1 Pennington Biomedical Research Center, Baton Rouge, Louisiana
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ABSTRACT |
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The hormone resistin is a member of a novel family of cysteine-rich secreted proteins associated with pulmonary inflammation (FIZZ3) and expressed in the murine small bowel and adipose tissue (1). Resistin was downregulated in the mouse by thiazolidinediones (TZDs), which are agonists for the antidiabetic peroxisome proliferatoractivated receptor (PPAR-
), and was proposed to link obesity to diabetes (2). The latter findings were contradicted in rodent models of obesity in which PPAR-
agonists augmented expression of resistin (3). At the genic level, single nucleotide polymorphisms (SNPs) in noncoding regions of the human resistin gene were either not significantly associated with insulin resistance (4,5) or associated with an insulin sensitivity index in the case of a different promoter SNP from the one that we present in this article (6). Resistin expression in humans has been reported at low levels in the adipose tissue of some but not all humans (7,8), and its reduced expression has also been proposed as a hallmark of obesity (9). In another study, resistin mRNA was not related to insulin resistance when using RNA isolated from cultured adipocytes (10) but was upregulated by acute hyperglycemia in various mouse adipose depots (11). Using 32 adipose tissues and quantitative PCR, however, there was increased amount of resistin mRNA in abdominal depots compared with thigh depots, suggesting an increased risk for type 2 diabetes as a result of central obesity and higher resistin (12). The genomic organization and regulation of the murine and human resistin genes are divergent and may explain these discrepant findings (13).
Resistin was also reported as a cysteine-rich adipose tissuespecific secretory factor that blocks adipocyte differentiation (14). This finding provides an appealing functional role for resistin by which ectopic fat accumulation occurs as a result of impaired lipid storage in adipocytes. Indeed, failure of adipocyte differentiation has been proposed as a cause of type 2 diabetes (15), possibly through an ectopic overload of fatty acids and lipotoxicity of nonadipose tissues (16). A role for resistin could therefore be envisioned in the prediabetic syndrome of insulin resistance 1) by virtue of its ability to block adipocyte differentiation or 2) as a direct result of increased circulating levels and insulin resistance.
Cellular oxidative stress is the result of normal cellular processes and occurs at the level of the mitochondria when proton potential is high (17), when free radicals are generated at the plasma membrane (18), or when cells are exposed to environmental toxins (19). Cellular responses to limit oxidative stress are grouped into phase I and phase II enzymes (20), the latter including heme oxygenase and NAD(P)H:quinone reductase (EC 1.6.99.2). NAD(P)H:quinone oxidoreductase 1 (NQO-1), a 274amino acid flavoprotein, is a prototypical phase II enzyme that is induced by electrophiles such as tert-butyl hydroxyquinone and oxygen free radicals (21). NQO utilizes NAD(P)H as an electron donor and acts to reduce and detoxify quinones and their derivatives. NQO-1 knockout mice exhibit alterations in the intracellular redox state with a resulting phenotype of reduced visceral adipose tissue mass (22). Combined with a growing body of evidence linking insulin resistance and oxidative stress (23), there may be a prominent role for free radicals in the regulation of adipocyte function. Oxidative stress impairs insulin action in adipocytes in vivo (2426), and in vivo, treatment with the antioxidant lipoic acid improves insulin-stimulated glucose disposal (27). Given the putative role of resistin in insulin resistance, oxidative stress might also have an impact on endogenous expression of resistin in the adipocyte.
To understand better the role of resistin in human insulin resistance, we examined the promoter of resistin for the presence of functional mutations. We report the identification of an SNP in the proximal promoter of the human resistin gene. The possible association of this SNP with obesity phenotypes was evaluated in a cohort of 978 individuals. Analysis of the homozygous genotypes in cell models showed that this is a functional SNP that affects promoter activity and could therefore affect expression of the gene in vivo. This hypothesis was tested in a second cohort of 58 individuals in whom resistin mRNA was measured in subcutaneous fat and related to the -180 SNP. Given 1) the potential link between oxidative stress and insulin resistance and 2) a lack of interaction between resistin expression in human adipocytes in vitro and known regulators of adipocyte function (glucocorticoids, PPAR agonists, cytokines, etc.; data not shown), we next tested the hypotheses that there might be significant interactions among the -180 SNP, resistin mRNA, and oxidative stress in vivo using NQO mRNA as a measure of oxidative stress. The results presented here provide insights into a potential role for resistin in the pathophysiology of the human insulin resistance syndrome.
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RESEARCH DESIGN AND METHODS |
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Sequence alignments and algorithmic analyses.
