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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1717-1722
Copyright © 2003 by The Endocrine Society

Effect of the Pro12Ala Polymorphism in the Peroxisome Proliferator-Activated Receptor (PPAR) {gamma}2 Gene on the Expression of PPAR{gamma} Target Genes in Adipose Tissue of Massively Obese Subjects

M. Kolehmainen, M. I. J. Uusitupa, E. Alhava, M. Laakso and H. Vidal

Department of Clinical Nutrition and Food and Health Research Center (M.K., M.I.J.U.), University of Kuopio, FIN-70211 Kuopio, Finland; Departments of Surgery (E.A.) and Internal Medicine (M.L.), Kuopio University Hospital, Kuopio, Finland; and Institut National de la Santé et de la Recherche Médicale U449 (H.V.), Faculté de Médecine R.Laennec, 69372 Lyon Cedex 08, France

Address all correspondence and requests for reprints to: Marjukka Kolehmainen, M.D., Department of Clinical Nutrition, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: marjukka.kolehmainen{at}uku.fi.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The aim was to study the effect of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor (PPAR) {gamma}2 gene on the expression of PPAR{gamma} target genes in adipose tissue. Adipose tissue samples were collected from 30 massively obese subjects (10 men and 20 women) from omental, sc abdominal, and femoral depots. The mRNA expression of PPAR{gamma}1, PPAR{gamma}2, lipoprotein lipase, p85{alpha} phosphatidylinositol 3-kinase, and uncoupling protein 2 were quantified by reverse transcription-competitive PCR. The genotypes of Pro12Ala polymorphism were determined by single-strand conformation polymorphism analysis. The frequency of the Ala12 allele was 13.3% (8 Pro12Ala and 22 Pro12Pro). There were no differences in body weight, fat mass, and fasting serum leptin between the genotypes. The mRNA expression of p85{alpha} phosphatidylinositol 3-kinase was significantly lower in the omental fat of the Pro12Ala carriers than the Pro12Pro carriers (P < 0.01). It also appeared that PPAR{gamma}2 expression was higher in men with Ala12 allele (P < 0.01). Interestingly, particularly in women, the expression of both PPAR{gamma} splice variants was lower in omental than sc fat independently of the genotype (P < 0.05–0.01). The common Pro12Ala polymorphism of the PPAR{gamma}2 gene has minor influence on mRNA expression of PPAR{gamma} target genes in adipose tissue of obese subjects. Expression of both PPAR{gamma} splice variants is dependent on fat depot: omental fat shows lower mRNA levels, compared with sc fat depots.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PEROXISOME PROLIFERATOR-ACTIVATED receptor (PPAR) {gamma}2 is a nuclear receptor that is almost exclusively expressed in adipocytes (1, 2). PPAR{gamma}2 controls adipocyte differentiation (3) by an interplay with other transcription factors like the CCAAT/enhancer-binding proteins and adipocyte differentiation and determination factor 1/sterol regulatory element-binding protein 1c (4, 5). There are two PPAR{gamma} proteins ({gamma}1 and {gamma}2) that differ only in their N terminus region, with PPAR{gamma}2 having a stretch of 28 additional amino acids (6). This additional sequence is encoded by a unique exon B, and mRNA expression of the two isoforms is driven by an alternative promoter within the single PPAR{gamma} gene (5). PPAR{gamma}1 and PPAR{gamma}2 share the same ligand-binding domain and both are activated by long-chain fatty acid derivatives, prostanoids, and antidiabetic thiazolidinedione drugs (7, 8, 9). However, although PPAR{gamma}1 is expressed at high level in adipocytes like PPAR{gamma}2, it is also found in many nonadipose tissues including intestinal wall, liver, muscle, and macrophages (1, 2, 10), indicating that PPAR{gamma}1 is able to subserve additional roles except the regulation of adipogenesis. The unique N terminus part of PPAR{gamma}2 confers a higher ligand-independent activation of transcription (11), suggesting that PPAR{gamma}2 could be more potent than PPAR{gamma}1 to induce the expression of target genes in the absence of activating ligands.

The Pro12Ala polymorphism has recently been reported in the exon B of the PPAR{gamma}2 gene (12). The Ala12 variant has lower affinity in vitro to a PPAR-responsive element (PPRE) and reduced ability to transactivate PPRE-containing promoters (13). Therefore, one could expect that the Pro12Ala polymorphism might affect fat mass accumulation and insulin sensitivity in vivo (14, 15). Its frequency has been reported to vary from 10% to 18% in different populations, and conflicting results regarding its association with obesity or insulin resistance have been published (12, 13, 16, 17, 18, 19, 20, 21, 22). Thus, the Pro12Ala substitution may not be sufficient to significantly affect fat mass or body mass index (BMI) by itself but rather via interactions with other polymorphisms (17) or with particular physiological and nutritional conditions (23) that could contribute to pathological implications of PPAR{gamma}2 in human obesity (24).

