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Medical Department III (Y.B., B.E., N.K., M.B., M.S., P.K.); Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics (D.T., J.T.), University Hospital Leipzig; Junior Research Group N03 (J.B., M.B.), Interdisciplinary Center for Clinical Research Leipzig; and Department of Surgery (M.R.S.), University of Leipzig, D-04103 Leipzig, Germany
Address all correspondence and requests for reprints to: Michael Stumvoll, M.D., Medical Department III, University of Leipzig, Philipp-Rosenthal-Strasse 27, D-04103 Leipzig, Germany. E-mail: michael.stumvoll{at}medizin.uni-leipzig.de.
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Abstract |
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Design: The visfatin gene (PBEF1) was sequenced in DNA samples from 24 nonrelated Caucasian subjects. Identified genetic variants were used for association analyses of T2DM in a case-control study (503 diabetic subjects and 476 healthy controls) and T2DM-related traits in 626 nondiabetic subjects. The effect of genetic variation in the visfatin gene on its mRNA expression in a subgroup of 157 nondiabetic subjects with measurements of visfatin mRNA expression in visceral and sc fat depots was also analyzed.
Results: Seven single-nucleotide polymorphisms (SNPs) and one insertion/deletion were identified. Three SNPs (rs9770242, –948G
T, rs4730153) that were representatives of their linkage disequilibrium groups were genotyped in Caucasians from Germany with a wide range of body fat distribution and insulin sensitivity for association analyses. No association of T2DM with any of the genotyped SNPs or their haplotypes was found. However, the ratio of visceral/sc visfatin mRNA expression was associated with all three genetic polymorphisms (P < 0.05). Moreover, the –948GT variant was associated with 2-h plasma glucose and fasting insulin concentrations (P < 0.05) in nondiabetic subjects.
Conclusions: In conclusion, our data suggest that genetic variation in the visfatin gene may have a minor effect on visceral and sc visfatin mRNA expression profiles but does not play a major role in the development of obesity or T2DM.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Using a differential display method, visfatin was identified as a peptide predominantly expressed in visceral adipose tissue both in humans and mice (8). This peptide was previously described as a growth factor for early B cells called pre-B-cell colony-enhancing factor 1 (PBEF1) (9). Visfatin activated the insulin receptor in various insulin-sensitive cell types in vitro, and visfatin treatment of mice acutely lowered plasma glucose in vivo. Moreover, mice heterozygous for a loss-of-function mutation in the visfatin gene had higher plasma glucose levels compared with wild-type littermates. In humans, visceral fat mass estimated by computed tomography was strongly correlated with plasma visfatin levels, whereas only a weak relationship with sc fat was observed (8). These findings indicated that visfatin could play a role in the association between visceral obesity and increased metabolic risk.
Recently, we examined whether visfatin plasma concentrations (enzyme immunoassay) and mRNA expression (RT-PCR) in visceral and sc fat correlate with anthropometric and metabolic parameters in subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance. Visfatin plasma concentration correlated with the visceral visfatin mRNA expression and percent body fat and negatively with sc visfatin mRNA expression. In addition, there was a significant correlation between visceral visfatin gene expression and body mass index (BMI) as well as percent body fat (10). Therefore, in the present study, we investigated whether genetic variation within the visfatin gene (PBEF1) might be responsible for changes in visfatin gene expression and whether it might affect obesity/T2DM and pathophysiologically relevant traits in humans.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Five hundred three patients with T2DM and 626 healthy subjects were recruited at the University Hospital in Leipzig, Germany. BMI was calculated as weight divided by squared height. Waist and hip circumferences were measured, and waist-to-hip ratio (WHR) was calculated. The healthy subjects included 308 males and 318 females (mean age, 47.8 ± 14.6 yr; mean BMI, 27.4 ± 5.1; mean WHR, 0.96 ± 0.18), and patients with T2DM included 250 males and 253 females (mean age, 59.0 ± 8.2 yr; mean BMI, 30.0 ± 4.9; mean WHR, 1.11 ± 0.14) (the data represent mean ± SD). In addition, oral glucose tolerance tests and fasting plasma insulin measurements were performed in all nondiabetic subjects. The oral glucose tolerance test was performed according to the criteria of the American Diabetes Association (11). The test was carried out after an overnight fast with 75 g standardized glucose solution (Glucodex; Merieux Ltd., Montreal, Canada). Venous blood samples were taken at 0, 60, and 120 min for measurements of plasma glucose concentrations. One hundred fifty of 626 subjects had impaired glucose tolerance. Because impaired glucose tolerance is a T2DM predicting factor, only the remaining 476 subjects with normal glucose tolerance were included as healthy controls in the T2DM association study.
