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Unregulated Elevation of Glutamate Dehydrogenase Activity Induces Glutamine-Stimulated Insulin Secretion

Identification and Characterization of a GLUD1 Gene Mutation and Insulin Secretion Studies With MIN6 Cells Overexpressing the Mutant Glutamate Dehydrogenase

¹ Department of Bio-Signal Analysis, Yamaguchi University Graduate School of Medicine, Ube, Japan
² Matsuo Clinic of Pediatrics, Yamaguchi, Japan
³ Department of Reproductive, Pediatric and Infection Science, Yamaguchi University School of Medicine, Ube, Japan

	ABSTRACT

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Glutamate dehydrogenase (GDH) is important in normal glucose homeostasis. Mutations of GDH result in hyperinsulinism/hyperammonemia syndrome. Using PCR/single-strand conformation polymorphism analysis of the gene encoding GDH in 12 Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), we found a mutation (Y266C) in one PHHI patient. This mutation was not found in any of the control or type 2 diabetic subjects. The activity of the mutant GDH (GDH266C), expressed in COS-7 cells, was constitutively elevated, and allosteric regulations by ADP and GTP were severely impaired. The effect of the unregulated increase in GDH activity on insulin secretion was examined by overexpressing GDH266C in an insulinoma cell line, MIN6. Although glutamine alone did not stimulate insulin secretion from control MIN6-lacZ, it remarkably stimulated insulin secretion from MIN6-GDH266C. This finding suggests that constitutively activated GDH enhances oxidation of glutamate, which is intracellularly converted from glutamine to -ketoglutarate, a tricarboxylic acid cycle substrate, which thereby stimulates insulin secretion. Interestingly, insulin secretion is also exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) but not at higher glucose concentrations (8–25 mmol/l). Our results directly illustrate the importance of GDH in the regulation of insulin secretion from pancreatic ß-cells.

	INTRODUCTION

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The importance of GDH in normal glucose homeostasis in humans is also evident from the recent findings that mutations in the GLUD1 gene, which encodes GDH, cause hyperinsulinism/hyperammonemia (HI/HA) syndrome (3–5). HI/HA syndrome is characterized by hyperinsulinemic hypoglycemia accompanied by mild, asymptomatic hyperammonemia. Patients are usually diagnosed as having hypoglycemia, which frequently occurs after high-protein meals, several months after birth. Mutations were initially identified in exons 11 and 12 of the GLUD1 gene (3,4) and subsequently in exons 6 and 7 (5). All of the mutations identified in these patients impair allosteric inhibition by GTP and are therefore "gain of function" mutations. Hyperinsulinemia and hyperammomenia may be attributable to increased oxidative deamination of glutamate due to hyperactive GDH in pancreatic ß-cells and hepatocytes (3). More recently, it was suggested that mitochondrially derived glutamate potentiates insulin secretion, acting directly on insulin secretory granules (6). This theory assumes reverse flux through GDH in the direction of glutamate formation. However, the existence of this mechanism is controversial (7,8).

Herein, we examined the GLUD1 gene mutation in Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI). One mutation (Y266C) was identified in a patient with clinical features of HI/HA syndrome. This mutation gave the enzyme distinctive kinetic properties; basal enzymatic activity appeared to be higher and was not further activated or inhibited by ADP and GTP, respectively. By overexpressing this constitutively activated form of GDH in MIN6 cells, we directly examined the effect of altered GDH activity on the regulation of insulin secretion.

	RESEARCH DESIGN AND METHODS

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Study subjects.
The 12 patients with PHHI were part of a cohort we previously studied (9). Patients with mutations in the SUR1 gene were excluded, and none of the 12 had mutations of the Kir6.2 gene. No information was available regarding plasma ammonium levels for these patients at the initiation of this study. Patients with type 2 diabetes and nondiabetic control subjects were recruited from Yamaguchi University Hospital and affiliated hospitals. At the time of recruitment, informed consent was obtained from each individual according to a protocol approved by the Human Studies Committee of Yamaguchi University School of Medicine, and the investigation was performed in accordance with the guidelines expressed in the Declaration of Helsinki.

