Abstract
Recent reports have suggested that WNT signaling is an important regulator for adipogenesis or insulin secretion and might be involved in the pathogenesis of type 2 diabetes. To investigate possible roles of the WNT genes in conferring susceptibility to type 2 diabetes, we examined the association of the genes that encode members of the WNT family with type 2 diabetes in the Japanese population. First, 40 single-nucleotide polymorphism (SNP) loci within 11 WNT genes were analyzed in 188 subjects with type 2 diabetes (case-1) and 564 controls (control-1). Among them, six SNP loci exhibited a significant difference (P<.05) in the allele and/or genotype distributions between case and control subjects. These SNP loci were further analyzed in another set of case (case-2; n=733) and control (control-2; n=375) subjects to confirm their statistical significance. As a result, one SNP locus in the WNT5B gene was strongly associated with type 2 diabetes (χ2=15.6; P=.00008; odds ratio=1.74; 95% confidence interval 1.32–2.29). Expression of the WNT5B gene was detectable in several tissues, including adipose, pancreas, and liver. Subsequent in vitro experiments identified the fact that expression of the Wnt5b gene was increased at an early phase of adipocyte differentiation in mouse 3T3-L1 cells. Furthermore, overexpression of the Wnt5b gene in preadipocytes resulted in the promotion of adipogenesis and the enhancement of adipocytokine-gene expression. These results indicate that the WNT5B gene may contribute to conferring susceptibility to type 2 diabetes and may be involved in the pathogenesis of this disease through the regulation of adipocyte function.
Introduction
Type 2 diabetes (MIM 125853) is one of the most common diseases of middle age, and its prevalence continues to increase steadily in many countries, including Japan. The pathogenesis of type 2 diabetes is characterized by the reduction of insulin secretion combined with peripheral insulin resistance (Kahn 1998), but the detailed mechanism is still not well known.
To date, several genes have been reported as candidates for conferring susceptibility to type 2 diabetes, including peroxisome proliferator-activated receptor γ (PPARG [MIM 601487]) (Altshuler et al. 2000), calpain-10 (CAPN10 [MIM 605286]) (Horikawa et al. 2000), and adiponectin (APM1 [MIM 605441]) (Hara et al. 2002), although these genes can account for the condition in only a small percentage of all diabetic patients; most of the susceptibility genes for type 2 diabetes remain to be identified.
The WNT genes have been reported to play a pivotal role in embryonic development and oncogenesis. Recently, one isoform of the WNT family has been reported to play an important role in adipogenesis (Ross et al. 2000; Bennett et al. 2002), and low-density lipoprotein receptor–related protein 5 (LRP5 [MIM 603506]), which is known as a mediator for WNT signaling, has also been shown to be involved in glucose-induced insulin secretion (Fujino et al. 2003). Therefore, genes related to the WNT-signaling pathway could be considered as candidate genes for conferring susceptibility to type 2 diabetes. However, to our knowledge, no association studies that focus on the WNT genes have yet been reported.
In the present study, we demonstrate the results of a case-control association study for the WNT genes in Japanese subjects, and we provide evidence that the WNT5B gene (MIM 606361) is a good candidate for conferring susceptibility to type 2 diabetes.
Methods
DNA Preparation and SNP Genotyping
DNA samples were obtained from the peripheral blood of patients with type 2 diabetes who came regularly to the outpatient clinic of Shiga University of Medical Science, Tokyo Women’s Medical University, Juntendo University, Kawasaki Medical School, or Chibanishi Medical Hospital. The diagnosis of diabetes was determined according to the criteria of the World Health Organization. Type 2 diabetes was clinically defined as “gradual, adult onset of the disease.” The subjects who were positive for anti-glutamic acid decarboxylase antibody and the patients with mitochondrial disease (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes [MELAS {MIM 540000}]) and maturity-onset diabetes of the young (MODY [MIM 606391]) were excluded. Control subjects consisted of members of the general population (control-1; n=564) and another set of control subjects (control-2; n=375) who were recruited through several medical institutes in Japan. DNA extraction was performed using a standard phenolchloroform procedure. Written informed consent was obtained from each patient, and the protocol was approved by the ethics committees of the Institute of Physical and Chemical Research and by those of each institute involved in the study.
