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Divisions of Pediatric Endocrinology (S.K.) and Endocrinology, Metabolism, and Nutrition (M.J.C., R.S.B.), Mayo Clinic, Rochester, Minnesota 55905; and Department of Cell Biology and Diabetes Research and Training Center, Albert Einstein College of Medicine (P.E.S.), Bronx, New York 10461
Address all correspondence and requests for reprints to: Rebecca S. Bahn, M.D., Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905. E-mail: bahn.rebecca{at}mayo.edu.
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Abstract |
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(PPAR), preadipocyte factor-1, and TSH receptor (TSHr) genes in orbital adipose tissues from GO patients (n = 22) and normal individuals (n = 18) and in orbital preadipocyte cultures derived from GO patients (n = 6) and normal subjects (n = 3) using quantitative real-time RT PCR. We found increased leptin, adiponectin, PPAR, and TSHr expression in GO compared with normal orbital tissue samples, with positive correlations in the GO tissues between TSHr and leptin, adiponectin and PPAR. In vitro differentiation of GO and normal preadipocytes resulted in enhanced adiponectin, leptin, and TSHr expression, with greater expression of the latter two genes in the GO cultures. These results suggest that de novo adipogenesis within orbital tissues with parallel enhanced expression of TSHr may be important in the pathogenesis of GO, and that potential therapies for GO might include inhibition of the adipogenic pathway. |
Introduction |
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Volume measurements of the orbital adipose/connective tissue compartment and extraocular muscles using computerized tomography scans revealed enlargement of either or both in 87% of patients with clinically detectable GO (7). More recently, investigators suggested that GO can be divided into two subtypes. The first subtype is characterized by prominent extraocular muscle enlargement and diplopia. The second, without muscle enlargement, is associated with expansion of the orbital fat compartment and proptosis (8, 9). In another study, the degree of proptosis was found to correlate well with the volume of the orbital fat compartment (10). Taken together, these studies suggest that expansion of the orbital fat compartment represents a major component of the disease process. However, it is unclear whether this increased volume is secondary to enhanced adipogenesis within the orbit or to enlargement of existing orbital adipocytes.
Adipogenesis is a complex process accompanied by a dramatic and coordinated increase in the expression of several genes, including leptin, adiponectin (also known as adipocyte complement-related protein of 30 kDa), and peroxisome proliferator-activated receptor (PPAR), with a concurrent decrease in the expression of preadipocyte factor-1 (pref-1). We have shown previously that orbital preadipocytes can be induced in vitro to differentiate into adipocytes bearing functional TSHr (11). We undertook the current study to ascertain whether de novo adipogenesis might be similarly enhanced within the orbit in GO.
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Patients and Methods |
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Orbital adipose/connective tissue specimens were obtained from patients who underwent orbital decompression surgery for severe GO (n = 22) and from individuals with no history of Graves’ disease (n = 18). Four of the 22 patients with GO had optic neuropathy. The clinical characteristics of the GO patients are shown in Table 1
. All GO patients were euthyroid at the time of orbital surgery. Normal orbital tissues were retrieved at very early autopsy from patients whose corneas were being harvested for transplantation. The sex ratio, body weight, and body mass index were not significantly different between the GO patients and normal individuals. However, the latter were significantly older than the patients with GO (Table 2). These studies were reviewed and approved by the Mayo Clinic institutional review board.
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Orbital adipose/connective tissue was obtained during the course of transantral orbital decompression surgery for GO, placed in a sterile container on saline-soaked gauze, and transported at room temperature to our laboratory, where it was frozen at -70 C. Normal orbital tissue was frozen immediately upon harvesting.
Cell cultures
GO (n = 6) and normal orbital adipose tissue (n = 3) samples were minced and placed directly in plastic culture dishes, allowing preadipocyte fibroblasts to proliferate as described previously (12). Cells were propagated in medium 199 containing 20% fetal bovine serum (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 were 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 (11); cultures were changed to serum-free DMEM/Ham’s F-12 (1:1; Sigma-Aldrich Corp., St. Louis, MO) supplemented with biotin (33 µM), pantothenic acid (17 µM), transferrin (10 µg/ml), T3 (0.2 nM), insulin (1 µM), carbaprostacyclin (0.2 µM; Calbiochem, La Jolla CA), and, for the first 4 d only, dexamethasone (1 µM) and isobutylmethylxanthine (0.1 mM). The differentiation protocol was continued for 10 d, during which time the medium was replaced every 3–4 d. Undifferentiated cultures were derived from fibroblasts obtained from the same patient’s orbital tissues and were maintained for the same period of time in medium lacking several of the components necessary for complete adipocyte differentiation (i.e. carbaprostacyclin, dexamethasone, and isobutylmethylxanthine).
