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(PPAR), and Thyrotropin Receptor by PPAR Agonist in Human Orbital Preadipocyte Fibroblasts
Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic/Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Rebecca S. Bahn, M.D., Division of Endocrinology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905. E-mail: . bahn.rebecca{at}mayo.edu
Abstract
The symptoms and signs of Graves’ ophthalmopathy (GO) result from both an accumulation of hydrated hyaluronan in the orbital muscles and connective tissues and an expansion of the orbital adipose tissues. Recent studies have suggested a link between the stimulation of adipogenesis within the orbit in GO and the expression in these tissues of TSH receptor (TSHR), the putative orbital autoantigen. To further investigate this association, we treated orbital fibroblasts from patients with GO with rosiglitazone, a thiazolidinedione agonist of the PPAR
receptor that stimulates adipocyte differentiation. We found this compound to be a potent stimulator of functional TSHR expression as well as TSHR and PPAR mRNA levels in differentiated cultures. In addition, rosiglitazone treatment stimulated recruitment and differentiation of a subset of cells within these cultures into mature lipid-laden adipocytes.
These results suggest that TSHR expression in GO orbital preadipocyte fibroblasts is linked to adipogenesis, and that ligation of the PPAR receptor results in differentiation of these cells. It is possible that endogenous PPAR ligands play a role in stimulating orbital adipogenesis in GO, and that future treatments may be aimed at antagonism of various components of the PPAR signaling system.
THE SYMPTOMS AND signs of Graves’ ophthalmopathy (GO) can be explained mechanically by an increase in the volume of tissues found within the confines of the bony orbit. This change results from both an accumulation of hydrated hyaluronan in the orbital muscles and connective tissues and an expansion of the adipose tissues within the orbit. Because orbital adipocytes from patients with GO have been shown to express TSH receptor (TSHR) (1, 2), the putative orbital autoantigen involved in pathogenesis of this disease, recent studies have explored the link between adipogenesis and induction of TSHR expression in orbital preadipocyte fibroblasts. These studies revealed that cultured orbital preadipocytes can be induced in vitro to differentiate into adipocytes bearing functional TSHR (3, 4). In addition, we reported that both adipogenesis and TSHR expression are stimulated by treatment of cultures with IL-6 or the combination of IL6/IL-4 (5). In contrast, TGFß, interferon-, and TNF
inhibit TSHR expression, and the latter two cytokines also inhibit adipogenesis in these cells (6).
Adipogenesis is a complex process that involves the interplay of several transcription factors, including activation of the nuclear hormone receptor, PPAR (7, 8). Thiazolidinediones (TZD) represent a class of potent antidiabetic medications that have been shown to be potent agonists of this receptor (9, 10). In this study we treated orbital fibroblast cultures with rosiglitazone maleate, a newly developed TZD, to determine whether PPAR ligation might impact both adipogenesis and TSHR expression in these cells.
Materials and Methods
Cell culture and adipocyte differentiation
Orbital adipose/connective tissue explants were obtained from patients undergoing orbital decompression surgery for severe GO (n = 5). All patients had been treated previously for Graves’ hyperthyroidism with 131I or antithyroid medication and were euthyroid on thyroid hormone replacement. No patient had undergone orbital radiation therapy. Patient 2 had been taking oral corticosteroids until 4 months before orbital surgery, and patient 3 was taking prednisone (80 mg/d) at the time of decompression surgery performed to alleviate optic neuropathy. GO tissue samples were minced and placed directly in plastic culture dishes, allowing preadipocyte fibroblasts to proliferate as described previously (11). Cells were propagated in medium 199 containing 20% FBS (HyClone Laboratories, Inc., Logan, UT), penicillin (100 U/ml), and gentamicin (20 µg/ml) in a humidified 5% CO2 incubator at 37 C and maintained in 80-mm2 flasks with medium 199 containing 10% FBS and antibiotics.
