1 Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas
2 Endocrinology Section, Department of Medicine, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas
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ABSTRACT |
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Type 2 diabetes is characterized by obesity-related insulin resistance. Although most glucose uptake occurs in skeletal muscle in the postabsortive state (1), considerable evidence (2) suggests a role in this uptake for factors secreted from adipose tissue. Adiponectin (also known as AdipoQ, APM1, and Acrp30) (3) has emerged as one such factor. Low adiponectin levels precede and predict type 2 diabetes (4), and increasing levels of plasma adiponectin improve insulin sensitivity (5), probably by acting through AMP kinase to increase fatty acid oxidation (6,7). Consistent with these roles, plasma adiponectin levels are decreased with obesity (3), despite increased adipose mass.
Recently, two related but distinct receptors for adiponectin were identified and the genes cloned from a human skeletal muscle expression library by binding to globular adiponectin (8). The two receptors, ADIPOR1 and ADIPOR2, were predicted to contain seven transmembrane domains, share 67% identity with the mouse gene, and show marked conservation of the membrane-spanning domains from yeast to mammals (8). Although expressed ubiquitously, ADIPOR1 showed the highest expression in skeletal muscle, whereas ADIPOR2 was found most abundantly in liver (8). ADIPOR1 appears to primarily bind the globular form of adiponectin, in contrast to ADIPOR2, which primarily binds the full-length form (8). More recently, both ADIPOR1 and ADIPOR2 were found to be abundantly expressed in human and rat pancreatic ß cells (9), where expression was increased by exposure to the free fatty acid oleate (9).
Genetic variation in the adiponectin gene has been reported to be associated with obesity, insulin resistance, type 2 diabetes, and adiponectin levels in multiple studies (10). We hypothesized that variation in the adiponectin receptor genes would likewise contribute to the risk of type 2 diabetes by reducing insulin sensitivity. We selected the ADIPOR1 gene because of its ubiquitous expression, including high levels in skeletal muscle and pancreatic ß-cells, and the evidence of an important role of the globular subunit in mediating adiponectin action (6,11,12). ADIPOR1 is located at 1q32.1 and is significantly telomeric to the 1q21-q24 linkage signals. We screened the entire ADIPOR1 gene for mutations and investigated the relationship of this variation to insulin sensitivity and type 2 diabetes. To further explore the role of genetic variation of ADIPOR1 on adiponectin levels, we examined allele-specific expression in transformed lymphocytes and compared total mRNA expression among individuals.
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RESEARCH DESIGN AND METHODS |
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Mutations were detected in 40 Caucasians (24 with type 2 diabetes and 16 glucose-tolerant control subjects) and 24 African Americans (16 with type 2 diabetes and 8 glucose-tolerant control subjects). Case-control association studies with type 2 diabetes were conducted in 192 Caucasian subjects with type 2 diabetes and 192 Caucasian control subjects and in 269 African-American subjects with type 2 diabetes and 136 nondiabetic African-American subjects. To reduce costs associated with typing large numbers of samples in large numbers of single nucleotide polymorphisms (SNPs), we constructed pooled DNA samples from each population. The pools equally represented each DNA sample and were constructed in triplicate from the concentration-adjusted DNA for each individual sample. We tested five pools: Caucasian control subjects (n = 192), Caucasian case subjects (n = 192), African-American control subjects (n = 130), African Americans with type 2 diabetes and diabetic nephropathy (n = 150) (14), and African Americans with type 2 diabetes and normal urine tests (n = 125) (14). As a result of new recruitment and necessary sample substitutions after pools were constructed, the individual samples included in the pooled and individual typing were slightly different, as reflected in slightly different sample sizes.
Insulin sensitivity was tested in 126 Caucasian nondiabetic members from 26 families for whom measurements of Si were available (13). Allele-specific expression and mRNA expression levels were compared among 25 Caucasian (10 nondiabetic and 15 with type 2 diabetes) and 25 African-American (8 nondiabetic and 17 with type 2 diabetes) individuals.
SNP detection and typing.