Real-time RT-PCR
Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. cDNA was synthesized using 200 ng total RNA incubated with random hexamers, followed by a 100-µl RT reaction with 6.25 U Multiscribe Reverse Transcriptase (PE Applied Biosystems, Foster City, CA). Conditions used were 25 C for 10 min, 37 C for 60 min, and 95 C for 5 min.
Oligonucleotide primers and TaqMan probes were designed using the computer program Primer Express (PE Applied Biosystems). To avoid amplification of genomic DNA, one of the two primers or the probe for TSHr, leptin, PPAR, and Pref-1 was placed at the junction between two exons. For adiponectin, primers and probes could not be designed across an exon intron junction, and hence, no RT controls were used to detect amplification of genomic DNA. Expression of 18S ribosomal RNA (rRNA) was used to correct for differences in the amount of total RNA added to a reaction and to compensate for different levels of inhibition during RT of RNA and PCR. Primers and probes for TSHr and 18S rRNA were designed and supplied by PE Applied Biosystems (Assays-on-Demand and predeveloped TaqMan assay reagents, respectively). The PCR primers and probes for leptin, adiponectin, PPAR, and pref-1 are shown in Table 3.
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. A human liposarcoma cell line was used as the positive control for pref-1. Reaction mixture, without the cDNA, was used as the negative control in each run.
The standard curve method was used to quantify the expression of the various genes and 18S rRNA in each sample. The normalized results were expressed as the ratio of RNA (picograms) of the target gene to RNA (picograms) of 18S rRNA. For each experimental sample, a gene was considered not to be expressed if amplification was not detected by cycle 40. The relative expression levels of these genes in orbital adipose tissue specimens from patients with GO were compared with those in specimens from normal controls. The number of orbital tissue specimens examined differed slightly between the genes studied (TSHr: n = 18 for GO, n = 15 for normal subjects; leptin, adiponectin, and PPAR: n = 22 for GO, n = 18 for normal subjects; pref-1: n = 13 for GO, n = 8 for normal subjects;).
Statistical analysis
The Mann-Whitney rank-sum test was used to assess statistically significant differences between the groups. Additional analyses were performed using Spearman rank order correlation to study correlations between expression levels of TSHr and the various adipogenesis-related genes.
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Results |
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TSHr mRNA was amplified in each of the 18 GO orbital tissue specimens and in 14 of 15 normal orbital tissues. The expression of TSHr, normalized to 18S rRNA, was significantly higher in orbital adipose tissues from GO patients than in normal adipose tissues (P < 0.05; Fig. 1A). TSHr expression did not correlate with the duration of GO, type of prior GO treatment, smoking history, time since diagnosis of hyperthyroidism, or age of the individual. We found no difference in TSHr expression between males and females (P = 0.543). TSHr mRNA levels were significantly lower (100- to 1000-fold) in GO orbit than in normal thyroid tissue.
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, were amplified in each of the 22 GO orbital tissues and in the 18 normal orbital tissues. In contrast, pref-1 was amplified in only 11 of 13 GO tissues and 5 of 8 normal adipose tissues. As was found for TSHr, the expression of leptin, adiponectin, and PPAR, normalized to 18S rRNA, was increased in orbital adipose tissues from GO patients compared with normal adipose tissue samples (P < 0.001 for leptin and adiponectin; P < 0.003 for PPAR; Fig. 1
, B–D). The expression levels of the genes did not correlate with the duration of GO, type of prior GO treatment, smoking history, time since diagnosis of hyperthyroidism, or age of the individual. Because adiponectin serum levels are known to vary inversely with weight and to be greater in females than in males, it is important to note that the mean weights of the normal subjects and GO patients were not significantly different (P = 0.283), and that there was a nearly equal proportion of females in both groups (55% in normal subjects and 60% in GO). Results for pref-1 showed wide variability of expression both within and between the 13 GO and 8 normal adipose tissues examined (Fig. 1E); thus, we found no significant differences between these groups.
