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Am J Physiol Endocrinol Metab 293: E1069-E1077, 2007. First published July 31, 2007; doi:10.1152/ajpendo.00182.2007
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Analysis of N-glycan in serum glycoproteins from db/db mice and humans with type 2 diabetes

Naofumi Itoh,1 Shinji Sakaue,1 Hiroaki Nakagawa,2 Masaki Kurogochi,2 Hiroshi Ohira,1 Kisaburo Deguchi,2 Shin-Ichiro Nishimura,2 and Masaharu Nishimura1

1First Department of Medicine, Graduate School of Medicine; and 2Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo, Japan

Submitted 22 March 2007 ; accepted in final form 19 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glycosylation has an important role in regulating properties of proteins and is associated with many diseases. To examine the alteration of serum N-glycans in type 2 diabetes, we used the db/db mouse model. Serum N-glycans were fluorescence labeled and applied to HPLC. There were reproducible differences in N-glycan profiles between the db/db mouse model and the db/+ control. The structures of the oligosaccharides, which had changed in their amounts, were analyzed by a two-dimensional mapping method, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and exoglycosidase digestion. Those analyses revealed an increase in the N-glycans possessing {alpha}1,6-fucose in the serum of db/db mice. The level of {alpha}1,6-fucosyltransferase mRNA was increased in the liver of the db/db mice. The ratio of a biantennary N-glycan with {alpha}1,6-fucose to that without {alpha}1,6-fucose in the liver tissue of the db/db mouse was increased relative to the db/+ control. Next, we analyzed the serum N-glycan profile in human subjects with type 2 diabetes and found an increased amount of a biantennary N-glycan that had an {alpha}1,6-fucose with a bisecting N-acetylglucosamine. In conclusion, the increase in {alpha}1,6-fucosylation is a striking change in the serum N-glycans of the db/db mice, whereas the change in the fucosylation in humans with type 2 diabetes was small, albeit statistically significant. It is likely that the change is caused, at least partially, by the increase in the {alpha}1,6-fucosyltransferase mRNA level in the liver. The increased {alpha}1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 diabetes.

{alpha}1,6-fucosylation; {alpha}1,6-fucosyltransferase; liver

THE N-GLYCOSYLATION OF PROTEINS is one of the most common forms of posttranslational modification. The alteration in N-glycan structure can modify various functions of target glycoproteins (41). Certainly, genetic defects that affect glycan biosynthesis and structure are known to cause various diseases in humans called congenital disorders of glycosylation (12).

The changes in structures of glycans have also been shown in some acquired diseases. Alterations in glycans in cancer cells were shown and linked with cell adhesion, metastasis, and oncogenic transformation, and those in the endothelium of inflammation tissue were shown and linked with leukocyte homing (9). Moreover, the structural changes in the N-glycan of serum proteins are associated with some diseases, e.g., agalactosylation (35) and fucosylation (14) of IgG in rheumatoid arthritis (32) and {alpha}1,6-fucosylation of {alpha}-fetoprotein in hepatocellular carcinoma (3, 4). Those are suggested to be useful for clinical markers (2). However, little research has been performed on the N-glycans in the serum glycoprotein of patients with diabetes mellitus (DM). More than 30 years ago, McMillan (28) demonstrated that fucose content in the serum glycoproteins of patients with diabetes was increased and that this increase could not be explained by the elevated glycoprotein levels. Although N-glycan structures with increased fucosyl residues remained unknown in this study, the finding suggested that fucosylation is linked to the pathophysiology of DM.

In mammals, there are four different linkages in fucosyations of N-glycan: {alpha}1,2-linkage to the terminal galactose (Gal), {alpha}1,3- or {alpha}1,4-linkage to N-acetylglucosamine (GlcNAc) on the outer branch, and {alpha}1,6-linkage to the GlcNAc binding to protein (Fig. 1) (21). The role of each type of fucosylation is not clear yet, but these must be distinguished. Those fucosylations are catalyzed by different fucosyltransferases, which transfer fucose from guanine diphosphate-fucose to oligosaccharide chains linked to proteins or lipids (21). So far, two enzymes are known as {alpha}1,2-fucosyltransferases (FUT1 and -2) and six enzymes are known as {alpha}1,3/4-fucosyltransferases (FUT3, -4, -5, -6, -7, and 9). Previous analyses of mice with disrupted FUT4 and/or FUT7 genes showed reductions of E-, P-, and L-selectin ligand activities (18, 26) and reduction of atherosclerotic lesion size (17). {alpha}1,6-Fucosyltransferase (FUT8) is the only reported enzyme that catalyzes {alpha}1,6-fucosylation in mammals (29). Elevated expression of FUT8 was observed in human ovarian serous adenocarcinoma (40), hepatoma, and liver cirrhosis (34) and was linked to tumor size and lymph node metastasis in thyroid papillary carcinoma (20). Recently, the function of {alpha}1,6-fucosylation was analyzed using FUT8-deficient mice (45). The mice showed growth retardation and 70% lethality. The surviving mice had emphysema-like changes of the lung tissue due to the defect of transforming growth factor-beta1 receptor signaling. Thus, it is likely that altered fucosylations of proteins affect physiological functions.


