Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.
"However, unlike other hexokinases, it is not inhibited by its product glucose-6-phosphate and maintains high glycolytic flux in the presence of elevated glucose, coupling carbohydrate sensing to insulin secretion in the b-cell (Bedoya et al. 1986, Alvarez et al. 2002, Newsholme & Krause 2012). Alterations in the activity of important glycolytic enzymes such as GCK and phosphofructokinase can modulate GSIS, and this may lead to impaired glucose metabolism and insulin secretion (Nielsen et al. 1998, Westermark & Lansner 2003, Gloyn et al. 2005). Furthermore, chronic hyperglycaemic conditions, as observed in T2DM, can negatively regulate the expression of several important glucose metabolising b-cell genes including SLC2A2, GCK, Ca 2C channels and insulin transcription factors pancreatic and duodenal homoeobox 1 (Pdx1), neurogenic differentiation 1 (NeuroD1) and v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafA) (Cnop et al. 2005, Newsholme et al. 2010). "
[Show abstract][Hide abstract] ABSTRACT: Pancreatic β-cell function is of critical importance in the regulation of fuel homeostasis, and metabolic dysregulation is a hallmark of diabetes mellitus (DM). The β-cell is an intricately designed cell type that couples metabolism of dietary sources of carbohydrates, amino acids, and lipids to insulin secretory mechanisms, such that insulin release occurs at appropriate times to ensure efficient nutrient uptake and storage by target tissues. However, chronic exposure to high nutrient concentrations results in altered metabolism that impacts negatively on insulin exocytosis, insulin action, and may ultimately lead to development of DM. Reduced action of insulin in target tissues is associated with impairment of insulin signalling and contributes to insulin resistance (IR), a condition often associated with obesity, and a major risk factor for DM. The altered metabolism of nutrients by insulin sensitive target tissues (muscle, adipose, and liver) can result in high circulating levels of glucose and various lipids, which further impact on pancreatic β-cell function, IR, and progression of the metabolic syndrome. Here, we have considered the role played by the major nutrient groups, carbohydrates, amino acids, and lipids, in mediating β-cell insulin secretion, while also exploring the interplay between amino acids and insulin action in muscle. We also focus on the effects of altered lipid metabolism in adipose and liver resulting from activation of inflammatory processes commonly observed in DM pathophysiology. The aim of this review is to describe commonalities and differences in metabolism related to insulin secretion and action, pertinent to the development of DM.
Journal of Endocrinology 03/2014; 221(3). DOI:10.1530/JOE-13-0616 · 3.72 Impact Factor
"The combination of these two features indicates that GCK can maintain glycolytic flux in the face of high-glucose load (Fu et al., 2013). Interestingly, it has been suggested that changes in GCK function resulted in decreased GSIS that could possibly lead to DM, thus highlighting the importance of this metabolic step (Gloyn et al., 2005; Rorsman & Braun, 2013). In addition, another glycolytic enzyme, phosphofructokinase (PFK), is also an important regulatory site in glycolysis and is allosterically controlled by ATP levels "
[Show abstract][Hide abstract] ABSTRACT: Regulation of metabolic fuel homeostasis is a critical function of β-cells, which are located in the islets of Langerhans of the animal pancreas. Impairment of this β-cell function is a hallmark of pancreatic β-cell failure and may lead to development of type 2 diabetes mellitus. β-Cells are essentially "fuel sensors" that monitor and react to elevated nutrient load by releasing insulin. This response involves metabolic activation and generation of metabolic coupling factors (MCFs) that relay the nutrient signal throughout the cell and induce insulin biosynthesis and secretion. Glucose is the most important insulin secretagogue as it is the primary fuel source in food. Glucose metabolism is central to generation of MCFs that lead to insulin release, most notably ATP. In addition, other classes of nutrients are able to augment insulin secretion and these include members of the lipid and amino acid family of nutrients. Therefore, it is important to investigate the interplay between glucose, lipid, and amino acid metabolism, as it is this mixed nutrient sensing that generate the MCFs required for insulin exocytosis. The mechanisms by which these nutrients are metabolized to generate MCFs, and how they impact on β-cell insulin release and function, are discussed in detail in this article.
"In these instances other mutational mechanisms may be at play, for instance, mechanisms that effect enzyme stability [Burke et al., 1999] or binding with regulatory molecules such at GKRP [Arden et al., 2007; Gloyn et al., 2005] or the bifunctional enzyme PFK-2/FBPase-2 [Arden et al., 2007] or perhaps with an unknown endogenous allosteric activator [Gloyn et al., 2005]. For example, the V62M GCK- MODY mutation appears be paradoxically kinetically activating as opposed to inactivating [Gloyn et al., 2005], and has been shown to have a lack of inhibition by liver GKRP and a lack of activation by pharmacological GCK small molecular activators [Gloyn et al., 2005], although studies in min6 cells have suggested that the V62M GCK mutation may result in catalytic instability [Arden et al., 2007]. There is evidence that at least nine other GCK- MODY mutations affect enzyme stability from recombinant mutant GCK protein studies [Burke et al., 1999; Davis et al., 1999; Galan et al., 2006; Garcia-Herrero et al., 2007; Gloyn et al., 2005; Kesavan et al., 1997; Sagen et al., 2006]. "
[Show abstract][Hide abstract] ABSTRACT: Glucokinase is a key regulatory enzyme in the pancreatic beta-cell. It plays a crucial role in the regulation of insulin secretion and has been termed the glucose sensor in pancreatic beta-cells. Given its central role in the regulation of insulin release it is understandable that mutations in the gene encoding glucokinase (GCK) can cause both hyper- and hypoglycemia. Heterozygous inactivating mutations in GCK cause maturity-onset diabetes of the young (MODY) subtype glucokinase (GCK), characterized by mild fasting hyperglycemia, which is present at birth but often only detected later in life during screening for other purposes. Homozygous inactivating GCK mutations result in a more severe phenotype presenting at birth as permanent neonatal diabetes mellitus (PNDM). A growing number of heterozygous activating GCK mutations that cause hypoglycemia have also been reported. A total of 620 mutations in the GCK gene have been described in a total of 1,441 families. There are no common mutations, and the mutations are distributed throughout the gene. The majority of activating mutations cluster in a discrete region of the protein termed the allosteric activator site. The identification of a GCK mutation in patients with both hyper- and hypoglycemia has implications for the clinical course and clinical management of their disorder.
Human Mutation 11/2009; 30(11):1512-26. DOI:10.1002/humu.21110 · 5.14 Impact Factor
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