Sequences alignments between the mouse and human resistin promoter orthologous regions were performed with a web-based algorithm (saturn.med.nyu.edu/searching/prsa.cgi; Skirball Institute of Biomolecular Medicine, NY University School of Medicine, New York, NY). The human DNA sequences used were from the GenBank accession number AF352730, and the mouse sequence was extracted from the publication by Hartman et al. (28). Algorithmic analyses to identify predicted recognition binding sites for transcription factors in the promoter of resistin were performed with the TESS software (29).
The -180C>G promoter polymorphism.
The -180C>G SNP in the human resistin promoter was identified in the SNP database (Sanger Center, Cambridge, U.K.) with dbSNP accession number 273660. The positional assignment for this SNP was based on the assignment of the transcription start site at nucleotide 3,168 in the sequence with accession number AF352730.
Genotyping of individuals for the -180C>G SNP was performed on a LI-COR DNA Analyzer 4200 (Lincoln, NE). Amplicons were generated with the primers FZ3P1:5'-TTTTGTCATGTTTGCATCAGC-3' and FZ3P2:5'-GGGCTCAGCTAACCAAAT-3'. PCR conditions were as follows: one cycle at 95°C for 4 min, followed by 30 cycles, each consisting of denaturation step at 95°C for 1 min, an annealing step at 60°C for 1 min, and an extension step at 72°C for 1 min. The sequencing reactions were performed as prescribed by the manufacturer (LI-COR, Lincoln, NE) with the following primer tagged with IRDye 800 at 795 nm: 5'-gaccagtctctggacatgaa-3'.
Transfection reporter constructs.
Reporter constructs were generated by amplification of genomic DNA from two homozygous individuals (C/C and G/G) and cloning of the PCR amplicons into the pGL3-basic luciferase reporter vector (Promega, Madison, WI). The PCR primers were the FZ3P1 and FZ3P2 primers described above with the addition of leader sequences that corresponded to the SacI and XhoI restriction enzymes to facilitate directional cloning into the pGL3-basic vector. PCR products and a pGL3-basic vector plasmid preparation were treated with the two enzymes as prescribed by the manufacturer (New England Biolabs, Beverly, MA), purified from agarose gel slices, and incubated into a ligation reaction at 16°C overnight in the presence of T4 DNA ligase (New England Biolabs). After transformation of competent DH5 cells (GIBCO-BRL, Gaithersburg, MD), transformants were grown in LB/Amp media and preparations were made for the plasmids representing the two genotypes: -303/+27(C/C) and -303/+27(G/G). The genotypes of the two constructs were confirmed by bidirectional sequencing of the plasmids (Perkin Elmer, Wellesley, MA).
Cell culture and luciferase assay 3T3-L1 cells were differentiated by incubation for 48 h in the following reagents: DMEM (Cellgro; Fisher, Pittsburgh, PA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 830 nmol/l insulin, and 2 µmol/l dexamethasone. Subsequently, the dexamethasone was removed from the media and the cells were maintained in the differentiated state in the presence of insulin for an additional 10 days. The cells were initially cultured in plastic dishes (10-cm diameter) at 37°C in a humidified atmosphere with 5% CO2 in the air. For the transfections, differentiated (grown an additional 10 days after removal of dexamethasone) 3T3-L1 cells were grown to 85% confluence in six-well (34.8-mm diameter per well) plates (Corning, Corning, NY). Cells were transiently cotransfected with the constructs and ß-gal plasmids for 24 h in the absence of serum using the Geneporter2 transfection reagent (Gene Therapy Systems, San Diego, CA), as previously described (30). According to the manufacturer of the kit (Gene Therapy Systems), the transfection efficiencies of the 3T3-L1 cells were estimated at 3040% and 8090%, respectively, using a ß-gal reporter plasmid. The media were then supplemented with 20% FCS for 24 h. Cells were harvested using 1x Geneporter2 lysis buffer, and the lysates were assayed for luciferase and ß-galactosidase activities, as prescribed by the assay manufacturer (Promega, Madison, WI) in a luminometer (Zylux, Pforzheim, Germany). The pGL3-basic vector was used as negative control and the pGL3-promoter vector was used as a positive control.
Luciferase activity measurements were normalized to ß-galactosidase values. Luciferase activity was also measured for untransfected cells. Each transfection was carried out in duplicate, and the experiments were repeated a minimum of five times for each construct, in each cell line.
The mean luciferase relative activity for each construct was calculated as a fold increase over the negative control empty vector (pGL3-basic). The data presented represent the means of five duplicate independent transfection experiments per construct.
Human studies.
The Pennington Institutional Review Board approved these studies, and all participants provided written informed consent before participation. Vital signs, laboratory values, body fat, and visceral adipose tissue mass were measured as previously described (31).
Association study (cohort 1).
The population consisted of 978 individuals who enrolled in studies at the Pennington Biomedical Research Center during the previous 5 years. Each participant signed informed consent. Volunteers were excluded for a diagnosis of diabetes as defined by a fasting plasma glucose >126 mg/dl. Blood glucose and insulin values were available for 671 of these individuals. Body composition was determined by dual X-ray absorptiometry (DEXA), and visceral adipose tissue (cm2) was measured by single-slice computed tomography (CT) scanning as previously described (31).
Measurement of resistin mRNA in human adipose tissue (cohort 2). Cohort 2 included 58 healthy overweight and obese men and women. Each participant signed informed consent. Body composition was determined by DEXA, and visceral adipose tissue (kg) was measured by multislice CT scanning as previously described (31). Liver fat (X-ray attenuation value; Houndfield Units) was measured by CT scanning with splenic X-ray attenuation as an intrascan control. A low liver:spleen ratio is indicative of a fatty liver, whereas a high liver:spleen ratio is indicative of low fat in the liver (32). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as the product of fasting insulin (µU/ml) and glucose values (mmol) divided by 22.5 (33).
After local anesthesia (according to institutional guidelines), adipose tissue was collected by needle biopsy of the superficial subcutaneous adipose tissue lateral to the umbilicus. Total RNA was isolated from 50 mg of frozen tissue using Trizol (Life Technologies, Gaithersburg, MD). RNA was dissolved in Formazol and stored at -80°C. The concentration and quality of the RNA were determined spectrophotometrically. The TaqMan Real Time RT-PCR technique was used to measure quantitatively the levels of human resistin mRNA (Applied Biosystems, Foster City, CA). The primer probe set for human resistin was designed using the Primer Express software (Perkin Elmer) as follows: forward primer 5'-AGCCATCAATGAGAGGATCCA-3', reverse primer 5'-TCCAGGCCAATGCTGCTTA-3', and the probe 5' FAM-TCGCCGGCTCCCTAATATTTAGGGCA-bhq-13' (Biosearch Technologies, Novato, CA) that spans the exon 2/exon 3 boundary. The RT-PCR was carried out in one tube using the ABI PRISM 7700 SDS instrument (Applied Biosystems) using 10 ng of total RNA as the template. The master mix included 300 nmol/l primers, 100 nmol/l probe, reverse transcriptase MuLV, RNase inhibitor, and AmpliTaq GOLD (Applied Biosystems). The Ct value for every sample was measured in duplicate, and resistin mRNA levels were normalized to cyclophilin mRNA levels (cat. no. 4310883E; Applied Biosystems).
Measurement of NQO mRNA (cohort 2).
The same procedures as described above for resistin RNA were followed for the measurement of NQO mRNA using the following primers: forward primer 5'-TCATTCTCTGGCCAATTCAGAGT-3', reverse primer 5'-GGAGTGTGCCCAATGCTATATG-3', and the probe 5'FAM-TCTGTGGCTTCCAAGTCTTAGAACCTCAACTG-BHQ13' (Biosearch Technologies).
Statistical analyses.
Genotype relationships with clinical and laboratory variables were determined using one-way ANOVA, two-way ANOVA, or mixed models (JMP v 4.0; SAS Institute, Cary, NC). Multivariate regression was used to test the significance of the relationship of resistin mRNA to HOMA-IR and liver fat including body fat, visceral fat, and sex in the models. Sample means are presented unless otherwise noted; however, variables were log-normalized before analysis where necessary. The results of the general linear model are graphically presented as leverage plots that illustrate the residuals of the independent variable after adjusting for the other variables in the model. Analyses were repeated for cohort 2 using ethnicity (race) and sex as covariates or using the whites only (also adjusted for sex). The data could not be analyzed for the blacks alone because of their low numbers in cohort 2 (CC = 0, GC = 11, GG = 1).
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RESULTS |
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Functional properties of the -180C>G SNP.
The impact of the -180C>G SNP on promoter activity of the corresponding region was assessed through two identical constructs (330 nt long) in the pGL3-basic vector that differed only by the polymorphism -303/+27(C/C) and -303/+27(G/G). The luciferase reporter gene was used to measure promoter activity. Differentiated 3T3-L1 cells were transiently cotransfected with the promoter constructs and control vectors. Luciferase values were averaged, and relative luciferase activity was calculated as a fold increase over the activity of the pGL3-basic vector. The -303/+27(G/G) construct had fourfold higher luciferase activity than the -303/+27(C/C) construct in the 3T3-L1 adipocytes (Fig. 1B).