SNP Genotyping
The SNPs for genotyping were selected from the IMS-JST (Institute of Medical Science–Japan Science and Technology Agency) Japanese SNP database (Hirakawa et al. 2002). First, each SNP locus within the WNT genes was genotyped by the invader assay, as described elsewhere (Ohnishi et al. 2001), for patients with type 2 diabetes (case-1; n=188; male:female ratio 101:87; mean±SD age [in years] 60.5±11.4; duration [in years] of diabetes 17.5±9.4; HbA1c [%] 7.5±1.32; fasting plasma glucose [FPG] [mg/dl] 150.5±46.2; mean±SD BMI [kg/m2] 23.5±3.4) and in the general population (control-1: n=564). After evaluation of the statistical data by use of four models (genotype distribution [2×3], allele frequency, dominant model, and recessive model), the SNPs that showed significant (P<.05) differences were further examined in another set of diabetic subjects (case-2; n=733; male:female ratio 437:296; mean±SD age [in years] 60.7±11.0; mean±SD duration [in years] of diabetes 12.8±9.9; mean±SD HbA1c [%] 7.2±1.2; mean±SD FPG [mg/dl] 159.5±42.5; mean±SD BMI [kg/m2] 23.2±3.4) and control subjects (control-2; n=375).
SNP Discovery in the WNT5B Gene
On the basis of GenBank information about DNA sequence in the genomic region that contains the WNT5B gene (GenBank accession number AC005182), we designed PCR primers to amplify appropriate fragments of genomic DNA. PCR and DNA sequencing were performed as described elsewhere (Saito et al. 2001), by study of 48 diabetic subjects.
Cell Culture and Real-Time Quantitative RT-PCR
A mouse 3T3-L1 cell line was obtained from the Health Science Research Resources Bank. Cells were grown to confluence and were induced to differentiate into adipocytes, according to methods described elsewhere (Kishida et al. 2001). Total RNA was extracted from mouse 3T3-L1 cells with Trizol reagent (Invitrogen) at different stages of adipocyte differentiation, and reverse transcription was performed using an oligo-dT primer. Expression of the endogenous Wnt5b gene during adipogenesis and expression of the mouse PPAR-γ (Pparg), mouse C/EBP-α (Cebpa), mouse adiponectin (apM1), and mouse leptin (Lep) genes in Wnt5b-overexpressing 3T3-L1 cells were examined at the indicated days, after induction of differentiation by real-time quantitative RT-PCR (Mx3000P Multiplex Quantitative PCR system [Stratagene]) with SYBR green detection. Results were normalized with mouse 36B4 (Arbp). Primers for the genes studied were as follows. For Wnt5b, sense 5′-CCA GTG CAG AGA CCG GAG ATG-3′, antisense 5′-GTT GTC CAC GGT GCT GCA GTT C-3′; for Arbp, sense 5′-CAA CGG CAG CAT TTA TAA CCC-3′, antisense 5′-CCC ATT GAT GAT GGA GTG TGG-3′; for Pparg, sense 5′-TTT GAA AGA AGC GGT GAA CCA C-3′, antisense 5′-ACC ATT GGG TCA GCT CTT GTG -3′; for Cebpa, sense 5′-AAA GCC AAG AAG TCG GTG GAC-3′, antisense 5′-GGT CAT TGT CAC TGG TCA ACT CC-3′; for apM1, sense 5′-GGG TGA GAC AGG AGA TGT TGG AAT G-3′, antisense 5′-GCC AGT AAA TGT AGA GTC GTT GAC G-3′; for Lep, sense 5′-TCC AGA AAG TCC AGG ATG ACA CCA A-3′, antisense 5′-TCA GCA TTC AGG GCT AAC ATC CAA C-3′. The amplifications were each performed in a 25-μl reaction volume that contained 1× EX Taq Buffer, 200 nM dNTP, 1/20,000 SYBR Green, 800 nM gene-specific primer, 0.05 U/ml EX Taq DNA polymerase, 2.75 ng/ml TaqStart antibody, and 5 ng of template DNA. The thermal profile was 50°C for 2 min, at 95°C for 10 min, followed by 40 cycles at 95°C for 30 s and at 60°C for 60 s.
Construction of Adenovirus Vector and Viral Infection
An adenovirus vector encoding the mouse Wnt5b gene was prepared using the Adenovirus Expression Vector Kit (TaKaRa). Forty-eight hours before the induction of differentiation, 3T3-L1 cells were transduced with a multiplicity of infection of 50 plaque-forming units/cell for 6 h, and samples were obtained at the indicated days after induction of differentiation.
Oil Red O Staining
At the indicated number of days after induction of differentiation, the cells were washed twice with PBS and then were fixed for 2 h with 3.7% formaldehyde. Fixed cells were incubated with oil red O for 15 min at room temperature. After the cells were washed once with water, the stained lipid droplets in the cells were visualized by light microscopy. For quantification, the dye was extracted with isopropyl alcohol, and the absorbance was measured at 540 nm.
