Physical Exercise–Induced Hypoglycemia Caused by Failed Silencing of Monocarboxylate Transporter 1 in Pancreatic β Cells
2007; Elsevier BV; Volume: 81; Issue: 3 Linguagem: Inglês
10.1086/520960
ISSN1537-6605
AutoresTimo Otonkoski, Hong Jiao, Nina Kaminen‐Ahola, Isabel Tapia‐Páez, Mohammed S. Ullah, Laura E. Parton, Frans Schuit, Roel Quintens, Ilkka Sipilä, Ertan Mayatepek, Thomas Meißner, Andrew P. Halestrap, Guy A. Rutter, Juha Kere,
Tópico(s)Hyperglycemia and glycemic control in critically ill and hospitalized patients
ResumoExercise-induced hyperinsulinism (EIHI) is a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion during anaerobic exercise or on pyruvate load. We aimed to identify the molecular basis of this novel disorder of β-cell regulation. EIHI mapped to chromosome 1 (LOD score 3.6) in a genome scan performed for two families with 10 EIHI-affected patients. Mutational analysis of the promoter of the SLC16A1 gene, which encodes monocarboxylate transporter 1 (MCT1), located under the linkage peak, revealed changes in all 13 identified patients with EIHI. Patient fibroblasts displayed abnormally high SLC16A1 transcript levels, although monocarboxylate transport activities were not changed in these cells, reflecting additional posttranscriptional control of MCT1 levels in extrapancreatic tissues. By contrast, when examined in β cells, either of two SLC16A1 mutations identified in separate pedigrees resulted in increased protein binding to the corresponding promoter elements and marked (3- or 10-fold) transcriptional stimulation of SLC16A1 promoter-reporter constructs. These studies show that promoter-activating mutations in EIHI induce SLC16A1 expression in β cells, where this gene is not usually transcribed, permitting pyruvate uptake and pyruvate-stimulated insulin release despite ensuing hypoglycemia. These findings describe a novel disease mechanism based on the failure of cell-specific transcriptional silencing of a gene that is highly expressed in other tissues. Exercise-induced hyperinsulinism (EIHI) is a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion during anaerobic exercise or on pyruvate load. We aimed to identify the molecular basis of this novel disorder of β-cell regulation. EIHI mapped to chromosome 1 (LOD score 3.6) in a genome scan performed for two families with 10 EIHI-affected patients. Mutational analysis of the promoter of the SLC16A1 gene, which encodes monocarboxylate transporter 1 (MCT1), located under the linkage peak, revealed changes in all 13 identified patients with EIHI. Patient fibroblasts displayed abnormally high SLC16A1 transcript levels, although monocarboxylate transport activities were not changed in these cells, reflecting additional posttranscriptional control of MCT1 levels in extrapancreatic tissues. By contrast, when examined in β cells, either of two SLC16A1 mutations identified in separate pedigrees resulted in increased protein binding to the corresponding promoter elements and marked (3- or 10-fold) transcriptional stimulation of SLC16A1 promoter-reporter constructs. These studies show that promoter-activating mutations in EIHI induce SLC16A1 expression in β cells, where this gene is not usually transcribed, permitting pyruvate uptake and pyruvate-stimulated insulin release despite ensuing hypoglycemia. These findings describe a novel disease mechanism based on the failure of cell-specific transcriptional silencing of a gene that is highly expressed in other tissues. Release of insulin from the pancreatic β cell is carefully controlled by metabolic demands, allowing maintenance of the circulating blood-glucose concentration within narrow physiological limits. It is therefore important that insulin release be strongly stimulated as glucose concentrations rise but also effectively switched off during fasting, to avoid hypoglycemia.1Rutter GA Visualising insulin secretion: the Minkowski Lecture 2004.Diabetologia. 