PCR/single-strand conformation polymorphism analysis.
All 13 coding exons of the human GLUD1 gene were examined by PCR/single-strand conformation polymorphism (SSCP) analysis. Each of the exons was PCR amplified from peripheral blood leukocyte DNA. Primers for exons 11 and 12 were synthesized according to Stanley et al. (3). Other primers were designed based on the public database sequences (10) (accession nos. X66300–X66309, X66311–X66319, and AL136982) (Table 1). SSCP analysis was performed at 15°C using a CleanGel DNA analysis kit and a Multiphor II electrophoresis system (Amersham Pharmacia Biotech, Tokyo) as previously described (9). Nucleotide changes were identified by direct sequencing of PCR products using a model 373A automated sequencer and BigDye terminators cycle sequencing kit (Applied Biosystems Japan, Tokyo).

The plasmids pcDNA3-hGDH and pcDNA3-hGDH266C were introduced into COS-7 cells by transfection with TransIT LT1 (PanVera, Madison, WI) according to the supplier’s protocol. A reporter plasmid, pcDNA3-lacZ, was cotransfected. Forty-eight hours after the transfection, cells were collected, suspended in PBS, and disrupted by sonification. After a brief centrifugation, the supernatant was used for GDH enzyme assay. ß-Galactosidase activity was also measured (11). In MIN6 cells, wild-type and mutant GDH were overexpressed using retrovirus-mediated gene transfer as described below. Samples for the GDH enzyme assay were prepared as for COS-7 cells.

GDH activity was measured as previously described (12) using a Beckman Coulter (Fullerton, CA) Spectrophotometer Model DU-640 at 25°C. The assay solution (1 ml) consisted of 10 mmol/l Tris-acetate (pH 8.0), 10 µmol/l EDTA, 100 µmol/l NADH, 50 mmol/l NH₄Cl, and 5 mmol/l -ketoglutarate. ADP or GTP was added to the solution at various concentrations. The reaction was started by adding cell extracts, and the decrease in absorbance at 340 nm was measured for 5 min. The activity was determined in duplicate or in triplicate for each sample. The half-maximal stimulatory concentration (SC₅₀) and the half-maximal inhibitory concentration (IC₅₀) were determined graphically.

Production of recombinant retroviruses and establishment of MIN6 cells stably expressing GDH266C.
The mutant GDH266C cDNA was inserted into the retrovirus vector pMX-puro (kindly provided by Dr. T. Kitamura, Institute of Medical Science, University of Tokyo) to make pMX-puro-GDH266C. Another retrovirus vector, pMX-puro-lacZ, which encodes the bacterial ß-galactosidase gene, was also constructed and used as a control. Recombinant retroviruses were produced by transfecting these recombinant retrovirus vectors into a transient retrovirus packaging cell line, Plat-E cells (13), using FuGENE 6 (Roche, Indianapolis, IN) according to the supplier’s protocol. Culture media containing infectious retrovirus particles were collected 48 h after transfection.

MIN6 cells (14) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, St Louis, MO) containing 25 mmol/l glucose and supplemented with 15% of heat-inactivated FCS, 72 µmol/l ß-mercaptoethanol, 50 unit/ml penicillin G, and 50 µg/ml streptomycin (DMEM-MING). MIN6 cells were infected with the recombinant retroviruses by incubating the cells with media containing the retroviruses for 24 h in the presence of 10 µg/ml polybrene (Sigma). Seventy-two hours after the initiation of infection, puromycin (2 µg/ml) was added to the culture medium, and MIN6 cells integrating the retroviral genome (MIN6-GDH266C and MIN6-lacZ) were selected.

Analysis of insulin secretion.
MIN6 cells overexpressing the mutant GDH via retrovirus-mediated gene transfer (MIN6-GDH266C) and control lacZ-overexpressing cells (MIN6-lacZ) were seeded onto 24-well plates at a concentration of 3.5 x 10⁵ cells/well and cultured in DMEM-MIN6 medium. Sixty hours later, insulin secretion was assayed by the static incubation method (15). In brief, after a 30-min preincubation in HEPES-balanced Krebs-Ringer bicarbonate buffer (KRBB) (10 mmol/l HEPES, 120 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l MgSO₄, 1.2 mmol/l KH₂PO₄, 20 mmol/l NaHCO₃, and 2 mmol/l CaCl₂, pH 7.4) supplemented with 0.5% BSA and 5 mmol/l glucose, the preincubation buffer was replaced with fresh HEPES-balanced KRBB containing 0.5% BSA and various concentrations of glutamine or glucose. After an additional 2-h incubation at 37°C, the buffer was collected, and immunoreactive insulin was measured by radioimmunoassay using rat insulin (Linco Research, St. Charles, MO) as a standard. The amounts of secreted insulin were corrected by the amounts of cell protein in each well.