To initiate adipocyte differentiation, orbital cells were grown to confluence in six-well plates in medium 199 with 10% FBS. Differentiation was carried out as reported previously (4); cultures were changed to serum-free DMEM/Ham’s F-12 (1:1; Sigma, St. Louis, MO) supplemented with biotin (33 µM), pantothenic acid (17 µM), transferrin (10 µg/ml), T3 (0.2 nM), insulin (1 µM), carbaprostacyclin (cPGI2; 0.2 µM; Calbiochem, La Jolla CA), and, for the first 4 d only, dexamethasone (1 µM) and isobutylmethylxanthine (IBMX; 0.1 mM). The differentiation protocol was continued for 10 d, during which time the medium was replaced every 3–4 d. For other experiments, fibroblasts derived from the same patients’ orbital tissues were maintained for the same period of time in medium lacking several of the components necessary for complete adipocyte differentiation (i.e. cPGI2, dexamethasone, and IBMX). These cultures are here referred to as nondifferentiated cultures. In a few instances, to examine morphological changes over time under differentiating and nondifferentiating conditions, cultures were extended to 21 d.
To study the effects of rosiglitazone maleate, pills containing this compound (GlaxoSmithKline, Philadelphia, PA) were crushed and dissolved in absolute ethanol [maximum, 0.47% (vol/vol)]. To evaluate the precise concentration of rosiglitazone maleate in the solution, each batch prepared as described above was subjected to HPLC, using pure rosiglitazone maleate (1 mg/ml ethanol) as the standard. Chromatography peaks were compared, and two runs per sample were analyzed.
The effect of rosiglitazone treatment was examined both in cells grown in differentiation medium (differentiated cultures) and in cells grown in medium lacking several of the components necessary for complete adipocyte differentiation (nondifferentiated cultures). Cultures of both types were treated with rosiglitazone (0.1, 1.0, 10, or 50 µM) for the entire 10-d differentiation period. Control cultures were grown for 10 d in the same medium, but containing vehicle [absolute ethanol, 0.47% (vol/vol)] without rosiglitazone maleate. On d 1, 4, 7, and 10 in culture, and occasionally also on d 12, 15, and 21, some cultures were examined by phase contrast microscopy using an Axiovert 35 light microscope (Carl Zeiss, Thornwood, NY) equipped with a Contax 167 MT camera. Parallel cultures were stained with Oil Red O and examined under light microscopy. Photographs were taken on Kodak Ektachrome Tungsten 64 color film (Rochester, NY).
Oil Red O staining
Orbital preadipocyte fibroblast cultures were plated in one-well culture chamber slides (Nalge Nunc International, Rochester, NY) in medium 199 containing 10% FBS, grown to confluence, and subjected to either the differentiation protocol or nondifferentiation conditions. Cells were washed twice with 1 x PBS, fixed in 10% formalin overnight at room temperature, and rinsed in 60% isopropanol before staining with filtered 0.21% Oil Red O in isopropanol/water for 1 h. Washed cells were exposed to Mayer’s hematoxylin solution (Sigma; MHS-32) for 5 min and rinsed with tap water before being visualized using an Axiovert (Carl Zeiss) light microscope and photographed at x32.
Analysis of TSH-dependent cAMP production
Cells were cultured as described for 10 d in six-well plates, then washed and preincubated in medium containing IBMX (1 mM; 1 ml/well) for 2 h at 37 C. After the addition of recombinant human TSH (3 x 10-7 M; Genzyme Corp., San Carlos, CA) to duplicate wells, incubation was continued for 2 h until terminated with the addition of hydrochloric acid (0.1 M; 1 ml/well) for 10 min (4). The culture media were subjected to centrifugation (600 x g), and cAMP production was measured using an acetylated procedure of a commercially available kit [cAMP (low pH) immunoassay, R\|[amp ]\|D Systems, Inc., Minneapolis, MN]. Each raw data point represents the mean of duplicate determinations measured 2 h after stimulation with recombinant human TSH.
Statistical analyses
For the purposes of statistical analyses, each cAMP value (whether obtained from differentiated or nondifferentiated cultures) was normalized to 100%, representing cAMP production by nonrosiglitazone-treated differentiated cells from the same patient, as measured in the same experiment (Table 1
). Each data point represents the mean ± SEM of two to five separate experiments, with each experiment using cells from different individuals. Statistical comparisons between groups were made using one-tailed t test for correlated samples.
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or TSHR mRNA
After experiments in vitro, cells were pelleted and stored frozen at -70 C. Total RNA was isolated directly from cultured cells using the Totally RNA Kit (Ambion, Inc., Austin, TX). Positive control RNA was prepared in the same manner from human thyroid (for TSHR) or abdominal adipose tissue (for PPAR). Negative control RNA was prepared from human Epstein-Barr virus-transformed B lymphocytes.