2004; 47: 1861-1872Crossref PubMed Scopus (66) Google Scholar β-cell metabolism has specific features to ensure the latter. First, β cells express the low-affinity glucose transporter GLUT22Thorens B Sarkar HK Kaback HR Lodish HF Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and β-pancreatic islet cells.Cell. 1988; 55: 281-290Abstract Full Text PDF PubMed Scopus (639) Google Scholar and glucose phosphorylating activity, glucokinase,3Iynedjian PB Mammalian glucokinase and its gene.Biochem J. 1993; 293: 1-13Crossref PubMed Scopus (273) Google Scholar to permit enhanced glucose metabolism as the concentration of the sugar changes over the physiological range.1Rutter GA Visualising insulin secretion: the Minkowski Lecture 2004.Diabetologia. 2004; 47: 1861-1872Crossref PubMed Scopus (66) Google Scholar Second, the activity of lactate dehydrogenase is unusually low.4Sekine N Cirulli V Regazzi R Brown LJ Gine E Tamarit-Rodriguez J Girotti M Marie S MacDonald MJ Wollheim CB et al.Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing.J Biol Chem. 1994; 269: 4895-4902Abstract Full Text PDF PubMed Google Scholar, 5Liang Y Bai G Doliba N Buettger C Wang L Berner DK Matschinsky FM Glucose metabolism and insulin release in mouse beta HC9 cells, as model for wild-type pancreatic beta-cells.Am J Physiol. 1996; 270: E846-E857PubMed Google Scholar, 6Zhao C Rutter GA Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 beta-cell lines.FEBS Lett. 1998; 430: 213-216Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Third, the expression of the plasma-membrane monocarboxylate transporter (MCT) MCT1 (SLC16A1 [MIM 600682]) as well as of other MCT isoforms7Price NT Jackson VN Halestrap AP Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past.Biochem J. 1998; 329: 321-328Crossref PubMed Scopus (295) Google Scholar is weak or undetectable in the β cell.8Zhao C Wilson MC Schuit F Halestrap AP Rutter GA Expression and distribution of lactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.Diabetes. 2001; 50: 361-366Crossref PubMed Scopus (110) Google Scholar This configuration has been proposed to reduce loss-of-glucose carbon as pyruvate 6Zhao C Rutter GA Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 beta-cell lines.FEBS Lett. 1998; 430: 213-216Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar and to avoid the unwanted stimulation of insulin release by lactate or pyruvate.9Ishihara H Wang H Drewes LR Wollheim CB Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells.J Clin Invest. 1999; 104: 1621-1629Crossref PubMed Scopus (156) Google Scholar We recently identified exercise-induced hyperinsulinism (EIHI) as a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion, particularly during anaerobic exercise.10Meissner T Otonkoski T Feneberg R Beinbrech B Apostolidou S Sipila I Schaefer F Mayatepek E Exercise induced hypoglycaemic hyperinsulinism.Arch Dis Child. 2001; 84: 254-257Crossref PubMed Scopus (44) Google Scholar, 11Otonkoski T Kaminen N Ustinov J Lapatto R Meissner T Mayatepek E Kere J Sipila I Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release.Diabetes. 2003; 52: 199-204Crossref PubMed Scopus (112) Google Scholar, 12Meissner T Friedmann B Okun JG Schwab MA Otonkoski T Bauer T Bartsch P Mayatepek E Massive insulin secretion in response to anaerobic exercise in exercise-induced hyperinsulinism.Horm Metab Res. 2005; 37: 690-694Crossref PubMed Scopus (25) Google Scholar Affected patients become hypoglycemic within 30 min after a short period of anaerobic exercise. This pathology might result from an inappropriate insulin release in response to exogenous catabolic metabolites such as lactate and pyruvate. Indeed, unlike unaffected individuals, patients responded to an acute exogenous pyruvate load with prompt insulin release.11Otonkoski T Kaminen N Ustinov J Lapatto R Meissner T Mayatepek E Kere J Sipila I Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release.Diabetes. 