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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0991
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5841-5847
Copyright © 2005 by The Endocrine Society

Genetic Variation in the Hypoxia-Inducible Factor-1{alpha} Gene Is Associated with Type 2 Diabetes in Japanese

Norihiro Yamada, Yukio Horikawa, Naohisa Oda, Katsumi Iizuka, Nobuyuki Shihara, Shoji Kishi and Jun Takeda

Department of Ophthalmology (N.Y., S.K.), Gunma University Graduate School of Medicine, 371-8511 Gunma, Japan; Laboratory of Medical Genomics, Biosignal Genome Resource Center (Y.H., N.S., J.T.), Institute for Molecular and Cellular Regulation, Gunma University, 371-8512 Gunma, Japan; Core Research for Evolutional Science and Technology (Y.H., K.I., N.S., J.T.), Japan Science and Technology Corporation, 332-0012 Kawaguchi, Japan; Department of Diabetes and Endocrinology (Y.H., J.T.), Gifu University School of Medicine, Gifu 501-1194, Japan; and Department of Internal Medicine (N.O.), Fujita Health University School of Medicine, 470-1192 Aichi, Japan

Address all correspondence and requests for reprints to: Yukio Horikawa, M.D., Ph.D., Department of Diabetes and Endocrinology, Gifu University School of Medicine, 1-1 Yanagido, Gifu-city, Gifu 501-1194, Japan. E-mail: yhorikaw{at}cc.gifu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context and Objective: Vascular endothelial growth factor plays a critical role both in neovascularization of proliferative diabetic retinopathy and in angiogenesis of islets in the pancreatic developmental stage in determining ß-cell mass and properties. Vascular endothelial growth factor mRNA levels increase as a result of increased transcriptional activation, mediated predominantly by hypoxia-inducible factor-1 {alpha} (HIF-1{alpha}) in response to hypoxia.

Design and Patients: In this study, we examined all regions of the HIF-1{alpha} to detect single-nucleotide polymorphisms (SNPs), evaluated the pattern of linkage disequilibrium to analyze haplotypes, and performed association studies in Japanese type 2 diabetes patients with or without retinopathy.

Results: A total of 35 SNPs were found in the gene, 27 of which were reported previously and eight of which were novel. Three of the 35 SNPs were located in coding regions, one in exon 2 (S28Y), and the others in exon 12 (P582S, A588T). The P582S HIF-1{alpha} mutation was associated with type 2 diabetes (P = 0.0028) by a consistently higher level of transcriptional activity than wild type, especially under hypoxic condition (P = 0.012), but no association with retinopathy was detected.

Conclusion: This is the first report that HIF-1{alpha} is associated with the occurrence of type 2 diabetes and suggests that the P582S HIF-1{alpha} mutation should be assessed in larger studies as a risk factor for type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
DIABETES LEADS TO specific microvascular complications of retinopathy, nephropathy, and neuropathy as well as increased risk of atherosclerosis, which may reflect underlying endothelial dysfunction. The risk of developing these complications is increased by poor glycemic control, but the relevance of genetic background is clearly established (1, 2, 3).

Diabetic retinopathy is a major cause of new-onset blindness among diabetic adults and is characterized by increased vascular permeability, tissue ischemia, and neovascularization. Neovascularization of the retina carries a high risk of blindness as a result of vitreous hemorrhage, fibrosis, and tractional retinal detachment. Vascular endothelial growth factor (VEGF) can stimulate angiogenesis, enhance collateral vessel formation, and increase permeability of the microvasculature (4). In diabetic proliferative retinopathy, VEGF plays a critical role in neovascularization and breakdown of the blood-retinal barrier characterized by hyperpermeability of retinal vessels. VEGF levels have been found to be markedly elevated in vitreous and aqueous fluids in the eyes of patients with proliferative diabetic retinopathy (PDR) (5). Furthermore, VEGF has been reported as a susceptibility gene for type 2 diabetes mellitus (T2DM) as well as diabetic retinopathy (6). VEGF is known to be a key factor in angiogenesis of islets in the pancreatic developmental stage in determining ß-cell mass and properties (7). In response to hypoxia, VEGF mRNA levels are increased by increased transcriptional activation, whereas overall protein synthesis is inhibited. This increase is mediated predominantly by hypoxia-inducible factor-1 (HIF-1) binding to a hypoxia response element located 1 kb upstream of the transcriptional start site of VEGF (8, 9).

