1 Department of Obstetrics and Gynecology and 2 Department of Genetics, Mersin University, School of Medicine, 33079, Mersin, Turkey
3 To whom correspondence should be addressed. Email: devrimertunc{at}hotmail.com
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Abstract |
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Key words:
glucose metabolism/polycystic ovary syndrome/PPAR-2 polymorphism/reproductive hormones
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Introduction |
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Peroxisome proliferator-activated receptors (PPARs) are mediators of the linkage between glucose and lipid metabolism. In cases of long-term glucose deprivation, PPARs are involved in supplying fatty acids and ketone bodies from adipose tissue as energy sources. Activated PPARs bind to the peroxisome proliferator response element and mediate the transcriptional effects of peroxisome proliferators. The peroxisome proliferation leads to an increase in the transcription of genes involved in peroxisomal and microsomal fatty acid oxidation (Rangwala and Lazar, 2004). Replacing glucose as a fuel by fatty acids or ketone bodies limits the need for de novo glucose synthesis and hence muscle protein catabolism. Another well-known action of PPAR-
is stimulation of fibroblast to differentiate into adipocytes, and stimulation of the adipocyte growth (Rangwala and Lazar, 2004
).
PPAR- is a transcription factor involved in adipogenesis and a functional receptor for thiazolidinediones which were introduced as insulin-sensitizing agents. A C to A nucleotide substitution in exon 2 results in an amino acid change, Pro to Ala at codon 12, and this is the most common variant of PPAR-
2 (Yen et al., 1997
). Clinical studies in humans have demonstrated a relationship between this polymorphism and alterations in body mass index and insulin sensitivity (Beamer et al., 1998
; Valve et al., 1999
), and a recent meta-analysis showed association of this variant with a decreased risk of type 2 diabetes (Altshuler et al., 2000
).
The PPAR-2 Pro12Ala polymorphism in women with PCOS has been investigated in several studies. Korhonen et al. (2003)
found that this polymorphism was less prevalent in women with PCOS. Two further studies compared the metabolic features of PCOS women according to the presence or absence of Pro12Ala, and established conflicting results (Hara et al., 2002
; Orio et al., 2004
). Besides conflicting results, the metabolic features of control women according to Pro12Ala polymorphism were not investigated in these two studies. For this reason, we aimed to compare the metabolic and androgenic profile of PCOS women with control group on the basis of Pro12Ala genotype.
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Materials and methods |
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The diagnosis of PCOS was based on the following criteria; sonographic polycystic ovary appearance, oligo/amenorrhea and clinical (a FerrimanGallwey score >8; acne, that persisted through the second decade of life or androgenetic alopecia) or biochemical hyperandrogenemia, and exclusion of non-classical congenital adrenal hyperplasia, thyroid dysfunction and hyperprolactinemia. Other exclusion criteria were any other systemic or endocrine disorder, including diabetes mellitus, and treatment with medications known to alter insulin hemodynamics or oral contraceptives within 3 months. The patients diagnosed as suffering from diabetes mellitus were not included in the study, to avoid confusing effects of diabetes on measures of insulin secretion and sex steroid levels. BMI was calculated as weight (kilograms) divided by height squared (meters). Waist and hip circumferences were registered and the waist/hip ratio (WHR) was calculated.
Primary outcome measures were glucose and insulin metabolism including fasting blood glucose, C-peptide and insulin levels and 75 g oral glucose tolerance test (OGTT). We also evaluated if the reproductive hormone levels would be different on the basis of PPAR-2 Pro12Ala genotype.
Biochemical and hormonal measurements
After an overnight fast, blood samples were collected for fasting glucose, insulin and C-peptide levels. Then all subjects underwent a 75 g OGTT. The degree of insulin resistance (IR) was estimated using homeostasis model assessment (HOMA) analysis as follows: Fasting serum insulin (µU/ml) x fasting plasma glucose (mmol/l) divided by 22.5. The HOMA method has been recently validated to be a good index of insulin resistance in subjects with a broad range of insulin sensitivity and has a good correlation with the insulin-mediated glucose uptake calculated by the euglycemic hyperinsulinemic glucose clamp (Bonora et al., 2000).