2003; 52: 199-204Crossref PubMed Scopus (112) Google Scholar This could be due to an overactivity of MCTs in the β-cell plasma membrane, which leads to the entry of pyruvate into the cell followed by its metabolism with a consequent increase in ATP production and insulin secretion. However, in earlier studies, no mutations were detected in the coding regions of eight MCT genes.11Otonkoski T Kaminen N Ustinov J Lapatto R Meissner T Mayatepek E Kere J Sipila I Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release.Diabetes. 2003; 52: 199-204Crossref PubMed Scopus (112) Google Scholar Therefore, we decided to map the disease gene, to identify the specific mutations, and to test their pathogenetic mechanisms in the β cell. Our study includes all 13 reported patients with EIHI. Twelve patients belong to two Finnish pedigrees. Their clinical phenotypes were described elsewhere.11Otonkoski T Kaminen N Ustinov J Lapatto R Meissner T Mayatepek E Kere J Sipila I Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release.Diabetes. 2003; 52: 199-204Crossref PubMed Scopus (112) Google Scholar However, DNA for linkage analysis was available from only 10 of the 12 (fig. 1). The fold response of insulin secretion to an intravenous bolus of sodium pyruvate provided the basis for differentiating affected and unaffected individuals. In all the unaffected individuals, this fold response has a value of 1; in the affected individuals, it has a range of 2–10 (fig. 1A).11Otonkoski T Kaminen N Ustinov J Lapatto R Meissner T Mayatepek E Kere J Sipila I Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release.Diabetes. 2003; 52: 199-204Crossref PubMed Scopus (112) Google Scholar One patient is from Germany; the pathophysiology of his insulin secretion during physical exercise has been studied in detail.12Meissner T Friedmann B Okun JG Schwab MA Otonkoski T Bauer T Bartsch P Mayatepek E Massive insulin secretion in response to anaerobic exercise in exercise-induced hyperinsulinism.Horm Metab Res. 2005; 37: 690-694Crossref PubMed Scopus (25) Google Scholar Genomic DNA was extracted from whole blood (PureGene [Gentra Systems]). A genomewide scan was performed with 430 microsatellite markers from the Linkage Mapping Set MD-10 (Applied Biosystems) on a MegaBace 1000 capillary sequencer (Molecular Dynamics). The alleles were visualized using Genetic Profiler 1.1 or 2.0 software (Molecular Dynamics). For fine mapping, five microsatellites were added to the highest peak region. Genotyping was performed at the Finnish Genome Center (genome scan) and Clinical Research Center, Karolinska Institutet (fine mapping). Parametric linkage analysis with use of GENEHUNTER (v. 2.1)13Kruglyak L Daly MJ Reeve-Daly MP Lander ES Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar was based on an autosomal dominant model with a penetrance of 0.999 for heterozygotes, disease-allele frequency of 0.001, and phenocopy rate of 0.001. The positions of marker loci were based on published maps (National Center for Biotechnology Information [NCBI]). One patient sample from both families and a control sample were sequenced in both directions for the 5′ and 3′ UTRs and 2-kb promoter region of the MCT1 gene (SLC16A1) and all exons and 1 kb of the promoter of the HMGCS2 gene. The primer sequences are available on request. Purified (PCR purification kit and gel-extraction kit [Qiagen]) PCR products were sequenced using ABI 377 or ABI 3100 (Applied Biosystems). The Staden package14Staden R The Staden sequence analysis package.Mol Biotechnol. 1996; 5: 233-241Crossref PubMed Scopus (1060) Google Scholar was employed for sequence alignment and comparison. Sequences were checked against the published genome (NCBI). To verify the frequency of the sequence changes in healthy individuals, 94 control samples from Finland and 32 control samples from Germany were sequenced. Tissues were from male C57BL/6 mice (aged 8–10 wks). Islets of Langerhans and pancreatic acini were collagenase isolated (for 3 min at 37°C) and were hand picked under a dissection microscope.15Van Lommel L Janssens K Tsukamoto K Quintens R Vander Mierde D Lemaire K Denef C Martens G Jonas J-C Pipeleers D et al.Probe-independent and direct quantification of insulin mRNA and growth hormone mRNA in enriched cell preparations.Diabetes. 