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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Advanced glycation endproducts (AGEs), which result from nonenzymatic reactions of glucose with tissue proteins, have been shown to accumulate on long-lived proteins in advanced aging and diabetes mellitus. Thus, AGEs have been implicated in some of the chronic complications associated with these disorders. In this study, we investigated the effects of the glucose-modified protein on the production of the potent bone resorption factors by cells derived from explants of human bone. AGEs stimulated the release of interleukin-6 (IL-6) in the culture supernatants from the bone-derived cells and increased the levels of IL-6 mRNA in the cells. By contrast, the levels of IL-11 in the culture supernatants were not altered by AGEs, and the other bone resorption factors IL-1α and IL-1β were undetectable (<1.0 pg/ml) either without or with the treatment of AGEs. Electrophoretic mobility-shift assays revealed that the transcription nuclear factor-κB, which is critical for the inducible expression of IL-6, was activated in the nuclear extracts from mouse osteoblastic MC3T3-E1 cells treated with AGEs. These results suggest that AGEs are involved in bone remodeling modulation by stimulating IL-6 production in human bone-derived cells.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Osteoporosis is a group of skeletal disorders characterized by a reduction in bone mass per unit of bone volume. It may occur if the rate of bone resorption exceeds that of bone formation, implying an uncoupling of the phases of bone remodeling.1 The incidence of osteoporosis increases dramatically with age, becoming widely prevalent in the elderly, in whom it has become a major public health problem.2 However, the precise mechanism of the disorder remains unresolved.

Recent studies have demonstrated a role for cytokines in the pathogenesis of osteoporosis. Interleukin-1 (IL-1),3,4 IL-6,5,6 IL-11,7 tumor necrosis factor-α (TNF-α),8,9 macrophage colony stimulating factor (M-CSF),10 and granulocyte macrophage colony stimulating factor (GM-CSF)11 have been shown to stimulate bone resorption. Such bone resorption cytokines are considered to play a pivotal role in the increased bone resorption in postmenopausal osteoporosis; IL-1, IL-6, TNF, M-CSF, and GM-CSF have been shown to be produced in greater abundance in the estrogen-deficient state,12–17 resulting in the stimulation of osteoclastogenesis.

Age-related osteoporosis affects the entire population of aging men and women. The reason for this pathological state is not well understood. Previous studies18–20 have suggested that humoral, nutritional, and metabolic changes in aging result in decreased bone formation as well as increased bone resorption. Diabetes mellitus has also been shown to be associated with osteopenia,21–23 although conflicting findings have been reported, especially in patients with non–insulin-dependent diabetes mellitus.23–25 Despite many investigations, there is no report regarding the involvement of cytokines in the pathogenesis of age- and diabetes-related osteoporosis.

Advanced glycation endproducts (AGEs) are the ultimate products of nonenzymatic glycation of proteins.26,27 The accumulation of AGEs on long-lived proteins have been shown to accelerate in advanced aging and diabetes mellitus.26,27 Therefore, AGEs are postulated to be linked to the development of some complications in aging and diabetes. Several studies have shown that AGE-modified proteins perturbed cellular functions, including cytokine and growth factor release in macrophages,28–30 increased permeability and procoagulant activity in endothelial cells,31 increased synthesis of extracellular matrix components in mesangial cells,32 and induction of macrophage migration.29 The cellular interactions of AGEs have been shown to be mediated by the receptor for AGE (RAGE), a member of the immunoglobulin superfamily.33,34

A recent study by Thomasek et al.35 has demonstrated that AGEs accumulated on collagen derived from cortical bones of diabetic or aged rats. This observation led us to hypothesize that AGEs, developed on collagenous matrix of bone in diabetes and aging, could cause uncoupling of the phases of bone remodeling, resulting in osteoporosis. In the present investigation, we examined the effects of AGEs on the production of the cytokines to stimulate bone resorption by human bone-derived cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Preparation of glucose-modified protein

AGE-bovine serum albumin (AGE-BSA) was prepared as described previously.36 BSA (essentially fatty acid free; Sigma, St. Louis, MO, U.S.A.) was incubated with 250 mM glucose-6-phosphate (G-6-P) at 37°C for 8 weeks in the presence of 1.5 mM phenylmethylsulfonyl fluoride and 0.5 mM EDTA in phosphate-buffered saline (PBS). Unincorporated G-6-P was removed by extensive dialysis against PBS. BSA was incubated in parallel without G-6-P, as unmodified proteins. The glucose-modified BSA, but not the unmodified BSA, has the characteristics of AGE-proteins in terms of specific absorption and fluorescence spectra.27