	RESULTS

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PCR/SSCP analysis of the human GLUD1 gene.
Human mitochondrial GDH is encoded by 13 exons of the GLUD1 gene, which is located at 10q23.3. Several related pseudogenes (GLUDP1–5) have been recognized (10,16). Among them, the GLUDP2 and -3 genes contain three exons (exons 2–4) that are highly homologous to the GLUD1 gene (10). Due to the extensive similarity of intron sequences surrounding these exons among the GLUD1 gene and its pseudogenes, we carefully chose the primer sequences so that these GLUD1 gene exons could be specifically PCR amplified from genomic DNA (data not shown).

We identified three nucleotide substitutions in 12 Japanese patients with PHHI. Two of those were silent mutations: CTA (Leu) to CTG (Leu) at codon 261 and GTG (Val) to GTC (Val) at codon 496. Altogether, 5 of 12 PHHI patients (42%) and 50 of 94 control subjects (53%) were heterozygotes of CTA/CTG at codon 261. Although they were easily recognized on SSCP analysis and were confirmed by sequencing, the CTA/CTA homozygote and the CTG/CTG homozygote were barely distinguishable by SSCP patterns. Therefore, we did not determine the frequency of this single nucleotide polymorphism. One PHHI patient was a heterozygote at codon 496 (GTG/GTC). The frequency of this variation was not determined in control subjects. The third nucleotide variation, TAT to TGT at codon 266, was found in one patient with PHHI. This nucleotide substitution changes Tyr266 to Cys, and the patient was a heterozygote. This mutation was not found in 79 patients with type 2 diabetes or in 94 control subjects.

Clinical characteristics of the patient with the Y266C mutation and studies of her family.
The female patient with the Y266C mutation was 21 years of age at the time of this study. She was born at 37 weeks and 5 days of gestation as the second of four daughters of nonconsanguineous parents. Pregnancy and delivery were normal. Her birth weight was 2,700 g. Her first seizure occurred at 7 months of age and was not responsive to anticonvulsants. Hypoglycemia (1.7 mmol/l) was disclosed at age 13 months, when a seizure occurred just after milk feeding. Oral administration of leucine (150 mg/kg) induced hypoglycemia, and diazoxide treatment was started under a diagnosis of leucine-sensitive hypoglycemia. After the initiation of diazoxide treatment, no hypoglycemic episodes were documented up to the time of this study, i.e., at 21 years of age. She has no family history of hypoglycemia, and her parents and three sisters were healthy. Her plasma ammonium levels, measured during this study, were mildly elevated (128 µmol/l, normal 12–84). Her parents and two of her sisters did not have the Y266C mutation, and their plasma glucose and ammonium levels were normal (data not shown).

Characterizations of GDH266C expressed in COS-7 cells.
The wild-type and mutant GDH were expressed in COS-7 cells by transfection of pcDNA3-hGDH and pcDNA3-hGDH266C, respectively. A reporter plasmid pcDNA3-lacZ was cotransfected. GDH activities in the crude extracts of COS-7 cells transfected with pcDNA3-hGDH and pcDNA3-hGDH266C were 0.82 ± 0.22 and 3.27 ± 0.72 µmol · NADH · mg^–1 · protein · min^–1, respectively, when activity was measured without ADP or GTP in the reaction mixture (means ± SE, n = 3). Endogenous GDH activity in untreated COS-7 cells (0.028 µmol/l · NADH · mg^–1 · protein · min^–1) was <1/20 the total GDH activity in COS-7 cells transfected with pcDNA3-hGDH and thus negligible compared with the exogenous GDH expressed by the transfected plasmids. Therefore, the basal activity (activity in the absence of ADP or GTP) of GDH266C is elevated as compared with the wild-type enzyme. To exclude the possibility that this difference in the enzyme activity reflected the difference in the transfection efficiency, we measured ß-galactosidase activities in the same cell extracts, and the GDH activity was normalized by the ß-galactosidase activities. After the normalization, the basal activity of GDH266C was still 3.5 ± 1.2 times higher (means ± SE, n = 3) than that of wild-type GDH.