2006; 55: 3214-3220Crossref PubMed Scopus (44) Google Scholar Embryonic stem (ES) cells were obtained from L. Schoonjans (Thrombix, Center of Vascular Biology, Katholieke Universiteit). MIN6 cells were obtained from E. Yamato (Division of Stem Cell Regulation Research, Osaka University Medical School). Total RNA from tissues was isolated using TRIzol (Invitrogen) followed by cleanup with RNeasy (Qiagen). Total RNA from islets, acini, ES cells, and MIN6 cells was extracted using the Absolutely RNA microprep kit (Stratagene). RNA quality and quantity were assessed using the 2100 BioAnalyzer (Agilent Technologies) and the NanoDrop ND-1000 spectrophotometer. One microgram of total RNA was used for cDNA synthesis with the RevertAid H Minus M-MuLV Reverse Transcriptase kit (Fermentas). Quantitative PCRs (qPCRs) were performed using ABsolute QPCR mix (ABgene) on a Rotor-Gene 3000 (Corbett Research). Quantification of MCT1 mRNA-expression levels was normalized using polymerase II alpha (Polr2a) mRNA as internal reference. In each run, a MIN6 cell sample was used as a calibrator, and relative MCT1 mRNA expression for this sample was set at 1.0. Reaction conditions and sequences for primers and dual-labeled fluorescent probes, synthesized by Sigma Genosys, are available on request. Significance of differences in mRNA expression was assessed using the two-tailed unpaired Student's t-test. Standard curves were constructed by amplifying serial dilutions of untreated mhAT3F cDNA (50 ng to 0.64 pg) and by plotting cycle threshold (CT) values as a function of starting reverse-transcribed RNA, the slope of which was used to calculate relative expression of the target gene.16Parton LE Diraison F Neill SE Ghosh SK Rubino MA Bisi JE Briscoe CP Rutter GA Impact of PPARγ overexpression and activation on pancreatic islet gene expression profile analyzed with oligonucleotide microarrays.Am J Physiol Endocrinol Metab. 2004; 287: E390-E404Crossref PubMed Scopus (32) Google Scholar Bases from −1342 to +216A in the MCT1 promoter17Cuff MA Shirazi-Beechey SP The human monocarboxylate transporter, MCT1: genomic organization and promoter analysis.Biochem Biophys Res Commun. 2002; 292: 1048-1056Crossref PubMed Scopus (38) Google Scholar were amplified using primers 5′-ACT GTC TTC TAA TAT ACT GAA CTG TTA TTG G-3′ and 5′-TCG TTT GCT TGT TCC AGT ACC CAC GCA-3′. PCR was performed using genomic DNA as template. The single 1.5-kb band was gel extracted and cloned into the pGEM T-Easy vector (Promega). The promoter fragment was then excised by digestion with EcoRI and was subcloned into the CFP-N vector. HindIII and KpnI digestion of the resulting construct produced a 1,558-bp MCT1 promoter fragment, which was ligated into pGL3-Basic vector (Promega). A point mutation was introduced at position +163 of the MCT1 promoter, with use of the QuickChange kit (Stratagene). A 1,595-bp MCT1 promoter region containing the 25-nt duplication was cloned using patient fibroblast–derived genomic DNA, with use of primers 5′-GGATGCCACACTGTCTTCTAATA-3′ and 5′- CCTCGTTTGCTTGTTCCAGTA-3′. The presence of the desired mutations was confirmed by sequencing. MIN6 β cells were seeded in a 24-well plate at a density of 2×105 ml−1 and were cotransfected for 4 h with 800 ng pGL3-promoter vector containing either wild-type or mutant hSLC16A1 promoter sequences, as detailed above, with 100 ng pRL-SV40 (Promega) containing Rotylenchulus reniformis luciferase gene as a control for transfection efficiency, with 10 μg ml−1 LipofectAMINE in Opti-MEM I medium (Invitrogen). Cells were harvested 48 h after transfection and were assayed for luciferase activity with the Dual-Glow Luciferase Assay System (Promega). Promoter