Ablation of glucokinase-expressing tanycytes impacts energy balance and increases adiposity in mice.

OBJECTIVES: Glucokinase (GCK) is critical for glucosensing. In rats, GCK is expressed in hypothalamic tanycytes and appears to play an essential role in feeding behavior. In this study, we investigated the distribution of GCK-expressing tanycytes in mice and their role in the regulation of energy balance. METHODS: In situ hybridization, reporter gene assay, and immunohistochemistry were used to assess GCK expression along the third ventricle in mice. To evaluate the impact of GCK-expressing tanycytes on arcuate neuron function and mouse physiology, Gck deletion along the ventricle was achieved using loxP/Cre recombinase technology in adult mice. RESULTS: GCK expression was low along the third ventricle, but detectable in tanycytes facing the ventromedial arcuate nucleus from bregma -1.5 to -2.2. Gck deletion induced the death of this tanycyte subgroup through the activation of the BAD signaling pathway. The ablation of GCK-expressing tanycytes affected different aspects of energy balance, leading to an increase in adiposity in mice. This phenotype was systematically associated with a defect in NPY neuron function. In contrast, the regulation of glucose homeostasis was mostly preserved, except for glucoprivic responses. CONCLUSIONS: This study describes the role of GCK in tanycyte biology and highlights the impact of tanycyte loss on the regulation of energy balance.

Glucokinase Deficit Prevalence in Women With Diabetes in Pregnancy: A Matter of Screening Selection.

Introduction: The prevalence among pregnant women with diabetes of monogenic diabetes due to glucokinase deficit (GCK-MODY) varies from 0 to 80% in different studies, based on the chosen selection criteria for genetic test. New pregnancy-specific Screening Criteria (NSC), validated on an Anglo-Celtic pregnant cohort, have been proposed and include pre-pregnancy BMI <25 kg/m2 and fasting glycemia >99 mg/dl. Our aim was to estimate the prevalence of GCK-MODY and to evaluate the diagnostic performance of NSC in our population of women with diabetes in pregnancy. Patients and Methods: We retrospectively selected from our database of 468 diabetic pregnant patients in Sant'Andrea Hospital, in Rome, from 2010 to 2018, all the women who received a genetic test for GCK deficit because of specific clinical features. We estimated the prevalence of GCK-MODY among tested women and the minimum prevalence in our entire population with non-autoimmune diabetes. We evaluated diagnostic performance of NSC on the tested cohort and estimated the eligibility to genetic test based on NSC in the entire population. Results: A total of 409 patients had diabetes in pregnancy, excluding those with autoimmune diabetes; 21 patients have been tested for GCK-MODY, 8 have been positive and 13 have been negative (2 of them had HNF1-alfa mutations and 1 had HNF4-alfa mutation). We found no significant differences in clinical features between positive and negative groups except for fasting glycemia, which was higher in the positive group. The minimum prevalence of monogenic diabetes in our population was 2.4%. The minimum prevalence of GCK-MODY was 1.95%. In the tested cohort, the prevalence of GCK-MODY was 38%. In this group, NSC sensitivity is 87% and specificity is 30%, positive predictive value is 43%, and negative predictive value is 80%. Applying NSC on the entire population of women with non-autoimmune diabetes in pregnancy, 41 patients (10%) would be eligible for genetic test; considering a fasting glycemia >92 mg/dl, 85 patients (20.7%) would be eligible. Discussion: In our population, NSC have good sensitivity but low specificity, probably because there are many GDM with GCK-MODY like features. It is mandatory to define selective criteria with a good diagnostic performance on Italian population, to avoid unnecessary genetic tests.

α-cell glucokinase suppresses glucose-regulated glucagon secretion.