Human bone-derived cell culture

Bone samples were obtained from four patients who underwent surgery. The samples of normal cancellous bone were collected following hip replacement surgery or maxillodental surgery. Bone specimens were minced into small fragments and incubated with 2 mg/ml collagenase and 0.1 mg/ml DNAse I for 2 h at 37°C with vigorous shaking. Bone fragments were then cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS; Bioserum, Victoria, Australia), 15 mM HEPES, 50 μg/ml ascorbic acid, 100 U/ml penicillin, and 30 μg/ml kanamycin sulfate.37 The bone fragments were removed after bone cells migrated. The cells were grown to confluency and subcultured using 30 μg/ml Actinase E (Kaken, Tokyo, Japan) digestion. All experiments were conducted by using third to eighth passages of the cells. These cells were able to produce bone gla protein when they were treated with 1,25-dihydroxyvitamin D3 (data not shown).

Measurement of cytokines

Human bone-derived cells were plated onto a 24-well plate (2 × 104 cells/well) in DMEM supplemented with 10% FCS and cultured to be grown to a subconfluent state. After the cultures were washed with PBS, the cells were treated for 48 h with BSA or AGE-BSA in 0.3 ml DMEM supplemented with 0.1% FCS and 50 μg/ml ascorbic acid. Conditioned media were collected and centrifuged at 3000 rpm for 5 minutes to remove any particulate material.

Mouse calvaria-derived osteoblastic cell lines, MC3T3-E1,37 was obtained from the RIKEN Cell Bank (Tsukuba, Japan). These cells were plated onto a 24-well plate (5 × 104 cells/well) in α-MEM containing 10% FCS and cultured to subconfluency. The cells were treated for 48 h with BSA or AGE-BSA in 0.3 ml α-MEM supplemented with 0.1% FCS. Conditioned media were collected as described above.

Human IL-6 concentrations in the culture supernatants were determined by enzyme-linked immunosolvent assays from Genzyme (Cambridge, MA, U.S.A.). Human IL-1α, IL-1β, and IL-11 concentrations and mouse IL-6 concentrations were measured by specific enzyme-linked immunosolvent assays from R & D Systems (Minneapolis, MN, U.S.A.).

Alkaline phosphatase assay

Subconfluent bone-derived cells were treated with BSA or AGE-BSA for the indicated periods. After the incubation, the cells were washed three times with physiological saline and lysed in 0.1% Triton X-100 with sonication. Alkaline phosphatase activity in the lysate was determined spectrophotometrically using p-nitrophenol phosphate (PNP) (Sigma Chemical Co., St. Louis, MO, U.S.A.) as substrate, as described previously.38 The enzyme activity was expressed as nanomoles of PNP per 30 minutes per micrograms of DNA. DNA content was determined on aliquots of the cell lysates using a fluorometric method.39

Northern blot analysis and reverse transcriptase polymerase chain reaction

Total RNA was extracted from bone-derived cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method.40 Heat-denatured RNA was electrophoresed (10 μg/lane) in 1% agarose-2% formaldehyde gels. The gels were transferred to nylon membranes (Hybond N+; Amersham, Buckinghamshire, U.K.), and the membranes were baked at 80°C for 2 h. A32P-labeled cDNA probe for human IL-641 was prepared by a random priming method.42 Hybridization was carried out using rapid hybridization buffer (Amersham) according to the manufacture's protocol. The same filters were rehybridized with the32P-labeled cDNA probe for human glyceraldehyde-3 phosphate dehydrogenase (G3PDH; Clontech, Palo Alto, CA, U.S.A.).

In reverse transcriptase polymerase chain reaction (RT-PCR) analysis, total RNA (1 μg) from human bone-derived cells was used as a template for cDNA synthesis in a 30-μl volume containing the following reagents: 0.5 mM dNTP (Pharmacia, Piscataway, NJ, U.S.A.); 1.25 U/ml oligo (dT); 100 μg/ml BSA; 4 U/μl RNAse inhibitor (Promega, Madison, WI, U.S.A.); 20 U/μl Moloney murine leukemia virus reverse transcriptase (GIBCO BRL, Gaithersburg, MD, U.S.A.); 10 mM dithiothreitol; 3 mM MgCl2; 75 mM KCl; and 50 mM Tris-HCl (pH 8.3). The reaction was incubated at 37°C for 60 minutes. The PCR was carried out at a concentration of 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM dNTP, 0.8 μM each of 5′ and 3′ primers, and 0.025 U/μl Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT, U.S.A.). Analysis of the transcripts for RAGE was performed using the following amplification profile: a denaturation step at 96°C for 1 minute, annealing at 55°C for 45 s, and extension at 72°C for 2 minutes for 30 cycles. The primers used were those described previously.36 The PCR-amplified products were separated on an agarose gel and hybridized with32P-labeled oligonucleotide probe (5′-AACCGTAACCCTGACCTGT GAAGTC-3′) homologous to the middle region of the expected RAGE amplification product. The PCR products were sequenced to confirm their identity by the dideoxy chain termination method using an Applied Biosystems 373A automated DNA sequencer (Applied Biosystems, Foster City, CA, U.S.A.).