Statistical Analysis
The statistical analysis used in the association study to calculate the Hardy-Weinberg equilibrium (Nielsen et al. 1998), and the linkage disequilibrium (LD) coefficients (D′ and Δ) (Devlin and Risch 1995) were as described elsewhere. The analysis of haplotype structure was performed by estimation of haplotype phasing by use of the EM algorithm (Excoffier and Slatkin 1995) and by construction of haplotype blocks, as described elsewhere (Gabriel et al. 2002; Tanaka et al. 2003). To analyze interaction between SNPs, logistic regression analysis was performed. The probability (Pc) of an individual being a case subject rather than a control subject is assumed to be affected by a set of SNPs, according to the logistic model; for example, logit(P)=a0+a1x1+a2x2 for a single SNP. Here, we use a coding scheme x1=-1,0,1, and x2=-0.5,0.5,-0.5 for genotypes 1/1, 1/2, and 2/2, respectively, for representing an additive effect by x1 and a dominance/recessive effect by x2 (Cordell and Clayton 2002). The weights are estimated by the maximum-likelihood method and are tested by comparison with the null hypothesis logit(Pc)=a0 (constant). For multiple SNPs, interaction effects are also added, in addition to the main effects of additional SNPs, and are tested stepwise, to determine whether their effects are significant. The tests were performed using R. To evaluate population structures, Wright’s F statistics were calculated for 66 randomly selected SNPs. A single statistic (FST) between each group was calculated as described elsewhere (Weir 1996). To analyze the results of the oil red O staining, the statistical significance of the difference between the two groups was analyzed using the unpaired t test. P values <.05 were considered significant.
Results
Association Study
A total of 45 SNPs within 11 WNT genes were found in the JSNP database. Among them, 40 SNP loci within 11 WNT genes were successfully genotyped with the invader assay. As shown in table 1, six SNP loci (two in WNT2, two in WNT5A, and two in WNT5B) showed a significant (P<.05) association with type 2 diabetes, and these SNP loci were further analyzed in another set of case (n=733) and control (n=375) subjects. The results indicated that only two SNP loci in the WNT5B gene at chromosome 12p13.3 were associated with type 2 diabetes (intron 4+438, C/G, CC vs. CG+GG: χ2=15.6, P=.00008, OR 1.74, 95% CI 1.32–2.29; intron 4+6763, C/T, CC vs. CT+TT: χ2=8.3, P=.004, OR 1.46, 95% CI 1.13–1.88) (tables 2 and 3).
Table 1.
Association of the WNT Genes with Type 2 Diabetes and SNP Locations[Note]
| Gene and JSNP IDa | Modelb | χ2 | Pc | Chromosome | SNP Location(Major Allele/Minor Allele) |
| WNT2: | |||||
| IMS-JST000659 | R | 3.1 | .078 | 7 | Intron 2+4921 (C/T) |
| IMS-JST007915 | R | 3.7 | .054 | 7 | Intron 3+340 (A/C) |
| IMS-JST011331 | R | 3.9 | .049 | 7 | Intron 3+5839 (T/C) |
| IMS-JST035778 | R | 3.2 | .073 | 7 | Intron 3+11329 (T/A) |
| IMS-JST035780 | R | 2.4 | .120 | 7 | Intron 3+6954 (A/C) |
| IMS-JST057756 | D | 2.2 | .140 | 7 | Intron 3+17103 (T/C) |
| IMS-JST111067 | R | 7.8 | .0051 | 7 | 3′-flank+1629 (T/C) |
| IMS-JST160646 | R | .1 | .71 | 7 | 3′-flank+2696 (T/C) |
| WNT2B: | |||||
| IMS-JST068883 | Allele | 1.9 | .31 | 1 | Exon 6+870 (C/T) |
| IMS-JST147806 | Allele | 1.1 | .29 | 1 | Intron 2+13176 (C/A) |