Glucagon secretion by pancreatic α-cells is triggered by hypoglycemia and suppressed by high glucose levels; impaired suppression of glucagon secretion is a hallmark of both type 1 and type 2 diabetes. Here, we show that α-cell glucokinase (Gck) plays a role in the control of glucagon secretion. Using mice with α-cell-specific inactivation of Gck (αGckKO mice), we find that glucokinase is required for the glucose-dependent increase in intracellular ATP/ADP ratio and the closure of KATP channels in α-cells and the suppression of glucagon secretion at euglycemic and hyperglycemic levels. αGckKO mice display hyperglucagonemia in the fed state, which is associated with increased hepatic gluconeogenic gene expression and hepatic glucose output capacity. In adult mice, fed hyperglucagonemia is further increased and glucose intolerance develops. Thus, glucokinase governs an α-cell metabolic pathway that suppresses secretion at or above normoglycemic levels; abnormal suppression of glucagon secretion deregulates hepatic glucose metabolism and, over time, induces a pre-diabetic phenotype.

Nutrient sensing in pancreatic islets: lessons from congenital hyperinsulinism and monogenic diabetes.

Pancreatic beta cells sense changes in nutrients during the cycles of fasting and feeding and release insulin accordingly to maintain glucose homeostasis. Abnormal beta cell nutrient sensing resulting from gene mutations leads to hypoglycemia or diabetes. Glucokinase (GCK) plays a key role in beta cell glucose sensing. As one form of congenital hyperinsulinism (CHI), activating mutations of GCK result in a decreased threshold for glucose-stimulated insulin secretion and hypoglycemia. In contrast, inactivating mutations of GCK result in diabetes, including a mild form (MODY2) and a severe form (permanent neonatal diabetes mellitus (PNDM)). Mutations of beta cell ion channels involved in insulin secretion regulation also alter glucose sensing. Activating or inactivating mutations of ATP-dependent potassium (KATP ) channel genes result in severe but completely opposite clinical phenotypes, including PNDM and CHI. Mutations of the other ion channels, including voltage-gated potassium channels (Kv 7.1) and voltage-gated calcium channels, also lead to abnormal glucose sensing and CHI. Furthermore, amino acids can stimulate insulin secretion in a glucose-independent manner in some forms of CHI, including activating mutations of the glutamate dehydrogenase gene, HDAH deficiency, and inactivating mutations of KATP channel genes. These genetic defects have provided insight into a better understanding of the complicated nature of beta cell fuel-sensing mechanisms.

A dynamic pathway analysis approach reveals a limiting futile cycle in N-acetylglucosamine overproducing Bacillus subtilis.

Recent advances in genome engineering have further widened the gap between our ability to implement essentially any genetic change and understanding the impact of these changes on cellular function. We lack efficient methods to diagnose limiting steps in engineered pathways. Here, we develop a generally applicable approach to reveal limiting steps within a synthetic pathway. It is based on monitoring metabolite dynamics and simplified kinetic modelling to differentiate between putative causes of limiting product synthesis during the start-up phase of the pathway with near-maximal rates. We examine the synthetic N-acetylglucosamine (GlcNAc) pathway in Bacillus subtilis and find none of the acetyl-, amine- or glucose-moiety precursors to limit synthesis. Our dynamic metabolomics approach predicts an energy-dissipating futile cycle between GlcNAc6P and GlcNAc as the primary problem in the pathway. Deletion of the responsible glucokinase more than doubles GlcNAc productivity by restoring healthy growth of the overproducing strain.

Characterization of the heterozygous glucokinase knockout mouse as a translational disease model for glucose control in type 2 diabetes.

BACKGROUND AND PURPOSE: The global heterozygous glucokinase (GK) knockout (gk(wt/del)) male mouse, fed on a high-fat (60% by energy) diet, has provided a robust and reproducible model of hyperglycaemia. This model could be highly relevant to some facets of human type 2 diabetes (T2D). We aimed to investigate the ability of standard therapeutic agents to lower blood glucose at translational doses, and to explore the glucose-lowering potential of novel glucokinase activators (GKAs) in this model. EXPERIMENTAL APPROACH: We measured the ability of insulin, metformin, glipizide, exendin-4 and sitagliptin, after acute or repeat dose administration, to lower free-feeding glucose levels in gk(wt/del) mice. Further, we measured the ability of novel GKAs, GKA23, GKA71 and AZD6370 to control glucose either alone or in combination with some standard agents. KEY RESULTS: A single dose of insulin (1 unit·kg(-1)), metformin (150, 300 mg·kg(-1)), glipizide (0.1, 0.3 mg·kg(-1)), exendin-4 (2, 20 µg·kg(-1)) and GKAs reduced free-feeding glucose levels. Sitagliptin (10 mg·kg(-1)), metformin (300 mg·kg(-1)) and AZD6370 (30, 400 mg·kg(-1)) reduced glucose excursions on repeat dosing. At a supra-therapeutic dose (400 mg·kg(-1)), AZD6370 also lowered basal levels of glucose without inducing hypoglycaemia. CONCLUSION AND IMPLICATIONS: Standard glucose-lowering therapeutic agents demonstrated significant acute glucose lowering in male gk(wt/del) mice at doses corresponding to therapeutic free drug levels in man, suggesting the potential of these mice as a translatable model of human T2D. Novel GKAs also lowered glucose in this mouse model.