HIF-1, a transcription factor found in mammalian cells cultured under reduced oxygen tension, plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 acts as a heterodimer composed of a 120-kDa HIF-1{alpha} subunit complexed with a 91- to 94-kDa HIF-1ß subunit (10). Although HIF-1ß is ubiquitously expressed and maintained at constant cellular levels, the HIF-1{alpha} protein level and transcriptional activity are tightly regulated in response to oxygen levels. Thus, HIF-1 activity is controlled by the oxygen-regulated expression of the HIF-1{alpha} subunit (11). Under nonhypoxic conditions, HIF-1{alpha} is hydroxylated on proline residues by a family of oxygen-dependent prolyl hydroxylases. The hydroxylated prolines independently mediate high-affinity binding to the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin-protein ligase complex that ubiquitinates HIF-1{alpha}, thereby targeting it for degradation. The critical proline residues for VHL binding when hydroxylated are P402 and P564, both of which are located in the oxygen-dependent degradation domain (12, 13)

The HIF-1{alpha} gene is located at chromosome 14q21-q24, where the susceptibility locus to T2DM was localized in Finns (14). The predicted 826-amino-acid HIF-1{alpha} contains a basic helix-loop-helix-Per-Arnt-Sim (PAS) domain at its N terminus and a transactivation domain and transcriptional inhibitory domain at its C terminus (15). HIF-1 target genes include those for energy metabolism, iron homeostasis, angiogenesis, and cell proliferation and viability (16). Thus, HIF-1{alpha} activates many genes in the glycolysis system under conditions of hypoxia differently from insulin (17). In addition, the involvement of HIF-1 in the pathophysiology of human disease including myocardial ischemia, cerebral ischemia, retinal ischemia, pulmonary hypertension, preeclampsia, intrauterine growth retardation, and cancer has been reported (16). However, the correlations between HIF-1 and T2DM including complications remain to be elucidated.

In this study, we examined all regions of the HIF-1{alpha} gene in Japanese subjects to detect single-nucleotide polymorphisms (SNPs), evaluated the pattern of linkage disequilibrium (LD) to compose haplotypes in the gene, and performed association studies in T2DM patients. We identified a susceptibility coding SNP (cSNP) (P582S) and haplotype in the HIF-1{alpha} gene for T2DM. This is the first report suggesting that HIF-1{alpha} is associated with the occurrence of T2DM.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

