Congenital hyperinsulinism caused by hexokinase I expression or glucokinase-activating mutation in a subset of β-cells

Abstract

Congenital hyperinsulinism causes persistent hypoglycemia in neonates and infants. Most often, uncontrolled insulin secretion (IS) results from a lack of functional K(ATP) channels in all β-cells or only in β-cells within a resectable focal lesion. In more rare cases, without K(ATP) channel mutations, hyperfunctional islets are confined within few lobules, whereas hypofunctional islets are present throughout the pancreas. They also can be cured by selective partial pancreatectomy; however, unlike those with a K(ATP) focal lesion, they show clinical sensitivity to diazoxide. Here, we characterized in vitro IS by fragments of pathological and adjacent normal pancreas from six such cases. Responses of normal pancreas were unremarkable. In pathological region, IS was elevated at 1 mmol/L and was further increased by 15 mmol/L glucose. Diazoxide suppressed IS and tolbutamide antagonized the inhibition. The most conspicuous anomaly was a large stimulation of IS by 1 mmol/L glucose. In five of six cases, immunohistochemistry revealed undue presence of low-K(m) hexokinase-I in β-cells of hyperfunctional islets only. In one case, an activating mutation of glucokinase (I211F) was found in pathological islets only. Both abnormalities, attributed to somatic genetic events, may account for inappropriate IS at low glucose levels by a subset of β-cells. They represent a novel cause of focal congenital hyperinsulinism.

FIG. 1.

Morphological mosaicism of the islets. Intraoperative diagnosis using frozen sections stained with toluidine blue is based on the coexistence of small islets containing β-cells with scanty cytoplasm (A) and hyperplastic islets containing cytoplasm-rich β-cells (B). Representative sections from the pancreas of case 3. Scale bar, 50 µm.

FIG. 2.

Effects of glucose and drugs acting on K_ATP channels on insulin secretion by normal (closed circles) and pathological pancreas (open circles) from each of the six studied cases. Normal tissue was not available for case 4. The concentration of glucose (G) was increased from 1 to 15 mmol/L, and 100 μmol/L diazoxide (Dz) and 100 μmol/L tolbutamide (Tolb) were added, as indicated at the top of the figures. Forskolin (Fk, 1 μmol/L) was present throughout to activate cAMP formation.

FIG. 3.

Effects of stepwise increases and decreases in glucose concentration (G in mmol/L) on insulin secretion by normal (closed circles) and pathological pancreas (open circles) from each of the six studied cases. There was no period in G0 at the start or the end of the experiment with tissue from cases 1 and 2. Normal tissue was not available for case 4. The low insulin content of normal fragments from case 6 makes the results uncertain. Forskolin (Fk, 1 μmol/L) was present throughout.

FIG. 4.

Effects of 25 μmol/L tolbutamide (Tolb) and 5 mmol/L inosine on insulin secretion by normal (closed circles) and pathological pancreas (open circles) from three cases. The experiments were started in the absence of glucose (G0), which was added at 1 mmol/L (G1) at 60 min. Forskolin (Fk, 1 μmol/L) was present throughout.

FIG. 5.

Effects of 10 mmol/L lactate, pyruvate, and phenylpyruvate (Phenylpyr) on insulin secretion by normal (closed circles) and pathological pancreas (open circles) from three cases. The experiments were performed in the presence of 1 mmol/L glucose (G1). Forskolin (Fk, 1 μmol/L) was present throughout.

FIG. 6.

A: Presence of HK-I in islets of the pathological region in pancreas of case 3. Hyperfunctional islets were positive for HK-I immunostaining, whereas hypofunctional islets (highlighted by a dotted line) were negative in an adjacent normal lobule. Scale bar, 200 µm. Immunolabeling of insulin (B and C) and HK-I (D and E) in the same islets from the normal (B and D) and pathological (C and E) regions of the pancreas of case 1. HK-I is present in β-cells of pathological islets and is absent in β-cells of normal islets. Scale bar, 50 µm. Note that in the exocrine pancreas, centro-acinar cells and vessel walls are more strongly labeled than acinar cells (also see Supplementary Fig. 2).

FIG. 7.

Electropherograms identifying a GCK mutation in case 6. RNA was extracted from paraffin-embedded slices containing hyperfunctional or hypofunctional islets and was reverse-transcribed. cDNA was then amplified and sequenced in both forward and reverse directions as shown. Both wild-type and mutated cDNA (Ile211Phe) were found in the pathological region, whereas only wild-type cDNA was identified in the normal region.