Until now, most of the studies have investigated the association between the Pro12Ala PPAR{gamma}2 polymorphism and highly complex traits such as obesity, fat mass, body weight changes, or insulin sensitivity (13, 16, 17, 18, 19, 21, 22). In the present work, we decided to directly investigate the influence of the Pro12Ala polymorphism on the regulation of the expression of known target genes of PPAR{gamma} in adipose tissue. Adipose tissue biopsies were taken from different fat depots in 30 massively obese subjects (22 Pro12Pro and 8 Pro12 Ala). The mRNA expression levels of known target genes of PPAR{gamma} [uncoupling protein (UCP)-2, lipoprotein lipase (LPL), p85{alpha} regulatory subunit of phosphatidylinositol-3 kinase (PI3K)], and PPAR{gamma}1 and {gamma}2 were determined by reverse transcription (RT)-competitive PCR (RT-cPCR).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Thirty massively obese subjects (10 men and 20 women) participated in the study. The subjects went through medical examinations before a gastric-banding operation for weight reduction. Twenty-two subjects (6 men and 16 women) had normal fasting plasma glucose concentrations, and 8 (4 men and 4 women) had been previously diagnosed as having type 2 diabetes. Two of the subjects had a cardioselective ß-blocking medication (atenolol). The nondiabetic subjects were submitted to a 2-h oral glucose tolerance test (OGTT) with a glucose load of 75 g before the banding operation.

All subjects received both oral and written information on the study, and they signed a written consent. The study was approved by the Ethics Committee of Kuopio University Hospital and the University of Kuopio.

Research design

The subjects visited the outpatient clinic of Kuopio University Hospital before the gastric-banding operation (84–168 d before), when body composition was determined by a bioelectrical impedance method (Bioelectrical Impedance, Body comp II, version 1.5; RJL Systems Inc., Detroit, MI) in fasting condition. Adipose tissue samples for the determination of mRNA concentrations were taken during the gastric-banding operation from abdominal sc, omental, and femoral sc adipose tissue under general anesthesia. The samples were immediately frozen in liquid nitrogen and stored at -70 C for later analyses. Five of the subjects denied the donation of femoral sc samples because of inconvenience in taking care of the incision.

Biochemical measurements

Fasting blood samples for the analyses of serum glucose, leptin, and free fatty acid concentrations were obtained in the morning after 12-h fasting. Serum glucose was analyzed by a glucose dehydrogenase method (Merck \|[amp ]\| Co., Darmstadt, Germany). Serum free fatty acids were measured by a turbidometric analyser (Kone Ltd., Espoo, Finland). Plasma glucose and insulin were determined from fasting blood samples taken before OGTT and 120 min after the glucose load. Plasma glucose concentration was analyzed using the glucose oxidase method (Daiichi Co., Kyoto, Japan). Commercial RIA kits were used for the analysis of plasma insulin (Phasedeph insulin RIA 100, Pharmacia Diagnostics, Uppsala, Sweden) and leptin (Linco Research, Inc., St. Charles, MO).

Screening of the Pro12Ala polymorphism in exon B of the PPAR{gamma} gene

DNA was prepared from the peripheral blood leukocytes by the salting-out method (25). Exon B of the PPAR{gamma} 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, and variants were detected by single-strand conformation polymorphism analysis as described previously (17).

Preparation of total RNA and quantitation of target mRNAs

For total RNA preparation, adipose tissue samples were crushed in liquid nitrogen. Total RNA from the frozen powder was prepared using the RNeasy total RNA kit (QIAGEN, Hilden, Germany). The amount of total RNA was quantified spectrophotometrically at 260 nm. The ratio of absorption (260/280 nm) of all preparations was between 1.8 and 2.0. Total RNA was suspended in water and stored at -80 C.

The mRNA levels of PPAR{gamma}1, PPAR{gamma}2, LPL, p85{alpha}PI3K, and UCP2 were quantified by RT-cPCR. Detailed description of the method has been published previously (26). The construction of the competitor molecules, validation of the assays, and sequences of the primers have been already reported (1, 27, 28, 29).

