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1 and 2 Messenger Ribonucleic Acid Expression in Human Adipocytes
University of Cambridge, Departments of Medicine and Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge, United Kingdom CB2 2QR; and Department of Medicine, Indiana University School of Medicine (R.C.), Indianapolis, Indiana 46202
Abstract
Both genetic and pharmacological studies raise the possibility that a primary increase in the amount or activity of peroxisomal proliferator-activated receptor 
(PPAR) in adipocytes could play a role in common types of human obesity. Using real-time RT-PCR assays we examined the relationship between body mass index (BMI) and PPAR isoform expression in freshly isolated human adipocytes. There were no consistent differences in the expression of either PPAR1 mRNA or PPAR2 mRNA between omental and sc adipocytes. In a group of 17 subjects (BMI range, 17–34 kg/m2) there was a strong and highly significant inverse correlation (r = -0.68; P < 0.005) between PPAR1 mRNA expression in adipocytes and BMI, whereas no significant relationship was apparent for PPAR2. In an independent study PPAR1 mRNA levels were decreased (1.1 ± 0.1 vs. 3.7 ± 0.8 arbitrary units; P < 0.01) in adipocytes from morbidly obese (BMI, 50.6 ± 14.1 kg/m2) vs. lean (BMI, 21.1 ± 1.0 kg/m2) subjects. In contrast, there was a significant increase in the expression of PPAR2 mRNA levels between the morbidly obese and lean groups (1.7 ± 0.2 vs. 1.1 ± 0.2 arbitrary units; P < 0.05). Treatment of isolated human adipocytes with TNF
resulted in a significant decrease in both PPAR1 and PPAR2 mRNA levels [40.6 ± 5.5% relative to control (P = 0.01) and 60.9 ± 24.8% (P = 0.02) respectively]. The strong inverse relationship between BMI and PPAR1 expression in human adipocytes is striking and may represent part of an autoregulatory mechanism restraining the expansion of individual adipocytes in states of positive energy balance. On the other hand, the increase in PPAR2 observed in adipocytes of morbidly obese individuals suggests a potential pathogenic effect of this isoform in promoting fat acquisition. Although an autocrine effect of the enhanced TNF secretion seen with increasing obesity might play a role in the changes in PPAR1, this would not provide an explanation for the different relationship of PPAR2 to adiposity. The significance of the divergent effect of human adiposity on the two isoforms will require a greater understanding of the differential properties of the two isoforms and of the differences in the functions of their respective regulatory elements.
PEROXISOME PROLIFERATOR-ACTIVATED receptor- (PPAR) is a nuclear hormone receptor that is highly expressed in adipose tissue (1). Both genetic and pharmacological evidence suggests that PPAR plays a key role in adipogenesis and the control of insulin sensitivity. For example, forced expression of PPAR in fibroblasts leads to the development of an adipocyte phenotype (2), and activation of PPAR in preadipocytes by agonists such as the thiazolidinedione class of insulin-sensitizing compounds promotes adipogenesis both in vitro (3) and in vivo (4). Furthermore, Barak et al. (5) and Rosen et al. (6) have provided direct evidence that PPAR is required for the formation of adipocytes in vivo. Given the role of PPAR in adipogenesis, it is possible that an increase in the expression or activity of PPAR in fat cells could contribute to the development of human obesity. In this regard, Ristow et al. (7) have reported an association between a rare Pro115Gln variant in the N-terminal ligand-independent activation domain of PPAR and severe human obesity. This mutation resulted in enhanced PPAR activity resulting from its interference with an inhibitory MAPK site.
PPAR is expressed as two major isoforms, 1 and 2, generated from the same gene by alternate promoter usage and RNA splicing (8, 9). A third mRNA species, PPAR3, results in the production of a protein identical to PPAR1 (10). PPAR2 is identical to 1 apart from an extra 30 N-terminal amino acids. Despite these structural differences, no clear functional differences between these two isoforms have been identified.
Previous studies of the relationship between adiposity and human adipose tissue PPAR expression have yielded conflicting results. Vidal-Puig et al. (1) showed that the PPAR2/1 mRNA ratio was positively correlated with body mass index (BMI). In contrast two independent reports did not describe any correlation between total PPAR mRNA expression in white adipose tissue and BMI (11, 12). Adipose tissue is composed of a number of cell types expressing PPAR, including adipocytes, immature preadipocytes at several stages of differentiation, and monocytes. Therefore, important observations concerning adipocyte PPAR expression and obesity may have been overlooked. Using a highly sensitive real-time PCR assay, we have examined the relationship between PPAR1 and -2 mRNA expression in freshly isolated mature adipocytes and BMI. In addition, we have examined the effects of TNF, a cytokine known to inhibit adipogenesis (13) and promote insulin resistance (14), on adipocyte PPAR1 and -2 mRNA expression.