For sex steroids, blood was drawn within 7 days of menstruation in spontaneously menstruating women. In 39 women with long-lasting oligo-amenorrhea, blood was drawn at the 7th day of progesterone withdrawal bleeding to avoid the effect of progesterone on measured parameters. We used 10 mg medroxyprogesterone acetate (Farlutal®, Deva, Ist, TR) for 10 days for this purpose. Serum was obtained from each subject for measurement of total testosterone (total T), 17-estradiol (E2), dehydroepiandrosterone sulfate (DHEAS), androstenedione (AS), 17
-hydroxyprogesterone (17
-OHP), and sex hormone binding globulin (SHBG). Free testosterone index (FTI) was calculated by the equation: Total testosterone (nmol/l) x 100 divided by sex hormone binding globulin (nmol/l). This value correlates quite well with the free testosterone level measured by equilibrium dialysis and appears to be useful to assess the clinical androgen status in women, except in pregnancy (Vermeulen et al., 1999
).
Assay methods
Plasma glucose levels were determined by the glucose-oxidase method immediately after blood samples were obtained. Blood samples for hormones were centrifuged immediately, and serum was stored at 20 °C until assayed. E2, total T, DHEAS and insulin levels were determined by competitive electro-chemoluminescent immunoassay method by auto-analyzer (Elecys 2010 RDM, Germany). FSH and LH levels were determined by sandwich electro-chemoluminescent immunoassay method by the same auto-analyzer. AS, C-peptide, IGF-1 and SHBG levels were determined by competitive enzyme-signed chemoluminescent immunoassay (Immulite 1000 Biodpc, Los Angeles, CA).
Detection of the Pro12Ala polymorphism in the PPAR-2 gene
DNA was prepared from peripheral blood leukocytes by proteinase Kphenolchloroform extraction method. Exon B of the PPAR- gene was amplified by PCR with the forward primer 5'-GACAAAATATCAGTGTGAATTACAGC- 3' and the reverse primer 5'-CCCAATAGCCGTATCTGGAAGG- 3' (product size, 167 bp). PCR was performed in a 6 ml volume containing 50 ng genomic DNA, 3 pmol of each primer, 10 mmol/l TrisHCl (pH 8.8), 50 mmol/l KCl, 1.5 mmol/l MgCl2, 0.1% Triton X-100, 100 mmol/l deoxy (d)-NTP, 0.25 U DNA polymerase (Dynazyme DNA Polymerase, Finnzymes, Espoo, Finland) and 0.55 mCi [32P]dCTP. PCR conditions for exon B were denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s and annealing at 66 °C for 1 min, with a final extension at 72 °C for 6 min. Variants were detected by single strand conformation polymorphism analysis. PCR products were first diluted 4- to 10-fold with 0.1% SDS and 10 mmol/l ethylenediamine tetra-acetate and then mixed (1:1) with loading dye mix (95% formamide, 20 mmol/l ethylenediamine tetraacetate, 0.05% bromophenol blue, and 0.05% xylene cyanol). After denaturing at 98 °C for 3 min, samples were immediately placed on ice. Two microliters of each sample were loaded onto nondenaturing polyacrylamide gels (acrylamide/N,N9-methylene-bis-acrylamide ratio, 49:1; 6%) containing 10% glycerol. Samples were run at temperatures that were shown to discriminate among the variants in the previously sequenced (Kretz et al., 1989
) samples of exon B (3738 °C). The gel was dried and autoradiographed overnight at 70 °C with intensifying screens.
Statistical analysis
All statistical analyses were performed by using SPSS v.12 (SPSS Inc., Chicago, IL) for Windows. Comparisons were made by a two-tailed analysis of variance (ANOVA). The homogeneity of the variances was evaluated by Levene's test. Logarithmic or squared-root transformations were applied before ANOVA, to ensure homogeneity of variances, as needed. When adjustments for covariates were required, comparisons were performed by ANCOVA analysis. Binary data were analyzed by Fischer exact test. A P-value at or below 0.05 was considered as statistically significant.