A total of 440 patients with T2DM [245 males and 195 females; age at testing, 60.5 ± 11.4 yr; duration, 11.4 ± 9.1 yr; body mass index (BMI), 23.9 ± 4.3 kg/m2; maximum BMI, 27.5 ± 4.6 kg/m2; glycosylated hemoglobin (HbA1c), 7.9 ± 3.8%; total cholesterol (T-chol), 0.52 ± 0.11 mmol/liter; high-density lipoprotein (HDL), 0.13 ± 0.04 mmol/liter; triglyceride (TG), 0.39 ± 0.25 mmol/liter) and 572 controls (231 males and 342 females; age at testing, 67.3 ± 6.5 yr; BMI, 23.0 ± 2.9 kg/m2; HbA1c, 5.0 ± 0.4%) were examined for an association study. The diagnosis of T2DM was based on medical records or 75-g oral glucose tolerance test according to the criteria of the Japan Diabetes Society (18). One hundred eighty-two T2DM patients with retinopathy [80 males and 102 females; PDR, n = 117; pre-PDR, n = 29; and simple diabetic retinopathy, n = 36; age at testing, 61.9 ± 11.2 yr; duration, 13.0 ± 9.7 yr; BMI, 23.7 ± 3.9 kg/m2; maximum BMI, 27.9 ± 6.6 kg/m2; HbA1c, 7.6 ± 1.9%; T-chol, 0.52 ± 0.14 mmol/liter; HDL, 0.12 ± 0.04 mmol/liter; TG, 0.41 ± 0.20 mmol/liter; hypertension (HT) drug use, 94 (–)/88(+); insulin therapy, 105(–)/77(+)] and 125 without retinopathy (47 males and 78 females; age at testing, 62.9 ± 11.6 yr; duration, 11.0 ± 8.6 yr; BMI, 23.7 ± 4.8 kg/m2; maximum BMI, 26.1 ± 6.6 kg/m2; HbA1c, 6.8 ± 1.4%; T-chol, 0.50 ± 0.10 mmol/liter; HDL, 0.10 ± 0.04 mmol/liter; TG, 0.36 ± 0.22 mmol/liter; HT drug use, 99(–)/26(+); insulin therapy, 89(–)/36(+)]were examined by an association study with special reference to retinopathy. An additional 88 patients diagnosed with PDR by an ophthalmologist were included in the patients with retinopathy. Control subjects were recruited on the following criteria: 60 or more years of age, no past history of diagnosis of diabetes mellitus, HbA1c less than 5.6%, and no familial history of diabetes mellitus in second-degree relatives. The study was approved by the Ethics Committee of Gunma University and Fujita Health University School of Medicine upon written, informed consent of each subject.

SNP identification in the HIF-1{alpha} gene

Genomic DNA was extracted from samples of whole blood using QIAamp DNA blood kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Sixteen of the random control samples (32 alleles) were used to detect SNPs in the HIF-1{alpha} gene. Primers for PCR experiments were designed by Primer 3 (available from http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) on the basis of the genomic contig sequence (GenBank accession number NT_026437) of the HIF-1{alpha} region. The mixture for PCR was 20 µl in 10 ng template DNA, 0.5 mM of each dNTP, 2.5 pmol of each forward and reverse primer, 0.5 U ExTaq polymerase (Takara, Kyoto, Japan), and 2 µl of 10x PCR buffer. The reaction conditions were an initial denaturation step of 95 C for 3 min and a subsequent 40 cycles of reaction at 94 C for 30 sec, 52–63 C for 30 sec, and 72 C for 1 min, and a final extension step of 72 C for 10 min. A 3-µl aliquot from each reaction was assayed on a 1% agarose gel to confirm the product, and the remainder was purified using MultiScreen Filtration System (Millipore, Billerica, MA) with Sephadex G-75 (Amersham Biosciences, Piscataway, NJ). Each PCR product was subjected to cycle sequencing with BigDye Terminator cycle sequencing FS (Applied Biosystems, Foster City, CA) using each forward and reverse primer. Reaction products were purified by ethanol precipitation and sequenced by ABI PRISM 3100 or 3700 sequencer. Results were processed with Autoassembler version 2.1 (Applied Biosystems) to compare sequences.

Mutation screening and genotyping of frequent polymorphisms in the HIF-1{alpha} gene

We examined all of the coding regions of the HIF-1{alpha} gene in 96 of the 440 T2DM patients (59 males and 37 females; age, 58.6 ± 12.1 yr; age at diagnosis, 44.2 ± 12.7 yr; duration, 14.4 ± 9.2 yr; BMI, 23.7 ± 3.8 kg/m2; maximum BMI, 28.9 ± 4.5 kg/m2; HbA1c, 7.3 ± 1.5%) and 96 of the 576 control subjects (35 males and 61 females; age, 67.6 ± 5.8 yr; BMI, 22.9 ± 2.8 kg/m2; HbA1c, 4.9 ± 0.3%). Twenty-four frequent SNPs were examined in the 440 T2DM patients and 572 controls by direct sequencing or TaqMan assay (Applied Biosystems). PCR was performed in a total volume of 5 µl, which contained 2.5 ng DNA, 1x TaqMan Universal PCR Master Mix, with each primer at a concentration of 900 nM and each probe at a concentration of 200 nM. Thermal cycling conditions were as follows: 50 C for 2 min and 95 C for 10 min to activate the amperase uracil-N-glycosylase and AmpliTaq Gold enzyme, respectively, followed by 40 cycles of 92 C for 15 sec and 52–63 C for 1 min. The fluorescence level was measured with an ABI PRISM 7900HT sequence detector (Applied Biosystems), resulting in clear identification of three genotypes.