Figure 1
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  		Fig. 1. The 4 types of N-glycan fucosylation in mammals. N-glycans are transferred to asparagines (Asn) on nascent polypeptides in the endoplasmic reticulum (ER) and modified by many glycosidases and glycosyltransferases in the ER and Golgi apparatus. (Mannose)3N-acetylglucosamine (GlcNAc)2Asn-peptide is the common structure of N-glycan. Mannose, GlcNAc, galactose, sialic acid, and fucose residues can be added to the structure. Fucose can be added in {alpha}1,2-, {alpha}1,3/4-, and {alpha}1,6-linkages by the corresponding fucosyltransferases.

 
In patients with type 1 DM and urinary albumin excretion, an increase in {alpha}1,3-fucosylation of {alpha}1-acid glycoprotein was reported (36). An increase in {alpha}1,3-fucosylated N-glycans of {alpha}1-acid glycoprotein from patients with type 2 DM has been shown, but the increase was not statistically significant (16). However, it is unknown whether the change in {alpha}1,3-fucosylation is most prominent and whether the change in N-glycosylation of {alpha}1-acid glycoprotein is most important in type 1 and type 2 DM among the changes in N-glycan profile. In those studies, the N-glycans of one serum protein were examined. To clarify the most striking changes in the structure and amount of N-glycans, it is necessary to perform a comprehensive analysis of serum without targeting a specific glycoprotein.

The purpose of this study is to examine the changes in the structure and amount of N-glycan of serum glycoproteins in db/db mice, a model of type 2 DM with obesity. In addition, we compared the serum N-glycan profiles between human subjects with type 2 DM and controls.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and materials. Trypsin, sodium cyanoborohydride, and beta-galactosidase (bovine testes) were purchased from Sigma-Aldrich (St. Louis, MO), {alpha}-chymotrypsin and pronase from Calbiochem (Darmstadt, Germany), peptide N-glycosidase F from Hoffman-La Roche (Basel, Switzerland), beta-galactosidase (jack bean) and glucose oligomers (4–20) from Seikagaku (Tokyo, Japan), 2-aminopyridine from Wako Pure Chemicals (Osaka, Japan), and 2,5-dehydroxybenzoic acid (DHB) and {alpha}-cyano-4-hydroxycinnaminic acid (CHCA) from Bruker Daltonics (Bremen, Germany). Bio-Gel P-4 (200–400 mesh) was obtained from Bio-Rad Laboratories (Hercules, CA), Sephadex G-15 from Amersham Biosciences (Uppsala, Sweden), an octadecyl-bonded silica (ODS) column (ShimPack HRC-ODS, 6.0 mm id x 150 mm) from Shimadzu (Kyoto, Japan), and an amide column (TSKgel Amide 80, 4.6 mm id x 250 mm) from Tosoh (Tokyo, Japan).

Animals. Male C57BL/KsJ db/db mice and age-matched db/+ mice were purchased from Charles River Japan (Tokyo, Japan). This animal study was approved by the Animal Care and Use Committee at Hokkaido University Graduate School of Medicine. The animals were housed on a 12:12-h light-dark lighting regimen (lights on at 6 AM) and had free access to food and water. After 2 wk of acclimatization, the 9-wk-old mice were weighed and bled via the tail vein in the nonfasted state. The blood glucose levels were determined with an Accu-Chek meter (Roche Diagnostics, Basel, Switzerland). The animals were anesthetized with diethyl ether and pentobarbital sodium (50 mg/kg ip). The blood samples were collected by cardiac puncture with 22-gauge needles, and the animals were killed by exsanguination. The samples were centrifuged and the resultant sera stored at –20°C until analyses.