We designed 16 sets of primers for amplicons of 300550 bp using Primer 3 and WAVEMAKER software version 4.0 (Transgenomic, Omaha, NE) to screen the eight exons (including the untranslated exon 1), 1,200 bp of the 5' flanking region, and the 960-bp 3' untranslated region, spanning 18,826 bp of genomic DNA. We screened all putative functional regions of the gene, including each exon and 100200 bp of the flanking intronic sequence, 1,200 bp of the 5' flanking sequence, and 960 bp of the 3' flanking sequence. Mutations were detected using the Transgenomic WAVE HT DNA Fragment Analysis System (Transgenomic), and altered migration was confirmed by bidirectional sequence analysis (15) using infrared dyelabeled primers and GR4200 Sequencers (LI-COR Biotech, Lincoln, NE). SNPs were typed by Pyrosequencing (PSQ96; Pyrosequencing, Uppsala, Sweden) using the manufacturers protocols, except that a universal sequence was appended to one sequence-specific primer and amplification was performed in the presence of the universal biotinylated primer. Typing of pooled DNA samples was performed by Pyrosequencing using Allele Quantification software (Pyrosequencing) as described by others (16,17). Each pool was constructed in triplicate and analyzed in duplicate. Additionally, at least 92 individual samples were tested for each ethnic group to determine linkage disequilibrium (LD) and Hardy Weinberg equilibrium. If the SD of six measures was not <2%, if the SNP was nonsynonymous, or if the difference in pooled frequencies exceeded 5%, individual samples were typed instead. The 5-bp insertion/deletion variant was typed using infrared fluorescent primers and separated on acrylamide gels using the LICOR GR4200 sequencers and read with SAGAGT software (LICOR Biotech). All SNPs were in Hardy-Weinberg equilibrium (P > 0.05).
Analysis of mRNA expression in transformed lymphocytes.
Total RNA was isolated from Epstein-Barr virustransformed lymphocytes grown to 0.51.0 x 106 cells/ml as described previously (18). Allele-specific expression of SNP16 in the 3' untranslated region was quantified using allele quantification software (SNP Software AQ; Pyrosequencing), the RT-PCR product, and the DNA primers for SNP16. Ratios were compared to DNA from the same individuals. ADIPOR1 mRNA levels were measured using total transformed lymphocyte RNA, primers designed using Primer Express software (Applied Biosystems, Foster City, CA), and real-time PCR performed using the SYBR green real-time PCR reagents kit according to the manufacturers protocol (Applied Biosystems). Reactions were performed in triplicate and detected on a Rotor-Gene RG 3000 (Corbett Research, Sydney, AU) and standardized to 18 S RNA.
Statistical analysis.
Allelic association was tested separately for each ethnic group using the Fishers exact test. Significance in pooled DNA samples was judged using the statistics described by Risch and Teng (19). The role of ADIPOR1 SNPs in family members was tested using general linear models as described previously (20). Pairwise linkage (D' and r2) was calculated from combined case and control population data using the expectation maximization algorithm separately for each ethnic group. Allele-specific expression was compared to DNA in the same individuals using the same primers by paired Wilcoxons signed-rank test to correct for unequal amplification, and expression ratios were corrected by dividing the observed RNA ratio by the observed DNA ratio. Individual variation in gene expression was compared using the unpaired t test in ln-transformed ADIPOR1to18 S mRNA ratios or by Mann-Whitney U test on nontransformed data. Analyses were performed in SPSS for Windows version 11.0 or 12.1 (SPSS, Chicago, IL).
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RESULTS |
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To test a role of ADIPOR1 in insulin sensitivity, we selected six common SNPs and one rare SNP to type in individuals of Northern European descent who had undergone evaluation of insulin sensitivity (Si) using frequently sampled intravenous glucose tolerance tests. Under a general linear model with Si as the dependent variable, BMI and age as covariates, and controlling for family membership, sex, and glucose tolerance status, no SNP had a significant impact on either Si or insulin secretion measured as disposition index. In an exploratory study, SNPs 28, 23, 3, and 9 were associated with BMI as the dependent variable in interaction with pedigree membership (P = 0.018 to P = 0.003) when age, sex, and diagnosis were included in the model.