In a subgroup of 322 nondiabetic subjects, body fat content and insulin sensitivity were measured. Percent body fat was measured by dual x-ray absorptiometry. Insulin sensitivity was assessed with the euglycemic, hyperinsulinemic clamp method as previously described (12, 13).
In addition, paired samples of visceral and sc adipose tissue were obtained from a subgroup of 189 Caucasian men (n = 95) and women (n = 94), who underwent open abdominal surgery for gastric banding, cholecystectomy, weight reduction surgery, abdominal injuries, or explorative laparotomy. The ages ranged from 24–86 yr and BMI from 20.8–54.1 kg/m2. Thirty-two subjects had type 2 diabetes. Thirty-one of 157 nondiabetic subjects had impaired glucose tolerance. All subjects had a stable weight with no fluctuations of more than 2% of the body weight for at least 3 months before surgery. Patients with severe conditions including generalized inflammation or end-stage malignant diseases were excluded from the study. Samples of visceral and sc adipose tissue were immediately frozen in liquid nitrogen after explantation. In addition, plasma visfatin concentrations were measured in all 189 subjects.
All studies were approved by the ethics committee of the University of Leipzig, and all subjects gave written informed consent before taking part in the study.
Assays
Plasma visfatin concentrations were measured in triplicate with a human visfatin enzyme immunometric assay (Phoenix Pharmaceuticals, Belmont, CA) as described in detail elsewhere (10).
Analysis of human visfatin gene expression
Human visfatin gene expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using the TaqMan assay, and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany). Total RNA was isolated from paired sc and visceral adipose tissue samples using TRIzol (Life Technologies, Inc., Grand Island, NY), and 1 mg RNA was reverse transcribed with standard reagents (Life Technologies). Two microliters of each RT reaction was amplified in a 26-ml PCR by using the Brilliant SYBR Green QPCR Core Reagent Kit from Stratagene (La Jolla, CA) according to the manufacturer’s instructions. Samples were incubated in the ABI PRISM 7000 sequence detector for an initial denaturation at 95 C for 10 min, followed by 40 PCR cycles, each cycle consisting of 95 C for 15 sec, 60 C for 1 min, and 72 C for 1 min. The primer sequences are available upon request. SYBR Green I fluorescence emissions were monitored after each cycle. Expression of human visfatin and human 36B4 mRNA were quantified by using the second-derivative maximum method of the TaqMan Software (Applied Biosystems), determining the crossing points of individual samples by an algorithm that identifies the first turning point of the fluorescence curve. Human visfatin mRNA expression was calculated relative to 36B4, which was used as an internal control because of its resistance to hormonal regulation (14). Amplification of specific transcripts was confirmed by melting curve profiles (cooling the sample to 68 C and heating slowly to 95 C with measurement of fluorescence) at the end of each PCR. The specificity of the PCR was further verified by subjecting the amplification products to agarose gel electrophoresis.
Sequencing of the visfatin gene
To identify genetic variants, all 11 exons (the total length of visfatin mRNA is 2376 bp; GenBank accession no. NM_005746), including intron/exon splicing sites, the 5' region (
1500 bp upstream of the first translation initiation site), and the 3' untranslated region (UTR), were sequenced in DNA samples from 24 nonrelated Caucasian subjects from whom paired samples of visceral and sc adipose tissue were collected. Sequencing was performed using the Big Dye Terminator (Applied Biosystems) on an automated DNA capillary sequencer (ABI PRISM 3100 Avant; Applied Biosystems). Sequence information for all oligonucleotide primers used for variant screening is available upon request.
Genotyping of visfatin single nucleotide polymorphisms (SNPs)
Genotyping of rs9770242 and –948GT polymorphisms was done using the TaqMan allelic discrimination assay (Custom TaqMan SNP Genotyping Assay; Applied Biosystems). Oligonucleotide sequences are available upon request. Genotyping of rs4730153 was done using C_2673294_10 Assays-on-Demand SNP Genotyping Products (Applied Biosystems). The TaqMan genotyping reaction was amplified on a GeneAmp PCR system 9700 (95 C for 10 min and then 95 C for 15 sec and 62 C for 1 min for 38 cycles), and fluorescence was detected on an ABI PRISM 7000 or ABI PRISM 7700 sequence detector (Applied Biosystems). To assess genotyping reproducibility, a random, approximately 10% selection of the sample was genotyped again in both SNPs; all genotypes matched initial designated genotypes.