activities were expressed as relative light units normalized against control reporter (R. reniformis luciferase) activity in each extract, as described elsewhere.18Ullah MS Davies AJ Halestrap AP The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1α-dependent mechanism.J Biol Chem. 2006; 281: 9030-9037Crossref PubMed Scopus (582) Google Scholar EMSA was performed according to standard protocols.19Sambrook J Russell DW Gel retardation assays for DNA-binding proteins: molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York2001: 17.13-17.17.Google Scholar In brief, 10-μg aliquots of MIN-6 nuclear extracts were preincubated with 0.5 μg of poly (dI-dC) and 1 μl of 100 mM dithiothreitol (DTT) in a 30-μl reaction with a 4× buffer—containing 80 mM HEPES (pH 7.6), 2 mM EDTA, 200 mM NaCl, 20% glycerol, 1.2 mg BSA, and 40 mM DTT—for 15 min at room temperature. 32P-end–labeled double-stranded wild-type or mutant probes were added, and the mixture was incubated for 15 min at room temperature. In competition assays, cold probes were incubated with 100- and 200-molar excess for 15 min at room temperature before the addition of the labeled probes. The samples were electrophoresed on 4.5% nondenaturing polyacrylamide gels in 1× TBE at 240 V in a cold room. The radioactive pattern was visualized by autoradiography and was quantified by PhosphorImager scanning (Fuji Photo). Oligonucleotides were normal probes (5′-GCC CGG CCA GAC AAA GTG GTG AGC TGC GAC-3′ and 5′-GTC GCA GCT CAC CAC TTT GTC TGG CCG GGC-3′) and mutant probes (5′-GCC CGG CCA GAC AAA ATG GTG AGC TGC GAC-3′ and 5′-GTC GCA GCT CAC CAT TTT GTC TGG CCG GGC-3′). Primary fibroblast cell lines were generated from skin-biopsy samples of one patient from each family and from four healthy age-matched control individuals. Steady-state levels of MCT1 and MCT4 protein and mRNA were measured by western blotting18Ullah MS Davies AJ Halestrap AP The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1α-dependent mechanism.J Biol Chem. 2006; 281: 9030-9037Crossref PubMed Scopus (582) Google Scholar and quantitative RT-PCR, respectively. Total RNA was isolated from cultured fibroblasts (TRI Reagent [Sigma]) and was DNAse treated (DNA-free [Ambion]) as recommended by the supplier. First-strand cDNA was synthesized from 2 μg total RNA. TaqMan primers and probes for quantitative RT-PCR were kindly donated by AstraZeneca (R&D Charnwood). The dual-labeled fluorogenic probes consisted of oligonucleotides with the 5′ reporter dye 6-carboxyfluorescein (FAM) and the 3′ Black Hole Quencher 1 dye (BHQ1). Primer and probe sequences are available on request. Lactate transport into the fibroblasts was determined by measurement of the decrease in intracellular pH with use of the fluorescent pH-sensitive dye 2′–7′-bis (carboxyethyl)-5–6-carboxy-fluorescein (BCECF) as described elsewhere.20Kirk P Wilson MC Heddle C Brown MH Barclay AN Halestrap AP CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression.EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (475) Google Scholar We diagnosed EIHI for 13 patients, 12 from two families and one unrelated patient. A total of 430 microsatellite markers were genotyped in 10 affected and 9 unaffected individuals (fig. 1A). Linkage analysis with a dominant model revealed the strongest evidence of linkage on chromosome 1, with total maximum LOD score 2.53 (fig. 1B). Five markers were added to the linkage peak, with an increase of maximum LOD score to 3.6 (fig. 1C). Four adjacent markers showed significant linkage by single-point analysis. A haplotype formed by markers D1S250, D1S534, D1S498, and D1S1595 was present in all affected but no unaffected individuals of the larger family. Nor was it present in the other pedigree, suggesting that the families have independent mutations. Several candidate genes with potential relevance were present within the linkage peak. We initially elected three of them for sequencing. The PHGDH gene, which encodes