Subjects and Methods
Subjects and sample acquisition
Omental and sc adipose tissue biopsies were obtained from patients undergoing elective open-abdominal surgery at Addenbrooke’s Hospital, Cambridge (AHC) and Indiana University/St. Vincent’s Hospital (IU). All patients were fasted for 12 h preoperatively, and all underwent general anesthesia. None was undergoing surgery for malignancies, and none of the patients had diabetes or severe systemic illness. Approval was obtained from the institutional review boards at IU and Cambridge Local Research Ethics Committee, and all patients involved gave informed consent. The patients from AHC included 17 subjects (8 men and 9 women; age, 55 ± 16 yr; BMI range, 17.1–34 kg/m2). Of this group, 11 subjects were lean (mean BMI, 20.9 ± 2.8 kg/m2), and 6 were overweight or obese (mean BMI, 29.1 ± 2.8 kg/m2). Patients from IU included 11 subjects, all of whom were morbidly obese (2 men and 9 women; age, 40 ± 8 yr, mean BMI, 50.6 ± 14.1 kg/m2).
Adipocyte isolation and cell culture
Adipose tissue biopsies were placed in normal saline and immediately processed (transport time to the laboratory was 5 min). The adipose tissue was diced finely and digested in a collagenase solution [Hanks’ balanced salt solution containing 3 mg/ml type II collagenase (Sigma, St. Louis, MO) and 1.5% BSA] for 1 h in a shaking water bath at 37 C. After digestion, the mature adipocytes were separated from the stromo-vascular cells by centrifugation (10 min, 1500 x g) of the digestion mixture over dionyl-phthalate oil. The floating layer of adipocytes was then decanted into a sterile tube, and either RNA was immediately extracted or cells were prepared for culture. Adipocytes were cultured in Costar six-well plates (Corning, Inc., Corning, NY) under standard conditions (37 C, 5% CO2) in serum-containing medium [DMEM/Ham’s F-12 (1:1), 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin] in the presence or absence of 10 ng/ml TNF. After 24 h, the adipocytes were isolated by centrifugation (10 min, 1500 x g) over dionyl-phthalate oil, and RNA was extracted as outlined below.
RNA extraction
Total RNA was extracted using the RNeasy mini extraction kit (QIAGEN, West Sussex, UK) according to the manufacturer’s recommendations. RNA samples were quantified by spectrophotometry. RNA integrity was assessed by agarose gel electrophoresis and ethidium bromide staining. The RNA samples were then diluted as appropriate in RNase-free water and stored at -80 C until use.
Quantitation of PPAR1 and PPAR2 mRNA expression by real-time quantitative PCR
Total RNA (5 µg) was reverse transcribed for 1 h at 37 C in a 25-µl reaction containing 1x RT buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol), 2.5 µg random hexamers, 2 mM deoxy-NTPs, and 200 U Moloney murine leukemia virus reverse transcriptase (Promega Corp., Southampton, UK). Reactions in which RNA was omitted served as negative controls.
A reaction containing 5 µg adipocyte total RNA was also included as a standard. After first strand cDNA synthesis, this standard was serially diluted 1:2 in deoxyribonuclease-free water to generate a standard curve for the PCR analysis.
Oligonucleotide primers and TaqMan probe for PPAR1 and -2 were designed using Primer Express, version 1.0 (Perkin-Elmer/PE Applied Biosystems, Foster City, CA) and sequences from the GenBank database (accession no. X90563 and U63415). For quantitation of PPAR1 and -2 isoforms, the same reverse primer and fluorogenic probe, but different forward primers, were used. The sequences were as follows: PPAR1 forward, 5'-GTGGCCGCAGAAATGACC-3'; PPAR2 forward, 5'-GATACACTGTCTGCAAACATATCACAA-3'; reverse, 5'-CCACGGAGCTGATCCCAA-3'; and probe, 5'-AGAGATGCCATTCTGGCCCACCAACTT-3'. The TaqMan probe was labeled at the 5' end with the reporter dye FAM and at the 3' end with the quencher TAMRA. Oligonucleotide primers and TaqMan probe for the glyceraldehyde-3-phosphate dehydrogenase internal control were purchased from Perkin-Elmer/PE Applied Biosystems.