GDH is known to be regulated by various allosteric regulators. We examined whether the activity of GDH266C can be regulated by ADP, an allosteric activator, or GTP, an allosteric inhibitor. As shown in Fig. 1, ADP activated wild-type GDH (3.32 ± 0.12 µmol/l · NADH · mg^–1 · protein · min^–1 at 200 µmol/l ADP), with a SC₅₀ of 11 µmol/l. On the other hand, the activity of GDH266C was not increased by ADP (3.45 ± 0.50 µmol/l · NADH · mg^–1 · protein · min^–1 at 200 µmol/l ADP). Similarly, although GTP inhibited the wild-type GDH activity, with a IC₅₀ of 0.65 µmol/l, the activity of GDH266C was not inhibited by GTP at a concentration as high as 100 µmol/l. The activities of wild-type GDH and GDH266C at 100 µmol/l GTP were 2.7 ± 0.9 and 96.6 ± 1.6% of those without GTP, respectively.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Kinetic characteristics of GDH266C expressed in COS-7 cells. GDH activities in the crude extract of COS-7 cells were measured in the presence of various concentrations of ADP (A) or GTP (B). Relative activities at each concentration of allosteric effectors were plotted as ratios to the activity measured in the absence of the effectors. Experiments were repeated three times, and means ± SE are presented. , COS-7 transfected with pcDNA3-hGDH (wild type); , COS-7 cells transfected with pcDNA3-hGDH266C (mutant).

Determined in crude cell extracts (Table 2), basal (in the absence of ADP) and inhibited (in the presence of GTP) GDH activities in MIN6-GDH266C were 20 times higher than the basal GDH activity in MIN6-lacZ. The values were even higher than activated MIN6-lacZ GDH activity (measured in the presence of 200 µmol/l ADP). GDH activity in MIN6-GDH266C was doubled in the presence of 200 µmol/l ADP, probably because intrinsic MIN6 GDH was activated. These results suggest that in MIN6-GDH266C, GDH activity is constitutively elevated and minimally regulated by allosteric regulators such as ADP and GTP.

View larger version (26K): [in this window] [in a new window]   FIG. 2. Glutamine- and glucose-stimulated insulin secretion. MIN6-GDH266C () and MIN6-lacZ cells () were incubated in the presence of various concentrations of glutamine (A) or glucose (B) for 2 h, and insulin released into the incubation buffer was measured. Each assay was performed in duplicate or triplicate wells, and values are means ± SE of three (0.1 and 0.2 mmol/l glutamine), four (0.5 and 10 mmol/l glutamine), five (1 and 5 mmol/l glutamine) (A), and seven (2 mmol/l glucose) to nine experiments (other glucose concentrations) (B). *P < 0.05, **P < 0.03, and ***P < 0.01 in comparison between MIN6-GDH266C and MIN6-lacZ (unpaired Student’s t test).

	DISCUSSION

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The nearly total insensitivity of GDH266C to GTP and ADP contrasts with the kinetic characteristics of other mutants that were recently identified in patients with HI/HA syndrome. In previous studies, the IC₅₀ elevation for GTP was 2- to 10-fold, with the IC₅₀ being <2 µmol/l (3–5), and activation by ADP was mostly intact. The difference is probably attributable to the nature of the mutations. In addition, it may also be explained by the fact that the previous studies were performed using patients’ lymphoblasts in which both mutant and wild-type GDH existed at a 1:1 ratio, and therefore the effects of mutations on kinetic properties were less prominent.

Despite the differences in the kinetic properties of the enzyme, the clinical characteristics of our patient and the previously reported patient with the same mutation (5) were very similar to those of patients with other mutations. It was recently reported that there is a genotype-phenotype correlation in regard to plasma ammonium levels but not the severity of hypoglycemia (5). Hypoglycemia in our patient was medically manageable with diazoxide, and the plasma ammonium level was only mildly elevated (128 µmol/l, normal 12–84).