Chronic glucokinase activator treatment at clinically translatable exposures gives durable glucose lowering in two animal models of type 2 diabetes.

BACKGROUND AND PURPOSE: Pharmacological activation of glucokinase (GK) lowers blood glucose in animal models and humans, confirming proof of concept for this mechanism. However, recent clinical evidence from chronic studies suggests that the glucose-lowering effects mediated by glucokinase activators (GKAs) are not maintained in patients with type 2 diabetes (T2D). Existing preclinical data with GKAs do not explain this loss of sustained glucose-lowering efficacy in patients. Here, we have assessed the effects of chronic (up to 11 months) treatment with two different GKAs in two models of T2D. EXPERIMENTAL APPROACH: Two validated animal models of T2D, insulin-resistant obese Zucker rats and hyperglycaemic gk(wt/del) mice, were treated with two different GKAs for 1 or 11 months respectively at exposures that translate to clinical exposures in humans. Blood glucose, cholesterol, triglycerides and insulin were measured. GKA pharmacokinetics were also determined. KEY RESULTS: Treatment with either GKA provided sustained lowering of blood glucose for up to 1 month in the Zucker rat and up to 11 months in hyperglycaemic gk(wt/del) mice, with maintained compound exposures. This efficacy was achieved without increases in plasma or hepatic triglycerides, accumulation of hepatic glycogen or impairment of glucose-stimulated insulin secretion. CONCLUSIONS AND IMPLICATIONS: Chronic treatment with two GKAs in two animal models of diabetes provided sustained lowering of blood glucose, in marked contrast to clinical findings. Therefore, either these animal models of T2D are not good predictors of responses in human T2D or we need a better understanding of the consequences of GK activation in humans.

Glucokinase activation ameliorates ER stress-induced apoptosis in pancreatic ß-cells.

The derangement of endoplasmic reticulum (ER) homeostasis triggers ß-cell apoptosis, leading to diabetes. Glucokinase upregulates insulin receptor substrate 2 (IRS-2) expression in ß-cells, but the role of glucokinase and IRS-2 in ER stress has been unclear. In this study, we investigated the impact of glucokinase activation by glucokinase activator (GKA) on ER stress in ß-cells. GKA administration improved ß-cell apoptosis in Akita mice, a model of ER stress-mediated diabetes. GKA increased the expression of IRS-2 in ß-cells, even under ER stress. Both glucokinase-deficient Akita mice and IRS-2-deficient Akita mice exhibited an increase in ß-cell apoptosis, compared with Akita mice. ß-cell-specific IRS-2-overexpressing (ßIRS-2-Tg) Akita mice showed less ß-cell apoptosis than Akita mice. IRS-2-deficient islets were vulnerable, but ßIRS-2-Tg islets were resistant to ER stress-induced apoptosis. Meanwhile, GKA regulated the expressions of C/EBP homologous protein (CHOP) and other ER stress-related genes in an IRS-2-independent fashion in islets. GKA suppressed the expressions of CHOP and Bcl2-associated X protein (Bax) and protected against ß-cell apoptosis under ER stress in an ERK1/2-dependent, IRS-2-independent manner. Taken together, GKA ameliorated ER stress-mediated apoptosis by harmonizing IRS-2 upregulation and the IRS-2-independent control of apoptosis in ß-cells.

iPSC-derived ß cells model diabetes due to glucokinase deficiency.

