1 Department of Medicine, Redland Hospital, Cleveland, Queensland 4163; 2 Department of Medicine, University of Queensland, and 3 Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Woolloongabba, Queensland 4102; and 4 Greenslopes Private Hospital, Greenslopes, Queensland 4120, Australia
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ABSTRACT |
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Glucocorticoids are
pivotal for adipose tissue development. Rodent studies suggest that
corticosteroid-binding globulin (CBG) modulates glucocorticoid action
in adipose tissue. In humans, both genetic CBG deficiency and
suppressed CBG concentrations in hyperinsulinemic states are associated
with obesity. We hypothesized that CBG deficiency in humans modulates
the response of human preadipocytes to glucocorticoids, predisposing
them to obesity. We compared normal preadipocytes with subcultured
preadipocytes from an individual with the first ever described complete
deficiency of CBG due to a homozygous null mutation. CBG-negative
preadipocytes proliferated more rapidly and showed greater peroxisome
proliferator-activated receptor--mediated differentiation than
normal preadipocytes. CBG was not expressed in normal human
preadipocytes. Glucocorticoid receptor number and binding
characteristics and 11
-hydroxysteroid dehydrogenase activity were
similar for CBG-negative and normal preadipocytes. We propose that the
increased proliferation and enhanced differentiation of CBG-negative
preadipocytes may promote adipose tissue deposition and explain the
obesity seen in individuals with genetic CBG deficiency. Furthermore,
these observations may be relevant to obesity occurring with suppressed
CBG concentrations associated with hyperinsulinemia.
adipose tissue; glucocorticoid; human; obesity; peroxisome
proliferator-activated receptor-
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INTRODUCTION |
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GLUCOCORTICOIDS ARE PIVOTAL for adipose tissue development. Endogenous or exogenous glucocorticoid excess is characterized by increased adipose tissue mass, especially centrally, with an increase in total fat cell number (19). Glucocorticoids act directly on adipose tissue. In vitro glucocorticoids increase both lipoprotein lipase activity in adipose tissue (27) and preadipocyte differentiation in a dose-dependent manner (13). Glucocorticoid receptors (GR) are present in human adipose tissue, with a greater GR density in visceral compared with subcutaneous adipose tissue (23, 29). Preadipocytes also express GRs, with regional and gender differences in GR complement (16).
Recent studies of lean and obese Zucker rats suggest that glucocorticoid action in adipose tissue may be modulated by corticosteroid-binding globulin (CBG). CBG is a 383-amino acid member of the serine protease inhibitor family of proteins. It is secreted by hepatocytes and binds over 90% of circulating cortisol under normal conditions. CBG constitutes a greater proportion of the total protein in rat white adipose tissue than in other tissues, including the liver (11). Furthermore, obese rats have less CBG in plasma and white adipose tissue than lean rats, and there is less CBG in visceral adipose tissue than in subcutaneous adipose tissue (12).
In humans, plasma CBG levels are inversely correlated with body mass and body mass index (20), and genetic CBG deficiency appears to be associated with obesity. An Italian-Australian family with a complete loss of function (null) mutation of the CBG gene, caused by a premature stop codon, has recently been characterized (34). Plasma CBG was undetectable by radioimmunoassay for three individuals homozygous for the null mutation, with ~50% normal CBG levels for null heterozygotes. Interestingly, individuals homozygous for the null mutation were relatively obese compared with other family members (34). A previous report of complete CBG deficiency described a boy, born to parents who were first cousins, who came to medical attention because he was obese. He was assessed as being CBG deficient on the basis of a lack of cortisol binding in serum and low total serum cortisol but with normal free cortisol levels (31). There are two CBG mutations associated with reduced cortisol-binding efficiency. With the CBG Lyon mutation, the homozygous individual was obese (9), but there was no comment on the weight of the individuals with the transcortin Leuven CBG variant (35).
We hypothesized that CBG is an important modulator of cortisol action
in preadipocytes and that CBG deficiency is associated with a change in
glucocorticoid response in human preadipocytes that predisposes to
obesity. We aimed to compare glucocorticoid-dependent activities of
preadipocytes from an individual who is homozygous for the CBG null
mutation (34) with those of normal preadipocytes. We
compared rates of replication and differentiation capacity of these
cells. Upon finding differences in these activities, we studied whether
human preadipocytes expressed CBG, and we compared GR characteristics
and 11-hydroxysteroid dehydrogenase (11
-HSD) activity as possible
mediators of the differences found.
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METHODS |
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Subjects and sample preparation.
Abdominal subcutaneous fat of ~1 cm3 was obtained (with
informed consent) by biopsy through a 1-cm periumbilical incision from a 57-yr-old male [body mass index (BMI) 39.4, waist 125 cm]
homozygous for the CBG null mutation. As controls, abdominal
subcutaneous adipose tissue samples of ~1 cm3 were
collected (with informed consent) during elective surgery from
Caucasion male subjects (see Table 1 for
age, BMI, and waist data). It was important to compare samples from the
same gender and site, because there are regional differences in rates
of differentiation (1) and 11-HSD activity
(6) and regional and gender differences in GR
(16). All subjects provided informed written consent. The
Greenslopes Private Hospital Ethics Committee and the Princess Alexandra Hospital Research Ethics Committee provided ethical approval
for the work. The normal subjects were not specifically tested to
ensure that they were not CBG deficient. However, our work and the work
of others suggest that severe mutations of the CBG gene are quite rare
(26).