| IMS-JST082135 | Allele | 1.6 | .20 | 1 | Intron 5+425 (A/G) |
| IMS-JST147811 | D | 1.7 | .18 | 1 | Intron 2+23287 (C/T) |
| IMS-JST147812 | R | 2.4 | .12 | 1 | Exon 6+224 (G/A) |
| WNT3: | |||||
| IMS-JST169568 | Allele | .9 | .34 | 17 | Intron 2+2715 (C/A) |
| WNT3A: | |||||
| IMS-JST153590 | R | .4 | .52 | 1 | Exon 4+665 (C/T) |
| WNT4: | |||||
| IMS-JST015559 | D | .9 | .32 | 1 | Intron 2+1784 (C/T) |
| IMS-JST015560 | D | .3 | .59 | 1 | Intron 2+2108 (A/G) |
| IMS-JST015561 | D | .3 | .56 | 1 | Intron 2+2134 (G/C) |
| IMS-JST015562 | Allele | .5 | .45 | 1 | Intron 2+2469 (G/A) |
| IMS-JST119480 | D | .5 | .45 | 1 | Intron 4+388 (C/T) |
| WNT5A: | |||||
| IMS-JST086337 | D | 3.9 | .049 | 3 | Exon 4+2328 (G/A) |
| IMS-JST128940 | D | .7 | .40 | 3 | Intron 3+1875 (T/C) |
| IMS-JST128941 | R | 4.9 | .026 | 3 | Intron 3+1798 (C/T) |
| WNT5B: | |||||
| IMS-JST024407 | R | 1.6 | .20 | 12 | Intron 4+1453 (T/G) |
| IMS-JST024409 | R | 5.0 | .025 | 12 | Intron 4+6763 (C/T) |
| IMS-JST024410 | Allele | .5 | .47 | 12 | Intron 4+7770 (C/T) |
| IMS-JST024411 | D | 1.4 | .24 | 12 | Intron 4+7976 (T/C) |
| IMS-JST012095 | R | .4 | .53 | 12 | Intron 5+905 (G/A) |
| IMS-JST012098 | 2×3 | 3.1 | .22 | 12 | Intron 5+4083 (G/T) |
| IMS-JST012099 | R | 1.2 | .27 | 12 | Intron 5+4148 (A/G) |
| IMS-JST024404 | R | 8.5 | .0036 | 12 | Intron 4+438 (C/G) |
| IMS-JST012100 | R | 1.7 | .19 | 12 | Intron 5+4214 (T/C) |
| IMS-JST161961 | Allele | .6 | .41 | 12 | Intron 1+11798 (C/T) |
| IMS-JST168889 | D | 1.3 | .25 | 12 | Exon 1+234 (G/A) |
| WNT9A: | |||||
| IMS-JST153589 | D | 1.7 | .19 | 1 | Exon 2+58 (G/A) |
| WNT10B: | |||||
| IMS-JST092651 | R | .7 | .38 | 12 | Exon 5+348 (G/A) |
| IMS-JST138743 | Allele | 1.4 | .23 | 12 | 3′-flank+244 (G/A) |
| WNT11: | |||||
| IMS-JST091808 | R | .2 | .64 | 11 | Exon 3+89 (G/A) |
| WNT16: | |||||
| IMS-JST159943 | R | 1.2 | .26 | 7 | Intron 3+5525 (A/G) |
| IMS-JST159946 | D | .4 | .51 | 7 | Intron 3+2747 (T/C) |
Note.— In this study, 188 cases and 564 controls were examined.
JSNP IDs selected from the IMS-JST Web site.
D = dominant model; R = recessive model; Allele = allele frequency; 2×3 = genotype distribution (2×3).
The smallest P value among the four models is presented.
Table 2.
Genotype and Allele Frequencies of the SNPs in WNT5B
|
Analysis of Intron 4+438 (C/G) |
|||||||
| No. (%) of Subjects |
No. (%) of Chromosomes |
HWE Testa |
|||||
| Population | CC | CG | GG | C | G | χ2 | P |
| Case-1 (n=188) | 89 (47.3) | 75 (40) | 24 (12.7) | 253 (67.3) | 123 (32.7) | 1.6 | .20 |
| Control-1 (n=562) | 199 (35.4) | 280 (50.0) | 83 (14.6) | 678 (60.3) | 446 (39.7) | .9 | .32 |
| Case-2 (n=733) | 304 (41.4) | 331 (45.1) | 98 (13.5) | 939 (64.1) | 527 (35.9) | .3 | .60 |
| Control-2 (n=345) | 100 (29.0) | 192 (55.6) | 53 (15.4) | 392 (56.8) | 298 (43.2) | 6.2 | .013 |
|
Analysis of Intron 4+6763 (C/T) |
|||||||
| No. (%) of Subjects |
No. (%) of Chromosomes |
HWE Testa |
|||||
| CC |
CT |
TT |
C |
T |
χ2 |
P |
|
| Case-1 (n=188) | 103 (54.8) | 69 (36.7) | 16 (8.5) | 275 (73.1) | 101 (26.9) | .8 | .36 |
| Control-1 (n=558) | 253 (45.3) | 253 (45.3) | 52 (9.4) | 759 (68.0) | 357 (32.0) | .9 | .32 |
| Case-2 (n=696) | 344 (50.0) | 282 (40.5) | 70 (9.5) | 970 (69.7) | 422 (30.3) | 1.2 | .28 |
| Control-2 (n=366) | 147 (40.1) | 183 (50.0) | 36 (9.9) | 477 (65.2) | 255 (34.8) | 3.8 | .052 |
Hardy-Weinberg equilibrium test.
Table 3.
