1 Program for Developmental and Reproductive Biology, Biomedicum, Helsinki, and the Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland
2 Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland
3 Department of Medical Genetics, University of Helsinki, Helsinki, Finland
4 Department of General Pediatrics, University Childrens Hospital, Düsseldorf, Germany
5 Department of Biosciences at Novum and Clinical Research Center, Karolinska Institute, Stockholm, Sweden
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ABSTRACT |
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Congenital hyperinsulinism (CHI) is a heterogeneous disorder characterized by severe hypoglycemia due to dysregulated insulin secretion. Most typically, nonketotic hypoglycemia manifests soon after birth or in infancy and may be severe enough to cause neurological damage (1). Until now, CHI has been shown to be caused by mutations in four genes linked with the stimulus-secretion coupling of pancreatic ß-cells. These include SUR1 and Kir6.2, which encode the two subunits of the ß-cell ATP-sensitive potassium channels. In addition, activating mutations in the glucokinase and glutamate dehydrogenase genes may also cause CHI, which is often less severe and may manifest later than the disease caused by ATP-sensitive potassium channel mutations (2).
We recently described an apparently new form of hyperinsulinemic hypoglycemia specifically associated with physical exercise in two adolescents (exercise-induced hyperinsulinism [EIHI]) (3). In these individuals, strenuous physical exercise caused an inappropriate burst of insulin release that predisposed to hypoglycemia. There was no apparent fasting hypoglycemia in the resting state.
In vitro studies have demonstrated that insulin release from the ß-cells is not stimulated by exogenous lactate and pyruvate, apparently due to the negligible transport of these monocarboxylates across the ß-cell membrane (4). We developed a hypothesis for the pathogenesis of EIHI based on aberrant responsiveness of insulin release to the increased levels of circulating lactate and/or pyruvate during exercise.
We have recently identified 10 additional cases of EIHI from two families. In this study, we report the autosomal-dominant inheritance of EIHI and clinical metabolic tests linking the disorder with abnormal transport or metabolism of pyruvate in the insulin-producing cells. In addition, we demonstrate that based on mutational analysis of the obvious candidate genes, the monocarboxylate transporters (MCTs) have normal coding sequences.
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RESEARCH DESIGN AND METHODS |
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Physical exercise test.
The test was based on a 10-min bicycle exercise, which began 60 min after the intravenous pyruvate test. The patients were asked to perform moderately strenuous bicycle exercise for 10 min. The aim was to increase the pulse rate to an age-adjusted target of 220 minus age in years. Blood test for glucose, insulin, pyruvate, and lactate were taken at -10, 0, 5, 10, 15, 20, 25, 30, 35, 40, 50 and 60 min. Samples for stress hormones (growth hormone, cortisol, and glucagon) were taken at -10 and 20 min. Heart rate and respiratory frequency were recorded throughout the test.
Sequencing of MCT genes.
Two patient samples (female index case of family 2 and the previously reported male case [3]) and one control sample were used for screening mutations in the promotor region of the MCT1 gene and coding regions of the genes MCT1MCT8 and CD147. PCR assays of genes MCT1, MCT3, and MCT6MCT8 (with known genomic organization) were carried out in 50-µl reactions containing 3050 ng genomic DNA from lymphoblasts, 1x PCR buffer (10 mmol/l Tris-HCl, 1.5 mmol/l MgCl2, 150 mmol/l KCl, and 0.1% Triton X-100), 160 mol/l dNTPs, 0.6 mol/l of each primer, 0.6 units DNA polymerase (DyNAzyme II; Finnzymes, Espoo, Finland), and 04% DMSO. The primer sequences used for sequencing of MCT family gene exons are available from T.O. upon request. Some poorly amplifying parts of coding regions were amplified by using DNA polymerase AmpliTaq Gold (Perkin Elmer, Roche Molecular Systems) and the high-fidelity enzyme DyNAzyme EXT (Finnzymes). To amplify the MCT2, MCT4, and MCT5 cDNAs, RNA was extracted (RNeasy Mini Kit; Qiagen, Valencia, CA) from lymphoblasts, and RT-PCR (Perkin Elmer) was carried out using the manufacturers protocol. The primers were designed using the Primer3 program (http://www-genome.wi.mit.edu/genome_software/other/primer3.html). The samples were denaturated for 2 min at 94°C, followed by 3540 cycles each of 35 s at 94°C, 35 s at 5562°C, and 1 min at 72°. Elongation was performed for 8 min at 72°C. Purified (PCR purification kit, gel extraction kit; Qiagen) PCR products were sequenced using ABI 377 and ABI 3100. Coding regions of the genes were inspected by direct sequencing of the genomic and RT-PCR products, and some parts of coding regions were cloned (TOPO TA Cloning Kit, pCR 2.1-TOPO vector; Invitrogen) before sequencing. Sequencing was routinely done in both directions, with some rare exceptions in which the unidirectional results left no uncertainty.
