FS suffered frequent, severe hypoglycemia. Three hypoglycemia screens indicated definite HI (blood glucose <2.6 mM, plasma insulin >3 mU/l; ref. 20
) as did the glucose requirement of 8 mg/kg/min (although many hyperinsulinemic infants require more than 15 mg/kg/min; ref. 18
). The episode of hypoglycemia that followed the glucagon test was not attributable to HI but rather suggested impaired fatty acid oxidation. (The pathogenesis of hypoglycemia in FAOD is probably multifactorial. NEFA cannot be used and blood concentrations of ketone bodies are low, so glucose utilization is increased; however, in addition, gluconeogenesis is probably impaired because acetyl-CoA concentrations in the liver are low.) Treatment with diazoxide and chlorothiazide led to a dramatic improvement in glucose homeostasis, providing further evidence that HI was the main cause of hypoglycemia.
The possibility of impaired fatty acid oxidation was investigated by examination of the blood acylcarnitine profile. All samples showed an elevated concentration of a carnitine species with an m/z
ratio of 304. This could be due to a hydroxybutyryl-carnitine or isomeric carnitine species. A similar peak has been reported in children with ketosis, so we had to prove that FS had levels of this compound that were higher than those seen in ketosis even when FS had been fed and was not ketotic (Table ). A number of identities for the “hydroxybutyrylcarnitine” were considered along with theories as to why it might be accumulating. The hypotheses were accumulation of (a) L
-3-hydroxybutyrylcarnitine due to SCHAD deficiency; (b) 3-hydroxyisobutyrylcarnitine due to 3-hydroxyisobutyryl-CoA deacylase deficiency; or (c) D
-3-hydroxybutyrylcarnitine due to a ketone body utilization defect (KBUD). The SCHAD hypothesis was consistent with a NEFA/D
-3-hydroxybutyrate ratio toward the upper limit of normal. The deacylase hypothesis seemed unlikely upon comparison of FS with the case described previously (25
). Plasma concentrations of D
-3-hydroxybutyrate did not suggest a KBUD.
Rather than prove that the compound in the blood was L
-3-hydroxybutyrylcarnitine and that the urine contained excess L
-3-hydroxybutyrate (which is indistinguishable from D
-3-hydroxybutyrate on simple organic acid analysis), it was decided to move directly to an assay of SCHAD. SCHAD is expressed in many tissues (26
) and has previously been assayed in fibroblasts. Clear evidence of reduced SCHAD activity was seen in fibroblasts from FS. There was no impairment in hydrogenation of 3-ketooctanoyl-CoA, a medium-chain substrate, in keeping with our organic acid and acylcarnitine analyses but at variance with the evidence of impairment in medium-chain 3-hydroxyacyl-CoA dehydrogenase activity in an SCHAD-knockout mouse and in another SCHAD-deficient patient (10
There was high residual SCHAD activity in the patient’s fibroblasts, which was surprising, as Western blots indicated that the fibroblasts contained no immunoreactive protein. There was no fall in residual enzyme activity in the patient’s fibroblasts after immunoprecipitation of residual SCHAD protein. The residual activity could be due to several enzymes: (a) Trifunctional protein HAD activity. This is unlikely because this enzyme has low activity toward acetoacetyl-CoA (27
) and residual SCHAD activity was detectable after immunoprecipitation of SCHAD protein from an LCHAD-deficient cell-line. (b) Peroxisomal L
- and D
-3-hydroxyacyl-CoA dehydrogenases (28
). (c) Presence of type II 3-hydroxyacyl-CoA dehydrogenase (29
) or human brain multifunctional dehydrogenase (30
) in fibroblasts. (d) Short-chain 2-methy -hydroxyacyl-CoA dehydrogenase (31
). Using 2-methylacetoacetyl-CoA as substrate, activity of this enzyme was low (0.32 mU/mg protein in control fibroblasts, 0.42 mU/mg in FS). Because it is only half as active toward straight chain as toward 2-methylacyl-CoA esters (31
), it is unlikely, however, that this enzyme was responsible for the residual activity.
Given that the residual activity was substantially reduced when a mitochondrial preparation was used, it seems likely that it was due to peroxisomal enzyme(s).
