Nicholas D. Tribble

The P446L variant in GCKR associated with fasting plasma glucose and triglyceride levels exerts its effect through increased glucokinase activity in liver

Genome-wide association studies have identified a number of signals for both Type 2 Diabetes and related quantitative traits. For the majority of loci, the transition from association signal to mutational mechanism has been difficult to establish. Glucokinase (GCK) regulates glucose storage and disposal in the liver where its activity is regulated by glucokinase regulatory protein (GKRP; gene name GCKR). Fructose-6 and fructose-1 phosphate (F6P and F1P) enhance or reduce GKRP-mediated inhibition, respectively. A common GCKR variant (P446L) is reproducibly associated with triglyceride and fasting plasma glucose levels in the general population. The aim of this study was to determine the mutational mechanism responsible for this genetic association. Recombinant human GCK and both human wild-type (WT) and P446L-GKRP proteins were generated. GCK kinetic activity was observed spectrophotometrically using an NADP+-coupled assay. WT and P446L-GKRP-mediated inhibition of GCK activity and subsequent regulation by phosphate esters were determined. Assays matched for GKRP activity demonstrated no difference in dose-dependent inhibition of GCK activity or F1P-mediated regulation. However, the response to physiologically relevant F6P levels was significantly attenuated with P446L-GKRP (n = 18; P ≤ 0.03). Experiments using equimolar concentrations of both regulatory proteins confirmed these findings (n = 9; P < 0.001). In conclusion, P446L-GKRP has reduced regulation by physiological concentrations of F6P, resulting indirectly in increased GCK activity. Altered GCK regulation in liver is predicted to enhance glycolytic flux, promoting hepatic glucose metabolism and elevating concentrations of malonyl-CoA, a substrate for de novo lipogenesis, providing a mutational mechanism for the reported association of this variant with raised triglycerides and lower glucose levels.

Activating glucokinase (GCK) Mutations as a cause of medically responsive congenital hyperinsulinism: prevalence in children and characterisation of a novel GCK mutation

OBJECTIVE Activating glucokinase (GCK) mutations are a rarely reported cause of congenital hyperinsulinism (CHI), but the prevalence of GCK mutations is not known. METHODS From a pooled cohort of 201 non-syndromic children with CHI from three European referral centres (Denmark, n=141; Norway, n=26; UK, n=34), 108 children had no K(ATP)-channel (ABCC8/KCNJ11) gene abnormalities and were screened for GCK mutations. Novel GCK mutations were kinetically characterised. RESULTS In five patients, four heterozygous GCK mutations (S64Y, T65I, W99R and A456V) were identified, out of which S64Y was novel. Two of the mutations arose de novo, three were dominantly inherited. All the five patients were medically responsive. In the combined Danish and Norwegian cohort, the prevalence of GCK-CHI was estimated to be 1.2% (2/167, 95% confidence interval (CI) 0-2.8%) of all the CHI patients. In the three centre combined cohort of 72 medically responsive children without K(ATP)-channel mutations, the prevalence estimate was 6.9% (5/72, 95% CI 1.1-12.8%). All activating GCK mutations mapped to the allosteric activator site. The novel S64Y mutation resulted in an increased affinity for the substrate glucose (S(0.5) 1.49+/-0.08 and 7.39+/-0.05 mmol/l in mutant and wild-type proteins respectively), extrapolating to a relative activity index of approximately 22 compared with the wild type. CONCLUSION In the largest study performed to date on GCK in children with CHI, GCK mutations were found only in medically responsive children who were negative for ABCC8 and KCNJ11 mutations. The estimated prevalence (approximately 7%) suggests that screening for activating GCK mutations is warranted in those patients.

Permanent neonatal diabetes mellitus caused by a novel homozygous (T168A) glucokinase (GCK) mutation: initial response to oral sulphonylurea therapy.

OBJECTIVE To evaluate the clinical response to sulphonylurea treatment in a child with a homozygous T168A GCK (glucokinase) mutation, causing permanent neonatal diabetes mellitus (PNDM). STUDY DESIGN Oral glibenclamide was given for 3 months. Pancreatic beta cell function was assessed by a glucagon stimulation test. Mutant and wild-type (WT) GCK were characterized. RESULTS Sulphonylurea treatment resulted in a 12-fold increase in basal and stimulated C-peptide levels. HbA1c levels were reduced from 9.4% to 8.1% on a reduced insulin dose (0.85 to 0.60 U/kg/day). Mutant T168A-GST-GCK showed reduced kinetic activity (0.02 fold) compared to WT. CONCLUSIONS Sulphonylureas can close the adenosine triphosphate (ATP)-sensitive potassium channel and elicit insulin secretion, but the ATP generated from metabolism is insufficient to fully restore insulin secretory capacity. Nonetheless, sulphonylurea treatment should be tried in patients with GCK-PNDM, particularly those with mutations resulting in less severe kinetic defects, in whom improved glycemic control may be obtained with lower doses of insulin.