The antisense RNA probe for PPAR was transcribed from a 431-bp PCR product with a T7 phage promoter at its 3'-end in the presence of T7 RNA polymerase (10 U) and [32P]UTP (50 µCi) for labeling. The resulting high specific activity probe encompassed nucleotides 27–412 containing two exons of the human cDNA sequence (12). This probe was designed to protect both PPAR1 (the 328-nucleotide predominant product) and PPAR2 (a 385-nucleotide product).
The antisense RNA probe for TSHR was transcribed from a 320-bp PCR product with a T7 phage promoter at its 3'-end in the presence of T7 RNA polymerase (10 U) and [32P]UTP (50 µCi) for labeling. The resulting high specific activity probe encompassed nucleotides 576–873 (exons 6–9) of the human TSHR cDNA sequence (13) and was designed to detect both the 2.4-kb intact TSHR, protecting a product of 298 nucleotides, and the 1.3-kb variant form, protecting a product of 217 nucleotides.
The antisense RNA probe for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was generated from pTRI-GAPDH human antisense control template (Ambion, Inc., Austin, TX). This probe was designed to protect a 154-nucleotide fragment of GAPDH mRNA.
Total RNA (80 µg for cells, 5 µg for thyroid or adipose tissue) was combined with 300,000 cpm PPAR and 3,000 cpm GAPDH probe in hybridization buffer, denatured at 95 C, and hybridized at 45 C for 16 h. Nonhybridized total RNA and probe were digested for 1 h at 37 C with ribonuclease A (0.175 U) and ribonuclease T1 (25 U; RNase Protection Kit, Roche Molecular Biochemicals, Indianapolis, IN). Samples were subsequently digested with proteinase K (50 µg) in the presence of 0.5% SDS and extracted with phenol/chloroform/isoamyl alcohol. The resulting ethanol-precipitated protected fragments were resuspended in loading buffer and resolved on a denaturing polyacrylamide gel (5% acrylamide/8 M urea).
Results
Effect of rosiglitazone treatment on adipocyte differentiation
These studies were carried out to assess and document the morphological changes in GO orbital cells treated with rosiglitazone compared with untreated cells. We studied both cells grown in adipocyte differentiation medium and cells grown for the same period of time under nondifferentiating conditions. Whether treated with rosiglitazone or not, a subset of the orbital cells grown in the differentiation medium lost their stellate (fibroblastic) morphology after 1–2 d in culture and converted to a spherical (adipocytic) shape with discrete vacuoles apparent under phase contrast in the cytoplasm. The total percentage of differentiated cells showing this type of morphological change in the treated cultures was somewhat greater (10–20%) than that in untreated cultures (5–10%). Similarly, the percentage of these cells containing lipid droplets staining positively with Oil Red O (representing triglyceride accumulation) was greater in the treated than in the untreated cultures. These droplets became apparent after about 7–10 d in differentiation culture and increased in number and size throughout the entire culture period. The most striking morphological feature observed was that the stained lipid droplets within individual adipocytes were generally significantly larger and more numerous in the treated than in the untreated differentiated cultures (Fig. 1, A and B). We interpreted these findings to suggest that rosiglitazone treatment of cells grown under these conditions stimulates both the number of cells within the cultures undergoing adipogenesis and the degree of triglyceride accumulation found in those cells.
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, C and D). This was most apparent after d 10 and increased progressively through d 21. We interpreted these findings to suggest that rosiglitazone itself stimulates adipogenesis in nondifferentiated cells. Effect of rosiglitazone treatment on functional TSHR expression
Treatment of orbital fibroblasts with rosiglitazone maleate (0.1–50 µM) during the 10-d differentiation period resulted in stimulation of TSH-dependent cAMP (2.6- to 4.7-fold) compared with control differentiated values. Peak stimulation was apparent at a dose of 10 µM after 10 d in culture (Fig. 2). The magnitude of stimulation increased progressively during the 10-d period (data not shown). Similarly, rosiglitazone treatment of nondifferentiated orbital fibroblasts resulted in measurable stimulation of TSH-dependent cAMP production (0.6- to 3.70-fold) compared with control differentiated values, with peak stimulation also apparent at 10 µM after 10 d in culture (Fig. 3). However, absolute levels of cAMP production were generally lower in the nondifferentiated cultures (with or without rosiglitazone treatment) than in parallel rosiglitazone-treated differentiated cultures (Table 1). Although the magnitude of stimulated cAMP production varied considerably from patient to patient, absolute levels of cAMP production in nondifferentiated cultures that were treated with between 1 and 50 µM rosiglitazone generally reached or exceeded those in untreated differentiated cultures from the same patients (Table 1).