Nuclear extraction and electrophoretic mobility-shift assay

MC3T3-E1 cells were treated with BSA or AGE-BSA in the serum-free medium, and the nuclear extracts were prepared by the methods of Schreiber et al.43 Electrophoretic mobility-shift assay (EMSA) was performed using a gel shift assay kit from Stratagene (La Jolla, CA, U.S.A.). The sequence of the double-stranded oligonucleotides used to detect the DNA binding activities of nuclear factor-κB (NF-κB) was 5′-GATCGAGGG GACTTTCCCTAGC 3′.44 Five micrograms of nuclear proteins were incubated with 500 pg of32P-labeled oligonucleotides for 30 minutes at room temperature, and the samples were loaded onto 5% nondenaturing acrylamide gels, according to the manufacture's protocol. In some experiments, nuclear extracts were incubated with either unlabeled oligonucleotides or rabbit polyclonal IgG against p50 or p65 (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) before the incubation with32P-labeled oligonucleotides. After electrophoresis, gels were exposed to X-ray films (X-Omat, Kodak, Rochester, NY, U.S.A.).

Statistics

All values were shown as means ± SE. When the significant difference was discussed, unpaired Student's t-test or Welch's t-test was employed.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Effects of AGEs on the production of cytokines by human bone-derived cells

Human bone-derived cells were cultured with BSA or AGE-BSA (1000 μg/ml) in the presence of 0.1% FCS for 48 h, and the concentrations of IL-6, IL-α, IL-1β, and IL-11 in the culture supernatants were determined. Table 1 shows that the bone-derived cells released significant amounts of IL-6 and IL-11 in this experimental condition. The treatment with AGE-BSA increased the IL-6 concentrations to 2.7-fold above that with BSA. In contrast, AGE-BSA induced no significant change in the IL-11 concentrations in the culture supernatants. Neither IL-1α nor IL-1β concentrations in the culture supernatants were at detectable levels (<1.0 pg/ml) even when the bone-derived cells were treated with or without AGE-BSA. As shown in Fig. 1, AGE-BSA increased the IL-6 concentrations in the culture supernatants of the bone-derived cells in a dose-dependent manner. In this experiment, the concentrations of BSA (including AGE-BSA and unmodified BSA) were kept at 1000 μg/ml by adjustment with unmodified BSA. AGE-BSA, at concentrations of 500 and 1000 μg/ml, induced a significant increase in the IL-6 concentrations.

Table Table 1. Effects of AGEs on IL-6, IL-1α, IL-1β, and IL-11 Release by Human Bone-Derived Cells
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Figure FIG. 1. Dose response of the effects of AGE-BSA on IL-6 concentrations in the culture supernatants from human bone-derived cells. The cells were cultured in DMEM–0.1% FCS containing 50 μg/ml ascorbic acid with various concentrations of AGE-BSA for 48 h. In this experiment, the concentrations of BSA (including AGE-BSA and unmodified BSA) were always set at 1000 μg/ml. After the incubation, IL-6 concentrations in the culture supernatants were determined. Bars represent means ± SE in triplicate assay. *p < 0.001, significant difference from cultures not treated with AGE-BSA.

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To examine whether AGE-BSA increases the levels of IL-6 mRNA in the human bone-derived cells, Northern blot analysis was employed (Fig. 2). The treatment with AGE-BSA for 24 h increased the IL-6 mRNA levels which was dependent on the amounts of AGE-BSA added. By contrast, expression of G3PDH mRNA appeared not to be affected by AGE-BSA.

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Figure FIG. 2. Northern blot analyses of IL-6 mRNAs in human bone-derived cells. The cells were treated for 24 h with various concentrations of AGE-BSA. Total RNA (10 μg) was electrophoresed, transferred to nylon membranes, and hybridized to specific probes for human IL-6 and G3PDH. Arrows indicate the positions of the mRNAs for IL-6 and G3PDH. The data are typical of four experiments.