Diabetes is a disorder characterized by loss of ß cell mass and/or ß cell function, leading to deficiency of insulin relative to metabolic need. To determine whether stem cell-derived ß cells recapitulate molecular-physiological phenotypes of a diabetic subject, we generated induced pluripotent stem cells (iPSCs) from subjects with maturity-onset diabetes of the young type 2 (MODY2), which is characterized by heterozygous loss of function of the gene encoding glucokinase (GCK). These stem cells differentiated into ß cells with efficiency comparable to that of controls and expressed markers of mature ß cells, including urocortin-3 and zinc transporter 8, upon transplantation into mice. While insulin secretion in response to arginine or other secretagogues was identical to that in cells from healthy controls, GCK mutant ß cells required higher glucose levels to stimulate insulin secretion. Importantly, this glucose-specific phenotype was fully reverted upon gene sequence correction by homologous recombination. Our results demonstrate that iPSC-derived ß cells reflect ß cell-autonomous phenotypes of MODY2 subjects, providing a platform for mechanistic analysis of specific genotypes on ß cell function.

Evolution of hepatic glucose metabolism: liver-specific glucokinase deficiency explained by parallel loss of the gene for glucokinase regulatory protein (GCKR).

BACKGROUND: Glucokinase (GCK) plays an important role in the regulation of carbohydrate metabolism. In the liver, phosphorylation of glucose to glucose-6-phosphate by GCK is the first step for both glycolysis and glycogen synthesis. However, some vertebrate species are deficient in GCK activity in the liver, despite containing GCK genes that appear to be compatible with function in their genomes. Glucokinase regulatory protein (GCKR) is the most important post-transcriptional regulator of GCK in the liver; it participates in the modulation of GCK activity and location depending upon changes in glucose levels. In experimental models, loss of GCKR has been shown to associate with reduced hepatic GCK protein levels and activity. METHODOLOGY/PRINCIPAL FINDINGS: GCKR genes and GCKR-like sequences were identified in the genomes of all vertebrate species with available genome sequences. The coding sequences of GCKR and GCKR-like genes were identified and aligned; base changes likely to disrupt coding potential or splicing were also identified. CONCLUSIONS/SIGNIFICANCE: GCKR genes could not be found in the genomes of 9 vertebrate species, including all birds. In addition, in multiple mammalian genomes, whereas GCKR-like gene sequences could be identified, these genes could not predict a functional protein. Vertebrate species that were previously reported to be deficient in hepatic GCK activity were found to have deleted (birds and lizard) or mutated (mammals) GCKR genes. Our results suggest that mutation of the GCKR gene leads to hepatic GCK deficiency due to the loss of the stabilizing effect of GCKR.

Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice.

Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. (13)C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

Characterization of the gene expression profile of heterozygous liver-specific glucokinase knockout mice at a young age.

In the liver, glucokinase (GCK) facilitates hepatic glucose uptake during hyperglycemia and is essential for the regulation of a network of glucose-responsive genes involved in glycolysis, glycogen synthesis, and lipogenesis. To better understand the consequences of changes in response to a liver-specific deficiency of GCK function, we examined the expression profiles of genes involved in glucose metabolism in the liver, pancreas, muscle and adipose tissue in heterozygous liver-specific Gck knockout (Gck(w/-)) mice. Our results showed that with the development of a liver GCK deficiency, significant decreases in the mRNA levels for insulin receptor and Glut2 were observed in the liver, and HkII in muscle, while glucagon mRNA increased markedly in the pancreas. The levels of circulating glucagon hormone levels increased with increased mRNA levels. Depite a decrease in muscle HkII levels, the hexokinase activity level did not change. Our findings suggest that in liver-specific Gck(w/-) mice, peripheral tissues use different strategies to tackle with hyperglycemia even at a young age. By identifying the specific changes that occur in different tissues at an early stage of glucokinase deficiency, potentially we can develop interventions to prevent further progression to diabetes.

Long-term renal changes in the liver-specific glucokinase knockout mouse: implications for renal disease in maturity-onset diabetes of the young 2.

To investigate the functional and structural renal changes in a long-term liver-specific glucokinase (gck) knockout mouse, a model was developed of maturity-onset diabetes of the young (MODY2). Hemizygous gck knockout mice, gck(w/-) groups, were compared at 6, 10, and 14 months with their age-matched normal littermates, gck(w/w) groups. To examine changes, we compared body weight, fasting blood glucose, serum insulin, and creatinine levels, as well as 24-h urine samples that were collected for urine volume and protein analysis between the 2 groups. Renal tissues were collected and stained with hemotoxylin-eosin and periodic-acid Schiff for light microscopic observation. The expression of renal transforming growth factor ß1 (TGF-ß1) was determined by Western blot. Our results show that fasting blood glucose levels were significantly higher in gck(w/-) mice compared with gck(w/w) mice (P < 0.01) for all age groups. Compared with age-matched gck(w/w) mice, 10-month old gck(w/-) mice have significantly elevated body weights (P < 0.01) and protein contents (P < 0.001). A gradual increase in mesangial matrix and a thickening of the glomerular basement membrane was observed in gck(w/-) mice at 10 and 14 months. The levels of renal TGF-ß1 expression are increasing in both gck(w/-) and gck(w/w) mice. Our results indicate that renal changes occur in the liver-specific gck knockout mouse model of MODY2 and suggest that TGF-ß1 may play a key role in pathogenesis of these renal changes.

Maturity onset diabetes of the young and pregnancy.

Three and a half decades after the clinical description of "Maturity Onset Diabetes of the Young" (MODY), and despite its low prevalence, important knowledge has been gathered concerning its genetic basis, molecular pathways, clinical phenotypes and pharmacogenetic issues. This knowledge has proved to be important not only for the attention of subjects carrying a mutation but also for the insight provided in Type 2 diabetes mellitus. In recent years, a shift from the term "MODY" to "monogenic diabetes" has taken place, the latter term being a better and more comprehensive descriptor. We stick to the "old" term because information on other types of monogenic diabetes and pregnancy is scarce. In this review we perform an overview of the entity, the prevalence rates reported in women with gestational diabetes mellitus and the specific impact of each type on pregnancy outcome.

The "other" forms of diabetes in children.

The large majority of children with diabetes seen by any pediatrician or in any pediatric diabetes clinic will fall into the two major categories of type 1 (T1DM) and, more recently in adolescents, type 2 (T2DM). But one has only to look at the diabetes classifications currently proposed by the American Diabetes Association or the World Health Organization to see that these forms account for only two of the many forms of diabetes listed. The goal of this chapter is to discuss, very briefly, the other classes of diabetes which occur in children and are of importance to physicians caring for adolescents.

Permanent neonatal diabetes caused by a homozygous nonsense mutation in the glucokinase gene.

Glucokinase deficiency is an unfrequent cause of permanent neonatal diabetes (PND), as only seven patients have been reported, either homozygous for a missense or frameshift mutation or compound heterozygous for both of them. We report here the first known case caused by a homozygous nonsense mutation (Y61X) in the glucokinase gene (GCK) that introduces a premature stop codon, generating a truncated protein that is predicted to be completely inactive as it lacks both the glucose- and the adenosine triphosphate-binding sites. The proband, born to consanguineous parents, was a full-term, intra-uterine growth-retarded male newborn who presented with a glycaemia of 129 mg/dL (7.16 mmol/L) on his second day of life, increasing thereafter up to 288 mg/dL (15.98 mmol/L) and 530 mg/dL (29.41 mmol/L) over the next 24 h, in the face of low serum insulin (<3 muIU/mL; <20.83 pmol/L). He was put on insulin on the third day of life. Insulin has never been discontinued since then. The patient was tested negative for anti-insulin and islet cell antibodies at age 5 months. His father had non-progressive, impaired fasting glucose for several years. The mother was found to be mildly hyperglycaemic only when her glucose was checked after the child was diagnosed. In conclusion, biallelic GCK loss should be considered as a potential cause of PND in children born to consanguineous parents, even if they are not known to be diabetic at the time of PND presentation.