Association of the SNPS in WNT5B with Type 2 Diabetes
|
Analysis of Intron 4+438 (C/G) |
|||||
| Population | Model | χ2 | P | OR | 95% CI |
| Case-1 versus control-1 | CC vs. CG+GG | 8.5 | .0036 | 1.64 | 1.17–2.29 |
| Case-2 versus control-2 | CC vs. CG+GG | 15.6 | .00008 | 1.74 | 1.32–2.29 |
|
Analysis of Intron 4+438 (C/G) |
|||||
| Model |
χ2 |
P |
OR |
95% CI |
|
| Case-1 versus control-1 | CC vs. CT+TT | 5.0 | .025 | 1.46 | 1.05–2.04 |
| Case-2 versus control-2 | CC vs. CT+TT | 8.3 | .004 | 1.46 | 1.13–1.88 |
To exclude the possibility that this result instead reflected the association of other genes located near the WNT5B gene with type 2 diabetes, we performed LD mapping around this locus, using 34 SNPs with allele frequencies of >0.1. The result indicated that the LD in this region extended to ∼20 kb upstream and 10 kb downstream of this SNP site (fig. 1). Hence, the crucial region for susceptibility to type 2 diabetes seemed to exist within this 30-kb LD block, which contained only the WNT5B gene. Therefore, we concluded that the WNT5B gene itself could explain the association of this locus with type 2 diabetes.
Figure 1.
LD mapping around the WNT5B gene. LD coefficients (D′ and Δ) between every two SNPs around the SNP in intron 4 (+438, C/G) were calculated. Minor allele frequencies of all SNPs used in this analysis are >10%. An asterisk (*) highlights the SNP in intron 4 (+438, C/G).
Next, we sought genetic polymorphisms in an entire region of the WNT5B gene. We identified 76 additional SNPs, for a total of 87 SNPs in this gene (fig. 2). We could not identify any variations within the coding region of the WNT5B gene. Among these 87 SNP loci, 45 SNP loci were successfully genotyped and were analyzed in the second set of case (case-2) and control (control-2) subjects. The result indicated that several of them were significantly associated with type 2 diabetes (tables 4–6); the association of SNPs at intron 3 and intron 4 with type 2 diabetes was especially strong (intron 3+1016: χ2=14.8, P=.0001, OR 1.73, 95% CI 1.31–2.29; intron 4+438: χ2=15.6, P=.00008, OR 1.74, 95% CI 1.32–2.29; intron 4+5776: χ2=15.9, P=.00007, OR 1.73, 95% CI 1.32–2.27).
Figure 2.
Polymorphisms identified in the WNT5B gene. The number sign (#) indicates the SNP in intron 4 (+438, C/G) that showed strong association. An asterisk (*) indicates SNPs selected from the IMS-JST Japanese SNP database.
Table 4.
Genotype Frequencies of SNPs in the WNT5B Gene
|
No. (%) of Subjects |
|||||
| Position (Major/Minor) | Population | Major Allele/Major Allele | Major Allele/Minor Allele | Minor Allele/Minor Allele | Total |
| SNP 33 | Case | 232 (33) | 327 (47) | 139 (20) | 698 |
| Intron 1+6974 (C/T) | Control | 102 (28) | 200 (55) | 64 (17) | 366 |
| SNP 51 | Case | 342 (49) | 298 (42) | 65 (9) | 705 |
| Intron 2+1079 (C/G) | Control | 149 (41) | 183 (50) | 34 (9) | 366 |
| SNP 55 | Case | 341 (49) | 297 (42) | 63 (9) | 701 |
| Intron 2+1636 (C/G) | Control | 142 (39) | 183 (50) | 38 (11) | 363 |
| SNP 57 | Case | 284 (40) | 318 (45) | 102 (15) | 704 |
| Intron 3+1016 (C/T) | Control | 95 (28) | 182 (54) | 61 (18) | 338 |
| SNP 58 | Case | 304 (41) | 331 (45) | 98 (14) | 733 |
| Intron 4+438 (C/G) | Control | 100 (29) | 192 (56) | 53 (15) | 345 |
| SNP 67 | Case | 291 (41) | 313 (45) | 99 (14) | 703 |
| Intron 4+5776 (G/C) | Control | 106 (29) | 194 (53) | 66 (18) | 366 |
| SNP 71 | Case | 344 (50) | 282 (41) | 70 (9) | 696 |
| Intron 4+6763 (C/T) | Control | 147 (40) | 183 (50) | 36 (10) | 366 |
| SNP 83 | Case | 587 (83) | 114 (16) | 4 (1) | 705 |
| Intron 5+4244 (A/C) | Control | 319 (88) | 41 (11) | 3 (1) | 363 |
Table 5.