phosphoglycerate dehydrogenase (MIM 606879), was located 580 kb from peak marker D1S534. The HMGCS2 gene, which encodes mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (MIM 600234) was 620 kb from D1S534, and the SLC16A1 gene, which encodes the MCT1 protein (MIM 600682), was located 6.2 Mb from the peak marker. We sequenced all three genes; no sequence variants were found in any exons of HMGCS2, but two variants in PHGDH—rs562038 and rs543703 in exon 1 and exon 11, respectively—were found in the EIHI-affected families. Sequencing of SLC16A1 was focused on the 5′ UTR and promoter regions (2 kb upstream of the transcription start site) because no mutations had been found previously in the coding region.11Otonkoski T Kaminen N Ustinov J Lapatto R Meissner T Mayatepek E Kere J Sipila I Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release.Diabetes. 2003; 52: 199-204Crossref PubMed Scopus (112) Google Scholar A previously unknown SNP (a G→A change) in exon 1 at position +163 relative to the transcription start site, was found in the affected individuals of pedigree 1 (fig. 2A). In a patient from pedigree 2, a 25-bp duplication was found between nucleotide positions −23 and −24. The duplicated sequence also contained an A→T change (fig. 2A and 2C). Sequencing of these putative mutations in at least 94 samples from Finland and Germany revealed not a single occurrence in healthy controls, excluding with >84% probability that they were polymorphisms with a frequency of ≥1%. In the German patient,10Meissner T Otonkoski T Feneberg R Beinbrech B Apostolidou S Sipila I Schaefer F Mayatepek E Exercise induced hypoglycaemic hyperinsulinism.Arch Dis Child. 2001; 84: 254-257Crossref PubMed Scopus (44) Google Scholar, 12Meissner T Friedmann B Okun JG Schwab MA Otonkoski T Bauer T Bartsch P Mayatepek E Massive insulin secretion in response to anaerobic exercise in exercise-induced hyperinsulinism.Horm Metab Res. 2005; 37: 690-694Crossref PubMed Scopus (25) Google Scholar several sequence variants were found, including a 2-bp insertion (fig. 2B). Database searching (TESS: Transcription Element Search System) revealed that the three putative mutations lie within the binding sites of several transcription factors (fig. 2A and 2B). A binding site for nuclear matrix protein 1 (YY1) was predicted at the point-mutation site in the mutant sequence of pedigree 1. The same point mutation disrupted the binding sites of two potential transcriptional repressors: albumin negative factor (ANF) and AML-1a (fig. 2A). The duplication variant introduced additional binding sites for the ubiquitous transcription factors simian-virus-40-protein-1 (Sp1), upstream stimulatory factor (USF), and myeloid zinc finger 1 (MZF1) (fig. 2A). The third mutation resulted in loss of a GATA-1–binding site (fig. 2B). These predictions were consistent with the hypothesis that the identified mutations affect regulation of the SLC16A1 gene. To establish potential functional consequences of the +163 G→A variant in pedigree 1, we performed EMSA. Distinct differences were seen in the protein-binding activity of this site in nuclear extracts, with binding to the common variant increasing progressively in MIN6, SyH-SY5Y, and HeLa cells (fig. 3). Moreover, in each cell type, a higher degree of binding to the mutated site was apparent, except for one low–molecular weight band that was lost in the mutated sequence in MIN6 cells (fig. 3). Competition assays confirmed the specificity of the probes. The EMSA result provided further evidence of altered transcriptional regulation introduced by the G→A mutation. Since it was not feasible to study SLC16A1 promoter activity in patient β cells, mRNA expression in skin fibroblasts from two EIHI-affected patients and four control individuals was assayed for two genes, SLC16A1 (which encode MCT1) and SLC16A3 (which encodes MCT4). As measured by quantitative RT-PCR (normalized against 18S RNA), MCT1 mRNA was two to three times