Statistical analyses
Before statistical analysis, nonnormally distributed parameters (glucose infusion rate, 2-h plasma glucose, fasting plasma insulin, and visceral and sc visfatin mRNA expression) were logarithmically transformed to approximate a normal distribution. Differences in genotype frequencies were compared using logistic regression between the diabetic cases and healthy controls. Multivariate linear relationships were assessed by a generalized linear model. In the additive model, homozygotes for the major allele (MM), heterozygotes (Mm), and homozygotes for the minor allele (mm) were coded to a continuous numeric variable for genotype (as 0, 1, or 2). A dominant model was defined as contrasting genotypic groups MM+Mm vs. mm, and the recessive model was defined as contrasting genotypic groups MM vs. Mm+mm. In haplotype analyses, groups of subjects carrying 2, 1, or 0 copies of the haplotype were compared. Statistical analyses were performed using the SPSS software package (SPSS, Inc., Chicago, IL) and the statistical analysis system of the SAS Institute (Cary, NC). P values of <0.05 were considered to be of statistical significance.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The visfatin gene was sequenced in 24 DNA samples to detect variation. Seven SNPs (three novel and four known database SNPs) and one ins/del variant were identified (Fig. 1
). Four SNPs were in the 5' region, a –1001TG (allele frequency of G = 0.31; rs9770242 in the NCBI SNP database, http://www.ncbi.nlm.nih.gov), a –948GT (T = 0.19), a –520GA (A = 0.02), and a –423AG (G = 0.31; rs1319501). Two SNPs were in introns: intron 6 (c.744 –87GA, A = 0.48; rs4730153) and intron 10 (c.1366 –8TC, C = 0.02). The ins/del was in the 3'UTR (c.1605 delT = 0.02). In addition one of the SNPs predicted silent substitution in exon 7 (c.903GA; TCGTCA301Ser; A = 0.41; rs11553095). Based on the genotypic concordance, several of these SNPs were in strong linkage disequilibrium (LD). LD (r2 and D') was calculated among the more common variants (minor allele frequency 
0.05) using the EMLD statistical program (https://epi.mdanderson.org/qhuang/Software/pub.htm) (15) (Table 1). Among these common variants, rs9770242 was in complete LD with rs1319501, and rs4730153 was in tight LD with rs11553095. Because SNPs in high genotypic concordance would provide the same information for association studies, only rs9770242 and rs4730153 were selected as representative variants for both LD groups and genotyped in all subjects for association analyses. In addition, the –948GT, which was unique among SNPs, was also selected for additional association analyses. The results of a Hardy-Weinberg equilibrium test were as follows: P = 0.81 for rs9770242, P = 0.33 for –948GT, and P = 0.53 for rs4730153.
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Visfatin genetic variants and type 2 diabetes.
Under logistic regression analysis, none of the representative SNPs was associated with type 2 diabetes in 503 cases and 476 healthy controls (with normal glucose tolerance) in the present study (Table 2). Moreover, using the PHASE version 2.1 software (16, 17), we identified four common haplotypes (frequency of each haplotype was >0.05) among the three different SNPs genotyped in all subjects involved in the study. These four haplotypes accounted for more than 95% of the all observed haplotypes: haplotype [T-G-A] (18%), [T-G-G] (56%), [G-G-A] (8%), and [G-T-A] (16%), where haplotypes are defined by the composition of alleles at each SNP in the order [rs9770242], [–948GT], and [rs4730153]. Each of the common haplotypes was analyzed for association with T2DM. However, none of the four haplotypes showed significant difference in frequency between diabetic and nondiabetic subjects (all P > 0.05; data not shown).
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). There was also no correlation between fasting plasma glucose and the analyzed SNPs. However, fasting plasma insulin and 2-h plasma glucose were significantly associated with –948GT in both additive and recessive modes of inheritance (Table 3). Subjects carrying the T allele (T/T homozygotes and G/T heterozygotes) had lower mean fasting plasma insulin and 2-h plasma glucose than G/G homozygotes (Table 3). Moreover, the T/T homozygotes showed a lower mean percentage of body fat and a higher mean glucose infusion rate than the carriers of the G allele, but this association did not quite reach statistical significance (P = 0.06 for percent body fat and P = 0.09 for glucose infusion rate; Table 3). Effects of the T allele in –948GT were reflected in the haplotype analysis as well. Carriers of the [G-T-A] haplotype (with two copies or one copy of the haplotype) had a lower mean fasting plasma insulin and 2-h plasma glucose than [G-T-A] noncarriers (all P < 0.05 in additive mode of inheritance; Table 4). This was not surprising because the T allele of –948GT occurred only in the [G-T-A] haplotype. In addition, the [T-G-A] haplotype was significantly associated with 2-h plasma glucose and glucose infusion rate (P < 0.05; Table 4). Subjects with the [T-G-A] haplotype had a higher mean 2-h plasma glucose and a lower glucose infusion rate compared with [T-G-A] noncarriers.