Preparation and derivatization of the N-linked oligosaccharide moiety from serum and liver tissue. Extracted liver tissues from db/db or db/+ mice were immediately washed with PBS and heated in 0.1 M ammonium bicarbonate, pH 8.0, at 90°C for 20 min. The tissues were then homogenized with a Sonifier (Branson, Danbury, CT) and delipidated once with chloroform-methanol (2:1) and twice with chloroform. After being dried, 10 mg of the sample was used. The serum (50-µl samples) was used after being heated at 90°C for 10 min. After the digestion of the samples with 50 µg each of trypsin and {alpha}-chymotrypsin, N-linked oligosaccharides were released from peptides with 5 U peptide N-glycosidase F, and then the peptides were digested by 50 µg of pronase. Each step was done in 10 mmol/l ammonium bicarbonate, pH 8.0, at 37°C overnight. The oligosaccharides were purified on a Bio-Gel P-4 column (10 x 380 mm) with water and reductively aminated with 2-aminopyridine and sodium cyanoborohydride (15, 46). Pyridylaminated (PA) oligosaccharides were purified by gel filtration on a Sephadex G-15 column (10 x 380 mm) with 10 mmol/l ammonium bicarbonate. To release the sialic acids, they were heated for 1 h at 90°C with HCl (pH 2.0).

HPLC analysis of PA oligosaccharides. HPLC analysis was performed using the Hitachi L-7000 HPLC system (Hitachi High-Technologies, Tokyo, Japan). The PA oligosaccharide samples were analyzed on an ODS column first. Elution was performed at a flow rate of 1.0 ml/min at 55°C using a gradient system. Solvent A was 10 mmol/l sodium phosphate buffer (pH 3.8), and solvent B was 0.5% (vol/vol) 1-butanol added in solvent A. The column was equilibrated with a solvent [A:B = 80:20 (vol/vol)], and after injection the concentration of solvent B was increased linearly to 50% for 60 min. The eluted PA oligosaccharides were detected with a fluorescence spectrometer at the excitation wavelength of 320 nm and the emission wavelength of 400 nm. Then, the separated oligosaccharides at each peak from the ODS column were analyzed using an amide column at a flow rate of 1.0 ml/min at 40°C with solvent C [3% (vol/vol) acetic acid-triethylamine (pH 7.3)/acetonitrile 35:65] and solvent D [3% (vol/vol) acetic acid-triethylamine (pH 7.3)/acetonitrile 50:50]. The column was initially equilibrated only with solvent C, and elution was performed using a linear gradient to solvent [C:D = 40:60 (vol/vol)] in 30 min. The PA oligosaccharides were monitored by fluorescence as well. The elution position of each PA oligosaccharide on each column peak was converted to a glucose unit (GU) value, which is the elution position relative to that of a PA isomaltooligosaccharide ladder (42), to increase the reproducibility of the glycan profiling. The relative amount of PA oligosaccharide was calculated on the basis of the peak area analyzed by a software program associated with the HPLC system.

beta-Galactosidase digestion. The PA oligosaccharides isolated by HPLC (30 pmol) were treated with 2 mU beta-galactosidase (jack bean) or 5 mU beta-galactosidase (bovine testes) in 0.1 M citrate-phosphate buffer (pH 4.0) at 37°C for 15 h. Then the mixture was heated at 90°C for 10 min. After centrifugation at 3,500 rpm, the supernatant was analyzed.

PA oligosaccharide analysis by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The PA oligosaccharides separated by HPLC were subjected to Ultraflex time-of-flight (TOF)/TOF mass spectrometry (Bruker Daltonics) equipped with a reflector and controlled by the Flexcontrol 2.1 software package (Bruker Daltonics). As matrices, DHB and CHCA were used. Matrix solutions were prepared as follows: DHB (10 mg) was dissolved in water (1 ml), and CHCA was prepared as a saturated solution in 3:1 (vol/vol) of acetonitrile-water. A few picomoles of oligosaccharides fractionated on HPLC were dissolved in 1 µl of water. Matrix solution (0.5 µl) was spotted on an Anchorchip plate (Bruker Daltonics), and 1 µl of the sample solution was added, dried at room temperature, and then subjected to matrix-assisted laser desorption ionization-TOF mass spectrometry (MALDI-TOF MS).