The -180C>G SNP and human fatness.
To explore the effects of the -180C>G SNP on human physiology, we genotyped 978 individuals (cohort 1; Table 1) with known body composition phenotypes (percentage fat and visceral adipose tissue by CT scanning). Genotype frequencies (CC = 49.8; CG = 40.9; GG = 9.3) were in Hardy Weinberg equilibrium. The G/G genotype of the -180C>G SNP was significantly associated with higher BMI and percentage body fat in white men only but not in women or in blacks of any sex (Table 1). However, after Bonferroni corrections for multiple testing were performed, this association was not significant (Table 1). No significant effects of the -180C>G SNP were found on fasting insulin values, with or without adjusting for the major effects of sex, body fat, and visceral fat.
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Interactions among the -180C>G SNP, oxidative stress, and insulin resistance.
We next tested the hypothesis that the SNP and oxidative stress interacted with respect to insulin resistance (measured by HOMA-IR) in cohort 2 (Fig. 2B). There was a significant relationship among the -180 SNP, oxidative stress, and HOMA-IR (genotype, P = 0.6; NQO, P = 0.004; NQO x genotype, P = 0.03). Because of the presence of two ethnic groups and sexes in the dataset (cohort 2), data were subsequently adjusted using ethnicity and sex as covariates (Fig. 2B). The interactions among genotype, NQO mRNA, and HOMA-IR remained significant (genotype, P = 0.72; NQO, P = 0.004; genotype x NQO, P = 0.03). The same analysis was performed for whites only (adjusted for sex) with essentially the same results (genotype, P = 0.46; NQO, P = 0.005; NQO x genotype, P = 0.04).
Resistin mRNA is related to insulin resistance and hepatic fat accumulation.
To test the hypothesis that high resistin mRNA (irrespective of genotype) was related to the insulin resistance syndrome and ectopic fat storage, we constructed multivariate models with insulin resistance (as measured by HOMA-IR) and the amount of liver fat (as measured by the liver:spleen ratio) as dependent variables in cohort 2. We found that high resistin expression was proportionally related to insulin resistance (P = 0.003) and to ectopic/hepatic fat (P = 0.008), after adjusting for the effects of body fat and visceral adiposity. This effect was present after applying to the data additional adjustments for ethnicity and sex in the case of HOMA-IR (P = 0.02; Fig. 3A) and liver fat (P = 0.008; Fig. 3B). The same analysis was also performed in whites only, adjusted for sex, and the effect remained significant for both measures (HOMA-IR, P = 0.01; liver fat, P = 0.003).
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DISCUSSION |
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-180C>G SNP and obesity phenotypes.
The possible association of the -180C>G SNP with obesity/diabetes phenotypes was examined in a cohort of 978 individuals (cohort 1). G/G homozygous male individuals (i.e., individuals with the high promoter activity genotype) were significantly more obese, by BMI and percentage body fat, than allozygotes, but the significance was eliminated after ethnicity and multiple comparisons were adjusting for. A 5' variant in human resistin, -420C>G, was recently reported to be associated with obesity (35). This variant is the same as our -180C>G. The discrepancy in the numbering relates to the numbering schema; they counted as nucleotide "-1" the one upstream of the "ATG" translation initiator, whereas we counted as nucleotide "-1" the one upstream of the transcription start site. Engert et al. (35) presented significant associations of the "G" allele (i.e., the combined C/G and G/G genotypes) with increased BMI, body weight, body fat mass, and waist circumference, again in men only. This is consistent with our unadjusted-for-multiple-comparisons findings (although in our cohort, the G/G genotype had a significant effect on its own), but these data do not represent conclusive evidence for an association with obesity. Two additional publications have also reported the -180C>G SNP (referred to as -179C>G SNP) but did not find any associations of this SNP with obesity (36,37) or in linkage disequilibrium with other SNPs in the gene. Taken together the findings of the four reports (3537), there is an overall consensus that the -180C>G SNP is not associated with obesity (when considering each genotype on its own), but there may be populations or yet-to-be-determined circumstances in which the G/G genotype might predispose for higher percentage body fat.
A functional role for the -180C>G SNP in vivo.