Estimation of haplotype frequencies and evaluation of pattern of LD in the HIF-1{alpha} gene

Haplotypes and haplogenotypes were inferred by the expectation-maximization method by Haploview (http://www.broad.mit.edu/personal/jcbarret/haploview) and PHASE 2.1.1 (http://www.stat.washington.edu/stephens), respectively. The coefficients for LD, D', and r2 value were estimated by GOLD software (available from http://www.well.ox.ac.uk/asthma/GOLD).

Cloning of human HIF-1{alpha} and variants

A cDNA identical to HIF-1{alpha} was retrieved from a human islet cDNA library and subcloned in pENTR/D-TOPO (Invitrogen, Carlsbad, CA) after amplification with Pfu (Stratagene, La Jolla, CA) and the primer set 5'-CACCATGGAGGGCGCCGGCGGCGCGAAC-3' and 5'-TCAGTTAACTTGATCCAAAGCTCTG-3' and transferred for expression to pcDNA3.2-DEST (Invitrogen). The P582S mutation was introduced by QuikChange site-directed mutagenesis kit (Stratagene) with pENTR/D-TOPO wild-type HIF-1{alpha} as template and confirmed by sequencing.

Functional analysis of the identified HIF-1{alpha} mutation

The reporter construct VEGF promoter-pGL3 was prepared by cloning the human VEGF gene promoter (–1180 to ~+338) into the firefly luciferase reporter vector pGL3-Basic (Promega, Madison, WI) (17). HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum and transfected using ExGen 500 (Fermentas, St. Leon-Rot, Germany) with various amounts of the reporter and test plasmid constructs. Transcriptional activity was normalized with a cotransfected control thymidine kinase (TK)-regulated Renilla luciferase vector, pRL-TK (Promega). The transactivation activity of wild-type and mutant proteins was measured using the Dual-Luciferase Reporter Assay System (Promega). After 6 h transfection, the cells were incubated 24 h under normoxic (21% O2) or hypoxic (1% O2) conditions using Anaero Pack (Mitsubishi Gas Chemical, Tokyo, Japan) before analysis of reporter gene activity.

Statistical analyses

Statistical difference in allele frequencies between T2DM and control groups or between T2DM with and without retinopathy groups was assessed by {chi}2 test, and other categorical clinical variables were compared using t test or logistic regression analysis adjusted for relevant covariates. Statistical analysis was performed with StatView 5.0 software (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Identification of polymorphisms in the HIF-1{alpha} gene

Sixteen individuals were examined for sequence variations in 38 kb of the 78-kb region of the HIF-1{alpha} gene including all 15 exons (NT_026437, nucleotides 61222006–61300408). A total of 35 SNPs were found in the gene; the locations of these SNPs are shown in Fig. 1Go in relation to the genomic structure of the HIF-1{alpha} gene. All SNPs were in Hardy-Weinberg equilibrium, and 27 were reported in the Institute of Medical Science-Japan Science and Technology Agency SNP database (http://snp.ims.u-tokyo.ac.jp/index.html) or in the National Center for Biotechnology Information SNP database (http://www.ncbi. nlm.nih.gov/SNP/). Eight SNPs (SNP-6, SNP-12, SNP-18, SNP-19, SNP-31, SNP-32, SNP-33, and SNP-34) were novel and had not been reported. Three of the 35 SNPs were located in coding regions, one in exon 2 (S28Y), and the other SNPs in exon 12 (P582S, A588T) (Table 1Go).



