Cell Biology Assessment of Glucokinase Mutations V62M and G72R in Pancreatic -Cells: Evidence for Cellular Instability of Catalytic Activity

Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Oxford, England, United Kingdom
Diabetes (Impact Factor: 8.1). 08/2007; 56(7):1773-82. DOI: 10.2337/db06-1151
Source: PubMed


Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R), the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of green fluorescent protein (GFP) GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity-to-GK immunoreactivity ratio and GK activity-to-GK mRNA ratio but not GK immunoreactivity-to-GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) increased GK activity for wild type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycemia in subjects with V62M and G72R mutations.

Download full-text


Available from: James A Shaw
  • Source
    • "Thus, the molecular mechanism leading to their diabetes remains unexplained. For some mutations, a cellular instability or defect in S-nitrosylation has been indicated [24] [25] [26]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.
    Full-text · Article · Jul 2012 · Biochimica et Biophysica Acta
  • Source
    • "Decreased S 0.5 , Decreased ATP K m , Decreased K cat MODY c.130G4A p.Gly44Ser [Gloyn et al., 2004] Increased S 0.5 , decreased hill number, decreased ATP K m , Decreased K cat MODY c.157G4T p.Ala53Ser [Davis et al., 1999] Kinetics same as wild type [Miller et al., 1999] Thermolabile [Gloyn et al., 2004] Near normal kinetics MODY c.182A4C p.Tyr61Ser [Estalella et al., 2007] Increased S 0.5 , decreased ATP K m MODY c.185T4C p.Val62Ala [Gloyn et al., 2004] Increased S 0.5 , decreased hill number, decreased K cat , decreased ATP K m MODY c.184G4A p.Val62Met [Arden et al., 2007; Gloyn et al., 2004, 2005] Decreased S 0.5 , decreased hill number, decreased thermostability, decreased K cat Catalytic instability demonstrated in min6 cells HH c.191C4A p.Ser64Tyr [Christesen et al., 2008] Decreased S 0.5 , decreased hill number, decreased K cat , increased ATP K m HH c.194C4T p.Thr65Ile [Gloyn et al., 2003, 2004; Heredia et al., 2006a] Decreased S 0.5 , decreased hill number, decreased K cat , increased ATP K m HH c.203G4T p.Gly68Val [Wabitsch et al., 2007] Decreased S 0.5 , decreased hill number, increased K cat , decreased ATP K m MODY c.208G4A p.Glu70Lys [Burke et al., 1999; Davis et al., 1999; Gloyn et al., 2004; Kesavan et al., 1997] Increased S 0.5 , decreased hill number, decreased K cat Clonal cell line study showed mutant to be thermostable Exon 3 MODY c.214G4A p.Gly72Arg [Arden et al., 2007; Gloyn et al., 2004; Sagen et al., 2006] Decreased S 0.5 , decreased hill number, slightly decreased thermostability, decreased K cat , increased ATP K m "
    [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.
    Full-text · Article · Nov 2009 · Human Mutation
  • Source
    • "(C, D) KA-PFK-2/FBPase-2 and rat liver glucokinase were expressed in MIN6 cells by incubation with adenoviral vectors for 6 h. After overnight culture, glucokinase activity was determined in the sonicated supernatant (C) and insulin secretion (D) was determined at 5 or 25 mM glucose in the absence or presence of 10 μM GKA (glucokinase activator)[27]. n = 1 based on three replicates within an individual experiment representative of two experiments. Results are expressed as a percentage of the control. "
    [Show abstract] [Hide abstract]
    ABSTRACT: PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.
    Full-text · Article · May 2008 · Biochemical Journal
Show more