In the reflector mode of MALDI-TOF MS, ions generated by a pulsed UV laser beam (nitrogen laser, {lambda} = 337 nm, 5 Hz) were accelerated to a kinetic energy of 23.5 kV. Metastable ions generated by laser-induced decomposition of the selected precursor ions were analyzed without any additional collision gas. In the MALDI-TOF/TOF mode, precursor ions were accelerated to 8 kV and selected in a timed ion gate. The fragments were further accelerated by 19 kV in the LIFT cell, and their masses were analyzed after the ion reflector passage. Masses were automatically annotated by using the FlexAnalysis 2.1 software package (Bruker Daltonics) (22).

Determination of N-glycan structure. Oligosaccharide structure was suggested by comparison of its elution position with data reported under the same analytical conditions [two-dimensional (2D) mapping] (http://www.glycoanalysis.info/) (42). Code numbers of oligosaccharide structures described in this manuscript are derived from these references. They consist of four numbers; the first numeral indicates the number of antennae of N-glycan. The second and third digits of "1" or "0" indicate the presence or absence of {alpha}1,6-fucose residue bonded to the reducing end and bisecting the GlcNAc residue in that order. The fourth numeral represents the serial number of PA oligosaccharides. The structures suggested by 2D mapping were confirmed by mass values obtained using MALDI-TOF MS.

Analysis of {alpha}1,6-fucosyltransferase gene expression. The mRNA level of FUT8 was analyzed by quantitative real-time RT-PCR. Total RNA was extracted from the liver, epididymal adipose tissue, and kidney of 9-wk-old db/db mice and db/+ mice using the RNeasy Mini Kit or the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). The RNA samples were evaluated using the 2100 Bioanalyzer and RNA 6000 Nano Assay Kit (Agilent Technologies, Palo Alto, CA). The RNA with >1.0 of 28S:18S rRNA ratios and with a low baseline between 18S rRNA and 5S rRNA peaks on the electropherograms was accepted. Total RNA (500 ng) was reverse transcribed with Superscript II Reverse Transcriptase and oligo (dT) primers (Invitrogen, Carlsbad, CA) for first-strand cDNA synthesis. Aliquots of cDNA were subjected to PCR amplification using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. TaqMan gene expression assays (Applied Biosystems) were used as the primers and FAM-labeled probe. Amplification was carried out with the 7300 Real-Time PCR System (Applied Biosystems). All reactions were triplicate determinations. Quantification of each mRNA level was performed by Sequence Detection Software version 1.3 (Applied Biosystems). The level of each FUT8 mRNA was normalized to the level of 18S rRNA analyzed using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems).

Study participants. We recruited humans with type 2 DM from patients who were treated at Hokkaido University Hospital. The diagnosis of type 2 DM was confirmed for all patients from the clinical records, using the World Health Organization criteria (1). Control subjects were recruited from individuals who requested annual medical checkups. They underwent a standard 75-g oral glucose tolerance test, and individuals with normal glucose tolerance (fasting plasma glucose <110 mg/dl and 2-h values <140 mg/dl) were included in the study. All of the subjects were screened through history, blood test, and urinalysis. Individuals with a history of acute inflammation, hepatitis, pregnancy, or prior malignancy were excluded. Those with impaired renal or liver function and overt proteinuria were also excluded. This study was approved by the Hokkaido University's institutional review board, and all participants gave written, informed consent. Twenty patients with type 2 DM and 18 controls were studied. Blood samples were collected after overnight fasting for N-glycan analysis and measurement of plasma glucose level and glycated hemoglobin. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Obesity was defined as BMI ≥25 kg/m2, in accordance with the recommendation of Japan Society for the Study of Obesity (10).

Statistical analyses. Data are presented as means ± SD. An unpaired t-test or Mann-Whitney's U-test was used to examine comparisons between groups when equal variance was shown by the F-test or when equal variance was not shown, respectively. Sex distribution and the percentage of obesity between groups were compared with the {chi}2-test. In all tests, P values of <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of the animals. The average body weight of the db/db mice was heavier than that of the db/+ mice (db/db: 41.5 ± 0.4 g, n = 13; db/+: 26.4 ± 0.3 g, n = 13; P < 0.001). The blood glucose levels of all db/db mice were >25.1 mmol/l, and those of 7 db/db mice could not be measured due to their exceeding the upper limit (33.3 mmol/l) of the blood glucose analyzer. The mean blood glucose level of db/+ control mice was 10.6 ± 0.6 mmol/l. The increased weight and glucose levels of the db/db mice were similar to those previously reported (19).

