Phenotypic Characterization of Congenital Hyperinsulinism Due to Novel Activating Glucokinase Mutations

Abstract

The importance of glucokinase (GK) in the regulation of insulin secretion has been highlighted by the phenotypes of individuals with activating and inactivating mutations in the glucokinase gene (GCK). Here we report 10 individuals with congenital hyperinsulinism (HI) caused by eight unique activating mutations of GCK. Six are novel and located near previously identified activating mutations sites. The first recognized episode of hypoglycemia in these patients occurred between birth and 24 years, and the severity of the phenotype was also variable. Mutant enzymes were expressed and purified for enzyme kinetics in vitro. Mutant enzymes had low glucose half-saturation concentration values and an increased enzyme activity index compared with wild-type GK. We performed functional evaluation of islets from the pancreata of three children with GCK-HI who required pancreatectomy. Basal insulin secretion in perifused GCK-HI islets was normal, and the response to glyburide was preserved. However, the threshold for glucose-stimulated insulin secretion in perifused glucokinase hyperinsulinism (GCK-HI) islets was decreased, and glucagon secretion was greatly suppressed. Our evaluation of novel GCK disease-associated mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response.

Article highlights: Our evaluation of six novel and two previously published activating GCK mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. These studies provide insights into the pathophysiology of GCK-hyperinsulinism and the dual role of glucokinase in β-cells and α-cells to regulate glucose homeostasis.

Figure 1

Schematic of GK. Location of activating mutations in relation to the catalytic site (glucose) and allosteric site (GKA) in the open conformation based on GK structure model 3VEV.

Figure 2

Pedigrees of eight families with HI associated with mutations in GCK. Arrows indicate probands. Circles, females; squares, males; diamonds, multiple individuals of unspecified sex. Black shapes, hypoglycemia diagnosed; gray shapes, hypoglycemia suspected. n/M, mutation positive; n/n mutation negative. In family no. 2, (*) indicates carriers of a novel intronic GCK variant (c.45 + 4t>a).

Figure 3

Effect of GCK-HI associated GCK mutations on enzyme activity. WT GK, eight GCK-HI mutants, and a MODY2 mutant were expressed and GST-GCK was purified. Glucose dose-dependent GCK enzyme activity was evaluated based on NADP⁺/NADPH-coupled enzyme assay. The vertical lines indicate glucose S_0.5 values.

Figure 4

GKRP inhibition of GK activity in the p.V91L and p.S69_E70insVPL GCK-HI mutants. GK enzyme activity of GK mutants (p.V91L and p.S69_E70insVPL) was assessed in the presence of different molar ratio of GK to GKRP (1:1 and 1:2) at glucose (G) concentrations of 3 mmol/L (A and D), 6 mmol/L (B and E), and 12 mmol/L (C and F). The error bars represent ± SEM. Statistics were calculated using single-factor ANOVA (n = 4–5). *P < 0.001; #P < 0.05 compared with WT.

Figure 5

Functional evaluation of insulin secretion of islets isolated from GCK-HI patients. Perifusion of primary islets isolated from surgical pancreatic specimens from GCK-HI patients (W99C, S69_E70insVPL, or R447L) to assess insulin release compared with normal control subjects. Stimulated with glucose ramp (0–25 mmol/L) and KCl (30 mmol/L) (A), AAM ramp (0–12 mmol/L) and KCl (30 mmol/L) (B), or glyburide (0.3 μmol/L) (C). Glucose threshold is indicated by dashed vertical lines.

Figure 6

Glucagon and insulin secretion in GCK-HI islets in presence of amino acids and glucose. Glucagon and insulin release were measured simultaneously in perifused normal human islets and islets isolated from GCK-HI patients (p.W99C, p.S69_E70insVPL, or p.R447L). Each perifusion used 1,000 islets, and data are presented at ng/100 islets/min for insulin and pg/100 islets/min for glucagon. Islets were perifused first with AAM (4 mmol/L) alone and then in combination with increasing glucose (G) concentration steps as denoted in WT control (A), p.S69_E70insVPL (B), p.W99C (C), and p.R447L (D).