Abstract
The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.
Highlights
Intermediate elevations of the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD)/ glutamate dehydrogenase (GDH) molar ratio did not affect the Km for ␣-ketoglutarate. This may explain why the inhibitory effect of SCHAD on GDH was only found in islets, but not in kidney and liver, because islets contained the highest ratio of SCHAD to GDH in both gene expression and enzyme activity. These studies of mice with disruption of the SCHAD gene demonstrate that they have a dysregulation of insulin secretion similar to the phenotype of hyperinsulinemic hypoglycemia reported in children with recessive inactivating mutations of HADH
These findings indicate that hyperinsulinism in SCHAD-deficient mice is caused by loss of a “moonlighting” function of the SCHAD protein, which normally provides a direct inhibitory effect on GDH enzymatic activity in pancreatic -cells (Fig. 7)
A total of five families has been reported with hyperinsulinism associated with SCHAD deficiency, and we are aware of three additional unpublished cases [2,3,4, 23]
Summary
SCHAD (hadh) Gene Targeting—hadhϪ/Ϫ mice were generated by homologous recombination in R1 mouse embryonic stem (ES) cells. RT-PCR and Western Blot Analysis—For RT-PCR analysis, total RNA was isolated from fresh tissue, including pancreatic islets, liver, kidney, and brain using TRIzol (Invitrogen). Western blots were performed on whole tissue protein extracts (pancreatic islets, liver, kidney, and brain) to detect SCHAD. SCHAD and GDH Gene Expression—Total RNA were isolated from islets, liver, and kidney using the TRIzol (Invitrogen) method. GDH and SCHAD Enzyme Activity—Tissue (freshly isolated islets, liver, and kidney) was homogenized in phosphate buffer (5 mM Na2HPO4, 5 mM K2HPO4, 1 mM EDTA, 1% Triton X-100, pH 7.4). Liver tissue (0.5 g wet weight) from hadhϩ/ϩ and hadhϪ/Ϫ mice was homogenized using 20 volumes of mammalian protein extraction reagent (M-PER, Pierce) and centrifuged at 2000 ϫ g for 10 min. Islets from hadhϪ/Ϫ and hadhϩ/ϩ mice were homogenized using PBS buffer plus 1% Triton.
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