Oligosaccharide alteration of the db/db mouse serum. A comparison of serum N-glycan profiles from db/db and db/+ mice was performed with HPLC using an ODS column. Typical HPLC elution patterns of the PA oligosaccharides from db/+ and db/db mouse sera are shown in Fig. 2. Elution patterns from db/+ or db/db mice were almost uniform. Three peaks whose GU values were 11.2, 13.2, and 14.0, referred to as peaks D, E, and F, respectively, were larger in db/db mice than in db/+ mice. The relative amounts of PA oligosaccharides from peaks D, E, and F in db/db serum were approximately twice those in db/+ serum (peak D: db/+ 2.4 ± 0.44%, db/db 4.4 ± 0.46%, P < 0.001; peak E: db/+ 3.2 ± 0.65%, db/db 5.7 ± 0.91%, P < 0.001; peak F: db/+ 10.0 ± 1.3%, db/db 22.4 ± 3.8%, P < 0.001; Fig. 3). These results showed that at least three kinds of N-glycans were increased in the serum of db/db mice. Conversely, the other three peaks whose GU values were 8.1, 9.7, and 10.3, referred to as peaks A, B, and C, respectively, in the serum of db/db mice were significantly decreased compared with those in the serum of db/+ mice (peak A: db/+ 12.4 ± 1.6%, db/db 9.1 ± 1.7%, P < 0.001; peak B: db/+ 13.1 ± 1.5%, db/db 9.8 ± 1.7%, P < 0.001; peak C: db/+ 43.2 ± 1.7%, db/db 34.4 ± 2.6%, P < 0.001; Fig. 3).


Figure 2
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  		Fig. 2. Representative chromatograms of pyridylaminated (PA) oligosaccharides. A: a chromatogram was derived from the serum of a db/+ mouse on an octadecyl-bonded silica (ODS) column. B: a chromatogram from the serum of a db/db mouse. Six main peaks were named peaks a–f in order from early to late elutions.

 

Figure 3
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  		Fig. 3. Relative amounts of each fraction referred to as peaks a–f. PA oligosaccharides derived from the serum of db/+ (n = 13) or db/db (n = 13) mice were subjected to HPLC using an ODS column. The area under the curve (AUC) of each peak on the chromatogram divided by the AUC of all peaks was compared between groups of mice. The differences between db/+ mice and db/db mice were statistically significant in all comparisons. {circ}, db/+; bullet, db/db.

 
Structures of N-glycans changed in serum of db/db mice. We investigated the structures of N-glycans whose amounts changed in the serum of db/db mice, using 2D mapping and mass value. First, each PA oligosaccharide corresponding to peaks A–F was isolated using an ODS column and was subjected to HPLC analysis using an amide column. Although the PA oligosaccharides included in peaks B, D, and E were divided into two or three components on an amide column, the largest components, which accounted for >80% of the peaks on ODS column HPLC, were considered to represent the peaks B, D, and E. The GU values of each peak on ODS and amide columns are shown in Table 1. Next, each PA oligosaccharide of peaks A–F isolated on the ODS column was subjected to MALDI-TOF MS. The mass-to-charge ratio (m/z) values are also shown in Table 1. From the database on the web (http://www.glycoanalysis.info/), we estimated the structures of the N-glycans. With this method, we identified peak D as code no. 310.18 (Table 1). Although the PA oligosaccharides 300.18 and 300.19 were considered as possible structures of peak A, 300.19 is an artificial PA oligosaccharide obtained from beta-galactosidase and beta-N-acetylhexosaminidase digests of sialylated tetra-antennary oligosaccharides (Fig. 4) (31). Oligosaccharide 300.19 has no beta1,2-linked GlcNAc residue, which must be added to the {alpha}1,3-linked mannose of core structure in the initial step of N-glycan biosynthesis (27). Thus, the structure of peak A was identified as 300.18. As possible structures of peak C, 200.13 and 200.4 were selected. However, the coinjection of peak C and 200.4 on HPLC revealed that peak C was coeluted with 200.4. Peak F was identified with 210.4 but not 210.13 in the same way.


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  		Table 1. GU, mass, and estimated PA oligosaccharide from each peak in db/db mice

 

Figure 4
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  		Fig. 4. Proposed structures of PA oligosaccharides in db/db mouse serum. They were estimated using two-dimensional (2D) mapping, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and beta-galactosidase digestion. The code numbers of PA oligosaccharides from peaks b and e had not been reported previously. The PA oligosaccharides from peak b were named as 200.32 (top) and 200.33 (bottom) and those from peak e as 210.32 (top) and 210.33 (bottom).

