycolytic enzyme glucokinase. In addition, we include the regulation of GLUT 2, which facilitates the final step in the transport of glucose out of the liver and into the bloodstream.

Regulation of glucose production by the liver.

Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing

Nutrient homeostasis is known to be regulated by pancreatic islet tissue. The function of islet beta-cells is controlled by a glucose sensor that operates at physiological glucose concentrations and acts in synergy with signals that integrate messages originating from hypothalamic neurons and endocrine cells in gut and pancreas. Evidence exists that the extrapancreatic cells producing and secreting these (neuro)endocrine signals also exhibit a glucose sensor and an ability to integrate nutrient and (neuro)hormonal messages. Similarities in these cellular and molecular pathways provide a basis for a network of coordinated functions between distant cell groups, which is necessary for an appropriate control of nutrient homeostasis. The glucose sensor seems to be a fundamental component of these control mechanisms. Its molecular characterization is most advanced in pancreatic beta-cells, with important roles for glucokinase and mitochondrial oxidative fluxes in the regulation of ATP-sensitive K+ channels. Other glucose-sensitive cells in the endocrine pancreas, hypothalamus, and gut were found to share some of these molecular characteristics. We propose that similar metabolic signaling pathways influence the function of pancreatic alpha-cells, hypothalamic neurons, and gastrointestinal endocrine and neural cells.

https://diabetes.diabetesjournals.org/content/diabetes/50/1/1.full.pdf

Glucose Sensing in Pancreatic β-Cells: A Model for the Study of Other Glucose-Regulated Cells in Gut, Pancreas, and Hypothalamus

Conversion of glucose into glycogen is a major pathway that contributes to the removal of glucose from the portal vein by the liver in the postprandial state. It is regulated in part by the increase in blood-glucose concentration in the portal vein, which activates glucokinase, the first enzyme in the pathway, causing an increase in the concentration of glucose 6-P (glucose 6-phosphate), which modulates the phosphorylation state of downstream enzymes by acting synergistically with other allosteric effectors. Glucokinase is regulated by a hierarchy of transcriptional and post-transcriptional mechanisms that are only partially understood. In the fasted state, glucokinase is in part sequestered in the nucleus in an inactive state, complexed to a specific regulatory protein, GKRP (glucokinase regulatory protein). This reserve pool is rapidly mobilized to the cytoplasm in the postprandial state in response to an elevated concentration of glucose. The translocation of glucokinase between the nucleus and cytoplasm is modulated by various metabolic and hormonal conditions. The elevated glucose 6-P concentration, consequent to glucokinase activation, has a synergistic effect with glucose in promoting dephosphorylation (inactivation) of glycogen phosphorylase and inducing dephosphorylation (activation) of glycogen synthase. The latter involves both a direct ligand-induced conformational change and depletion of the phosphorylated form of glycogen phosphorylase, which is a potent allosteric inhibitor of glycogen synthase phosphatase activity associated with the glycogen-targeting protein, GL [hepatic glycogen-targeting subunit of PP-1 (protein phosphatase-1) encoded by PPP1R3B]. Defects in both the activation of glucokinase and in the dephosphorylation of glycogen phosphorylase are potential contributing factors to the dysregulation of hepatic glucose metabolism in Type 2 diabetes.

Glucokinase and molecular aspects of liver glycogen metabolism.