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Results |
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Anthropometric characteristics and metabolic profile are summarized in Table I. The women in PCOS and control groups did not differ significantly in age and WHR. Women with Pro12Ala had significantly higher BMI than the women without this polymorphism (P<0.05). Although the groups did not differ significantly in fasting blood glucose (P>0.05, Table I), striking differences were encountered in the mean fasting insulin levels across the groups. PCOS women without Pro12Ala had the highest mean insulin level (P<0.05). Although the mean insulin levels of control women without Pro12Ala and PCOS women with Pro12Ala did not differ, they had significantly higher mean insulin levels than control women with Pro12Ala (P<0.05). The same tendency was observed with regard to the C-peptide concentrations (Table I). The PCOS patients without Pro12Ala had the highest degree of IR (HOMA), whilst control women without Pro12Ala and PCOS women with Pro12Ala had moderate degrees of IR (Table I). Although 1 h levels of OGTT in both groups of PCOS women were higher than control group, the PCOS women without Pro12Ala had highest degree of glucose intolerance as demonstrated by 2 h OGTT level. The mean 2 h OGTT levels in control women differed also significantly on the basis of PPAR-2 genotype (Table I).
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Discussion |
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The association of BMI with PPAR-2 Pro/Ala polymorphism is complex and controversial. In previous reports concerning Pro/Ala polymorphism in PCOS, no association was found between Pro/Ala variant and BMI (Hara et al., 2002
; Orio et al., 2004
). However, the mean BMI of the subjects in these studies were >30 kg/m2. The findings of two studies are very interesting and worth mentioning. Although there was no association between Pro/Ala genotype and BMI in lean (<25 kg/m2) and obese (>30 kg/m2) subjects, the carriers for Pro/Ala genotype with a BMI between 25 and 30 kg/m2 had higher BMI than non-carriers (Kao et al., 2003
). The BMI of our patients were also within these ranges and we found higher mean BMI in Pro/Ala carriers in both controls and women with PCOS. Furthermore, in a longitudinal study, subjects were followed from birth up to 40 years, and it was found that Ala12 allele of PPAR-
2 gene (either Pro/Ala or Ala/Ala) was associated with higher ponderal index at birth and higher weight gain after the second decade of life, but normal weight gain between 7 and 20 years (Pihlajamaki et al., 2004
). A recent meta-analysis with 19 136 subjects showed that Pro/Ala genotype was associated with higher BMI, and this association was more obvious in subjects with a BMI >27 kg/m2 (Masud and Ye, 2003
).
These data suggest that the function of PPAR-2 Pro12Ala polymorphism may be regulated by age and weight. The disparate results about the association of Pro12Ala and BMI might be genetic and environmental interactions that could modify the function of this polymorphism. Actually, a strong interaction was found between dietary polyunsaturated/saturated fatty acid ratio and the Pro/Ala polymorphism for BMI (Luan et al., 2001
). So, the difference in BMI that we observed in our study may be dependent on dietary habits of Turkish population that contains high rates of saturated fats. Furthermore, we observed that PPAR-
2 polymorphism was associated with increased BMI, without any effect on WHR. The current studies reported either an increased (Kao et al., 2003
) or similar (Beamer et al., 1998
; Frederiksen et al., 2002
) WHR in women PPAR-
2 polymorphism when compared with controls. Valve et al. (1999)
reported that Ala/Ala polymorphism, but not Pro/Ala, was associated with a symmetrical increase in waist and hip. Interestingly, troglitazone that acts through PPAR-
has been reported to result in decreased intra-abdominal fat mass, without any effect on subcutanous fat in patients with non-insulin dependent diabetes mellitus (NIDDM) (Kelly et al., 1999
). A definite causal role of PPAR-
2 polymorphism in causing obesity remains to be established by more detailed characterization of the body fat distribution and genotype of affected individiuals.
We found significantly lower mean fasting insulin and C-peptide levels in patients with Pro12Ala polymorphism. Furthermore, when a comparison was made within the groups, carriers for this polymorphism were less insulin-resistant (HOMA) and less glucose-intolerant, as demonstrated by 2 h OGTT values. However, presence of this polymorphism alone seemed not to be sufficient to explain the abnormalities in glucose metabolism in PCOS, when we compared Pro12Ala-positive women in both groups (Table I). Similar results were attained by Hara et al. (2002). They found that PCOS patients with Pro/Ala genotype had significantly lower fasting insulin levels and were significantly less insulin-resistant, although 2 h glucose levels did not differ. In contrast, it has also been suggested that there is no association between Pro12Ala and these parameters in both control group and PCOS women, separately (Orio et al., 2004
). A similar conflict also exists about the association of Pro12Ala and insulin-resistance in normal subjects (Beamer et al., 1998
; Altshuler et al., 2000
; Gonzalez Sanchez et al., 2002
; Buzzetti et al., 2004
).