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  		FIG. 1. Polymorphisms of HIF1A identified in this study. Nucleotide indicates the location of the SNP relative to the A of ATG of the initiator Met of HIF1A (GenBank no. NT_026437).

 

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  		TABLE 1. Polymorphisms identified in HIF1A region in this study

 
Evaluation of pattern of LD in the HIF-1{alpha} gene

Twenty-three SNPs were used to define haplotypes and evaluate the pattern of LD in the 440 T2DM patients and 572 control subjects. As shown in Fig. 2Go, one LD block appears in this region in both groups. The five SNPs at position SNP-22 (g.25200), SNP-23 (g.25299), SNP-24 (g.27009), SNP-26 (g.34942), SNP-27 (g.35074), and the three SNPs at position SNP-14 (g.45483), SNP-16 (g.46572), and SNP-17 (g.46820) are in complete LD.



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  		FIG. 2. Pairwise LD in HIF1A evaluated by r2. Pairwise LD was determined using 276 marker pairs. Color gradations from red (perfect LD, i.e. r2 = 1) to blue (no LD, i.e. r2 = 0) reflect the degree of the observed LD. The upper triangle shows LD pattern estimated with 440 T2DM patients, and the lower triangle shows that with 576 controls (CONT).

 
Mutation screening and association study of genetic variation of the HIF-1{alpha} gene in T2DM patients

All exons were examined in 96 T2DM patients and 96 control subjects. We found a total of three cSNPs (S28Y, P582S, and A588T), of which S28Y is novel. We then performed an association study using possible pairwise haplotypes in T2DM patients and controls. Ten SNPs (SNP-30, -2, -3, -4, -25, -7, -18, -20, -13, and -28) were used to define haplotypes. The other SNPs were excluded because of the rarity of minor alleles. We found that a haplotype comprising SNP-25 and SNP-13, which are in strong LD, represents significant susceptibility to T2DM at a P value of 10–11 based on a two by four {chi}2 test, and also after multiple adjustment. The 1-1/2-2 haplogenotype comprising these two SNPs was associated with significantly decreased risk of T2DM [T2DM, 25.5%; control, 32.0%; odds ratio (OR) = 0.73; 95% confidence interval (CI), 0.55–0.96; 1 – ß = 53%). The 2-2/2-2 haplogenotype also was associated with decreased risk (T2DM, 2.8%; control, 5.1%; OR = 0.51; 95% CI, 0.27–1.08), but the difference was not significant because of the small sample. As shown in Table 2Go, the P582S mutant allele was found with significantly less frequency in T2DM patients than in control subjects (P = 0.0028; 1 – ß = 52%) and also was significant after adjustment for sex, age, and BMI by logistic regression analysis (P = 0.0048). Interestingly, the P582S mutant allele is completely assigned on the 2-2 haplotype of SNP-25 and SNP-13, which was observed more frequently in control subjects with strong statistical significance (Fig. 3Go). Accordingly, the mutant allele S582 is a representative SNP of the 2-2 haplotype, which contributes to decreased risk of T2DM. Indeed, if a dominant model is assumed, the S582 HIF-1{alpha} mutation is associated with decreased risk of T2DM (OR = 0.57; 95% CI, 0.37–0.88; P = 0.010).


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  		TABLE 2. Association study using frequent SNPs in HIF1A in patients with T2DM and controls

 


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  		FIG. 3. Haplotype features in an LD block between SNP-25 and SNP-13 with frequencies. The numbers 1 and 2 indicate major allele and minor allele, respectively. CONT, Control.