Analysis of SNPs in the WNT5B Gene with Type 2 Diabetes
| χ2 for |
P for |
|||||||
| Position | 2×3a | Allele | Major/Majorversus Others | Minor/Minorversus Others | 2×3a | Allele | Major/Majorversus Others | Minor/Minorversus Others |
| Intron 1+6974 | 5.9 | .4 | 3.2 | .92 | .053 | .516 | .073 | .34 |
| Intron 2+1079 | 6.4 | 3.4 | 5.9 | .001 | .041 | .063 | .015 | .97 |
| Intron 2+1636 | 8.8 | 6.7 | 8.8 | .61 | .013 | .0099 | .0031 | .43 |
| Intron 3+1016 | 14.8 | 11.9 | 14.8 | 2.19 | .0006 | .0006 | .0001 | .14 |
| Intron 4+438 | 15.8 | 10.4 | 15.6 | .77 | .0004 | .0013 | .00008 | .38 |
| Intron 4+5776 | 16.1 | 13.6 | 15.9 | 2.88 | .0003 | .0002 | .00007 | .09 |
| Intron 4+6763 | 9.4 | 4.5 | 8.3 | .01 | .0092 | .034 | .004 | .91 |
| Intron 5+4244 | 4.8 | 3.1 | 4.0 | .25 | .092 | .077 | .046 | .62 |
Genotype distribution (2×3).
Table 6.
Analysis of SNPs in the WNT5B Gene with Type 2 Diabetes
|
OR (95% CI) of |
|||
| Position | Allele | Major/Majorversus Others | Minor/Minorversus Others |
| Intron 1+6974 | 1.06 (.89–1.27) | 1.29 (.98–1.70) | .85 (.61–1.18) |
| Intron 2+1079 | 1.20 (.99–1.45) | 1.37 (1.06–1.77) | 1.01 (.65–1.56) |
| Intron 2+1636 | 1.28 (1.06–1.55) | 1.47 (1.14–1.91) | 1.18 (.77–1.81) |
| Intron 3+1016 | 1.39 (1.15–1.67) | 1.73 (1.31–2.29) | 1.30 (.92–1.84) |
| Intron 4+438 | 1.35 (1.13–1.63) | 1.74 (1.32–2.29) | 1.18 (.82–1.69) |
| Intron 4+5776 | 1.41 (1.17–1.69) | 1.73 (1.32–2.27) | 1.34 (.95–1.89) |
| Intron 4+6763 | 1.23 (1.01–1.48) | 1.46 (1.13–1.88) | .98 (.64–1.49) |
| Intron 5+4244 | .73 (.52–1.04) | .69 (.47–1.00) | 1.46 (.33–6.56) |
We then examined haplotype structure using the EM algorithm, which indicated that five SNPs with the allelic frequency of >0.15 in the WNT5B gene, including intron 4+438 C/G, constituted one haplotype block and that five common haplotypes could cover >90% of the population (fig. 3A). Subsequent association studies for each haplotype with type 2 diabetes identified a significant association of haplotypes 1 and 2 with type 2 diabetes. However, the associations of these haplotypes were not stronger than those found at the single locus.
Figure 3.
A, Analysis of haplotype structure and estimated haplotype frequencies within the WNT5B gene. 56 = intron 3+439; 57 = intron 3+1016; 58 = intron 4+438; 67 = intron 4+5776; 71 = intron 4+6763. a, Haplotype 1/others: χ2=4.41; P=.036; OR 1.24, 95% CI 1.01–1.52. b, Haplotype 2/others: χ2=5.59; P=.018, OR .77, 95% CI .62–.96. B, Plots of LD coefficients (Δ) between the SNP at intron 4+438 and other SNPs within the WNT5B gene.
We further evaluated interactions among the SNPs used in this study by logistic regression analysis, as described in the “Methods” section, and we identified significant interaction between the SNP at intron 4+438 of the WNT5B gene and the SNP at 3′-flanking+1629 of the WNT2 gene (recessive for the WNT5B SNP, plus additive for the WNT2 SNP with interaction; P=.007, combined empirical P=.000035; OR 2.22; 95% CI 1.58–3.11). We could not identify any significant interaction between other combinations of the WNT genes.
Expression of Wnt5b during Adipogenesis
To learn the possible involvement of the WNT5B gene in the pathogenesis of type 2 diabetes, we first investigated the expression pattern of the mouse Wnt5b gene during adipogenesis, by quantitative real-time PCR. As shown in figure 4, expression of the Wnt5b gene was most abundant at 2 d after induction of differentiation, followed by a rapid decrease in response as a result of further cell differentiation into adipocytes.
Figure 4.
Expression pattern of the Wnt5b gene in 3T3-L1 cells during differentiation. Quantitative real-time PCR was used to evaluate the amount of Wnt5b mRNA. Total RNA was extracted from 3T3-L1 cells at 0, 1, 2, 3, 4, 5, 6, 7, and 10 d after induction of differentiation.