higher in both patients than it was in any control, whereas MCT4 mRNA levels were similar in patients and controls. To determine whether the increase in SLC16A1 mRNA in patient fibroblasts led to increased expression of MCT1 and monocarboxylate transport activity, western blotting and transport assays were performed (fig. 4). There was no evidence that the patient fibroblasts showed greater expression or activity than did the control fibroblasts. This lack of correlation between levels of MCT1 mRNA and MCT1 protein is well established in a variety of tissues and is thought to result from posttranscriptional regulation involving the unusually long (1.6 kb) 3′ UTR of MCT1 mRNA.21Halestrap AP Price NT The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.Biochem J. 1999; 343: 281-299Crossref PubMed Scopus (1020) Google Scholar, 22Halestrap AP Meredith D The SLC16 gene family—from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond.Pflugers Arch. 2004; 447: 619-628Crossref PubMed Scopus (751) Google Scholar In most tissues, such as fibroblasts, the expression of MCT1 protein may be relatively insensitive to the level of MCT1 mRNA, which is controlled to a much greater extent by translational mechanisms. However, in the β cell, where the basal expression of MCT1 mRNA is extremely low, MCT1 protein expression may respond significantly to the increased mRNA production that results from the activated promoter in the patients. The potential functional relevance of the above findings for β cells was tested by measuring MCT1 mRNA levels in a number of mouse tissues, including purified pancreatic acini and islets, clonal murine β cells (MIN6), and α cells (αTC1-9) (fig. 5A and 5B). Compared with all other tissues, MCT1 mRNA was very low or absent in β cells and islets, consistent with differences reported elsewhere in lactate-transport activity4Sekine N Cirulli V Regazzi R Brown LJ Gine E Tamarit-Rodriguez J Girotti M Marie S MacDonald MJ Wollheim CB et al.Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing.J Biol Chem. 1994; 269: 4895-4902Abstract Full Text PDF PubMed Google Scholar and MCT1 levels8Zhao C Wilson MC Schuit F Halestrap AP Rutter GA Expression and distribution of lactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.Diabetes. 2001; 50: 361-366Crossref PubMed Scopus (110) Google Scholar in pancreatic β cells. Transfection of MIN6 cells with an MCT1 promoter-luciferase reporter construct including the nucleotides from −1342 to +216 revealed a 3.5-fold (P<.001) increase in promoter activity after the G→A mutation at position 163 (fig. 5C, mutation P1). A similar (two-to-threefold) increase in the MCT1 promoter activity induced by this mutation was seen when the same promoter-luciferase reporter constructs were expressed in L6 myoblasts and HeLa and CHO cells (data not shown). Transfection of the MIN-6 cells with a promoter construct, including the 25-nt duplication found in pedigree 2, resulted in an even more dramatic (10-fold, P<.001) stimulatory effect on transcription (fig. 5C, mutation P2). We identify here linkage to mutations in the SLC16A1 gene in two pedigrees of patients with EIHI. Whereas the linkage interval was relatively large, we failed to detect mutations in other likely potential genes in this region, but we demonstrated instead that two of the three SLC16A1 gene mutations lead to profound increases in the transcription of this gene. These changes are likely to lead to increased levels of MCT1 protein, and hence transport activity, selectively in β cells where, in healthy subjects, MCT1 levels are extremely low. The present study thus suggests that human β cells, like those of rodents,4Sekine N Cirulli V Regazzi R Brown LJ Gine E Tamarit-Rodriguez J Girotti M Marie S MacDonald MJ Wollheim CB et al.Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing.J Biol Chem. 1994; 269: 4895-4902Abstract Full Text PDF PubMed Google Scholar, 8Zhao C Wilson MC Schuit F Halestrap AP Rutter GA Expression and distribution of lactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.Diabetes. 