For each mRNA assay, a specific RT reaction was performed from 0.1 µg total RNA with 2.5 U thermo-stable reverse transcriptase (Tth DNA polymerase, Promega Corp., Charbonnier, France) and with a specific antisense primer under conditions that warrant optimal synthesis of first-strand cDNA (26). After amplification by PCR in the presence of known amounts of a specific competitor cDNA, the PCR products were analyzed in denaturing 4% acrylamide gel (Ready-mix, Pharmacia) using an ALF Express DNA sequencer (Pharmacia). The target mRNA concentration was calculated at the competition equivalence point as previously reported (26).

Statistical analyses

All calculations were performed using the SPSS/WIN program (version 10.0, SPSS, Inc., Chicago, IL). Results are given as means and SEM unless otherwise stated. Nonparametric Mann-Whitney test was used for studying the difference in mRNA level between groups. After division into the groups of polymorphism, differences between the adipose tissue depots were analyzed using nonparametric Wilcoxon paired sample t test. When analyzing solely gender differences, an independent sample t test was used. The correction of Bonferroni was used to take into account the effect of multiple comparisons by multiplying each test value by 3 according to the number of adipose tissue sites, i.e. omental, sc abdominal, and sc femoral region.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Characteristics of the subjects

In the group of 30 morbidly obese subjects, the frequency of the Ala-allele was found to be 13.3% (8 Pro12Ala and 22 Pro12Pro). None of the subjects had the Ala12Ala genotype. There were no significant differences between the Pro- or Ala-allele carriers regarding body weight, BMI, fat mass, fasting serum leptin, and free fatty acids (Table 1Go). Eight type 2 diabetic subjects had similar anthropometric parameters to the nondiabetic subjects (data not shown), but their fasting glucose level was significantly higher (10.9 ± 1.3 vs. 6.1 ± 0.4 mmol/liter, P = 0.001). When the type 2 diabetic subjects were excluded from the analysis, the subjects of the Pro12Pro group were characterized by a significantly higher fasting glycemia than the Pro12Ala carriers. There was no significant difference in the glucose response during the OGTT between the groups of PPAR{gamma} genotype (Table 1Go).


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  		Table 1. Basic characteristics of subjects with the Pro12Pro or Pro12Ala genotypes of the PPAR{gamma}2 gene

 
Expression of PPAR{gamma} and its target genes in adipose tissue

Table 2Go shows the mRNA levels (expressed as absolute values, in attomole per microgram total RNA) of PPAR{gamma}1 and PPAR{gamma}2, LPL, p85{alpha}PI3K, and UCP2 after an overnight fast, in three different fat depots according to the genotype group. There were no significant differences in the basal expression levels of the investigated mRNAs that could be related to the PPAR{gamma} genotype, with the exception of a significantly lower expression (about 40% reduction) in the mRNA abundance of p85{alpha}PI3K in the omental fat of the Pro12Ala carriers (Table 2Go). There was also a trend for a higher expression of PPAR{gamma}2 mRNA in the sc depots in the Pro12Ala; however, the differences did not reach significance (P = 0.083 in abdominal sc depot). Diabetic patients did not differ significantly from nondiabetic subjects in terms of expression results, and exclusion of the type 2 diabetic subjects from the analyses did not change the results (data not shown).


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  		Table 2. PPAR{gamma}, LPL, p85 {alpha}PI 3-kinase, and UCP2 depot-related mRNA concentrations (amol/µg total RNA) in subjects with the Pro12Pro or Pro12Ala genotypes of the PPAR{gamma}2 gene

 
Interestingly, a reduction in the expression level of both PPAR{gamma} variants ({gamma}1 and {gamma}2) was observed in the omental fat when compared with the sc depots (both abdominal and femoral) independently of the PPAR{gamma} genotype (Table 2Go). Reduced mRNA abundance in the omental depot was also found for LPL, but the difference was significant for the Pro12Pro carriers only. A similar trend was observed in the Pro12Ala group (omental vs. abdominal P = 0.161; omental vs. femoral P = 0.091). Because gender may influence the expression of LPL between fat depots in obese subjects, we investigated whether this apparent reduction of PPAR{gamma} and LPL mRNA levels in omental depot could reflect a gender-related effect. Table 3Go shows that obese women were characterized by higher expression levels of PPAR{gamma}2 and LPL mRNAs in the two sc fat depots. In men, there was no regional difference in the expression of these mRNAs, and the values were similar to those found in the omental fat of the women (Table 3Go). Data on type 2 diabetic patients did not modify these results either (data not shown).