Mammalian glucokinase was identified 30 years ago as a distinct form of hexokinase in rat liver. The hexokinases (ATP: hexose 6phosphotransferases, EC 2.7.1.1) constitute a family of evolutionarily and stucturally related enzymes present in eukaryotic cells from yeast to mammals. In the cells of higher organisms, the physiologically significant substrate for these enzymes is Dglucose. The reaction catalysed by the hexokinases, ATP + Dglucose -+ ADP+ D-glucose 6-phosphate, is the first and obligatory step for glucose utilization after transport of the sugar into the cell. Mammalian tissues contain four different hexokinases which can be isolated by conventional protein separation techniques and for which cDNAs have been cloned. The isoenzymes of the rat have been designated hexokinases type I-IV or A-D in order of increasing negative net charge. The subject of this Review is hexokinase type IV or D, usually called glucokinase. Glucokinase stands apart from all the other hexokinases by a number of criteria. The first and most striking is its low affinity for glucose. The enzyme is half-saturated with glucose at 6 mM, compared with Km values in the micromolar range for the three other mammalian hexokinases. This feature led to the discovery of the enzyme and underlies its key role in the physiology of glucose homeostasis. The second hallmark ofmammalian glucokinase is its highly typical tissue distribution. The glucokinase gene is transcribed and the mRNA translated into active enzyme only in hepatocytes and insulin-secreting f-cells of the pancreatic islets of Langerhans, reflecting the great functional specialization of this isoenzyme. A third outstanding feature is the developmental and multihormonal regulation of the enzyme, illustrated most dramatically by the transcriptional induction of the glucokinase gene by insulin in the liver. The distinctive kinetics of glucokinase, its tissue-specific expression and its hormonal regulation were recognized within a few years of the discovery of the enzyme. However, our understanding of these particular characteristics has remained superficial until recently. The main reason for limited progress was the difficulty of purifying the enzyme, hence of raising specific antibodies or obtaining peptide sequence for the isolation of cDNA clones. Once this obstacle was surmounted, studies on glucokinase became a very fertile field of research. The most recent and medically rewarding outcome of this research has been the discovery of mutations of the glucokinase gene as the cause of one subtype of non-insulin-dependent diabetes mellitus (NIDDM). The purpose of this article is to review the recent developments on the structure and function of the glucokinase gene and its gene products, as they relate to our understanding of blood glucose homeostasis. The reader interested in historical perspectives and a complete background on the biochemistry of glucokinase should refer to the classical reviews of Walker [1], Weinhouse [2] and Colowick [3]. Recent commentaries on topical aspects are also available [4-8]. The glucokinase gene was first cloned from the rat and the structure of the gene in this species can serve as the standard of reference (Figure 1 a). The most remarkable feature is the presence of alternative promoters, responsible for the initiation of transcription at different sites on the DNA in hepatic and endocrine cells. The first clue to the existence of cell-type-specific promoters came from the sequences oftwo quasi-full-length cDNAs isolated from rat liver and insulinoma libraries [9,10]. The sequences were essentially identical for more than 2000 nucleotides starting from the 3' ends of the cDNAs, but segments of approximately 100 nucleotides at the 5' ends were found to differ entirely. The 5' specific sequences ofthe cDNAs were mapped to widely separated sites in genomic DNA by Magnuson and co-workers [10,11]. These investigators further identified nine exons, numbered 2-10 in the transcription unit, whose assembly gives rise to the common sequence found in the liver and insulinoma-derived cDNAs. The leader exon encoding the 5' end of the hepatic mRNA, termed exon 1L, was contained in a phage A clone which also carried the common exons 2-4. The leader exon for the 5' end of the insulinoma mRNA, termed exon 1lf in reference to the fl-cells of the islets of Langerhans, was localized in a different phage clone with non-overlapping genomic DNA. It was therefore concluded that the liver-specific exon IL was contiguous to the body of the structural gene, whereas the islet-type exon Ifi was located at an unspecified distance further upstream. The intervening sequence between the two leader exons has yet to be mapped accurately. Several attempts to isolate rat genomic DNA clones for the entire region have remained unsuccessful in my laboratory, perhaps suggesting unusual features of this DNA. In any event, more than 22 kb ofDNA separate the two leader exons in the rat gene (Figure la). The fact that the upstream exon lfl is used exclusively in isletderived cells, and the downstream exon 1L exclusively in liver, was established by primer extension and nuclease protection experiments and further confirmed by reverse transcription and PCR [10]. It should be noted that the two tissue-specific exons 1 of the glucokinase gene specify not only the 5' untranslated regions of the islet and liver mRNAs, but also their initial 45 nucleotides of protein coding sequence. It follows that the rat islet and liver glucokinase enzymes will differ in primary structure by 15 amino acids (including initiator Met) at the N-terminal ends of the molecules, for a total sequence of 465 amino acid residues. In addition to the differential splicing ofleader exons associated with the cell-specific control of transcription initiation, other modes of alternative splicing are known to affect glucokinase transcripts. An optional cassette exon has been identified in the rat gene between the originally described exons 1 L and 2 (Figure la). This cassette exon, termed exon 2A, is retained in a minor fraction of glucokinase mRNA in rat liver [12]. Alternative

Mammalian glucokinase and its gene.

Glucokinase, a key regulatory enzyme of glucose metabolism in mammals, provides an interesting model of tissue-specific gene expression. The single-copy gene is expressed principally in liver, where it gives rise to a 2.4-kilobase mRNA. The islets of Langerhans of the pancreas also contain glucokinase. Using a cDNA complementary to rat liver glucokinase mRNA, we show that normal pancreatic islets and tumoral islet cells contain a glucokinase mRNA species approximately 400 nucleotides longer than hepatic mRNA. Hybridization with synthetic oligonucleotides and primer-extension analysis show that the liver and islet glucokinase mRNAs differ in the 5' region. Glucokinase mRNA is absent from the livers of fasted rats and is strongly induced within hours by an oral glucose load. In contrast, islet glucokinase mRNA is expressed at a constant level during the fasting-refeeding cycle. The level of glucokinase protein in islets measured by immunoblotting is unaffected by fasting and refeeding, whereas a 3-fold increase in the amount of enzyme occurs in liver during the transition from fasting to refeeding. From these data, we conclude (i) that alternative splicing and/or the use of distinct tissue-specific promoters generate structurally distinct mRNA species in liver and islets of Langerhans and (ii) that tissue-specific transcription mechanisms result in inducible expression of the glucokinase gene in liver but not in islets during the fasting-refeeding transition.

https://www.pnas.org/content/pnas/86/20/7838.full.pdf

Paul-Richard Pilot

Papers

Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans