Limits...
Extremes of clinical and enzymatic phenotypes in children with hyperinsulinism caused by glucokinase activating mutations.

Sayed S, Langdon DR, Odili S, Chen P, Buettger C, Schiffman AB, Suchi M, Taub R, Grimsby J, Matschinsky FM, Stanley CA - Diabetes (2009)

Bottom Line: Kinetic analysis of the enzymes included determinations of stability, activity index, the response to glucokinase activator drug, and the effect of glucokinase regulatory protein.Diazoxide treatment was effective in child 3 but ineffective in child 1 and only partially effective in child 2.Allosteric responses to inhibition by glucokinase regulatory protein and activation by the drug RO0281675 were impaired by the ins454A but unaffected by the M197I mutation.

View Article: PubMed Central - PubMed

Affiliation: Clinical Translational Research Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

ABSTRACT

Objective: Heterozygous activating mutations of glucokinase have been reported to cause hypoglycemia attributable to hyperinsulinism in a limited number of families. We report three children with de novo glucokinase hyperinsulinism mutations who displayed a spectrum of clinical phenotypes corresponding to marked differences in enzyme kinetics.

Research design and methods: Mutations were directly sequenced, and mutants were expressed as glutathionyl S-transferase-glucokinase fusion proteins. Kinetic analysis of the enzymes included determinations of stability, activity index, the response to glucokinase activator drug, and the effect of glucokinase regulatory protein.

Results: Child 1 had an ins454A mutation, child 2 a W99L mutation, and child 3 an M197I mutation. Diazoxide treatment was effective in child 3 but ineffective in child 1 and only partially effective in child 2. Expression of the mutant glucokinase ins454A, W99L, and M197I enzymes revealed a continuum of high relative activity indexes in the three children (26, 8.9, and 3.1, respectively; wild type = 1.0). Allosteric responses to inhibition by glucokinase regulatory protein and activation by the drug RO0281675 were impaired by the ins454A but unaffected by the M197I mutation. Estimated thresholds for glucose-stimulated insulin release were more severely reduced by the ins454A than the M197I mutation and intermediate in the W99L mutation (1.1, 3.5, and 2.2 mmol/l, respectively; wild type = 5.0 mmol/l).

Conclusions: These results confirm the potency of glucokinase as the pancreatic beta-cell glucose sensor, and they demonstrate that responsiveness to diazoxide varies with genotype in glucokinase hyperinsulinism resulting in hypoglycemia, which can be more difficult to control than previously believed.

Show MeSH

Related in: MedlinePlus

Calculated thresholds for glucose-stimulated insulin release (GSIR) in activating and inactivating mutations of glucokinase. Thresholds are plotted against the inverse of the mutant enzyme activity index relative to wild-type (WT) enzyme. Because relative expression of the glucokinase forms is affected by enzyme affinity for glucose and the ambient glucose concentration, the wild-type enzyme dominates the estimated threshold for the heterozygous inactivating defects, but the mutant enzyme dominates the threshold for activating mutations. Thus, the threshold for inactivating mutations plateaus at ∼7 mmol/l, whereas the calculated threshold for severe activating mutations approaches zero as the relative activity increases. For purposes of consistency, all kinetic data in the figure are from the laboratory of F.M.M. Threshold and activity indexes were calculated per Gloyn et al. (24). ●, glucokinase hyperinsulinism mutations; ○, MODY2 mutations; X, wild type. GK, glucokinase.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2682682&req=5

Figure 5: Calculated thresholds for glucose-stimulated insulin release (GSIR) in activating and inactivating mutations of glucokinase. Thresholds are plotted against the inverse of the mutant enzyme activity index relative to wild-type (WT) enzyme. Because relative expression of the glucokinase forms is affected by enzyme affinity for glucose and the ambient glucose concentration, the wild-type enzyme dominates the estimated threshold for the heterozygous inactivating defects, but the mutant enzyme dominates the threshold for activating mutations. Thus, the threshold for inactivating mutations plateaus at ∼7 mmol/l, whereas the calculated threshold for severe activating mutations approaches zero as the relative activity increases. For purposes of consistency, all kinetic data in the figure are from the laboratory of F.M.M. Threshold and activity indexes were calculated per Gloyn et al. (24). ●, glucokinase hyperinsulinism mutations; ○, MODY2 mutations; X, wild type. GK, glucokinase.

Mentions: Figure 5 shows the effects of different glucokinase activating mutations on predicted glucose threshold for insulin release, based on the relative activity indexes of the expressed proteins in vitro. Cases reported to be diazoxide unresponsive (ins454A and Y214C) have very low glucose thresholds, as does the A456V mutation, which in one patient was not completely responsive to diazoxide. In contrast, the better responses to diazoxide in child 2 and child 3 correlate with higher calculated glucose thresholds. It should be noted that the range of plasma glucose levels in our three patients and in the reports of other glucokinase hyperinsulinism cases (Table 3) tends to be higher than their predicted glucose thresholds (Fig. 5). This may partly reflect the effects of counterregulatory responses to hypoglycemia. An additional potential problem in correlating data on glucokinase kinetics with clinical features in patients is that some of the changes in enzyme properties exert opposing effects. One example of this phenomenon is the V62M mutation, which has been associated with MODY2 diabetes (36). When expressed in vitro, this mutation has an increased activity index consistent with causing hypoglycemia, rather than diabetes. The increased instability of this mutant form of glucokinase may counterbalance its enhanced activity and explain why the mutation results in a net loss of function in vivo (36). Similarly, although the ins454A, W99L, and M197I mutations have increased activity indexes, they also have slightly reduced stability. Moreover, the impact of the reduced affinity of M197I for ATP in vivo is uncertain. Given these problems, efforts to understand the in vivo and in vitro phenotypes of glucokinase hyperinsulinism mutations will require that accurate data be obtained on the clinical features of affected individuals. For example, these data should especially include careful documentation of the ability of diazoxide treatment to completely normalize plasma glucose levels and accurate estimates of the in vivo glucose “set point,” as illustrated in Figs. 1 and 2.


Extremes of clinical and enzymatic phenotypes in children with hyperinsulinism caused by glucokinase activating mutations.

Sayed S, Langdon DR, Odili S, Chen P, Buettger C, Schiffman AB, Suchi M, Taub R, Grimsby J, Matschinsky FM, Stanley CA - Diabetes (2009)

Calculated thresholds for glucose-stimulated insulin release (GSIR) in activating and inactivating mutations of glucokinase. Thresholds are plotted against the inverse of the mutant enzyme activity index relative to wild-type (WT) enzyme. Because relative expression of the glucokinase forms is affected by enzyme affinity for glucose and the ambient glucose concentration, the wild-type enzyme dominates the estimated threshold for the heterozygous inactivating defects, but the mutant enzyme dominates the threshold for activating mutations. Thus, the threshold for inactivating mutations plateaus at ∼7 mmol/l, whereas the calculated threshold for severe activating mutations approaches zero as the relative activity increases. For purposes of consistency, all kinetic data in the figure are from the laboratory of F.M.M. Threshold and activity indexes were calculated per Gloyn et al. (24). ●, glucokinase hyperinsulinism mutations; ○, MODY2 mutations; X, wild type. GK, glucokinase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682682&req=5

Figure 5: Calculated thresholds for glucose-stimulated insulin release (GSIR) in activating and inactivating mutations of glucokinase. Thresholds are plotted against the inverse of the mutant enzyme activity index relative to wild-type (WT) enzyme. Because relative expression of the glucokinase forms is affected by enzyme affinity for glucose and the ambient glucose concentration, the wild-type enzyme dominates the estimated threshold for the heterozygous inactivating defects, but the mutant enzyme dominates the threshold for activating mutations. Thus, the threshold for inactivating mutations plateaus at ∼7 mmol/l, whereas the calculated threshold for severe activating mutations approaches zero as the relative activity increases. For purposes of consistency, all kinetic data in the figure are from the laboratory of F.M.M. Threshold and activity indexes were calculated per Gloyn et al. (24). ●, glucokinase hyperinsulinism mutations; ○, MODY2 mutations; X, wild type. GK, glucokinase.
Mentions: Figure 5 shows the effects of different glucokinase activating mutations on predicted glucose threshold for insulin release, based on the relative activity indexes of the expressed proteins in vitro. Cases reported to be diazoxide unresponsive (ins454A and Y214C) have very low glucose thresholds, as does the A456V mutation, which in one patient was not completely responsive to diazoxide. In contrast, the better responses to diazoxide in child 2 and child 3 correlate with higher calculated glucose thresholds. It should be noted that the range of plasma glucose levels in our three patients and in the reports of other glucokinase hyperinsulinism cases (Table 3) tends to be higher than their predicted glucose thresholds (Fig. 5). This may partly reflect the effects of counterregulatory responses to hypoglycemia. An additional potential problem in correlating data on glucokinase kinetics with clinical features in patients is that some of the changes in enzyme properties exert opposing effects. One example of this phenomenon is the V62M mutation, which has been associated with MODY2 diabetes (36). When expressed in vitro, this mutation has an increased activity index consistent with causing hypoglycemia, rather than diabetes. The increased instability of this mutant form of glucokinase may counterbalance its enhanced activity and explain why the mutation results in a net loss of function in vivo (36). Similarly, although the ins454A, W99L, and M197I mutations have increased activity indexes, they also have slightly reduced stability. Moreover, the impact of the reduced affinity of M197I for ATP in vivo is uncertain. Given these problems, efforts to understand the in vivo and in vitro phenotypes of glucokinase hyperinsulinism mutations will require that accurate data be obtained on the clinical features of affected individuals. For example, these data should especially include careful documentation of the ability of diazoxide treatment to completely normalize plasma glucose levels and accurate estimates of the in vivo glucose “set point,” as illustrated in Figs. 1 and 2.

Bottom Line: Kinetic analysis of the enzymes included determinations of stability, activity index, the response to glucokinase activator drug, and the effect of glucokinase regulatory protein.Diazoxide treatment was effective in child 3 but ineffective in child 1 and only partially effective in child 2.Allosteric responses to inhibition by glucokinase regulatory protein and activation by the drug RO0281675 were impaired by the ins454A but unaffected by the M197I mutation.

View Article: PubMed Central - PubMed

Affiliation: Clinical Translational Research Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

ABSTRACT

Objective: Heterozygous activating mutations of glucokinase have been reported to cause hypoglycemia attributable to hyperinsulinism in a limited number of families. We report three children with de novo glucokinase hyperinsulinism mutations who displayed a spectrum of clinical phenotypes corresponding to marked differences in enzyme kinetics.

Research design and methods: Mutations were directly sequenced, and mutants were expressed as glutathionyl S-transferase-glucokinase fusion proteins. Kinetic analysis of the enzymes included determinations of stability, activity index, the response to glucokinase activator drug, and the effect of glucokinase regulatory protein.

Results: Child 1 had an ins454A mutation, child 2 a W99L mutation, and child 3 an M197I mutation. Diazoxide treatment was effective in child 3 but ineffective in child 1 and only partially effective in child 2. Expression of the mutant glucokinase ins454A, W99L, and M197I enzymes revealed a continuum of high relative activity indexes in the three children (26, 8.9, and 3.1, respectively; wild type = 1.0). Allosteric responses to inhibition by glucokinase regulatory protein and activation by the drug RO0281675 were impaired by the ins454A but unaffected by the M197I mutation. Estimated thresholds for glucose-stimulated insulin release were more severely reduced by the ins454A than the M197I mutation and intermediate in the W99L mutation (1.1, 3.5, and 2.2 mmol/l, respectively; wild type = 5.0 mmol/l).

Conclusions: These results confirm the potency of glucokinase as the pancreatic beta-cell glucose sensor, and they demonstrate that responsiveness to diazoxide varies with genotype in glucokinase hyperinsulinism resulting in hypoglycemia, which can be more difficult to control than previously believed.

Show MeSH
Related in: MedlinePlus