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  		Table 3. PPAR{gamma}, LPL, p85{alpha}PI 3-kinase, and UCP2 mRNA concentrations (amol/µg total RNA) in subjects with the Pro12Pro or Pro12Ala genotypes of the PPAR{gamma}2 gene according to the gender

 
Table 3Go shows mRNA levels of the target genes in men and women according to their PPAR{gamma} genotype. Although the low number of Ala-allele carriers in each group (n = 4) limited the power of statistical analyses, the data showed that PPAR{gamma} and LPL mRNA expression levels were higher in the sc depots of the women, independently of PPAR{gamma} genotype (Table 3Go). Furthermore, the expression of PPAR{gamma}2 was significantly higher in the abdominal sc fat among men carrying the Ala allele, compared with the Pro12Pro carriers (Table 3Go). A similar trend was also observed in the femoral sc fat of the Pro12Ala men and the two sc depots of the Pro12Ala women. This suggests that there was a tendency for a higher expression of PPAR{gamma}2 in the sc fat depots among the Pro12Ala carriers (P = 0.056 when corrected with gender). Finally, there was a tendency for a reduced p85aPI3K mRNA expression in the omental fat of the Pro12Ala carriers in both genders (Table 3Go).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our aim was to examine whether the Pro12Ala polymorphism of PPAR{gamma}2 could affect its transcriptional activity in vivo by comparing the expression levels of a group of target genes in adipose tissue samples from morbidly obese subjects with either the Pro12Pro or the Pro12Ala genotype. To our knowledge, this work represented one of the first studies on direct functional genomics that has been so far performed in vivo in human fat. As target genes of PPAR{gamma}2, we focused our investigations on LPL, UCP2, p85{alpha}PI3K, and PPAR{gamma}1. The LPL gene was one of the first target genes of PPAR{gamma} to be described, and functional PPREs have been identified in its promoter region (30). LPL catalyzes the hydrolysis of circulating triglycerides, a limiting step for the deposition of fatty acids in the adipocytes. The expression of the gene encoding p85{alpha}PI3K has recently been shown to be increased by thiazolidinediones in human adipose tissue (31). The induction of this important mediator of the insulin-signaling pathway increases the antilipolytic action of insulin in adipocytes (32). The transcription of the gene-encoding UCP2 has also been shown to be activated by PPAR{gamma} ligands in human adipocytes (31, 33). UCP2 is also regarded as an important protein in obesity, potentially implicated in the control of thermogenesis (34). Finally, PPAR{gamma}2 has been reported to be able to induce the mRNA expression of PPAR{gamma}1 in the early stage of adipogenesis (35), suggesting that PPAR{gamma}1 is a possible target of PPAR{gamma}2 in adipocytes.

We did not find significant difference in anthropometric and metabolic parameters of the morbidly obese subjects carrying either the Proline or the Alanine at position 12 of PPAR{gamma}2. This result is in agreement with some studies (19, 36, 37), but in other studies the Ala allele has been associated with higher BMI and increased weight gain (16, 17, 21, 38, 39) but also better insulin sensitivity and a lower BMI in some studies (13). With the limited number of subjects in our study, it was not possible to observe the differences in these kinds of associations. It should be noted that highly complex traits such as obesity, fat mass, body weight changes, or insulin sensitivity results from integrated regulatory networks that may therefore result in difficulties to assign a functional role to the genetic variation in PPAR{gamma}2. In addition, the Pro12Ala variation may not be sufficient to significantly affect directly these general phenotypes but rather via the interactions with other genes (17) or with particular physiological and nutritional conditions (23) that may contribute to the role of PPAR{gamma}2 in human obesity. On the other hand, because PPAR{gamma}2 is a nuclear receptor controlling the expression of specific target genes in adipose tissue, the primary consequence of a functional mutation could be a modification in the expression levels of these target genes. Our results demonstrated that there are no major differences in the mRNA expression levels of the target genes of PPAR{gamma} in human fat depots according to the genotype at position 12 of PPAR{gamma}2. Reduced LPL activity in postheparin plasma was recently shown to be associated with the Ala12 allele (40). This was not, however, seen at the level of adipose tissue gene expression in our study. The only significant difference was a lower expression of p85{alpha}PI3K mRNA in the omental fat depot and a tendency for a higher expression of PPAR{gamma}2 itself in the sc fat depots in the subjects from the Pro12Ala group. The biological significance of these differences remains to be determined. They might have, however, some relationship with the responsiveness to insulin in the fat depots of the Pro12Ala carriers who have been demonstrated to be more sensitive to insulin in some studies, particularly in the Finnish population (13).