We analyzed our data to determine whether correlations exist between the expression of TSHr and that of any of the adipogenesis-related genes. Within the GO tissues, we found significant positive correlations between expression levels of TSHr and leptin (P < 0.001; r = 0.775; Fig. 2A), TSHr and adiponectin (P < 0.004; r = 0.652; Fig. 2B), and TSHr and PPAR (P < 0.001; r = 0.757; Fig. 2C). In contrast, no correlations were found between expression levels of pref-1 and any of the other genes studied in GO tissues. Additionally, no correlations existed in normal orbital tissues between the expression of TSHr and any of the other genes studied.
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TSHr mRNA expression increased upon differentiation in each of the six GO cultures (mean, 11.9-fold; range, 4.2- to 44.4-fold; P = 0.002) and in two of three normal orbital cultures (mean, 1.6-fold; range, 0.9- to 2-fold; Fig. 3). The degree of increase in expression of TSHr was significantly greater in GO cultures compared with normal cultures (P = 0.024). Similarly, leptin mRNA expression increased upon differentiation in all GO (mean, 16.1-fold; range, 3.7- to 40.3-fold; P = 0.002) and normal cultures (mean, 3.2-fold; range, 2.1- to 5.3-fold), and the degree of increase in expression of this gene was also significantly higher in GO cultures than in normal cultures (P = 0.048). Adiponectin expression also increased upon differentiation in both GO (mean, 12.9-fold; range, 1.4- to 60-fold; P = 0.002) and normal (mean, 9.1-fold; range, 2.3- to 22.6-fold) preadipocyte cultures. However, the degree of increase in expression of this gene was not significantly different between GO and normal cultures.
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(GO: mean, 2.2-fold; range, 0.9- to 7.6-fold; P = 0.394; normal: mean, 1.8-fold; range, 1.2- to 2.1-fold) or pref-1 (GO: mean, 0.89-fold; range, 0.1- to 3-fold; normal: mean, 0.3-fold; range, 0.04- to 0.9-fold) after differentiation in either GO or normal cultures (Fig. 3). Further, the degree of change in TSHr after differentiation did not correlate significantly with any of the adipogenesis-related genes in either GO or normal cultures. However, in GO cultures, we noted a trend toward significance in the correlation between changes in TSHr and adiponectin (r = 0.829; P = 0.0583) and between changes in TSHr and PPAR (r = 0.829; P = 0.0583; data not shown). |
Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Although we and others have found TSHR expression levels in thyroid and GO orbital adipose tissues to be the highest measured (5, 6), TSHR mRNA is detectable in adrenal, kidney, thymus (15), pituitary (16), human abdominal adipose tissue (17), skin, liver, leukocytes, lymph nodes, brain, gut, and pancreas (18). The finding of low level TSHR expression in diverse tissues suggests that extrathyroidal TSHR expression may not have physiological relevance in normal individuals. However, in the setting of Graves’ disease with circulating antibodies and T cells directed against this antigen, there may be autoimmune reactivity directed against TSHr in adipose and connective tissues throughout the body (19). Patients in whom GO or pretibial dermopathy become clinically apparent might represent a subset affected by a combination of environmental and local factors (20, 21). Potentially important factors might include gravitational dependency and trauma, smoking, anatomic constraints of the bony orbit, dysfunction of efferent lymphatic channels, and elaboration within these tissues of particular cytokines. These anatomic regions appear to be particularly susceptible to various cytokine effects due to site-specific phenotypic characteristics of fibroblasts (22).
Adipose tissue volume increases through a combination of increased cell number (hyperplasia) and expanded cell size (hypertrophy). New adipocyte formation, or adipogenesis, plays an on-going role in adipose tissue enlargement throughout life (23). Sorisky and colleagues (24) demonstrated that cultures derived from human orbital adipose/connective tissue contain adipocyte precursor cells (comprising 5–10% of the total), capable of differentiating into lipid-filled adipocytes when cultured under conditions known to stimulate adipogenesis in fibroblasts from other sites. Adipogenesis is a complex process associated with activation of several adipocyte-specific genes, including leptin, adiponectin, and PPAR, and inhibition of the preadipocyte gene pref-1. PPAR is a nuclear hormone receptor that is highly expressed in adipose tissue (25, 26). The activation of this receptor is critical for the complex process of adipogenesis, and several ligands of this receptor that have profound effects on this process as well as on insulin sensitivity have been developed (27, 28). Leptin is a protein produced and secreted exclusively by mature adipocytes (29). Similarly, adiponectin is a recently identified, adipose tissue-derived, soluble protein produced solely by mature adipocytes (30). This protein has important metabolic effects related to whole body insulin sensitivity and also possesses antiatherogenesis properties. Serum levels of adiponectin decrease with obesity and are higher in females than in males. Both adiponectin expression and secretion are stimulated by activators of PPAR (31).