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). As for the –948GT variant, subjects homozygous for the T allele had a lower mean log visceral/sc ratio of visfatin mRNA than subjects with the G allele (G/G+G/T). As expected, also the [G-T-A] haplotype carrying the T allele of –948GT was associated with log visceral/sc visfatin mRNA levels. Subjects carrying two copies of the [G-T-A] haplotype had a lower mean log visceral/sc ratio of visfatin mRNA (–18.04 ± 19.09) than noncarriers (0.58 ± 1.09) or subjects with only one copy of [G-T-A] (0.53 ± 0.26; P < 0.05 after adjusting for age, sex, BMI, percent body fat, and WHR). Furthermore, increased log visceral/sc ratio of visfatin mRNA was seen in subjects with the [T-G-G] haplotype (2.62 ± 2.23) when compared with noncarriers (–0.43 ± 0.54 in subjects with one copy of the haplotype and –4.6 ± 4.11 in noncarriers; P < 0.05).
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T, and rs4730153) genotypes with log visceral/sc mRNA ratio were found only in the group of old subjects (>56 yr; all P < 0.05 after adjusting for sex, BMI, percent body fat, and WHR). The genotype effects were consistent with those found in the entire cohort. We also performed statistical analyses excluding subjects with impaired glucose tolerance, but the results remained unchanged (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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PBEF1 was therefore analyzed as a candidate gene for human T2DM and/or obesity. The human PBEF1 maps to a region on chromosome 7q22.2 previously found to be linked to insulin resistance syndrome-related phenotypes in nondiabetic Mexican-Americans (19) and to BMI in a combined analysis of genome-wide linkage scans for BMI from the National Heart, Lung, and Blood Institute Family Blood Pressure Program (20).
In the present study, we report four previously known and four novel genetic variants that were identified by sequencing the entire gene and the flanking 5' region. Although visceral obesity is a strong predictor of insulin resistance and type 2 diabetes (1, 2, 3) and visceral visfatin gene expression correlates with BMI and percent body fat in humans, our association analysis of three representative SNPs and their haplotypes in PBEF1 suggest that this gene by itself does not appear to be a major susceptibility gene for T2DM or obesity. Alternatively, because only 24 individuals were sequenced for variant detection, it is possible that a rare T2DM or obesity causative variant is present in PBEF1 but was not detected. However, among 157 nondiabetic subjects with visfatin mRNA measurement in visceral and sc fat tissue, all three genetic variants were significantly associated with the visceral/sc ratio of visfatin expression. Consistent with the previously described positive correlation of visceral/sc visfatin mRNA expression with percent body fat, the –948GT variant also showed borderline association with percent body fat. With increased visceral visfatin mRNA expression one would expect to observe higher plasma visfatin levels. In obese KKAy mice, doubling of plasma visfatin levels led to a reduction of plasma glucose and insulin concentrations (8). In our study, in contrast, the T allele of the –948GT variant was significantly associated with lower visceral/sc visfatin mRNA expression and reduced 2-h plasma glucose and fasting plasma insulin concentrations. Because the T allele was present only in the [G-T-A] haplotype, this haplotype was also associated with the visceral/sc ratio of visfatin expression as well as 2-h plasma glucose and fasting plasma insulin concentrations. This was also the only haplotype that was associated with both visfatin expression and clinical characteristics.
The physiological mechanism by which the –948GT polymorphism affects changes in glucose metabolism, body fat content, and visceral/sc ratio of visfatin mRNA expression is unclear at the moment. Compared with adipose tissue of lean mice, adipose tissue of obese mice contains increased amounts of proinflammatory cytokines such as TNF-
or IL-6. These cytokines increase mRNA levels of visfatin (21), which may explain increased mRNA levels of visfatin in visceral fat. However, association of –948GT with visceral/sc ratio of visfatin mRNA levels was present even after adjustment for percent body fat and WHR and thus was independent of effects of body fat content. Consistent with this, no differences in association were found between nonobese and obese groups. In contrast, the observed association was dependent on age and suggests that age-related factors other than obesity may still be involved. Although visfatin has been suggested to play a physiological role in glucose homeostasis, studies investigating visfatin effects on obesity are inevitable to identify potential mechanisms linking body fat content to visfatin gene expression. The c.903GA variant was in tight LD with rs4730153 and thus likely associated with visfatin mRNA levels. c.903GA is positioned within exon 7 but does not predict an amino acid substitution (301Ser); therefore, we do not believe this SNP is a functional variant within PBEF1 but rather assume that it is in high LD with a nearby functional variant. Two other SNPs (rs9770242 and –948GT) associated with visfatin mRNA expression are located in the putative promoter region. Conceivably, because activity or expression of transcription factors differs among depots, SNPs in the promoter region may alter the expression of the target gene in a depot-specific manner.