 
Similar haplotypes were also identified at significant levels in patients with and without retinopathy, but none reached statistical significance after multiple adjustment (data not shown). We also performed an association study using single polymorphisms in patients with and without retinopathy, resulting in identification of two significant SNPs (SNP-15 and -29 at P values of 0.033 and 0.045, respectively), although these were also not statistically significant after multiple adjustment and adjustment for sex, age, duration, HbA1c, BMI, T-chol, TG, presence of medicine for HT, and presence of insulin therapy by logistic regression analysis (data not shown).

Hypoxia-dependent transactivation of polymorphic HIF-1{alpha}

The transcriptional activity of the S582 HIF-1{alpha} mutant was then compared with that of wild type under normoxic or hypoxic conditions. The HIF-1{alpha} vectors were transfected into HEK293 cells with a firefly luciferase reporter gene regulated by human VEGF gene promoter and with a control TK-regulated Renilla luciferase vector. The mutant S582 HIF-1{alpha} showed a hypoxia-dependent increase in transcription activity as did the wild-type HIF-1{alpha}. The results show consistently higher HIF-1{alpha} transcription activity in cells transfected with mutant S582 than with wild-type HIF-1{alpha}. However, enhanced transactivation capacity of the S582 HIF-1{alpha} mutant was observed with statistical significance only under hypoxic condition (P = 0.012) (Fig. 4Go).



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  		FIG. 4. Transactivation capacity of mutant S582 HIF-1{alpha}. Transcription activity with no vector, empty vector, wild type, and P582S HIF-1{alpha} (500 ng) was analyzed by cotransfection assay using reporter vector VEGF promoter-pGL3 (200 ng/35-mm well) and a Renilla-luciferase (25 ng/35-mm well) as internal control. The average of three independent experiments is shown (bar, ±SD).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The estimated prevalence of diabetes in Japan is 13 million, 90% with T2DM as a result of both environmental and genetic factors. So far, only genetic variants of the Calpain-10 gene (19), PPAR{gamma} gene (20), and Kir 6.2 gene (21) have been reported to be related to the occurrence of T2DM in large studies, and genes including angiotensin converting enzyme (ACE), aldose reductase (AR), nitric oxide synthase (NOS), VEGF, and paraoxonase 1 (PON1) etc. have been reported to contribute to the development of diabetic retinopathy (22).

We previously compared the expression profile of 1000 mRNAs of rat pancreatic islets with those of rat retina, resulting in the identification of 123 commonly expressed genes. These genes are candidates in both development of T2DM and diabetic retinopathy (23). One of them, VEGF, was reported to be a susceptibility gene for both onset of diabetes and diabetic retinopathy (6, 7), and HIF-1{alpha} was reported to induce expression predominantly of VEGF and other important genes involved in glucose metabolism under hypoxic condition, leading us to examine the correlation between HIF-1 and T2DM and retinopathy more closely.

In this study, we examined 38 kb covering the entire coding region of the HIF-1{alpha} gene in 16 Japanese subjects and identified a total of 35 genetic variations. Thirty-two SNPs were identified in noncoding regions, and three SNPs were identified in coding regions. We defined haplotypes by all possible pairs of 10 SNPs, based on the LD pattern estimated using the frequent SNPs, and examined the associations with T2DM and retinopathy. The haplotype comprising SNP-25 and SNP-13 was most significantly associated with T2DM. cSNP-10 (P582S) was significantly associated with decreased risk of T2DM. Interestingly, the rare allele cSNP-10, resulting in S582, was completely assigned on the 2-2 haplotype comprising SNP-25 and SNP-13, which was observed at significantly higher frequency in control subjects. Thus, the S582 HIF-1{alpha} mutant could be protective against the onset of T2DM.

The progress of diabetic retinopathy is known to reflect the duration of diabetes, control of blood glucose level, insulin dosage, and blood pressure. The percentage of patients treated with insulin and hypertension drugs was higher in the group with retinopathy, consistent with previous studies, although the P582S HIF-1{alpha} mutation was not associated with onset of diabetic retinopathy even after logistic regression analysis.