Transport of pyruvate and lactate dehydrogenase activity in fibroblasts.
Small skin biopsies were taken from the ventral arm of a patient with EIHI (index case of family 1) and an age- and sex-matched healthy control subject. Fibroblasts were grown out of the biopsies in RPMI-1640 medium (Gibco, Paisley, Scotland) supplemented with 30% FCS (Gibco) at 37°C in a CO2 incubator. Transport of radioactively labeled pyruvate was studied in batches of 104 cells plated in 96-well plates between passages 3 and 5. The cells were preincubated for 30 min in Krebs-Ringer bicarbonate buffer (KRB; 140 mmol/l NaCl, 3.6 mmol/l KCl, 0.5 mmol/l NaH2PO4, 0.5 mmol/l MgSO4, 1.5 mmol/l CaCl2, 2 mmol/l NaHCO3, 10 mmol/l HEPES [pH 7.4], and 0.1% BSA). Experiments were initiated by replacing the buffer with fresh KRB containing 1 µCi/ml (0.05 µCi/well) of [1-14C]L-pyruvate (NEN Life Science Products, Boston, MA). After 130 min incubation at 37°C, the cells were immediately washed twice with KRB, detached with trypsin-EDTA (Gibco), and mixed with Optiphase Hisafe liquid scintillation solution (Wallac, Turku, Finland). The incorporated radioactivity was counted in a ß-counter (Wallac). Alternatively, the cells were incubated for 5 min in KRB containing different concentrations (0.12510 mmol/l) of cold Na-pyruvate (Gibco) and 1 µCi/ml of [1-14C]L-pyruvate.
Lactate dehydrogenase (LDH) activity was measured from the confluent fibroblast cultures. Cells were washed with PBS, harvested, and, after three freeze-thaw cycles, centrifuged for 15 min at 15,000g. Supernatants were filtered with PD-10 columns (Pharmacia Amersham Biotec), and enzymatic activity was measured with a spectrophotometer in 1-ml cuvettes containing 100 mmol/l lactate, 600 mmol/l Tris [pH 8.5], 0.1 mmol/l NAD+, and sample. Protein concentration was measured with a Bio-Rad DC Protein Assay kit.
Statistics.
Significance of the observed differences between patients and control subjects was tested with one-way ANOVA for repeated measures and Students unpaired t test. Logarithmic transformation was used to normalize the distribution of serum insulin values. P < 0.05 was regarded as significant.
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RESULTS |
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Pyruvate test.
The intravenous pyruvate test was performed to test the hypothesis that exercise-associated hypoglycemia is associated with an abnormal ß-cell response to circulating pyruvate. The injected sodium pyruvate bolus was well tolerated by both patients and control subjects. No side effects were reported. Blood glucose levels remained normoglycemic in all cases, although a slightly lower resting level and a small decrease after the injection was evident in the patients. As expected, the blood pyruvate concentration increased rapidly after the injection to levels comparable with those measured after physical exercise. However, peak pyruvate concentration at 1 min after injection was nearly twice as high in the patients as in control subjects (282 ± 43 vs. 155 ± 20 µmol/l, P < 0.05) (Fig. 3). Thereafter, pyruvate concentrations decreased similarly in both groups. Lactate levels were not different between the groups at 0, 5, and 10 min. The most dramatic difference was observed in the serum insulin levels (Table 2 and Fig. 3). There was no response whatsoever in the control subjects after the pyruvate injection. However, the patients insulin levels increased significantly already at 1 min after injection, and the highest concentrations were measured at 3 min. The 1 + 3-min peak response was 5.4 ± 0.9-fold in the patients, as compared with 0.94 ± 0.08-fold in control subjects (P < 0.001) (Fig. 3).
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Pyruvate transport and LDH activity in fibroblasts.
Transport of labeled pyruvate was studied in early-passage cultured skin fibroblasts of the index patient of family 1 and a young healthy control subject. As expected, rapid pyruvate transport could be recorded. No kinetic differences were detected between patient and control cells (data not shown). To identify a possible difference in the affinity of the pyruvate transport system, the amount of labeled pyruvate transported into the cell during 5 min was recorded in the presence of increasing concentrations of unlabelled pyruvate. Again, no differences could be detected in the responses of patient and control cells (not shown). Multiple LDH measurements were done, and the specific activity in control cells was repeatedly 1015% higher than in patient-derived cells.