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Next, the effects of AGE-BSA on IL-6 production were examined in the bone-derived cells from four patients. As shown in Table 2, the production of IL-6 was detected in all the bone-derived cells, although its concentrations were variable. The treatment with AGE-BSA consistently increased the IL-6 concentrations about 2- to 3-fold in the culture supernatants from each of the cells.

Table Table 2. Effects of AGEs on IL-6 Release by Bone-Derived Cells from Four Patients
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Effects of AGEs on alkaline phosphatase activity and DNA contents of human bone-derived cells

In the next experiments, we examined the effects of AGEs on DNA contents and alkaline phosphatase activity in human bone-derived cells. The treatment with AGE-BSA for 48 h had no apparent effect on DNA contents per well in each of the bone-derived cells from four patients, compared with the BSA treatment. In addition, the AGE-BSA treatment also exerted no significant effect on alkaline phosphatase activity in those cells. Exposure of the bone-derived cells to AGE-BSA for longer periods of 8 days, also had no effects on alkaline phosphatase activity and DNA contents (data not shown).

Expression of RAGE mRNA in human bone-derived cells

Since various biological effects of AGEs have been shown to be mediated by RAGE,33,34 we examined whether human bone-derived cells express mRNA for RAGE. RT-PCR reaction using specific primers for human RAGE36 was followed by hybridization with32P-labeled oligonucleotide probe homologous to the middle region of the expected amplified product. The result demonstrated appearance of the amplified fragment of 480 bp. There was no amplified fragment by direct PCR of total RNA without RT (Fig. 3). Next, the 480 bp fragment was sequenced by the dideoxy chain termination method. It revealed that the nucleotide sequence of the RT-PCR products was completely concordant with the reported sequence of human RAGE.34 Thus, the PCR products obtained were found to be specific for human RAGE mRNA.

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Figure FIG. 3. Identification of mRNA for receptor for AGEs (RAGE) in human bone-derived cells by RT-PCR. Total RNA was isolated from the bone-derived cells. RNA (1 μg) was reverse-transcribed into cDNA. PCR reactions were performed using specific primers for human RAGE. The PCR products were run on 1.5% agarose gel and hybridized with32P-labeled oligonucleotide probe homologous to the middle region of the expected 480 bp RAGE amplified product. RT, reaction with reverse transcriptase. The predicted length of the PCR product (480 bp) is shown.

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Effects of AGEs on activation of NF-κB in mouse MC3T3-E1 cells

Recent observations45,46 have demonstrated that AGEs induced activation of the transcription nuclear factor NF-κB in endothelial cells. The NF-κB binding site is identified within the promoter region of the IL-6 gene, which is considered to be important for the transcriptional activation of the gene by IL-1, TNF, or lipopolysaccharide.47 Thus, we examined whether AGEs also activate NF-κB in bone-derived cells. For its purpose, mouse osteoblastic MC3T3-E1 cells were treated with BSA or AGE-BSA. The experiments revealed that the IL-6 concentrations in the culture supernatants of these cells were 50 ± 1.5 pg/ml, which were significantly (p < 0.001) increased to 87 ± 2.7 pg/ml by the treatment with AGE-BSA. Therefore, EMSA was performed using the nuclear extracts from MC3T3-E1 cells and the oligonucleotide probe specific for NF-κB binding. The results showed enhanced intensity of gel-retarded bands when the cells were treated for 4 h with AGE-BSA, compared with BSA (Fig. 4, lanes 1 and 2). The gel-retarded bands were specific for NF-κB, since it disappeared in the presence of excess unlabeled oligonucleotides (Fig. 4, lane 3) but not unrelated oligonucleotides (Fig. 4, lane 4). Addition of anti-p50 antibody or anti-p65 antibody partially supershifted the gel-retarded bands, in which the supershift by anti-p50 antibody was minor compared with that induced by anti-p65 antibody (Fig. 4, lanes 5 and 6). Addition of the both antibodies supershifted the retarded bands completely (Fig. 4, lane 7).

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Figure FIG. 4. Effects of AGE-BSA on DNA binding activity of NF-κB. Nuclear proteins were extracted from MC3T3-E1 cells treated with 1000 μg/ml of BSA (lane 1) or AGE-BSA (lanes 2–7) for 4 h. EMSA using the oligonucleotide probe specific for NF-κB binding was performed on nuclear extracts preincubated with no reagents (lanes 1 and 2), 50-fold excess of unlabeled NF-κB probe (lane 3), 50-fold excess of unrelated oligonucleotides (lane 4), anti-p50 antibody (lane 5), anti-p65 antibody (lane 6), or both anti-p50 and anti-p65 antibodies (lane 7).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