 
There was no PA oligosaccharide corresponding to peaks B and E in the database. The oligosaccharide of peak B had the same m/z value and GU value on the amide column as peak C. This result indicated that peak B consists of the same sugars as peak C. To examine the linkage between Gal and GlcNAc, isolated oligosaccharides of peaks B and C were digested with jack bean beta-galactosidase. It is known that the terminal disaccharide Galbeta1,4GlcNAc is more sensitive to beta-galactosidase from jack bean than the disaccharide Galbeta1,3GlcNAc (25). The digests were again analyzed by using an amide column. The GU value of the digested peak C oligosaccharide was 2 GU smaller than that of the undigested peak C oligosaccharide, as expected. However, the GU value of the digested peak B oligosaccharide was 1 GU smaller than that of the undigested peak B oligosaccharide (Fig. 5). This means that the oligosaccharide of peak B had one Galbeta1–3GlcNAc linkage, which is difficult to digest with jack bean beta-galactosidase. To verify the Galbeta1,3GlcNAc linkage, bovine testes beta-galactosidase that can digest both Galbeta1,3GlcNAc and Galbeta1,4GlcNAc was also used. The GU value of peak B was decreased by 2 GU as well as that of peak C after the digestion (Fig. 5). The coinjection of peaks B and C digested by bovine testes beta-galactosidase to an amide column resulted in the same elution position. Thus, the structure of peak B was suggested as a biantennary oligosaccharide, including a Galbeta1,3GlcNAc component shown in Fig. 4. Because peaks E and F also had the same m/z value and GU of an amide column, the analysis with beta-galactosidases was performed as described above. As a result, peak E also had one Galbeta1,3GlcNAc linkage (Fig. 4). To confirm the linkage position of fucose, MALDI-TOF/TOF MS analysis of peak E was performed. The signal of the fragment from the precursor ion (1,866 m/z) showed 446 (m/z), which indicated the structure with fucose-GlcNAc-PA. Accordingly, the structure of peak E was identified as the same as 210.4, except for a Galbeta1,3GlcNAc linkage (Fig. 4).


Figure 5
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  		Fig. 5. Chromatograms of PA oligosaccharides before and after digestion by beta-galactosidases (jack bean and bovine testes) on an amide column. a–c: undigested and digested peak b. d–f: undigested and digested peak c.

 
Thus, the structures of peaks A, B, and C were identical to those of peaks D, E, and F except for modification of the {alpha}1,6-fucose residue. Collectively, N-glycans with {alpha}1,6-fucose were increased in db/db mouse serum.

Level of {alpha}1,6-fucosyltransferase mRNA increased in the liver of db/db mice. Increases in peaks D, E, and F may be attributed to {alpha}1,6-fucosylation of peaks A, B, and C. We examined the mRNA expression of FUT8 in liver, epididymal adipose tissue, and kidney. Total RNAs were extracted from each tissue and subjected to RT-PCR analysis. As shown in Fig. 6, the expression levels of FUT8 mRNA were significantly increased in the liver from db/db mice. Compared with the heterozygote, the expression levels of FUT8 mRNA from the liver in db/db mice were ~1.7-fold higher (P < 0.01, each n = 6). In contrast to the liver, there were no differences in FUT8 mRNA expression between db/db and db/+ mice in adipose tissue and kidney (Fig. 6).


Figure 6
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  		Fig. 6. Levels of fucosyltransferase (FUT)8 mRNA. Total RNAs were extracted from the liver (A), epididymal fat (B), and kidney (C) of db/+ (n= 6) and db/db (n = 6) mice and subjected to RT-PCR analysis. Levels of FUT8 mRNA were normalized by those of 18S rRNA. The ratio of db/+ mice is considered to be 1.0. Data are means ± SD. *P < 0.05.

 
{alpha}1,6-Fucosylation of the N-glycan in the liver. Given the increased FUT8 mRNA level in the liver, the {alpha}1,6-fucosylation of the N-glycan in the liver tissue was analyzed. Homogenates of liver were subjected to HPLC using an ODS column and the ratio of the relative amounts of peaks F and C (F/C) were employed as the index of {alpha}1,6-fucosylation. In agreement with the increased FUT8 mRNA of the db/db mouse liver, the F/C ratio of the liver was increased in the db/db mouse (db/+ 0.456 ± 0.014, db/db 0.688 ± 0.095, P < 0.01, each n = 6; Fig. 7).


Figure 7
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  		Fig. 7. The relative amounts of peaks f and c (f/c) of liver tissue from db/+ (n = 6) and db/db (n = 6) mice. The liver from the mouse was homogenized and subjected to HPLC analysis using an ODS column. f/c was obtained, and the ratios were calculated. Data are means ± SD. *P < 0.01.