Sequencing of the Schad
gene from fibroblasts showed that FS is homozygous for a C773T mutation that leads to a change from proline to leucine at amino acid 258 (246 in the mature protein after cleavage of the mitochondrial targeting sequence); the parents are heterozygotes. Several lines of evidence indicate that C773T is responsible for the reduction in SCHAD activity and is a disease-causing mutation: (a) No other difference from the wild-type sequence was detected in the any of the eight exons of Schad
from FS. (b) The C773T mutation was not found in 200 control chromosomes, including 100 from individuals with the same ethnic background, indicating that the mutation is not a common polymorphism. (c) As shown in Figure , Pro258 is part of a highly conserved amino acid region, and the equivalent of Pro258 is present in SCHAD of all species investigated. (d) Analysis of the crystal structure of SCHAD reveals that Pro258 is the starting point of one of the α-helices (α12) of the C-terminal domain (32
). Barycki et al. argue that the orientation of these α-helices relative to one another is critical for enzyme function. Replacement of the proline at the starting point of an α-helix by leucine is likely to prevent normal protein folding. This could lead to rejection by the chaperonin system (leading to destruction of the nascent protein) or to synthesis of a protein with reduced catalytic activity.
The Western blots indicated reduced immunoreactive SCHAD protein in fibroblasts. This suggests that the P258L substitution alters the tertiary structure of the nascent enzyme to such a degree that it is not recognized by chaperonins and is destroyed. This phenomenon has been described for point mutations in short-chain acyl-CoA dehydrogenase (33
Additional evidence that the C773T mutation in the SCHAD coding sequence is responsible for the patient’s disease is provided by the functional assay after in vitro expression of the mutated protein. We included 81 nucleotides of the 5′UTR, in addition to the complete coding sequence, as a template for the expression of the SCHAD protein, to ensure protein formation starting from the translation start site. It has been shown that absence of the sequences upstream of the translation start codon may lead to aberrant choice of translation start sites by the in vitro expression system (34
). The SCHAD protein expressed from the wild-type construct had an apparent molecular mass of the expected size. The mutated sequence yielded a comparable amount of protein with a slightly lower apparent molecular mass as determined by SDS-PAGE. This is probably due to the P258L substitution The protein folding introduced by proline in the wild-type sequence cannot be undone by denaturing the protein, so the wild-type protein may exhibit a different migration pattern in a denaturing gel than the mutant protein. The complete absence of SCHAD activity of the in vitro synthesized protein harboring the P258L substitution further corroborates the link between the mutation and the patient’s disease.
The acylcarnitine profile in blood from FS was unique with regard to the presence of more than 0.6 μM of hydroxybutyrylcarnitine. However, it was also abnormal to the extent that the acetylcarnitine concentration was usually above the normal range. Comparison with other hyperinsulinemic children suggested that elevation of blood spot acetylcarnitine is caused by HI rather than being a specific effect of SCHAD deficiency. How could HI lead to an elevated concentration of acetylcarnitine in blood? Insulin leads to increased production of acetyl-CoA from glucose in muscle and it is likely that when acetyl-CoA is being produced by pyruvate dehydrogenase at a rate that exceeds its removal in the Krebs cycle, it is buffered by conversion to acetylcarnitine.
The hypoglycemia observed in FS was not typical of a FAOD; it was not easily provoked by a prolonged fast, but, rather, occurred in an unpredictable fashion, often 2–6 hours after a feed. On some occasions the plasma insulin level was elevated, but on others, it was normal and the NEFA/D
-3-hydroxybutyrate ratio was at the upper limit of normal (a supranormal value is typical of hypoglycemia due to a FAOD). Although it is conceivable that FS has two separate genetic defects, one in the Schad
gene and another causing HI, we believe it more likely that the absence of SCHAD activity in the β cell causes inappropriate insulin secretion; SCHAD is abundant in the β cell (3
) because it fulfills an important regulatory function.
The postulated role of lipid signaling in insulin secretion is shown in Figure (right); the pathway that triggers secretion via the increased ATP/ADP ratio is shown on the left (14
). Fatty acids are a major energy source for unstimulated islets. An early change caused by glucose is a shift from fatty acids to glucose as fuel. It is proposed that this occurs through conversion of glucose to malonyl-CoA, which inhibits carnitine palmitoyl transferase I (CPT I) and blocks the entry of long chain fatty acyl CoA (LCFA-CoA) into the mitochondrion. Thus LCFA-CoA is converted instead into diacylglycerol, triglycerides, fatty acids, and acylated proteins. LCFA-CoA or the complex lipids derived from them are potent regulators of enzymes, ion channels, and signal-transducing effectors. It is proposed that the complex lipids or acylated proteins augment insulin secretion by a KATP
-independent mechanism. Unequivocal evidence for such a lipid-linked signaling mechanism is lacking, but there is experimental evidence for a KATP
-independent mechanism that augments insulin secretion in response to glucose (13
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 (more ...)