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Proliferation assay. Confluent preadipocyte monolayers were detached with trypsin-Versene and replated at 1 × 103 cells/well (subconfluent) in 96-well plates in SCM for 16-20 h. The wells were washed with PBS, and then SCM, SCM + 10-500 nM cortisol, or SCM + 10-500 nM RU-486 was reapplied. After 48 h, preadipocyte cell number was assessed using a formazan colorimetric assay (Promega) as previously described (14). Briefly, the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added to each well at a concentration of 200 µg/ml. After incubation at 37°C for 4 h, absorbance at 490 nm was measured using a Bio-Rad 3550 microplate reader. The MTS proliferation assay was validated by confirming that the formazan absorbance vs. direct cell counts were linear over the range studied.
Differentiation.
Confluent monolayers of subcultured preadipocytes in 25-cm2
flasks were washed, and differentiation medium (DMEM-Ham's F-12 with
100 nM rosiglitazone, 0.25 mM IBMX for the first 2 days, 100 IU of
penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 100 nM cortisol, 0.2 nM triiodothyronine, 500 nM insulin, 17 µM pantothenate, 33 µM biotin, 10 µg/ml transferrin, and 15 mM HEPES) or a differentiation medium without rosiglitazone was applied. Rosiglitazone is a peroxisome proliferator-activated receptor- (PPAR
) activator that has been previously demonstrated to enhance the differentiation of normal human preadipocytes that have been grown
in SCM (1).
CBG gene expression.
Total RNA was extracted from confluent preadipocytes and stored at
70°C before being reverse-transcribed using the random hexamer
priming option of a commercially available kit (SUPERSCRIPT, Life
Technologies, Gaitherburg, MD). The integrity of the
reverse-transcription step was checked by glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) RT-PCR. A negative control, with no reverse
transcriptase added, was included for each RNA sample. Previously
published CBG (24) and GAPDH (8) primer
sequences and a second set of CBG primers we designed (sense
5'-GACAAGGGGAAGATGAACAC-3', antisense 5'-GCACAGCTTTATGGACCAC-3') were
obtained commercially. The PCR was performed in a Corbett Research
PC-90 microplate thermal sequencer with an annealing temperature of
55°C and 2 mM MgCl2, these parameters having been optimized for the primers by use of HepG2 cDNA as the positive control.
The PCR products were visualized under ultraviolet illumination after
electrophoresis on an ethidium bromide-labeled 2% DNA agarose gel.
GR assessment. Dexamethasone binding as a measure of GR number was assessed using a whole cell-binding assay, as previously described (16). Dexamethasone has minimal affinity for CBG (26). Briefly, 25-cm2 flasks of confluent cells were washed with PBS and preincubated in DMEM-Ham's for 30 min at 37°C. This was followed by a 60-min incubation with one of six serial dilutions of 0.78-25 nM tritiated dexamethasone ([3H]Dex; 70-110 Ci/mmol, Amersham Australia) or 0.78-25 nM [3H]Dex with a 250 times excess of unlabeled dexamethasone (Sigma-Aldrich) in DMEM-Ham's. After the cells were washed with ice-cold PBS, they were lysed, and an aliquot was taken from each flask to count bound [3H]Dex and for protein determination (3). A Scatchard plot provided the binding characteristics for each sample.
11-HSD activity.
Confluent preadipocytes in 25-cm2 flasks were washed with
PBS and DMEM-Ham's F-12, with 500 nM tritiated cortisol or cortisone applied in triplicate for a 6-h incubation. The cortisone and cortisol
in the media were measured by an improved method of HPLC, as previously
reported (22). The cells were washed with PBS and lysed by
sonication, and the protein was measured (3) to allow the
calculation of cortisol/cortisone interconversion in femtomoles per
milligram protein per hour. There were no measurable amounts of
cortisol or cortisone in the PBS washes or the cell lysates.
Statistical analysis. Comparisons of replication and G3PD activity used the Student's t-test (two-tailed). The statistical analyses were performed with the data analysis function of Microsoft Excel, version 5. Statistical significance was defined as P < 0.05.
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RESULTS |
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Proliferation.
The CBG-negative cells grew to confluence more quickly than normal
preadipocytes, requiring subculturing more frequently. For this reason,
the proliferation comparisons were done both with samples that had been
grown in vitro for the same period of time and with separate samples
that were the same passage number. The CBG-negative preadipocytes
proliferated more quickly than any of the normal preadipocyte samples
(P < 0.0001 for all; Fig. 1). Cortisol and the antiglucocorticoid
RU-486 had no influence on proliferation rates for CBG-negative or
normal preadipocytes in these short-term incubation studies (data not
shown).