We found significantly increased expression of all three gene markers of adipocyte differentiation (leptin, adiponectin, and PPAR) in orbital adipose tissue from patients with GO compared with normal orbital tissue. In addition, the expression of each of these genes correlated positively with TSHr gene expression. We reported previously that PPAR is expressed in orbital adipose tissue from GO patients and that both adipogenesis and PPAR are stimulated in cultures of GO orbital fibroblasts treated with a PPAR agonist (32). The potential clinical significance of this observation was suggested by investigators who reported the case of a patient with GO who experienced exacerbation of his eye disease, with expansion of the orbital fat, after treatment of diabetes mellitus type 2 with the PPAR agonist pioglitazone (33).
In the current study we found higher levels of leptin and adiponectin, genes produced exclusively by mature adipocytes, in uncultured orbital adipose tissue specimens from patients with GO compared with normal orbital tissue specimens. Although effects of prior treatment for GO cannot be entirely ruled out, these results suggest that there may be a relatively greater number of mature adipocytes in GO than in normal orbital tissues. This may result from the stimulation of adipogenesis in orbital preadipocytes by some unknown factor present in Graves’ disease. The positive correlations noted between TSHr and adipogenesis-related gene expression in uncultured GO tissues further suggest that the expanded orbital adipose tissue volume in GO stems from adipogenesis rather than lipid accumulation in existing mature adipocytes. That similar correlations were not found in normal orbital adipose tissues that had lower levels of TSHr, leptin, adiponectin, and PPAR further supports the concept that enhanced orbital adipogenesis is a component of the disease process in GO.
We found that GO preadipocytes respond more vigorously to culture conditions inducing differentiation than do normal orbital cultures, as measured by increased leptin and TSHr expression. A similar, but not statistically significant, trend was apparent for adiponectin expression in GO compared with normal orbital cultures. These in vitro observations parallel our findings of increased adipocyte and TSHr gene expression in GO orbital adipose tissue specimens compared with normal tissue samples. These results suggest that some circulating or local factor present in the orbit in GO, the effects of which are maintained in tissue culture, may render preadipocytes especially susceptible to differentiation. The nature of these putative stimulators is unclear and is the subject of current investigation.
Orbital fibroblasts derived from the connective tissue/adipose depot in the posterior orbit are a distinct subpopulation from those investing the extraocular muscles (34). The preadipocyte fibroblasts studied in this investigation are of the former variety, as they are derived from orbital fat removed in the course of orbital decompression surgery for GO. The fibroblasts investing the extraocular muscles do not differentiate into adipocytes in culture conditions similar to those we used in this study, but are similarly capable of glycosaminoglycan synthesis (35). This heterogeneity in orbital fibroblast phenotype may help to explain why some patients with GO experience predominant enlargement of the extraocular muscles with accumulation of glycosaminoglycans, whereas in other patients expansion of the orbital adipose tissues represents the major disease process (34).
In summary, de novo adipogenesis within the orbit, with parallel enhanced expression of TSHr in orbital adipocytes, may be of central importance in the pathogenesis of GO. This process would result in increased expression of TSHr in the orbit, perhaps allowing this receptor to act as an autoantigen there. In addition, the concomitant expansion of orbital adipose tissue volume would lead to the development of the clinical signs and symptoms of GO. Factors responsible for stimulation of orbital adipogenesis are unknown, but various cytokines or other immune factors known to be present in the GO orbit may be involved (20, 36). It is perplexing that this process appears clinically to be limited to the orbit, with similar, although not identical, histopathological changes found in the pretibial tissues of patients with pretibial dermopathy. This relative anatomic site specificity might be explained by local mechanical factors and by phenotypic characteristics of fibroblasts residing in these anatomic sites (22, 34). Finally, these findings suggest that inhibition of orbital adipogenesis by antagonism of various components of the adipogenic pathway might be of benefit in the treatment of GO.
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Footnotes |
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Abbreviations: FBS, Fetal bovine serum; GO, Graves’ ophthalmopathy; PPAR, peroxisome proliferator-activated receptor ; pref-1, preadipocyte factor-1; rRNA, ribosomal RNA; TSHr, TSH receptor.
Received August 21, 2003.
Accepted October 31, 2003.
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