 
Alteration in the serum N-glycan profile in human with type 2 diabetes. Next, we examined whether {alpha}1,6-fucosylation was increased in serum of humans with type 2 DM as observed in the db/db mouse. Characteristics of the patients with type 2 DM and the control subjects whose sera were analyzed are summarized in Table 2. The mean age of subjects in the DM group was older than that in the control group. Although there was no significant difference in BMI between the control and DM groups, the percentage of the subjects classified as obese tended to be higher in the DM group (65.0 vs. 33.3%, P = 0.051).


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  		Table 2. Characteristics of the controls and patients with type 2 DM

 
The PA oligosaccharides prepared from the human serum were subjected to HPLC using an ODS column. A typical elution pattern is shown in Fig. 8. The elution pattern of the human serum differed from that of the mouse serum. We referred to the main peaks as peaks I–VI, as indicated in Fig. 8. Using the 2D mapping method and m/z value, the structure of each peak was identified (Table 3 and Fig. 9). Because peak III was found to consist of two components, using HPLC with an amide column, and peaks III and IV were not separated well, correct quantifications of peak III and IV could not be performed with an ODS column. We compared the relative amounts of peaks II, V, and VI, whose structures had {alpha}1,6-fucose, between the control and DM groups. Although the amounts of peaks II and V were not increased in the DM group, that of peak VI was increased in the DM group (control 4.6 ± 0.7%, DM 5.8 ± 1.1%, P < 0.001; Fig. 10).


Figure 8
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  		Fig. 8. Typical HPLC elution pattern of the PA oligosaccharides derived from the human serum on an ODS column. The main peaks were named i–vi in order from early to late elutions.

 

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  		Table 3. GU, mass, and estimated PA oligosaccharide from each peak in humans

 

Figure 9
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  		Fig. 9. Proposed structures of PA oligosaccharides in human serum. They were estimated using 2D mapping and MALDI-TOF MS.

 

Figure 10
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  		Fig. 10. Relative amount of each fraction from peak ii (210.1; A), peak v (210.4; B), and peak vi (211.4; C). PA oligosaccharides derived from the sera of humans with type 2 diabetes (DM; n = 20) or control subjects (C; n = 18) were subjected to HPLC using an ODS column. The AUC of each peak on the chromatogram divided by the AUC of all peaks was compared. Data are means ± SD. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We showed that {alpha}1,6-fucosylation of N-glycan is increased in the serum protein of db/db mice, a model of type 2 DM with obesity. In addition, we also found an increase of an N-glycan with {alpha}1,6-fucose (211.4) in the serum of humans with type 2 DM, although the increase was small. To the best of our knowledge, the association between {alpha}1,6-fucosylation and type 2 DM has not previously been reported.

{alpha}1,6-Fucosylation regulates E-cadherin-mediated cell adhesion (13), {alpha}3beta1 integrin function (47), antibody-dependent cellular cytotoxicity of IgG (38, 39), liver lysosomal acid lipase activity (43), transforming growth factor-beta1 signaling (45), EGF receptor-mediated intracellular signaling (44), and protein secretion from the liver into the bile duct (33). Given those changes in protein properties by {alpha}1,6-fucosylation, the increase in {alpha}1,6-fucosylation may affect a variety of glycoprotein properties in type 2 DM. In the present study, proteins whose {alpha}1,6-fucosylation is increased in sera of db/db mice and humans with type 2 DM remain unknown. The identification of these proteins may give important information about pathophysiology of type 2 DM. The differences in {alpha}1,6-fucosylation of those identified proteins between control and type 2 DM samples may be larger than the differences observed in the present study, because the results from whole serum proteins are prone to conceal the differences of the specific proteins.

Our result was in agreement with the previous findings of increased fucose residues in the serum glycoproteins of patients with diabetes (28). Higai et al. (16) showed that {alpha}1,3-fucosylation of {alpha}1-acid glycoprotein tended to be increased in the serum of patients with type 2 DM. However, our results demonstrated that {alpha}1,3-fucosylation was a minor structure in glycoproteins in whole serum, as described previously (30), and showed no evidence of an increase in {alpha}1,3-fucosylation in whole serum of mice and humans with type 2 DM. This discrepancy seems to be due to differences in samples (isolated protein vs. whole serum). The comprehensive analysis in the present study proved that {alpha}1,6-fucosylation was more dominant than {alpha}1,3-fucosylation in the serum N-glycome and that an N-glycan with {alpha}1,6-fucose was significantly increased in the serum of humans with type 2 DM.