Partial and whole gene deletion mutations of the GCK and HNF1A genes in maturity-onset diabetes of the young.

AIMS/HYPOTHESIS: Heterozygous mutations of glucokinase (GCK) and hepatocyte nuclear factor-1 alpha (HNF1A; also known as hepatic transcription factor 1 [TCF1]) genes are the most common cause of MODY. Genomic deletions of the HNF1B (also known as TCF2) gene have recently been shown to account for one third of mutations causing renal cysts and diabetes syndrome. We investigated the prevalence of partial and whole gene deletions in UK patients meeting clinical criteria for GCK or HNF-1alpha/-4alpha MODY and in whom no mutation had been identified by sequence analysis. METHODS: A multiplex ligation-dependent probe amplification (MLPA) assay was developed using synthetic oligonucleotide probes for 30 exons of the GCK, HNF1A and HNF4A genes. RESULTS: Partial or whole gene deletions were identified in 1/29 (3.5%) probands using the GCK MLPA assay and 4/60 (6.7%) of probands using the HNF1A/-4A MLPA assay. Four different deletions were detected: GCK exon 2, HNF1A exon 1, HNF1A exons 2 to 10 and HNF1A exons 1 to 10. An additional Danish pedigree with evidence of linkage to HNF1A had a deletion of exons 2 to 10. Testing other family members confirmed co-segregation of the deletion mutations with diabetes in the pedigrees. CONCLUSIONS/INTERPRETATION: Large deletions encompassing whole exons can cause GCK or HNF-1alpha MODY and will not be detected by sequencing. Gene dosage assays, such as MLPA, are a useful adjunct to sequence analysis when a diagnosis of MODY is strongly suspected.

Glucokinase and IRS-2 are required for compensatory beta cell hyperplasia in response to high-fat diet-induced insulin resistance.

Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of beta cell-specific Gck (Gck(+/-)) causes impaired insulin secretion to glucose, although the animals have a normal beta cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked beta cell hyperplasia, whereas Gck(+/-) mice demonstrated decreased beta cell replication and insufficient beta cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet-fed Gck(+/-) mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet-fed wild-type mice compared with those fed standard chow and reduced expression in HF diet-fed Gck(+/-) mice compared with those of HF diet-fed wild-type mice. HF diet-fed Irs2(+/-) mice failed to show a sufficient increase in beta cell mass, and overexpression of Irs2 in beta cells of HF diet-fed Gck(+/-) mice partially prevented diabetes by increasing beta cell mass. These results suggest that Gck and Irs2 are critical requirements for beta cell hyperplasia to occur in response to HF diet-induced insulin resistance.

Prevalence and clinical phenotype of the p.Val226Met glucokinase gene mutation in French Canadians in Quebec, Canada.

Our objectives were to describe the clinical phenotype of maturity-onset diabetes of the young (MODY) type 2 in a group of French Canadians and estimate its prevalence in this population. Index cases were identified by an abnormal fasting blood glucose (FBG) upon metabolic evaluation for dyslipidemia. Mutational analyses confirmed that all probands and affected family members were positive for the same glucokinase mutation, p.Val226Met. The prevalence of this mutation was estimated from a representative sample of French Canadians. Eleven individuals in 5 different families were diagnosed with MODY 2. Four of the five families originated from the same region in Quebec. In affected children (n = 6), the median age at diagnosis was 7.6 years (range = 2.9-9.4). All were asymptomatic. The range of FBG was 4.4-7.0 mmol/L; 5 out of the 6 pediatric patients had normal FBG values during the course of follow-up. One child presented with consistently normal FBG. Four of the adults who screened positive for MODY 2 had been previously misdiagnosed with type 2 DM, and one female had a history of gestational DM. The estimated prevalence of heterozygotes for the p.Val226Met mutation in French Canadians was 0.057% (95%CI 0.01-0.32%). In conclusion, this report presents the first confirmed case of MODY 2 with persistently normal FBG. In children and adolescents, a normal FBG does not allow for the exclusion of a MODY 2 diagnosis. Our results are consistent with a founder effect for the p.Val226Met glucokinase gene mutation in Quebec, Canada.