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DISCUSSION |
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Insulin release from the normal adult ß-cell is quite unresponsive to acute changes in the extracellular concentrations of lactate or pyruvate. This has been considered to be a protective mechanism of the pancreatic ß-cell, allowing the organism to prevent undesired insulin secretion induced by pyruvate and lactate metabolism during exercise or catabolic states (4). The unresponsiveness is due to at least two mechanisms: very low or absent expression of the major MCT, MCT1 on the ß-cell membrane (8), and low activity of LDH in the ß-cell (9,10). These metabolic features result in the selective channelling of glycolytic metabolites into ß-cell mitochondria. Thus, an increased uptake of pyruvate could lead to an increased production of ATP, followed by increased insulin release. Furthermore, experimental in vitro overexpression of MCT1 in the ß-cell conferred sensitivity of insulin release to exogenous pyruvate but not lactate (4). To achieve lactate-induced insulin release, LDH also had to be overexpressed. This prompted us to develop a diagnostic test for EIHI based on pyruvate-induced insulin secretion. The observed clear difference in the pyruvate response between patients and control subjects would thus support the hypothesis that the major pathogenetic mechanism in EIHI involves monocarboxylate transport over the ß-cell membrane.
This hypothesis could naturally not be tested directly, since it is not possible to obtain ß-cells from the patients. We approached this question by sequencing the major candidate genes, encoding the known human MCTs (MCT1MCT8) (7) and the chaperone protein CD147, which is essential for the correct targeting of MCT proteins to the plasma membrane (11). To study the role of polymorphism in the MCT genes, we used single-strand confirmation polymorphism and direct sequencing of exons to detect sequence variation and segregation study within families as well as catalogues of known common single nucleotide polymorphisms to infer their significance. Any ambiguous results were followed-up until definitively solved. Our results showed that none of the observed polymorphisms was likely to be disease associated.
In addition, we studied pyruvate transport in cultured patient fibroblasts in order to detect a universal transport defect. The results suggested that monocarboxylate transport was normal in patients as assessed. However, this result does not rule out the possibility of a ß-cell-specific transport defect, possibly caused by mutations in a gene responsible for the cell-type-specific expression of a MCT. Nevertheless, other possibilities involving defects in pyruvate metabolism must be considered. Thus, we also studied LDH activity in the fibroblasts. This was slightly higher in control than in patient cells, but the small difference is most likely unrelated to the pathogenesis of EIHI.
Although not studied systematically yet, the lack of postprandial hypoglycemia and the normal glucose-induced insulin release in at least one of our patients, together with at least partial sensitivity to diazoxide, indicate that the ß-cell defect is between pyruvate and the potassium-sensitive ATP channel. If the production of pyruvate in ß-cells is normal in EIHI, one has to considerer mutations resulting in gain of function in pyruvate dehydrogenase or carboxylase, Krebs cycle, or mitochondrial respiratory chain. Furthermore, other enzymes affecting ATP/ADP or NAD/NADH ratios come into question, as described for glutamate dehydrogenase in the hyperinsulinism-hyperammonemia syndrome (12). At the moment, we do not have any clues to support any of these possibilities. The mild disease phenotype, as well as the normal levels of amino acids, ammonia, pyruvate, and lactate, rule out known enzyme defects in pyruvate metabolism (13).
After the identification of the first EIHI patient in Finland, we could rapidly detect 10 additional cases in two families simply by informing the local pediatric endocrinologists. This would suggest that the disease may not be exceedingly uncommon. Exercise-related hypoglycemic symptoms are likely not to be very rare. Since exercise-induced hyperinsulinism has previously not been considered and specifically sought, it may well be that many individuals carrying this trait have simply adapted to the relatively small handicap and not been diagnosed. It will be interesting to find out the prevalence of EIHI in a larger number of individuals with exercise-associated hypoglycemia. Further pathogenetic studies will become possible if the genetic mapping and identification of the disease gene through linkage disequilibrium analysis in the affected families turns out to be successful.
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ACKNOWLEDGMENTS |
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The authors are indebted to Professor Claes Wollheim for the pathogenetic ideas that resulted in these studies and to the European Network for Research into Hyperinsulinism (QLG1-2000-00513), supported by the European Union.
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FOOTNOTES |
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Received for publication 15 April 2002 and accepted in revised form 14 October 2002.
CHI, congenital hyperinsulinism; EIHI, exercise-induced hyperinsulinism; KRB, Krebs-Ringer bicarbonate buffer; MCT, monocarboxylate transporter.
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REFERENCES |
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