In the present study, the FUT8 mRNA level in the liver of db/db mice was about twofold higher compared with that in the liver of control db/+mice. Previously, the increased FUT8 mRNA level in the liver was also shown in patients with chronic hepatitis and liver cirrhosis (34). In the liver of patients with type 2 DM, various changes are caused by insufficient insulin action (37). Those include deterioration of many enzymes' activities related to glucose and lipid metabolism. Furthermore, accumulation of visceral fat accelerates the influx of fatty acid into the liver, causing steatosis (5). Those disorders may increase the FUT8 mRNA level.

Although the FUT8 enzyme activity could not be measured in the present study, we showed that the F/C ratio was increased in the liver tissue of the db/db mouse. This finding supported the increased activity of FUT8 in the liver to some extent. Considering that serum glycoproteins are synthesized mainly in the liver, the tissue most likely responsible for the increase in serum {alpha}1,6-fucosylation may be the liver. However, we must realize that mRNA level may not necessarily parallel the enzyme activity. FUT8 enzyme activity of tissues other than liver may be increased in the db/db mouse, even if the mRNA level is not increased.

The C57BL/KsJ db/db mouse has a mutation in the leptin receptor (Ob-R) gene (6, 23). This mutation results in a lacking of most of the intracellular domain of the long isoform of the receptor Ob-Rb. The db/db mouse is thought to become hyperphagic and obese with hyperleptinemia due to a functional defect in the aberrant Ob-Rb (7, 8). In addition to the mutant gene, the BL/Ks inbred strain background predisposes the mice to overt diabetes (24). The Ob-Rb receptor is expressed most highly in the hypothalamus. Although the expression of Ob-Rb was observed in some other tissues, defective leptin signaling in the hypothalamus is thought to be responsible for obesity of the db/db mouse (8). Accordingly, decreased Ob-Rb signaling in the hypothalamus may be associated with the increase in hepatic FUT8 mRNA via obesity or diabetes. However, Ob-Rb is also expressed by hepatocytes (11). The reduction of hepatic Ob-Rb signaling may be related to the increased FUT8 mRNA in the liver.

The db/db mouse is often used as a model of human type 2 DM with obesity, in which leptin resistance is one of the characteristics. However, because the mutation of the leptin receptor gene itself is extraordinarily rare in humans, it remained unclear whether the results of the db/db mouse were applicable to humans with type 2 DM with obesity. Therefore, we examined the serum of humans with type 2 DM and showed an increase of a biantennary N-glycan that had an {alpha}1,6-fucose with a bisecting GlcNAc in the patients with type 2 DM. This result was in agreement with that of db/db mouse in the way that {alpha}1,6-fucosylation was increased. However, the N-glycan profile of human serum protein was considerably different from that of mouse serum protein. Furthermore, the increased N-glycan in humans with type 2 DM was different from that in db/db mice, and the increased N-glycan in the patients with diabetes was limited to only one of the three analyzed N-glycans with {alpha}1,6-fucose. One reason for those differences may be the difference in species. In addition, the heterogeneous pathophysiology of human type 2 DM and the uniform pathophysiology of db/db mice may account for the difference. For example, although not all subjects with type 2 DM were obese, all db/db mice were obese. That difference may be attributed to the difference in the degree of disorders between db/db mice and subjects with type 2 DM, e.g., blood glucose level, body weight, and steatosis.

In conclusion, {alpha}1,6-fucosylation of serum N-glycans and FUT8 mRNA expression in the liver were increased in db/db mice. In addition, an N-glycan with {alpha}1,6-fucose was increased in the serum of humans with type 2 DM. The increased {alpha}1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 DM. It is likely that the increase in {alpha}1,6-fucosylation reflects some pathophysiology of type 2 DM. Further studies are needed to find factors correlating with increased {alpha}1,6-fucosylation in some patients with type 2 DM.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was partly supported by a grant for the National Project on "Functional Glycoconjugate Research Aimed at Developing New Industry" from the Ministry of Education, Culture, Sport, Science, and Technology of Japan.


   ACKNOWLEDGMENTS
 
We thank Dr. Noriko Takahashi of Nagoya City University for advice about PA oligosaccharide nomenclature.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Sakaue, First Dept. of Medicine, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan (e-mail: sakaue-s{at}med.hokudai.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
 GRANTS
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