The same P582S HIF-1{alpha} somatic mutation was found in one of five androgen-independent prostate cancer samples in a recent study, although the mutant was not functionally characterized (24). In addition, an HIF-1{alpha} polymorphism, P582S, was identified in a study of renal cell cancers, but the significance in renal cell cancer patients and controls differs in two studies (25, 26). Recently, a P582S HIF-1{alpha} mutation was identified in prostate cancer and was reported to enhance transcriptional activity as a result of increased stability under normoxic conditions, resulting in increased tumor microvessel density (27, 28). Proline 582 has not been identified as a site for HIF-1{alpha} hydroxylation and is not known to mediate VHL binding. Moreover, the substitution of serine for proline in this position does not appear to prevent VHL binding in vitro to a fragment of HIF-1{alpha} after hydroxylation at proline 564 (29) Thus, if proline 582 is not a target for hydroxylation, conformational changes induced by the proline to serine substitution could influence hydroxylation at other sites as well as degradation in vivo.

HIF-1{alpha} is an important inducing factor of VEGF, a key factor in angiogenesis of islets in the pancreatic developmental stage in determining ß-cell mass and properties (7), although a high expression level of HIF-1{alpha} is observed in hypoxic human adult pancreatic islets and is reported to be correlated with apoptosis (30). In the present study in HEK293 renal cells, the mutant S582 showed a consistently higher level of HIF-1{alpha} transcriptional activity than in wild type, and the enhanced transactivation capacity of the mutant was observed with statistical significance only under hypoxic conditions. Accordingly, polymorphism P582S, by enhancing the transcriptional activity of target genes, could be a protective factor against onset of T2DM by its activities in the pancreatic developmental stage. When the two groups with and without S582 are compared, there are no significant differences in age, BMI, HbA1C, and the presence or absence of insulin therapy (data not shown), partly because of the small number of samples. Functional analysis of the mutant protein S582 HIF-1{alpha} using ß-cell- or endothelium-derived cell lines might also be required. We did not detect a significant difference in P582S allele frequencies in patients with or without retinopathy, but additional studies with an increased number of patients classified with precise clinical information are required to assess the correlation of this variant with diabetic retinopathy.

It has been reported that acute intensive insulin therapy results in transcriptional activation of VEGF via p38 MAPK, phosphatidylinositol-3-kinase, and HIF-1{alpha}, producing paradoxical worsening of diabetic blood-retinal barrier breakdown (31). Thus, more attention should be paid to patients with the P582S HIF-1{alpha} mutant allele when they begin treatment with insulin therapy. This polymorphism should be further assessed in larger studies as a risk factor for the development of T2DM and as a biomarker for responses to specific therapies, antiangiogenic therapies in particular.


   Acknowledgments
 
We thank S. Oike, R. Kawakami, Y. Yaginuma, I. Uda, Y. Ibe, and T. Takahashi for assistance.


   Footnotes
 
This study was supported by Grant-in-Aid for Scientific Research and for Scientific Research on Priority Areas (C) Medical Genome Science from the Japanese Ministry of Science, Education, Sports, Culture, and Technology; a Health and Labor Science Research Grant for Research on Human Genome and Tissue Engineering from the Japanese Ministry of Health, Labor, and Welfare; and the Naito Foundation.

First Published Online July 26, 2005

Abbreviations: BMI, Body mass index; CI, confidence interval; cSNP, coding SNP; HbA1c, glycosylated hemoglobin; HDL, high-density lipoprotein; HIF-1{alpha}, hypoxia-inducible factor-1{alpha}; HT, hypertension; LD, linkage disequilibrium; OR, odds ratio; PDR, proliferative diabetic retinopathy; SNP, single-nucleotide polymorphism; T-chol, total cholesterol; T2DM, type 2 diabetes mellitus; TG, triglyceride; TK, thymidine kinase; VEGF, vascular endothelial growth factor; VHL, von Hippel-Lindau.

Received May 5, 2005.

Accepted July 18, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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