The lack of major differences in the expression of PPAR{gamma} target genes in adipose tissue of obese subjects carrying genetic variation in PPAR{gamma}2 gene contrasted with the major impact of the Pro12Ala substitution on PPAR{gamma} transcriptional function in in vitro experiments (13). This suggests that the presence of the two alleles can counteract the effect of the mutation. It might have been possible that subjects with the Ala12Ala genotype presented more important differences. According to an in vitro study (13), these subjects might not develop a massive state of obesity because of low transcriptional activity of PPAR{gamma}2. Therefore, they could have low or even only faint expression of PPAR{gamma}2 and reduced expression of PPAR{gamma} target genes, although association studies do not support this view (16, 17, 21, 38, 39). Unfortunately, we did not recruit subjects with the Ala12Ala genotype in the present study to verify this hypothesis. Alternatively, one could argue that the small sample size of the investigated group was a limiting factor in our study to reach significance. It should be kept in mind, however, that in this clinical setting and with the thorough molecular analysis of several gene expressions in biopsies from different fat depots, it was not possible to study a larger population. According to power estimations and with coefficient of variations of the RT-cPCR assays ranging from 7% to 15% (26), the size of the groups of polymorphism (22 Pro12Pro and 8 Pro12 Ala) were at the lower limit to enlighten significant differences if any. It appeared, therefore, that the common PPAR{gamma}2 polymorphism has no major impact on the expression of PPAR{gamma} target genes.

General anesthesia could have an effect on gene expression in adipose tissue, eventually masking the influence of the PPAR{gamma} polymorphism. Although we cannot exclude this hypothesis, it should be indicated that there are no clear data showing how anesthesia could affect gene expression in human adipose tissue, particularly the expression of PPAR{gamma} target genes. In addition, it is important to consider that the subjects from both groups of polymorphism underwent the same clinical intervention, with the same anesthesia procedure and identical biopsy technique.

In addition to the study of the influence of PPAR{gamma}2 polymorphism on target gene expression in adipose tissue, our study design allowed us to compare the mRNA expression levels of important genes in different fat depots in obese subjects. We found significantly higher expressions of PPAR{gamma}1, PPAR{gamma}2, and LPL in the sc than omental fat depot in women. This difference was not observed in men who had similar mRNA levels of these genes in the three investigated regions. As a consequence, the morbidly obese women were characterized by higher expression of PPAR{gamma} and LPL mRNAs than men in sc depots. Regarding the expression of LPL, gender- and depot-related differences have been previously reported in several studies (41, 42, 43, 44). For PPAR{gamma}, the obtained data were more original. A similar gender difference has been observed in a study in which PPAR{gamma} expression was investigated in sc abdominal adipose tissue samples only (2). However, in other studies involving lean and moderately obese subjects, such gender difference was not observed (1, 31). The reasons for higher expression of PPAR{gamma}1 and {gamma}2 mRNAs in the sc fat depots of morbidly obese women remain to be further investigated. However, this difference might not be caused by the difference in fat mass because the differences remained significant after correction with fat mass in the present study. In previous studies, such regional difference in PPAR{gamma} mRNA levels was not observed in obese subjects, but the number of subjects was limited and, more importantly, the genders were not separately analyzed (45, 46). Interestingly, the expression of other genes has been found to be characterized by a regional difference in women only (41, 42, 44, 47). The mechanism behind this gender difference is not yet clearly defined but may be involved in differential sensitivity to steroid hormones (42).

In conclusion, we demonstrate that the Pro12Ala polymorphism of PPAR{gamma}2 gene has a minor influence on mRNA expression of the PPAR{gamma} target genes in adipose tissue of obese subjects. Because the amino acid substitution markedly affects PPAR{gamma}2 transcriptional activity in vitro (13), the minor effect on gene expression observed in vivo suggests that the presence of the two alleles can counteract the impact of the mutation. Our study strongly suggests, therefore, that the common Pro12Ala polymorphism has little influence on adipocyte function, at least in the morbidly obese patients.


   Acknowledgments
 
We thank Raisa Valve, Ph.D., for screening the polymorphism in the study population. In addition, Paulette Vallier, Natalie Vega, Erja Kinnunen, Kaija Kettunen, and Irja Kanniainen are thanked for the skillful technical assistance.


   Footnotes
 
This work was supported by grants from the Academy of Finland; Research Council for Health; Jenny and Antti Wihuri Foundation, Finland; and Saastamoinen Foundation, Kuopio, Finland.

Abbreviations: BMI, Body mass index; LPL, lipoprotein lipase; OGTT, oral glucose tolerance test; PI3K, p85{alpha} regulatory subunit of phosphatidylinositol-3 kinase; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-responsive element; RT, reverse transcription; RT-cPCR, reverse transcription-competitive PCR; UCP, uncoupling protein.

Received April 17, 2002.

Accepted January 3, 2003.


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 Introduction
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 Results
 Discussion
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