Because the in vitro data suggested that the -180C>G SNP might affect expression of resistin, we tested this hypothesis in a cohort of 58 overweight and hyperinsulinemic individuals (cohort 2) by measuring resistin mRNA expression in abdominal subcutaneous adipose tissue. The data set was adjusted for ethnicity and sex, and no effects of the three genotypes were observed on BMI, fasting glucose, the liver:spleen ratio, fat mass, insulin levels, HOMA-IR, and visceral adiposity. In contrast, G/G homozygotes (i.e., individuals with the high promoter activity genotype) had significantly higher expression levels of resistin than allozygotes, confirming our hypothesis and recapitulating the in vitro data. This analysis was repeated for whites only, and G/G homozygotes still had significantly higher resistin mRNA levels compared with allozygotes. The association of the G/G genotype with high resistin mRNA remained significant even after multiple testing was adjusted for. Further experiments to determine the protein levels of resistin would be required to investigate whether the higher resistin mRNA levels in G/G homozygotes also translate to higher protein levels, thus confirming the physiological functionality of the SNP. For several individuals with high resistin mRNA, we measured resistin in a second adipose tissue sample separated from the first one by 8 weeks. Resistin mRNA was similar in the two samples (data not shown). As such, resistin mRNA expression is stable over time and the high expression in G/G homozygotes is not a transient event. However, although all C/C homozygotes had low resistin mRNA, not all G/G homozygotes exhibited high resistin mRNA. This was reflected by the bimodal distribution of resistin mRNA levels in cohort 2 (Fig. 2A, inset). We therefore hypothesized that there was an additional factor involved in the regulation of resistin other than the -180C>G genotype.
Interactions among the -180C>G SNP, oxidative stress, and resistin mRNA/HOMA-IR.
To test the hypothesis that oxidative stress was the additional factor involved in the regulation of resistin, we measured the effects of oxidative stress on resistin mRNA in vivo. Our rationale for measuring oxidative stress was based on the fact that previous studies have demonstrated a role for oxidative stress as an activator of the Sp-1 transcription factor, one of the transcription factors that might bind to the -180 GC SNP (38,39) and the putative role of oxidative stress in insulin resistance (reviewed by Evans et al. [23]). Oxidative stress was measured by the expression levels of NQO, a prototypical phase II antioxidant enzyme. In addition, NQO knockout mice exhibit alterations in the intracellular redox state and have reduced visceral adipose tissue mass (22).
There was no direct correlation between the -180C>G SNP and NQO mRNA. The causes of high oxidative stress in human fat cells could be the result of a number of factors, including high-fat diet, inactivity, smoking, and so forth. Analyses of the data showed a significant interaction among the -180C>G SNP, NQO mRNA, and resistin mRNA, but this interaction did not hold significance after ethnicity and sex were adjusted for, which could be due to the disproportionate frequencies of the genotype in the black individuals of this cohort. This possibility was confirmed when the white individuals only were examined (adjusted for sex), where the interaction among genotype, resistin mRNA, and NQO mRNA was significant.
The interaction among the -180C>G SNP, NQO mRNA, and HOMA-IR was also tested to examine for possible effects of the genotype on insulin resistance with relation to the levels of oxidative stress. There was a significant interaction illustrating an effect of the SNP alone and oxidative stress only when the GG genotype was present. These data suggest that the resistin -180C>G SNP and cellular oxidative stress are potentially related to the insulin resistance syndrome in Homo sapiens.
Resistin is directly and independently related to insulin resistance and liver fat.
We next tested the hypothesis that resistin mRNA levels (irrespective of genotype) were directly related to insulin action. We found that high resistin expression was proportionally related to insulin resistance, after adjusting for the effects of body fat and visceral adiposity. This effect was present after we adjusted further for ethnicity and sex and when we performed the analysis in white individuals only (also adjusted for sex).
Fatty liver is also a component of the insulin resistance syndrome (32) and contributes to increased hepatic glucose output. To test the relationship of resistin mRNA to fatty liver, we constructed a multivariate model with the amount of liver fat as the dependent variable and adjusted for the major effects of fatness and visceral adiposity. We found that resistin mRNA was directly and independently related to liver fat infiltration.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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The authors acknowledge the studies and principal investigators whose support of the PBRC database made the population studies possible: George Bray, Donna Ryan, Frank Greenway, Jennifer Lovejoy, and Mike Lefevre. We also acknowledge the expert database management provided by Julia Volaufova and Connie Murla.
Address correspondence and reprint requests to George Argyropoulos or Steve R. Smith, Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA 70808. E-mail: argyrog{at}pbrc.edu or smithsr@pbrc.edu
Received for publication December 4, 2002 and accepted in revised form March 19, 2003
CT, computed tomography; DEXA, dual X-ray absorptiometry; HOMA-IR, homeostasis model assessment of insulin resistance; NQO, NAD(P)H:quinone oxidoreductase; PPAR, peroxisome proliferatoractivated receptor; SNP, single nucleotide polymorphism; TZD, thiazolidinedione
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REFERENCES |
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