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TSHR mRNA levels were assessed by RPA after the 10-d differentiation period in untreated orbital cells from a patient with GO and in cells from this individual treated during differentiation with rosiglitazone (1 or 10 µM). In addition, we examined levels of TSHR mRNA in both rosiglitazone-treated and untreated GO orbital cells from another patient that were grown in medium lacking several of the components necessary for complete adipocyte differentiation (nondifferentiated cultures). Although TSHR gene expression was detectable in untreated differentiated cells, we found that treatment with rosiglitazone increased the TSHR mRNA levels in these cells (Fig. 4). In contrast, the nondifferentiated cells, whether treated with rosiglitazone or not, showed only very low levels of TSHR mRNA that were just at the detection level of the RPA. It was not possible, therefore, to determine whether rosiglitazone had any slight effect on TSHR mRNA levels in these nondifferentiated cells (data not shown).
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mRNA
PPAR mRNA levels were assessed by RPA after the 10-d differentiation period in untreated GO orbital cells and in cells treated for the entire period with rosiglitazone (10 µM). Although PPAR mRNA was clearly present in untreated differentiated cultures, we found stimulated expression of this transcript in orbital cultures that were exposed to rosiglitazone during differentiation (Fig. 5).
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Human adipose tissue is dynamic, retaining the lifelong potential for hyperplasia through cellular differentiation and growth (14). Preadipocytes or preadipocyte fibroblasts are precursor cells found in the stromal-vascular compartment of adipose tissues that are committed to the adipocyte lineage (15). Differentiation of these cells involves a complex regulatory pathway that is controlled by the coordinate expression of specific regulatory genes and several transcription factors. Included among these is PPAR, a nuclear hormone receptor that is highly expressed in adipose tissue (7, 8). This receptor recognizes several different ligands, and its activation induces a program of downstream genes essential for the differentiation of preadipocytes into mature adipocytes (16, 17).
We report here that rosiglitazone is a potent stimulator of adipogenesis, functional TSHR expression, and TSHR and PPAR mRNA levels in differentiated human orbital preadipocyte fibroblasts from patients with GO. The process of adipogenesis (as determined morphologically or by increased PPAR levels) was invariably associated with enhanced functional TSHR expression. This was true in the untreated differentiated cultures and in both the differentiated and undifferentiated cultures treated with rosiglitazone. The nondifferentiated cultures seem to be somewhat less sensitive to rosiglitazone in terms of stimulation of TSH-dependent cAMP production, as the curve is shifted slightly to the right in these cultures. However, the peak responses, in terms of fold stimulation as well as absolute value of cAMP, are similar in the differentiated and nondifferentiated cultures. The increase in functional TSHR expression was paralleled by an increase in TSHR mRNA levels in the differentiated cultures, whether treated with rosiglitazone or not. However, we were unable to determine whether TSHR mRNA levels were stimulated by rosiglitazone in the nondifferentiated cultures, because levels of this mRNA in those cultures were near or below RPA detection levels. However, no instance of enhanced TSHR functional expression occurred without increased adipogenesis, nor did adipocyte differentiation occur without a concomitant increase in functional TSHR expression.
It is unclear from these studies whether the increase in functional TSHR expression in the rosiglitazone-treated cultures is secondary to stimulation of adipogenesis through PPAR ligation or is a direct result of PPAR agonism by rosiglitazone or other factors present in the differentiating culture medium. However, as the magnitude of both adipogenesis (as determined by Oil Red O staining) and enhanced functional TSHR expression increased in parallel during the 10 d in differentiation culture (data not shown), it seems likely that PPAR receptor ligation per se is not directly responsible for stimulation of TSHR expression. If it were, one might expect peak levels of TSHR function to precede evidence of triglyceride accumulation, a late event in adipocyte differentiation. Finally, as fetal and neonatal human adipocytes have been shown to express significantly higher levels of TSHR than do normal adult adipocytes (18), it may be that increased TSHR expression is an important feature of newly differentiated adipocytes regardless of the stimulus to differentiation.
