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

Guidelines for the prevention and treatment of travelers' diarrhea: a graded expert panel report.

Background : Travelers' diarrhea causes significant morbidity including some sequelae, lost travel time and opportunity cost to both travelers and countries receiving travelers. Effective prevention and treatment are needed to reduce these negative impacts. Methods : This critical appraisal of the literature and expert consensus guideline development effort asked several key questions related to antibiotic and non-antibiotic prophylaxis and treatment, utility of available diagnostics, impact of multi-drug resistant (MDR) colonization associated with travel and travelers' diarrhea, and how our understanding of the gastrointestinal microbiome should influence current practice and future research. Studies related to these key clinical areas were assessed for relevance and quality. Based on this critical appraisal, guidelines were developed and voted on using current standards for clinical guideline development methodology. Results : New definitions for severity of travelers' diarrhea were developed. A total of 20 graded recommendations on the topics of prophylaxis, diagnosis, therapy and follow-up were developed. In addition, three non-graded consensus-based statements were adopted. Conclusions : Prevention and treatment of travelers' diarrhea requires action at the provider, traveler and research community levels. Strong evidence supports the effectiveness of antimicrobial therapy in most cases of moderate to severe travelers' diarrhea, while either increasing intake of fluids only or loperamide or bismuth subsalicylate may suffice for most cases of mild diarrhea. Further studies are needed to address knowledge gaps regarding optimal therapies, the individual, community and global health risks of MDR acquisition, manipulation of the microbiome in prevention and treatment and the utility of laboratory testing in returning travelers with persistent diarrhea

Inhibition of wild type, I436N and L315H glucokinase proteins by human GKRP.

<p>Data are shown as mean ±SEM, and were obtained from 4 independent measurements. Independent t-tests were used to ascertain differences between GKRP-mediated inhibition of both mutants versus that obtained with the wild-type GCK enzyme.</p

Clinical and biochemical parameters of Slovakian <i>GCK</i> mutation carriers.

<p>Data are presented as median values (range).</p>*<p>Data only available for 35 probands.</p>#<p>Data only available for 33 probands.</p>$<p>For the remaining 32 subjects, diabetes/impaired glucose tolerance was not detected prior to genetic testing.</p><p>BMI = body mass index, HbA1c = glycated hemoglobin A1c, HDL = High Density Lipoprotein, OHA = oral hypoglyceamic agents.</p

<i>GCK</i> mutations identified in Slovakian probands with a phenotype of GCK-MODY.

<p>All sequence information is based on GenBank reference sequence NM_000162.3. Nucleotide numbering reflects cDNA position, with +1 corresponding to the A of the major start codon of exon 1a (present in the pancreatic isoform). Y = yes, N = no.</p

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 β-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 βTC3 insulinoma cells. Expression of either mutant in βTC3 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

Multicenter initial guidance on use of antivirals for children with coronavirus disease 2019/Severe acute respiratory syndrome coronavirus 2

BackgroundAlthough coronavirus disease 2019 (COVID-19) is mild in nearly all children, a small proportion of pediatric patients develop severe or critical illness. Guidance is therefore needed regarding use of agents with potential activity against severe acute respiratory syndrome coronavirus 2 in pediatrics.MethodsA panel of pediatric infectious diseases physicians and pharmacists from 18 geographically diverse North American institutions was convened. Through a series of teleconferences and web-based surveys, a set of guidance statements was developed and refined based on review of best available evidence and expert opinion.ResultsGiven the typically mild course of pediatric COVID-19, supportive care alone is suggested for the overwhelming majority of cases. The panel suggests a decision-making framework for antiviral therapy that weighs risks and benefits based on disease severity as indicated by respiratory support needs, with consideration on a case-by-case basis of potential pediatric risk factors for disease progression. If an antiviral is used, the panel suggests remdesivir as the preferred agent. Hydroxychloroquine could be considered for patients who are not candidates for remdesivir or when remdesivir is not available. Antivirals should preferably be used as part of a clinical trial if available.ConclusionsAntiviral therapy for COVID-19 is not necessary for the great majority of pediatric patients. For those rare cases of severe or critical disease, this guidance offers an approach for decision-making regarding antivirals, informed by available data. As evidence continues to evolve rapidly, the need for updates to the guidance is anticipated