Hyperinsulinism in short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency reveals the importance of beta-oxidation in insulin secretion

Abstract

A female infant of nonconsanguineous Indian parents presented at 4 months with a hypoglycemic convulsion. Further episodes of hypoketotic hypoglycemia were associated with inappropriately elevated plasma insulin concentrations. However, unlike other children with hyperinsulinism, this patient had a persistently elevated blood spot hydroxybutyrylcarnitine concentration when fed, as well as when fasted. Measurement of the activity of L-3-hydroxyacyl-CoA dehydrogenase in cultured skin fibroblasts with acetoacetyl-CoA substrate showed reduced activity. In fibroblast mitochondria, the activity was less than 5% that of controls. Sequencing of the short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) genomic DNA from the fibroblasts showed a homozygous mutation (C773T) changing proline to leucine at amino acid 258. Analysis of blood from the parents showed they were heterozygous for this mutation. Western blot studies showed undetectable levels of immunoreactive SCHAD protein in the child's fibroblasts. Expression studies showed that the P258L enzyme had no catalytic activity. We conclude that C773T is a disease-causing SCHAD mutation. This is the first defect in fatty acid beta-oxidation that has been associated with hyperinsulinism and raises interesting questions about the ways in which changes in fatty acid and ketone body metabolism modulate insulin secretion by the beta cell. The patient's hyperinsulinism was easily controlled with diazoxide and chlorothiazide.

Figure 1

Analysis of butyl esters of carnitine and acylcarnitine species in a blood spot from FS. Peak identities are: m/z 218, free carnitine; 227, D9-carnitine; 260, acetyl-carnitine; 263, D3-acetylcarnitine; 277, D3-propionylcarnitine; 304, hydroxybutyrylcarnitine; 347, D3-octanoylcarnitine; 456, palmitoylcarnitine ; 459, D3-palmitoylcarnitine; and 482, oleoylcarnitine.

Figure 2

Western blot analysis of SCHAD in fibroblasts. Fibroblast homogenates (100 μg protein) were analyzed by SDS-PAGE, electroblotted, and visualized by reaction with rabbit anti-SCHAD and ECL+. The results were compared with purified pig heart SCHAD, bovine liver SCHAD, and a rat liver mitochondrial preparation (protein loadings shown).

Figure 3

Restriction fragment length analysis of DNA fragments containing nt 773 of the SCHAD coding sequence. DNA from FS, her parents, and controls was amplified using primers for exon 7 (Table 2). The PCR product was analyzed on a 2% agarose gel, either untreated (lane 1) or after exposure to ApaI for 2 hours (lanes 2–5). The C773T mutation abrogates an ApaI restriction site present in the normal sequence. ApaI digestion of the DNA fragment from controls yields two bands of 121 and 171 nt (asterisk). Exposure of the mutated sequence to ApaI yields a single band of 292 nt (arrow). Lane 1, patient DNA fragment (untreated); lane 2, DNA fragment of the father after ApaI treatment; lane 3, DNA fragment of the mother after ApaI treatment; lane 4, DNA fragment of the patient treated with ApaI; lane 5, DNA fragment of a control treated with ApaI. The pattern of the DNA fragments of the parents reveals the presence of both the normal and the mutated sequence.

Figure 4

Diagram of aligned SCHAD sequences from several evolutionarily distant species. Pro258 (in bold), which is replaced by Leu in the DNA of FS, is completely conserved in the SCHAD coding sequences from different species, including bacteria. Human, NP_005318; Pig, AAD20939; Caenorhabditis elegans, P41938; Clostridium acetobutylicum, AAA95971.

Figure 5

Expression of wild-type and C773T mutant SCHAD using a reticulocyte lysate system. (a) Western blot showing no protein with vector alone, immunoreactive SCHAD protein with an apparent molecular weight of 35 kDa with the wild-type SCHAD construct, and immunoreactive SCHAD protein with a slightly lower apparent molecular weight with the C773T mutant construct. (b) SCHAD enzyme activity obtained with expression of the wild-type and C773T mutant SCHAD proteins. Enzyme activity is equal to the nanomole substrate converted per microliter of reticulocyte lysate between 10 and 30 minutes’ incubation.

Figure 6

The two postulated pathways for β cell signaling (from ref. 14). PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; CL, citrate lyase; CPT1, carnitine palmitoyl transferase 1; m, mitochondrial; c, cytosolic; AcCoa, acetyl-CoA; LCFA-CoA, long-chain fatty acyl-CoA.