What effects would FAOD in general, and SCHAD deficiency in particular, have on the lipid signaling pathway? Any defect leading to accumulation of cytosolic LCFA-CoA and increased esterification could induce inappropriate insulin secretion. Accumulation of triglyceride, particularly in liver, occurs in LCHAD deficiency, in very long chain acyl-CoA dehydrogenase (VLCAD) deficiency, and in medium chain acyl-CoA dehydrogenase (MCAD) deficiency during fasting-induced decompensation (35
). However, our data do not suggest that these FAOD produce HI (Table ). It is possible that in SCHAD deficiency, accumulation of short chain acyl-CoA esters in the mitochondrion causes insulin secretion by inhibition of CPT I. The isoform of CPT I in the β cell is the same as that in the liver (36
). This enzyme, located in the outer mitochondrial membrane, has one inhibition site that faces the cytosol and is inhibited by dicarboxylic CoA esters (physiologically by malonyl-CoA) and a second inhibition site, facing the intermembrane space, that is inhibited by short chain mono-carboxylic CoA esters (37
). What would be the role of inhibition of CPT I by L
-3-hydroxybutyryl-CoA? We hypothesize that it is the cell’s ketone body–sensing mechanism. High levels of D
-3-hydroxybutyrate and acetoacetate in the blood will lead to a rise in intramitochondrial acetoacetyl-CoA via the ketone body utilization pathway. In a cell containing abundant SCHAD, operation of the enzyme in the “reverse” direction would lead to accumulation of L
-3-hydroxybutyryl-CoA. Inhibition of CPT I by L
-3-hydroxybutyryl-CoA would inhibit fatty acid oxidation and, in the β cell, lead to insulin secretion. In muscle, acetoacetate and 3-hydroxybutyrate inhibit fatty acid oxidation by a mechanism independent of malonyl-CoA (38
Our patient’s hypoglycemia responded to diazoxide, which inhibits insulin secretion by keeping KATP channels open. This does not disprove the hypothesis that HI in SCHAD deficiency is due to stimulation of the lipid-signaling pathway; the Prentki model suggests that large amounts of insulin are only secreted if both the KATP channel and the lipid-signaling pathway are activated.
There are significant differences between FS and previous putative cases of SCHAD deficiency. In contrast to the child reported by Tein et al. (6
), FS had no evidence of skeletal or cardiac myopathy and SCHAD activity was low in her fibroblasts. The cases reported by Bennett et al. in 1996 (7
) had a vigorous ketone response to hypoglycemia and medium- and long-chain 3-hydroxydicarboxylic acids in the urine. Neither of these features was present in FS. It is possible that some patients previously reported as SCHAD deficient (6
) were in fact deficient in other enzymes with SCHAD activity. However, the recently reported patient with G118A and C171A mutations in the Schad
gene, presented with fulminant liver failure at 3 years (10
); HI was not reported. This raises the possibility of phenotypic variation in SCHAD deficiency. The SCHAD-knockout mouse dies when subjected to a 10-hour fast (11
); our observations suggest that this could be due to hyperinsulinemic hypoglycemia.
We have checked acylcarnitine profiles in other children with HI and found no further cases with a raised hydroxybutyrylcarnitine, suggesting that SCHAD deficiency is a rare cause of HI. However, cases such as this provide important insight into normal biochemistry and physiology. In this case, the biochemistry of the β cell and the physiology of insulin secretion.
In summary, we describe here a new syndrome of hyperinsulinism and SCHAD deficiency that should be easily recognizable by analysis of acylcarnitine species and that responds well to treatment with diazoxide. It provides, to our knowledge, the first “experiment of nature” that links impaired fatty acid oxidation to HI and that provides support for the concept that a lipid signaling pathway (14
) is implicated in the control of insulin secretion. It raises the possibility that HI might contribute to hypoglycemia in other FAODs.