Article

Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young

The Children's Hospital of Philadelphia, Filadelfia, Pennsylvania, United States
Journal of Biological Chemistry (Impact Factor: 4.6). 05/2005; 280(14):14105-13. DOI: 10.1074/jbc.M413146200
Source: PubMed

ABSTRACT 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.

1 Follower
 · 
87 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age-at-diagnosis. The majority (28/30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI≤0.01). There was no correlation between CSG and RAI (r(2)=0.05, p=0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison of CSG with RSI detected a highly significant correlation (r(2)=0.74, p=0.002). We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity.
    Human Molecular Genetics 07/2014; · 6.68 Impact Factor
  • Source
    [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; DOI:10.1530/JOE-13-0616 · 3.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cryptosporidium parvum may cause virtually untreatable infections in AIDS patients, and is recently identified as one of the top four diarrheal pathogens in children in developing countries. Cryptosporidium differs from other apicomplexans (e.g., Plasmodium and Toxoplasma) by lacking many metabolic pathways including the Krebs cycle and cytochrome-based respiratory chain, thus relying mainly on glycolysis for ATP production. Here we report the molecular and biochemical characterizations of a hexokinase in C. parvum (CpHK). Our phylogenetic reconstructions indicated that apicomplexan hexokinases including CpHK were highly divergent from those of humans and animals (i.e., at the base of the eukaryotic clade). CpHK displays unique kinetic features that differ from those in mammals and Toxoplasma gondii (TgHK) in the preference towards various hexoses and its capacity to use ATP and other NTPs. CpHK also displays substrate inhibition by ATP. Moreover, 2-deoxy-D-glucose (2DG) could not only inhibit the CpHK activity, but also the parasite growth in vitro at concentrations nontoxic to host cells (IC50 = 0.54 mM). While the exact action of 2-deoxy-D-glucose on the parasite is subject to further verification, our data suggest that CpHK and the glycolytic pathway may be explored for developing anti-cryptosporidial therapeutics.
    Protist 09/2014; 165(5). DOI:10.1016/j.protis.2014.08.002 · 3.56 Impact Factor