The TZD class of orally active antidiabetic agents has been shown to enhance tissue insulin sensitivity, although the precise mechanism of action is unknown (19). These compounds are recognized as ligands for PPAR and are potent promoters of preadipocyte differentiation in several species (10, 20, 21, 22, 23). As insulin resistance in humans is most commonly associated with obesity, it seems paradoxical that these insulin-sensitizing agents would be stimulators of adipogenesis. However, evidence suggests that that clinical TZD treatment results primarily in an increase in the number of very small fat cells, thus promoting insulin sensitivity without increasing total body weight (10).
In this study rosiglitazone treatment of differentiated orbital preadipocyte fibroblasts resulted in a modest (
2-fold) increase in the percentage of cells showing morphological evidence of adipogenesis. However, there was apparent a striking increase in the size and number of Oil Red O-staining triglyceride droplets within individual cells after treatment with this compound. Therefore, it appears that rosiglitazone acts in orbital fibroblast cultures both to recruit cells into the adipocyte lineage and to stimulate more complete adipocyte differentiation of these cells. This results in an increase in both the number and size of adipocytes present in the cultures. Rosiglitazone also stimulated adipogenesis in nondifferentiated cultures, but the effect was more modest. However, such a change occurring within the orbit in vivo might be expected to result in an increase in the volume of the orbital adipose tissue compartment, leading to the development of proptosis and other clinical manifestations of GO.
Evidence suggests that adipose tissues from various species, ages of animals, or particular fat depots respond to TZD treatment in various different ways (10). For example, TZD treatment of the obese Zucker fa/fa rat (an animal model of obesity with insulin resistance) results in the recruitment of new, very small adipocytes with increased insulin sensitivity, rather than in the appearance of the larger, more lipid-laden cells seen in our orbital cultures (24). The reason for such differences in adipogenic response to TZD treatment is unknown. However, regional differences in the production and availability of natural ligands for PPAR have been demonstrated between various adipose tissue depots (25). It is possible that such differences contribute to depot-specific and age-related differences in cellular adipogenic potential. Similarly, these and other regional characteristics of fibroblasts (26) may contribute to the relatively site-specific clinical involvement of the orbit and pretibial skin in Graves’ disease.
It appears that all human adipose tissue expresses PPAR, regardless of its anatomical site of origin (7, 8). However, not all adipose tissue depots appear to be sensitive to the adipocyte differentiation-promoting effects of TZD. A study by Adams and colleagues (27) demonstrated that TZD stimulates the transcriptional activity of PPAR and promotes differentiation in preadipocytes derived from human sc tissues. However, these investigators reported that preadipocytes derived from the omentum are relatively refractory to the differentiation-promoting effects of TZD while expressing PPAR at similar levels. Our results document the expression of PPAR by human orbital preadipocytes and suggest that the orbit represents a fat depot with the capacity to respond to the adipogenic effects of TZDs.
In conclusion, our studies suggest that rosiglitazone treatment of orbital preadipocyte fibroblasts from patients with GO stimulates both adipocyte differentiation and levels of TSH-dependant cAMP production in these cells. This was true whether cells were grown in specific differentiation medium or in medium lacking particular components necessary for complete adipocyte differentiation. In addition, we demonstrated increased TSHR and PPAR mRNA levels in differentiated cells treated with rosiglitazone. Further studies are necessary to understand the relationship between adipogenesis and TSHR expression in the orbit. However, these results suggest that there may be a role for novel drugs aimed at the antagonism of various components of the PPAR signaling system (28, 29, 30) in the treatment or prevention of GO.
Acknowledgments
Footnotes
This work was supported in part by NIH Grant EYO8819 (to R.S.B.) from the National Eye Institute.
Abbreviations: cPGI2, Carbaprostacyclin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GO, Graves’ ophthalmopathy; IBMX, isobutylmethylxanthine; RPA, ribonuclease protection assay; TSHR, TSH receptor; TZD, thiazolidinediones.
Received September 21, 2001.
Accepted January 29, 2002.
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