AGEs have been shown to exert biological activities on various kinds of cells, such as macrophages, endothelial cells, mesangial cells, renal cell carcinoma cells, and fibroblasts.28–33,36,48 In the present study, we demonstrated for the first time that AGEs enhanced the production of the bone resorption factor IL-6 in normal human bone-derived cells. AGEs also increased the levels of IL-6 mRNA in these cells. By contrast, AGEs did not influence the production of another bone resorption cytokine, IL-11, in the bone-derived cells. The concentrations of IL-1α and IL-1β in the culture supernatants were undetectable (<1.0 pg/ml), even when the cells were incubated either with or without AGE-BSA. TNF-α mRNA was not detected in the bone-derived cells by RT-PCR analysis (our unpublished observation), which is consistent with the previous report.49 Thus, among various cytokines examined that are known to stimulate bone resorption, both IL-6 and IL-11 are produced in significant amounts by human bone-derived cells, but only IL-6 production is up-regulated by AGEs. In this relation, Girasol et al.7 have shown that IL-11 is essential for osteoclastogenesis in general, whereas IL-6 is important for osteoclastogenesis in an estrogen-deficient state.

It remains obscure how the signals of AGEs are conveyed into the cells. Several studies33,34,45,46 have revealed that RAGE has a central role in mediating the interactions of AGEs with cellular surfaces. In the present investigation, we showed for the first time that RAGE mRNA is expressed in human bone-derived cells. Therefore, it is possible that the binding of AGEs with the cellular surface RAGE leads to the expression of IL-6 genes in these cells. Recently, it has been shown that AGEs induce oxidant stress and thereby activate the transcription factor NF-κB in vascular endothelium.45,46 In this relation, we demonstrated that AGEs enhanced activation of NF-κB in mouse osteoblastic cells. EMSA using antibodies against p50 (NFKB1) or p65 (Rel A) revealed that at least these two proteins were involved in the NF-κB activation in the cells. It is also possible that other NF-κB/Rel family proteins, such as p52 (NFKB2), c-Rel, and Rel B, participate. The NF-κB binding site in the promoter region of the IL-6 gene has been shown to be important for the transcriptional regulation of the IL-6 gene,47 and in vivo targeting of p50 subunit resulted in reduced expression of the IL-6 gene.50 Thus, AGEs may stimulate IL-6 gene transcription in osteoblastic cells by activating NF-κB. In contrast, the IL-11 gene does not contain an NF-κB binding site in its promoter region.51 This seems to be consistent with our results that AGEs did not alter the concentrations of IL-11 in the culture supernatants from the bone-derived cells.

In age- and diabetes-related osteoporosis, decreased bone formation18,24 as well as increased bone resorption20,52 have been observed. In the present study, we failed to show that AGEs had direct influence on DNA contents and alkaline phosphatase activity of the human bone-derived cells. In this respect, AGEs appeared not to alter the proliferation and differentiation of the bone-derived cells. It is also possible that IL-6, whose release is stimulated by AGEs, has some effects on these cells in vivo, since soluble IL-6 receptor present in sera is able to cooperate with IL-6 in activating a gp130-mediated pathway.53 Alternatively, there is a possibility that AGEs exert their inhibitory effect on bone formation by indirect mechanisms. Recently, Fong et al. demonstrated that formation of AGEs on bone matrix inhibits the matrix-induced bone differentiation, probably involving alterations of binding sites for extractable proteins with bone inductive properties such as bone morphogenetic protein-2.54

It has been suggested that multifactorial changes are related to osteoporosis in advanced aging and diabetes mellitus.18–25 AGEs accumulate on long-lived extracellular matrix proteins in aging and diabetes.26,27 It has been demonstrated that the levels of collagen-linked fluorescence in cortical bones were increased in aged and diabetic rats, as a result of the formation of AGEs.35 In the present investigations, we have not directly demonstrated that AGEs induce bone resorption. However, the current study implies that AGEs formed on bone matrix proteins would induce the osteoblasts to produce IL-6, which in turn may stimulate osteoclastogenesis and thereby bone resorption, leading to osteoporosis unless bone formation is efficiently coupled. Thus, it introduces a potential mechanism for age- and diabetes-related osteoporosis, in which AGEs are crucial in triggering an uncoupling event in bone remodeling systems.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

We thank Dr. Yuko Takagaki (Kanagawa Dental College, Yokosuka, Japan) and Dr. Kazuo Hiroshima (Osaka National Hospital, Osaka, Japan) for providing us with normal human bone samples. We also thank Keiko Tsujii for her excellent secretarial assistance in preparing the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and by the Enami Memorial Foundation for Diabetes Research.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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