Effect of Overexpression of the Mouse Wnt5b Gene on Adipogenesis and Adipocyte Gene Expression
We next examined the effect of overexpression of the mouse Wnt5b gene on adipocyte differentiation. 3T3-L1 preadipocytes were infected with adenovirus vectors encoding mouse Wnt5b or lacZ 48 h before induction of differentiation, and cells then were induced to differentiate into adipocytes, as described in the “Methods” section. As shown in figure 5A and 5B, the cells overexpressing Wnt5b significantly accelerated differentiation of 3T3-L1 cells compared with control cells (the content of lipid droplets was 1.6-fold, 1.9-fold, and 1.7-fold at 4, 7, and 10 d, respectively; P<.05 vs. lacZ).
Figure 5.
Effects of Wnt5b gene overexpression in 3T3-L1 cells on adipogenesis. An adenovirus vector encoding the Wnt5b gene was infected into 3T3-L1 cells 48 h before induction of differentiation at a multiplicity of infection of 50. A, Lipid droplets stained with oil red O at 5 d after induction of differentiation. B, Quantitative analysis of oil red O staining. Values are mean±SE of five independent experiments. An asterisk (*) indicates P<.05 versus control cells (LacZ).
Expression of the Cebpa and Pparg genes, which are thought to play a key role in the adipogenesis process, was also elevated in the Wnt5b-overexpressing cells (fig. 6A and 6B). We further examined the expression of several adipocytokine genes in the same cells and identified that the expression of apM1 and Lep was significantly increased in Wnt5b-overexpressing cells compared with control cells (apM1 is expressed 2.0-fold and 1.5-fold, and Lep is expressed 1.6-fold and 1.7-fold, at day 3 and day 7, respectively [fig. 6C and 6D]), whereas the expression of Il-6 and Rstn was unchanged between these two cell lines (data not shown).
Figure 6.
Effects of Wnt5b gene overexpression in 3T3-L1 cells on adipocyte gene expression: the expression of Pparg (A), Cebpa (B), apM1 (C), and Lep (D) in Wnt5b overexpressing 3T3-L1 cells at 3 and 7 d after induction of differentiation. The results obtained from four independent experiments are presented. An asterisk (*) indicates that P<.05. A double asterisk (**) indicates P<.01 versus control cells (LacZ).
Discussion
In this study, we examined the association of the WNT genes with type 2 diabetes, and we identified the WNT5B gene at chromosome 12p13.3 as a new candidate for conferring susceptibility to type 2 diabetes. Recent cumulative evidence suggested that some members of the WNT family had remarkable functions in relation to the pathogenesis of type 2 diabetes. To date, the WNT family consists of 19 genes, including the WNT5B gene (details can be viewed on the Wnt Web site). Most WNT proteins are considered to be secreted from cells and to act through cell-surface receptors, namely Frizzled receptors and their coreceptors LRP5 and LRP6 (MIM 603507). The WNT1 gene (MIM 164820) has been reported to inhibit adipogenesis through the β-catenin pathway (Ross et al. 2000), and the WNT10B (MIM 601906) gene has also been shown to play an important role in an early stage of adipogenesis (Bennett et al. 2002). In addition, it has been reported that LRP5 plays some role in glucose-induced insulin secretion in the pancreatic islets and in cholesterol metabolism in the liver (Fujino et al. 2003). On the basis of these observations, the WNT genes seem to be worthy candidates for conferring susceptibility to type 2 diabetes, but, until now, the association of these genes with type 2 diabetes had not, to our knowledge, been examined.
In this study, we identified a significant association of SNP loci within the WNT5B gene with type 2 diabetes. Because we examined the association between 40 SNP loci in 11 WNT genes and type 2 diabetes, by use of four models for each locus, type 1 error due to multiple testing should be included. To eliminate the possibility of type 1 error, we adopted Bonferroni’s method to our overall analytical process, according to the following calculation: overall P values (P test-1× P test-2) were multiplied by the number of tests (40×4+6×4). The corrected P value obtained for a particular SNP within the WNT5B gene was .00005 (.0036×.00008×46×4); thus, the association of this locus with type 2 diabetes was thought to be statistically significant.
We further compared population structures among the case and control groups to confirm that our replication samples were appropriately collected for the study. The average values of FST between each group suggested that there was no population stratification within each group (average FST=0.0018, 0.0032, 0.0089, and 0.0014, calculated by comparison between case-1 and case-2, control-1 and control-2, case-1 and control-1, and case-2 and control-2, respectively).
Subsequent LD mapping revealed that the LD block of this locus contained only the WNT5B gene (fig. 1). Therefore, the WNT5B gene itself should be a candidate for conferring susceptibility to type 2 diabetes. Because the gene encoding APM1 type 2 receptor (ADIPOR2 [MIM 607946]), which was recently identified and considered to be a candidate gene for type 2 diabetes, was located next to the WNT5B gene, we also analyzed seven SNPs in ADIPOR2, but we could not identify any association between them and type 2 diabetes (P>.05; data not shown).