Using GDH266C, which has unique kinetic properties, as a tool, we examined the role of GDH in the regulation of insulin secretion and the pathophysiology of ß-cells from HI/HA syndrome patients. We established MIN6 cells stably overexpressing the mutant GDH and analyzed glutamine- and glucose-stimulated insulin secretion. GDH, upon stimulation by allosteric activators such as leucine, presumably enhances glutamate oxidation and thereby stimulates insulin secretion (1,2). Herein, we have directly shown for the first time that increased GDH activity confers on ß-cells the ability to secrete insulin in response to glutamine. GDH266C-overexpressing MIN6 cells secreted insulin in response to glutamine in a dose-dependent manner, whereas control ß-galactosidase–overexpressing MIN6 cells were unresponsive to glutamine stimulation. This observation clearly demonstrated the importance of GDH catalyzing oxidative deamination of glutamate for insulin secretion rather than glutamate formation from -ketoglutarate by reverse reaction (6). Enhanced insulin secretion in response to glutamine from MIN6-GDH266C is directly relevant to the phenotype of HI/HA syndrome patients—they tend to develop hypoglycemia after a high-protein meal (21).

Physiologically, glutaminolysis may play more important roles in insulin secretion at low glucose concentrations and contribute to interprandial, basal insulin secretion (8). In pancreatic islets, glucose increases GTP levels while decreasing ADP levels in a concentration-dependent manner (22–24). At low glucose concentrations, flux through the TCA cycle via glucose metabolism is low, whereas glutaminolysis is activated because relatively low intracellular GTP concentrations and high ADP concentrations activate GDH, leading to sustained basal insulin secretion (8). In MIN6-GDH266C, in which GDH activity is supraphysiologically elevated, insulin secretion was exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) compared with control MIN6-lacZ. This observation is consistent with a previous study (8) and may also explain the HI/HA syndrome patients’ fasting hypoglycemia. At higher glucose concentrations, flux through the TCA cycle is probably high enough to mask the effect of enhanced glutaminolysis on insulin secretion in MIN6-GDH266C cells.

Recently, glutamate was suggested to be a mediator of glucose-stimulated insulin secretion, acting directly on insulin secretory granules (6). According to this hypothesis, GDH catalyzes the formation of glutamate from -ketoglutarate rather than oxidative deamination of glutamate. However, our results from MIN6-GDH266C and previous evidence, including clinical data from HI/HA syndrome, do not support this hypothesis (3–5,7,8). Investigations to elucidate changes in intracellular glutamate concentrations and glutamine oxidation in MIN6-GDH266C will further clarify the role of GDH in the regulation of insulin secretion.

	ACKNOWLEDGMENTS

 
This study was partly supported by Grants-in-Aid for Scientific Research (12671113 and 11557012 to Y.T.) and for Creative Basic Research (10NP0201 to Y.O.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

We thank all of our patients, their family members, and physicians caring for the patients who participated in this study. We also thank Dr. T. Kitamura (Institute of Medical Science, University of Tokyo) for providing us with the retrovirus vector pMX-puro and the transient retrovirus packaging cell line, Plat-E, as well as Yukari Kora-Miura and Atsuko Tanimura for their expert technical assistance. We are grateful to Dr. K. Ariyoshi (Yamaguchi University) for his help in preparing recombinant retroviruses.

	FOOTNOTES

 
Address correspondence and reprint requests to Yukio Tanizawa, MD, PHD, Department of Bio-Signal Analysis, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami Kogushi, Ube, Yamaguchi 755-8505, Japan. E-mail: tanizawa{at}yamaguchi-u.ac.jp.

Received for publication 26 August 2001 and accepted in revised form 6 November 2001.

DMEM, Dulbecco’s modified Eagle’s medium; GDH, glutamate dehydrogenase; HI/HA, hyperinsulinism/hyperammonemia; IC₅₀, half-maximal inhibitory concentration; KRBB, Krebs-Ringer bicarbonate buffer; PHHI, persistent hyperinsulinemic hypoglycemia of infancy; SC₅₀, half-maximal stimulatory concentration; SSCP, single-strand conformation polymorphism; TCA, tricarboxylic acid.

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TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

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