SNP16 was both common and located in the 3' untranslated region and thus ideal to examine allele-specific expression. Among 16 heterozygous individuals, the G (major) allele was slightly but significantly overexpressed relative to DNA, independent of ethnicity and diagnosis (ratio 1.35, P = 0.001). Overall, ADIPOR1 mRNA levels (expressed as the ratio of ADIPOR1 mRNA to 18S RNA) varied widely, from 0.5 to 21, among individuals. Although expression in cell lines from 10 Caucasian control and 10 Caucasian diabetic individuals did not differ, cell lines from 20 African-American individuals with diabetes showed a 45% decrease in ADIPOR1 mRNA levels when compared with cell lines from 10 African-American control subjects without considering the underlying genotype (geometric mean ratio 8.1 vs. 4.5, P = 0.028 by Mann-Whitney U; ln-transformed ratios 2.10 ± 0.52 vs. 1.50 ± 0.82 [means ± SD] in control and diabetic samples, respectively).
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DISCUSSION |
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Among the two ethnic groups studied and using both our own screening and public databases, we were able to confirm 22 sequence variants. Only one SNP altered an amino acid (N44K in exon 2), but was not associated with type 2 diabetes. No other SNP was associated with type 2 diabetes or altered insulin sensitivity. We have >80% power to detect an 810% difference in allele frequency over the range of minor frequencies examined in this study among either Caucasian or African-American case-control studies. This difference is equivalent to an odds ratio of 1.7. Thus, we do not have adequate power to detect an association with odds ratios of 1.21.4, as observed in some confirmed diabetes genes, although no trend in this direction was observed. Surprisingly, four of seven SNPs tested showed an interaction with family membership to alter BMI. Although SNPs 23 and 28 are in strong LD (r2 >0.95), SNPs 3 and 9 have low levels of LD with each other and with SNPs 23 and 28 and thus are independent observations. A biological explanation for this observation is not obvious, and without the other factors in our model, we could not demonstrate a difference in BMI between genotypes among the 126 subjects studied.
Although we found no association of any variant with type 2 diabetes, we did find two lines of support for altered ADIPOR1 gene expression. We used transformed lymphocytes for these studies, a tissue that has been increasingly used as a surrogate for tissues not easily obtained, and for which levels of gene expression appear to be stable and heritable (24). SNP16 (rs1139646) in the 3' untranslated region showed a 35% increase in expression of the major allele (C) after correcting for unequal amplification. The 3' untranslated region may play a role in posttranscriptional regulation of gene expression, and a search for functional elements placed SNP16 in the consensus sequence for the internal ribosome entry site. These sites generally control translation in the 5' untranslated regions. That the altered allele ratios were seen in both Caucasian and African-American populations, despite much lower LD among African Americans, would support a direct role for SNP16 in the allele-specific expression. However, SNP16 was not associated with detectable physiologic effects, perhaps because of tissue-specific differences, inadequate power to detect subtle changes, or effects on a parameter that we did not measure. We also found that among African-American individuals, ADIPOR1 mRNA was reduced by 45% in cell lines from diabetic subjects compared with control subjects. Within each group, we observed large variations among individual levels of expression. We have observed similarly large differences for other genes (S.C.E. and H.W., unpublished data) and thus believe this is a biological property rather than a technical artifact. Nonetheless, these findings will require confirmation. Furthermore, we cannot determine from this study whether that decrease resulted from a cis-acting element in a regulatory region of the ADIPOR1 gene such as SNP16 or a promoter element well upstream of the gene or from a trans-acting variant in these individuals. However, the decrease is biologically plausible and might suggest a role for this gene or a gene regulating ADIPOR1 in the metabolic syndrome among African-American individuals. Studies of ADIPOR1 gene expression in muscle from African-American diabetic and control subjects might be particularly informative, as would an analysis of the 10 ADIPOR1 tagSNPs in nondiabetic African-American individuals characterized for insulin sensitivity.
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ACKNOWLEDGMENTS |
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We thank Terri Hale and Judith Cooper for their assistance with subject ascertainment and the nursing staff of the General Clinical Research Center for their support of clinical studies.
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FOOTNOTES |
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Address correspondence and reprint requests to Steven C. Elbein, MD, Professor of Medicine, Central Arkansas Veterans Healthcare System, Endocrinology 111J/LR, 4300 West 7th St., Little Rock, AR 72205. E-mail: elbeinstevenc{at}uams.edu
Received for publication March 4, 2004 and accepted in revised form April 23, 2004
LD, linkage disequilibrium; SNP, single nucleotide polymorphism
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REFERENCES |
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