On the basis of previous reports, expression of the WNT5B gene appears to be moderately detectable in adult prostate and fetal brain and weakly detectable in adult liver and kidney (Saitoh et al. 2001). It was also reported that the Wnt5b gene is involved in the differentiation and proliferation of several types of cells, such as chondrocytes (Yang et al. 2003), and that expression of the Wnt5b gene is transiently increased during myogenesis (Jamali et al. 2001). However the involvement of this gene in the pathogenesis of type 2 diabetes has not emerged thus far. To our knowledge, the present study is the first to demonstrate clear expression of the Wnt5b gene in 3T3-L1 adipocytes. It is interesting that expression of the Wnt5b gene was transiently increased at an early phase of adipogenesis, which suggests some role for this gene in the regulation of adipocyte differentiation.
In a previous report, other members of the WNT family, WNT1 and WNT10B, were shown to be capable of inhibiting adipocyte differentiation (Ross et al. 2000). We therefore examined a possible role of the Wnt5b gene in adipogenesis, using 3T3-L1 cells overexpressing the Wnt5b gene. The present study clearly demonstrates that overexpression of the Wnt5b gene in preadipocytes results in the promotion of adipocyte differentiation, when compared with the control cells. The expression of Pparg and Cebpa, which are essential transcription factors for adipogenesis, was also increased in Wnt5b-overexpressing 3T3-L1 cells. Therefore, it appears that the Wnt5b gene has an opposite effect on adipogenesis, when compared with WNT1 or WNT10B. We also identified that the expression of several anti-apoptotic genes, such as insulinlike growth factor-1 (Igf-1) and WNT1-inducible secreting protein-1 (Wisp1), was decreased in Wnt5b-overexpressing 3T3-L1 cells (Igf-1, 36% and 44%; Wisp1, 30% and 44% at day 0 and day 2, respectively; authors' unpublished data). Because these genes are thought to be a downstream target of the WNT-1/β-catenin–signaling pathway—which has an anti-apoptotic action by activation of Akt kinase (Su et al. 2002) and is reported to be upregulated by overexpression of the WNT1 gene in 3T3-L1 cells (Longo et al. 2002)—Wnt5b has the opposite effect of WNT1, in terms of the regulation of adipocyte function.
The mechanism by which the WNT5B gene can affect the susceptibility to type 2 diabetes remains uncertain. Differentiated adipocytes have been known to act as endocrine cells and are known to be capable of secreting several cytokines, so-called “adipocytokines.” These cytokines, which are produced and secreted by adipocytes, were thought to be involved in systemic insulin sensitivity. Therefore, we examined the expression of several adipocytokine genes in Wnt5b-overexpressing 3T3-L1 cells and identified that the overexpression of the Wnt5b gene resulted in significant increases in the expression of apM1 and Lep in 3T3-L1 cells. From these observations, we hypothesized that the difference in the expression or function of the WNT5B gene among the individuals could contribute to the susceptibility to type 2 diabetes through, at least in part, affecting adipocyte function, including the production of adipocytokines, although the elucidation of the precise mechanism requires further study.
Finally, it should be mentioned that the OR for the SNP associated with type 2 diabetes in this study was not very high, even when the association of the SNP was shown to be strong by χ2 test. This discrepancy is probably explained by the fact that multiple genetic factors are involved in susceptibility to type 2 diabetes in a complex manner, and effects of individual genes in a complex genetic and environmental background are often very small on their own. Therefore, evaluation of interactions among individual genes with and without main effects is necessary for full understanding of the involvement of genetic background for conferring susceptibility to type 2 diabetes. In the present study, we examined interaction between the SNPs among genes encoding the WNT family, and we identified significant interaction between WNT5B and WNT2 (MIM 147870). However, interaction with other gene families should be examined in the future, and those kinds of studies will contribute to further understanding of the mechanism of the genetic susceptibility to type 2 diabetes.
In conclusion, we have identified the WNT5B gene as a new candidate for conferring susceptibility to type 2 diabetes. Our results also suggest that the WNT5B gene plays an important role in the regulation of adipocyte function and might contribute to the pathogenesis of type 2 diabetes.
Acknowledgments
This work was supported by a grant from the Japanese Millennium Project. We thank Ms. Ohashi for her technical assistance.
Electronic-Database Information
The accession number and URLs for data presented herein are as follows:
- GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for WNT5B [accession number AC005182])
- Institute of Medical Science–Japan Science and Technology Agency (IMS-JST) Japanese SNP database, http://snp.ims.u-tokyo.ac.jp/ (for SNPs and primers)
- Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for type 2 diabetes, PPARG, CAPN10, APM1, LRP5, WNT5B, MELAS, MODY, LRP6, WNT1, WNT10B, ADIPOR2, and WNT2)
- Wnt Gene Web site, http://www.stanford.edu/~rnusse/wntwindow.html
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