We used the Transcription Element Search System (http://www.cbil.upenn.edu/tess) based on TRANSFAC, IMD, and CBIL-GibbsMat databases to examine transcriptional regulatory sequences surrounding rs9770242, –948GT, and rs1319501 genetic variants. The system did not reveal any known human transcriptional factor binding sites in the sequence surrounding rs9770242 and –948GT. In contrast, the region surrounding rs1319501 matches human transcriptional binding sites for Jun/activator protein 1 (AP-1) and retinoic acid receptor-1 (RAR-1). Jun/AP-1 is a potent human transcription factor, which is responsible for increased transcription of different cellular genes in response to tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and serum factors (22). RAR-1 is a member of the steroid hormone receptor superfamily and a negative regulator of AP-1-responsive genes, dependent on the DNA-binding domain of RAR and possibly via a complex with c-Jun (23). Because rs1319501 is in complete LD with rs9770242 and thus also associated with visceral/sc ratio of visfatin mRNA level, it would be worthwhile to further explore a possible functional role of this variant. Regarding rare genetic variants, the region surrounding c.1366 –8TC matches the human transcriptional binding site for a multifunctional transcriptional regulator Yin-Yang-1 (YY-1). YY-1 is a zinc finger protein that has been shown to activate, repress, or initiate transcription. The number of both cellular and viral genes that have YY-1 binding sites in their transcriptional regulatory regions emphasizes the importance of YY-1 as a transcriptional regulator (24).
In conclusion, we suggest that genetic variation in the PBEF1/visfatin does not have a major impact on diabetes development and/or obesity but may have a minor contribution in determining glucose homeostasis and visfatin mRNA expression. A mechanism by which altered levels of visfatin mRNA could result in changes in glucose parameters is unknown. Additional genetic studies in other populations, as well as functional studies, are inevitable to better define the relative importance of these genetic variants.
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Acknowledgments |
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Footnotes |
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Disclosure statement: All authors have read and approved submission of the revised manuscript to The Journal of Clinical Endocrinology & Metabolism. The manuscript has not been submitted for publication elsewhere while under consideration for The Journal of Clinical Endocrinology & Metabolism.
First Published Online April 24, 2006
Abbreviations: AP-1, Activator protein 1; BMI, body mass index; LD, linkage disequilibrium; RAR-1, retinoic acid receptor-1; SNP, single nucleotide polymorphism; T2DM, type 2 diabetes mellitus; UTR, untranslated region; WHR, waist-to-hip ratio; YY-1, Yin-Yang-1.
Received January 24, 2006.
Accepted April 19, 2006.
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References |
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coactivator 1ß (PGC-1ß), a novel PGC-1-related transcription coactivator associated with host cell factor. J Biol Chem 277:1645–1648This article has been cited by other articles:
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  P. Kovacs, M. Geyer, J. Berndt, N. Kloting, T. E. Graham, Y. Bottcher, B. Enigk, A. Tonjes, D. Schleinitz, M. R. Schon, et al. Effects of Genetic Variation in the Human Retinol Binding Protein-4 Gene (RBP4) on Insulin Resistance and Fat Depot Specific mRNA Expression Diabetes, December 1, 2007; 56(12): 3095 - 3100. [Abstract] [Full Text] [PDF]
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 A. J. Walley, A. I.F. Blakemore, and P. Froguel Genetics of obesity and the prediction of risk for health Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R124 - R130. [Abstract] [Full Text] [PDF]
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A. Lopez-Bermejo, B. Chico-Julia, M. Fernandez-Balsells, M. Recasens, E. Esteve, R. Casamitjana, W. Ricart, and J.-M. Fernandez-Real Serum Visfatin Increases With Progressive {beta}-Cell Deterioration. Diabetes, October 1, 2006; 55(10): 2871 - 2875. [Abstract] [Full Text] [PDF]
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S. D. Bailey, J C. Loredo-Osti, P. Lepage, J. Faith, J. Fontaine, K. M. Desbiens, T. J. Hudson, C. Bouchard, D. Gaudet, L. Perusse, et al. Common Polymorphisms in the Promoter of the Visfatin Gene (PBEF1) Influence Plasma Insulin Levels in a French-Canadian Population. Diabetes, October 1, 2006; 55(10): 2896 - 2902. [Abstract] [Full Text] [PDF]
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