Naturally Occurring Glucokinase Mutations Are Associated with Defects in Posttranslational S-Nitrosylation

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic beta-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in betaTC3 insulinoma cells. Expression of either mutant in betaTC3 cells did not affect the metabolic response to 5 mM glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion.

Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia.

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113–1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) –mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC50 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.

Insights Into the Pathogenicity of Rare Missense GCK Variants From the Identification and Functional Characterization of Compound Heterozygous and Double Mutations Inherited in Cis

OBJECTIVE To demonstrate the importance of using a combined genetic and functional approach to correctly interpret a genetic test for monogenic diabetes. RESEARCH DESIGN AND METHODS We identified three probands with a phenotype consistent with maturity-onset diabetes of the young (MODY) subtype GCK-MODY, in whom two potential pathogenic mutations were identified: [R43H/G68D], [E248 K/I225M], or [G261R/D217N]. Allele-specific PCR and cosegregation were used to determine phase. Single and double mutations were kinetically characterized. RESULTS The mutations occurred in cis (double mutants) in two probands and in trans in one proband. Functional studies of all double mutants revealed inactivating kinetics. The previously reported GCK-MODY mutations R43H and G68D were inherited from an affected father and unaffected mother, respectively. Both our functional and genetic studies support R43H as the cause of GCK-MODY and G68D as a neutral rare variant. CONCLUSIONS These data highlight the need for family/functional studies, even for previously reported pathogenic mutations.

The previously reported T342P GCK missense variant is not a pathogenic mutation causing MODY

Keywords Glucokinase.Glucokinase mutation.Hyperglycaemia.Missense mutation.MODY.Non-pathogenicAbbreviationsGCK GlucokinaseSNP Single nucleotide polymorphismTo the Editor: There is renewed interest in the difficulties ofinterpreting the role of missense variants identified in largescale medical resequencing projects made possible by nextgeneration sequencing technologies. To assist in ascribing arole in disease causation for these variants, a combination ofstatistics,bioinformatictoolsand,ultimately,functionalstudies,willbeneeded.MissensevariantsidentifiedinhighlypenetrantMendelian disorders where co-segregation studies can beperformed provide a model to explore the utility of these tools.The glycolytic enzyme glucokinase plays a key role inglucose stimulated insulin secretion and has been termed thepancreatic beta cell glucose sensor [1]. Heterozygous inacti-vating mutations in the GCK gene encoding glucokinasecause a subtype of MODY characterised by mild fastinghyperglycaemia (fasting plasma glucose 5.5–8 mmol/l,HbA

Glucokinase (GCK) and other susceptibility genes for β-cell dysfunction: the candidate approach

There are well-documented examples in the literature of where determining the genetic aetiology of a disorder has provided insights into important regulatory pathways and protein interactions, and, more recently, has led to improved treatment options for patients. The studies of monogenic forms of beta-cell dysfunction are no exception. Naturally occurring mutations in the gene for the beta-cell enzyme glucokinase (GCK) result in both hyper- and hypo-glycaemia. Over 200 mutations have been described, and careful study of the mutational mechanisms for a number of these has provided important insights into glucokinase regulation. Increased understanding of post-translational regulatory mechanisms holds the promise of novel pharmacotherapeutic options for the treatment of T2DM (Type 2 diabetes mellitus). It is well established that common genetic variation in genes involved in monogenic forms of beta-cell dysfunction contributes to susceptibility to T2DM. Recent genome-wide scans for association have identified a number of novel T2DM susceptibility genes which probably influence beta-cell mass and/or function. Their identification allows the investigation of the role of rare mutations in monogenic beta-cell dysfunction. Current results indicate the importance of these genes in pancreatic development and suggest that mutations which result in a severe functional defect could be lethal.

Functional characterisation of the glucokinase regulatory protein gene variant P446L shows diminished regulation by fructose-6 phosphate resulting in increased glucokinase activity

Prevalence of lipid abnormalities before and after the introduction of lipid modifying therapy among Swedish patients with type 2 diabetes and/or coronary heart disease (PRIMULA Sweden)In the ACTION (A Coronary disease Trial Investigating Outcome with Nifedipine GITS) trial, the benefits of adding nifedipine GITS to the treatment of patients with stable symptomatic coronary artery disease were particularly apparent in those with concomitant hypertension. This further analysis has assessed whether or not the addition of nifedipine GITS is particularly beneficial in the treatment of patients with the combination of diabetes mellitus and chronic stable angina.Different sets of risk factors for the development of albuminuria and renal impairment in type 2 diabetes : the Swedish National Diabetes register (NDR)