PCR was carried out in duplicate for each sample on an ABI 7700 sequence detection system (Perkin-Elmer/PE Applied Biosystems). Each 25-µl reaction contained 2 µl first strand cDNA, 1x PCR master mix, 300 nM of each forward and reverse primer, and 75 nM TaqMan probe. All reactions were carried out using the following cycling parameters: 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. After PCR, standard curves were constructed from the standard reactions for each target gene and internal control by plotting Ct values, i.e. the cycle number at which the fluorescence signal exceeds background, vs. log cDNA dilution. The Ct readings for each of the unknown samples were then used to calculate the amount of either target or internal control relative to the standard. For each sample, results were normalized by dividing by the amount of target by the amount of internal control [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. The expression levels of GAPDH in adipocytes did not show any correlation with BMI within this subject group. Because the amplification efficiencies of PPAR1 and -2 were equal, the 2/1 ratio could be calculated using the equation 2-
Ct, where Ct = 2 - 1 Ct. The intra- and interassay coefficients of variation were 0.03–2.21% and 0.03–2.42%, respectively.
Statistical analysis
Intraindividual comparisons were analyzed using the paired Wilcoxon nonparametric test. Interindividual comparisons were analyzed using the unpaired Wilcoxon test. Linear correlations were analyzed using the Spearman test. P < 0.05 was considered significant.
Results
PPAR1 and -2 mRNA expression in freshly isolated adipocytes
Adipocyte PPAR1 and -2 mRNA expression were quantified using real-time PCR. These assays were shown to be linear within the experimental range (Fig. 1
). The two PPAR isoforms were expressed at similar levels in freshly isolated adipocytes from lean subjects (AHC group; n = 11; BMI, 21.1 ± 1.0 kg/m2). There were no consistent differences in the expression of either PPAR1 mRNA (Fig 2A) or PPAR2 mRNA (Fig 2B) between omental and sc adipocytes isolated from each subject within the AHC group (n = 17).
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1 and -2 mRNA expression
In a group of 17 subjects (AHC group; BMI, range, 17–34 kg/m2) there was a strong and highly significant inverse correlation (r = -0.68; P < 0.005) between adipocyte PPAR1 mRNA expression and BMI (Fig 3A). No significant relationship was apparent for PPAR2 (Fig 3B).
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1 and -2 mRNA expression in lean and morbidly obese subjects
Subcutaneous adipocyte PPAR1 and 2 mRNA expressions were quantified in a group of lean (AHC group; n = 11; BMI, 21.1 ± 1.0 kg/m2) and morbidly obese (IU group; n = 11; BMI, 50.6 ± 14.1 kg/m2) subjects. PPAR1 mRNA levels were decreased (1.1 ± 0.1 vs. 3.7 ± 0.8 arbitrary units; P = 0.006) in adipocytes from morbidly obese compared with lean subjects, respectively (Fig 4A). In contrast, there was a significant increase in the expression of PPAR2 mRNA levels (1.7 ± 0.2 vs. 1.1 ± 0.2 arbitrary units; P = 0.02) in adipocytes from the morbidly obese compared with the lean subjects (Fig. 4B). As a result, the PPAR2/PPAR1 mRNA ratio was significantly higher in the morbidly obese compared with the lean group (3.71 ± 0.46 vs. 0.85 ± 0.19, respectively; P < 0.0001; Fig. 4C).
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on PPAR1 and -2 mRNA expression in cultured adipocytes
To identify a possible mechanism for the effects of obesity on adipocyte PPAR mRNA expression, the effect of TNF was investigated. Isolated sc adipocytes were cultured for 24 h with or without TNF (10 ng/ml). TNF resulted in a significant decrease in the expression of both PPAR1 (40.6 ± 5.5% decrease vs. control; n = 3; P = 0.01) and PPAR2 (60.9 ± 24.8% decrease vs. control; n = 3; P = 0.02; Fig. 5).
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Using real-time PCR we have measured the effects of BMI on the expression of both PPAR1 and PPAR2 mRNA in human adipocytes. We found that 1) there were no consistent differences in the expression of either PPAR1 mRNA or PPAR2 mRNA between omental and sc adipocytes; 2) PPAR1 mRNA expression is inversely correlated with adiposity across a range of BMI (17–34 kg/m2) and remains markedly decreased in morbidly obese compared with lean subjects; 3) in contrast, PPAR2 mRNA expression does not correlate with adiposity over a normal range of BMIs, but was significantly increased in morbidly obese compared with lean subjects; and 4) exposure of isolated adipocytes to TNF significantly decreases the mRNA expression of both isoforms.