2001; 50: 361-366Crossref PubMed Scopus (110) Google Scholar may be uniquely configured in this way. Low levels of plasma-membrane–lactate transport activity ensure that the uptake of lactate and pyruvate—and hence the inappropriate, and potentially fatal, secretion of insulin—does not occur during exercise. In patients with EIHI, selective increases in β-cell MCT1 expression may permit pyruvate or lactate to stimulate β-cell mitochondrial metabolism, leading to increased intracellular ATP/adenosine diphosphate ratios, the closure of KATP channels,23Bryan J Crane A Vila-Carriles WH Babenko AP Aguilar-Bryan L Insulin secretagogues, sulfonylurea receptors and K(ATP) channels.Curr Pharm Des. 2005; 11: 2699-2716Crossref PubMed Scopus (107) Google Scholar inhibition of adenosine monophosphate–activated protein kinase,24Salt IP Johnson G Ashcroft SJ Hardie DG AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release.Biochem J. 1998; 335: 533-539Crossref PubMed Scopus (317) Google Scholar plasma membrane depolarization, Ca2+ influx, and insulin exocytosis.1Rutter GA Visualising insulin secretion: the Minkowski Lecture 2004.Diabetologia. 2004; 47: 1861-1872Crossref PubMed Scopus (66) Google Scholar Given the very low lactate dehydrogenase (LDH) activity in β cells,6Zhao C Rutter GA Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 beta-cell lines.FEBS Lett. 1998; 430: 213-216Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar the very efficient pyruvate oxidation by β-cell mitochondria,23Bryan J Crane A Vila-Carriles WH Babenko AP Aguilar-Bryan L Insulin secretagogues, sulfonylurea receptors and K(ATP) channels.Curr Pharm Des. 2005; 11: 2699-2716Crossref PubMed Scopus (107) Google Scholar and the fact that overexpression of both MCT1 and LDH was required for a secretory response to lactate in isolated islets,9Ishihara H Wang H Drewes LR Wollheim CB Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells.J Clin Invest. 1999; 104: 1621-1629Crossref PubMed Scopus (156) Google Scholar we suspect that changes in circulating pyruvate, rather than lactate, are the principal trigger for insulin secretion in patients with EIHI.11Otonkoski T Kaminen N Ustinov J Lapatto R Meissner T Mayatepek E Kere J Sipila I Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release.Diabetes. 2003; 52: 199-204Crossref PubMed Scopus (112) Google Scholar Our data are consistent with silencing of the MCT1 promoter in β cells by inhibitory transcription factors that remain to be characterized. Likewise, duplication of a cis-acting region, as seen in one pedigree, may provide increased basal transcriptional activity. In any case, the resulting increase in transcription, from near zero to a significant level, seems likely to lead to an increase in MCT1 activity in the β cell. Since our normal MCT1-promoter construct shows significant but low activity in MIN6 cells, additional posttranscriptional inhibitory mechanisms are likely to exist to further inhibit expression of MCT1 protein. In conclusion, we describe here a novel mechanism for human disease. The failed silencing of the expression of a cell-membrane transporter (MCT1) in a specific cell type (pancreatic β cells) leads to an inappropriate physiological response in patients with EIHI, characterized by inappropriate insulin release during anaerobic exercise. This study was supported by the Foundation for Pediatric Research (to T.O.), Academy of Finland and Sigrid Juselius Foundation (to J.K. and T.O.), Swedish Research Council (to J.K.), and Juvenile Diabetes Research Foundation (JDRFI) (to T.O. and F.S.). M.S.U. was supported by an Industrial Studentship from AstraZeneca (R&D Charnwood, Loughborough). G.A.R. thanks the Wellcome Trust for a Programme Grant and Research Leave Fellowship and thanks the National Institutes of Health, JDRFI, and Medical Research Council (UK).
Referência(s)