Involvement of PPAR- in insulin sensitivity stems from the discovery that thiazolidinediones (TZD) are strong and specific activators of this PPAR isoform (Kliewer et al., 2001
). Although the exact mechanisms of TZDs in decreasing blood glucose and insulin resistance are obscure, it is certainly believed that it acts through PPARs, especially PPAR-
. Suggested mechanisms are increased glucose uptake from muscles and adipose tissue through increase in glucose transporter protein 4 (GLUT4), stimulation of phophatidyl-3-kinase, increased phosphorylation of insulin receptor substrates (Yki-Jarvinen, 2004
). PPAR-
is expressed mainly in white and brown adipose tissue. It is almost absent in skeletal muscle. How PPAR-
in adipose tissue induces glucose uptake and decrease insulin resistance remains to be elucidated, however, endocrine factors such as leptin, adiponectin and resistin may play a role (Stumvoll and Häring, 2002
; Ferre, 2004
). It is also possible that PPAR-
can directly regulate the genes involving in glucose metabolism, since a decrease in insulin resistance has also been observed in mice lacking adipose tissue after TZD treatment (Kim et al., 2000
).
In the light of previous findings, it appears possible that alterations in transcriptional activity of the Ala variant in adipocytes (where PPAR-2 is expressed) primarily enhance insulin's action on suppression of lipolysis, resulting in a decreased release of free fatty acids. Secondly, reduced availability of free fatty acids would then permit muscle to utilize more glucose and liver to suppress glucose production more efficiently upon insulin stimulation (Boden, 1997
). Furthermore, interestingly, it has been observed that increasing the circulating free fatty acids by lipid infusion results in a decrease of insulin concentration in subjects with Pro12Ala polymorphism (Stefan et al., 2001
).
Although there were striking differences in glucose metabolism on the basis of PPAR-2 genotype, Pro12Ala did not significantly affected reproductive hormones in both groups (Table II). Although SHBG tended to be higher, and DHEAS tended to be lower in women with Pro12Ala polymorphism, the difference did not reach statistical significance. Similarly, free testosterone levels and DHEAS in PCOS women with Pro12Ala were lower in the study of Hara et al. (2002)
, but it did not reach significance level either.
Although Pro12Ala polymorphism was more prevalent in control women in our study, no difference was observed when compared with women with PCOS. This is possibly due to small sample size of our study. However, our primary aim was to detect any difference in metabolic parameters and reproductive hormones. We achieved statistically significant differences for parameters of glucose metabolism, indicating a sufficient power of the study for these variables. The levels of DHEAS and SHBG tended also to be different on the basis of PPAR-2 genotype, but we did not reach a statistical significance. According to our results, however, to achieve a statistical significance level of 0.05 with 80% power, 5088 patients would be required to detect a difference for DHEAS, and even more patients for SHBG (StudySize v1.08). The main problem in these parameters was great individual variances.
In conclusion, both control women and women with PCOS had significant differences in glucose metabolism on the basis of PPAR-2 Pro12Ala polymorphism in our study. The divergence with other studies may stem from the age, BMI and dietary habits of selected populations. However, this genotype had consistently no effect on reproductive hormones. We think that this variant has no direct causal relationship with PCOS, as previously suggested (Korhonen et al., 2003
). However, this variant may alleviate chronic hyperinsulinemia and hyperglycemia in susceptible women for PCOS. Chronic hyperinsulinemia and hyperglycemia have been suggested to result in serine/threonine phosphorylation of insulin receptor beta-subunit and insulin receptor substrates (Dunaif et al., 1995
), and this unique alteration for PCOS has been suggested to be the underlying pathophysiological mechanism for insulin resistance and hyperandrogenism in women with PCOS (Venkatesan et al., 2001
). Thus, Pro12Ala variant may break the process that leads to PCOS in susceptible women.
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References |
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Submitted on November 23, 2004; resubmitted on December 23, 2004; accepted on January 10, 2005.
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