No significant differences were found in the mRNA expression of either PPAR1 or -2 between omental and sc adipocytes, suggesting that the previously reported regional differences in the metabolism and physiology of fat tissue between these depots (15) is not mediated by PPAR. In most tissues expressing PPAR, such as whole adipose tissue, PPAR1 is the dominant isoform (1, 16). We have also found that PPAR1 mRNA is expressed to a higher level compared with PPAR2 mRNA in undifferentiated preadipocytes (unpublished data). Our finding that the level of PPAR2 is similar to (lean subjects) or even greater than (obese subjects) that of PPAR1 mRNA in isolated human mature adipocytes is consistent with reports showing that PPAR2 mRNA expression is increased to a greater extent than PPAR1 during the process of adipogenesis (17). Furthermore, recent in vitro evidence clearly suggests that it is PPAR2 (not PPAR1) that plays a specific role in adipogenesis (18).
The highly significant inverse correlation between BMI and PPAR1 mRNA expression is in agreement with a previous report from our group showing that the expression of total PPAR in adipocytes was negatively correlated with BMI in a group of subjects with a similar range of BMI (19–30 kg/m2) (19). In contrast, other studies in whole adipose tissue from humans and rodents found no such correlation (1, 11, 12, 20). A possible explanation for these apparent conflicts may include the fact that previous studies examined the expression of PPAR mRNA in whole adipose tissue and not freshly isolated adipocytes. Adipose tissue is composed of a number of cell types expressing PPAR. These include mature lipid-laden adipocytes, and stromo-vascular cells such as preadipocytes, partially differentiated preadipocytes, monocytes, and macrophages. Both PPAR1 and -2 mRNA expression are increased during preadipocyte differentiation in vitro (17). Because PPAR expression is known to increase as preadipocytes progress through the differentiation pathway, a positive relationship between adiposity and PPAR expression would be predicted to occur in the differentiating preadipocytes. Thus, the failure to demonstrate a relationship between PPAR1 mRNA levels in whole adipose tissue and indexes of adiposity may be attributable to diametrically opposed relationships in mature adipocytes compared with preadipocytes. The underlying mechanism(s) for this phenomenon is unclear. As PPAR is a key regulator of genes involved in promoting lipid storage in adipocytes (21), the progressive reduction of adipocyte PPAR1 expression with increasing adiposity may represent an autoregulatory mechanism limiting the indefinite expansion of individual mature adipocytes as fat stores increase.
In contrast to PPAR1, adipocyte PPAR2 mRNA expression did not correlate with BMI over a normal range. However, PPAR2 mRNA was significantly increased in adipocytes isolated from a group of morbidly obese compared with lean subjects. This finding is consistent with other studies carried out in whole adipose tissue from humans and monkeys (1, 22). Our finding in freshly isolated mature adipocytes shows that the increase in PPAR2 expression in whole adipose tissue seen with increasing BMI may not be solely attributable to the increase in the percentage of preadipocytes undergoing differentiation.
To examine the mechanism underlying the differential effects of adiposity on PPAR1 and -2 in adipocytes, the effects of TNF were investigated. The expression of TNF in adipose tissue was increased in obese subjects (23, 24, 25). Exposure of isolated adipocytes to TNF in vitro resulted in a significant decrease in both PPAR1 and -2 mRNA and is therefore unlikely to be the mechanism explaining the differential effects of obesity on adipocyte PPAR1 and -2 expression.
In conclusion, the expression of the two major PPAR isoforms in human adipocytes shows a marked divergent response to increasing alterations in adipose mass. A clearer understanding of the mechanism(s) mediating these effects and their physiological relevance will require further investigation of the biological functions of these two isoforms and the regulatory elements controlling their expression.
Acknowledgments
We thank the patients who donated adipose tissue for these studies. We also gratefully acknowledge the surgeons at Addenbrooke’s Hospital and the surgical assistance of RoseMarie Jones, Margaret Inman, and John Huse and the support of the nurses and staff of the Indiana University General Clinical Research Center and St. Vincent’s Hospital surgical suite.
Footnotes
Address all correspondence and requests for reprints to: Prof. Stephen O’Rahilly, University of Cambridge, Departments of Medicine and Clinical Biochemistry, Addenbrooke’s Hospital, Hills Road, Cambridge, United Kingdom CB2 2QR. E-mail: .
This work was supported in part by a grant from the American Diabetes Association (to R.V.C.) and by Grant M01 RR-00750-28 from Indiana University General Clinical Research Center.
Abbreviations: AHC, Addenbrooke’s Hospital, Cambridge; BMI, body mass index; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IU, Indiana University/St. Vincent’s Hospital; PPAR, peroxisomal proliferator-activated receptor .
Received September 20, 2001.
Accepted May 20, 2002.
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