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Differentiation.
CBG-negative preadipocytes differentiated more readily than normal
preadipocytes in differentiation medium containing rosiglitazone. CBG-negative preadipocytes had visibly greater lipid accumulation and
had a 10-fold greater G3PD activity than similarly treated normal
preadipocyte samples (1,333 vs. 52, 68, 118, 142, 198, and 254 mU/mg
protein, or P = 0.005, 0.006, 0.006, <0.001, 0.001, 0.012, respectively; Fig. 2). In the
absence of rosiglitazone, CBG-negative and normal preadipocytes showed
similar low levels of G3PD activity (47 vs. 14, 36, and 68 mU/mg
protein).
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CBG gene expression.
There was no evidence of CBG mRNA in normal human preadipocytes. With
use of two different sets of CBG primers with expected PCR products of
247 and 296 base pairs, the PCR produced strong bands in the
HepG2-positive control lanes, but there were no bands in any
preadipocyte lane (Fig. 3).
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GR.
With use of dexamethasone whole cell-binding assays, the GR
characteristics were the same for the CBG-negative and normal preadipocytes. For three assays, including both strains of CBG-negative preadipocytes, the dissociation constants (Kd)
were 6.8, 9.5, and 12.1 nM vs. 5.1-12.6 nM for normal
preadipocytes (Fig. 4), and the maximal
binding capacities (Bmax) were 263, 488, and 603 fmol/mg
vs. 327-599 fmol/mg for normal preadipocytes (Fig.
5).
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11-HSD activity.
Type 1 11
-HSD activity was normal, with cortisone-to-cortisol
conversion for the two CBG-negative preadipocyte strains being 14 and
22 fmol · mg
protein
1 · h
1 vs.
normal preadipocytes 10, 10, 17, and 18 fmol · mg
1 · h
1
(Fig. 6). There was minimal
cortisol-to-cortisone conversion (type 2 11
-HSD activity) in
CBG-negative or normal cells.
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DISCUSSION |
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Preadipocytes from an individual with complete CBG deficiency had
increased proliferation and enhanced PPAR-mediated differentiation compared with normal preadipocytes. These characteristics may promote
adipose tissue deposition by increasing the number of preadipocytes and
their conversion to mature adipocytes. The observations may be relevant
to the role of CBG in obesity reported in genetic CBG deficiency
(34) and the potential role of suppressed CBG levels in
hyperinsulinemic humans (10).
The mechanism of hyperproliferation and accentuated
thiazolidinedione-stimulated differentiation in preadipocytes from
genetically CBG-deficient individuals is unclear, as CBG gene
expression was not detected in cultured human preadipocytes.
Furthermore, measures of GR binding and 11-HSD activity were normal.
CBG may be narrowly expressed in human tissues, with definite tissue expression demonstrated in hepatocytes and in placental syncytiotrophoblasts (24) where CBG may modulate glucocorticoid and progesterone tissue interactions (2). CBG has been reported in rodent adipose tissue by corticosterone binding but not gene expression (11, 12), and this may represent circulating CBG from plasma taken into cells. There is speculation in the literature regarding this for other cell types with putative CBG membrane receptors involved (32). The fact that corticosterone binding in rodent white adipose tissue mirrors plasma CBG levels (12) supports the idea that CBG may be sequestered in adipose tissue and/or taken up by preadipocytes/adipocytes. The facts that there is less CBG in the adipose tissue of obese rats than in that of lean rats, and that there are regional differences in CBG in adipose tissue, subcutaneous greater than visceral, strongly suggest that CBG is an important modulator of glucocorticoid activity in adipose tissue (12).
However, this does not explain why preadipocytes from a CBG-negative individual should respond differently than normal preadipocytes under identical in vitro conditions if preadipocytes do not express CBG. CBG influences the kinetic parameters of cortisol transport and clearance. In studies comparing subjects with normal or high CBG concentrations (using estrogen-containing oral contraceptives), increased CBG levels were associated with a reduced rate of cortisol clearance and an increased mass of circulating cortisol in a smaller volume of distribution (4). It may simply be that long-term growth in a CBG-deficient environment, in vivo, causes changes in preadipocyte growth and differentiation that are sustained in vitro for at least four passages. This is indirectly supported by the fact that there were no short-term differences in glucocorticoid responsiveness and no change in replication rate with additional glucocorticoid or with the antiglucocorticoid RU-486.
Alternatively, the lack of CBG during fetal development may modulate glucocorticoid action, causing permanent changes in gene expression in preadipocytes. CBG is present in a number of fetal tissues, with temporal and spatial changes in CBG localization suggesting that CBG influences steroid hormone activity in fetal tissues (33). Glucocorticoids are involved in gene programming, with one example being that of glucocorticoid exposure in late gestation causing permanent changes to rat hepatic phosphoenolpyruvate carboxykinase gene expression (25).
We looked for mechanisms that may be mediating the different
responsiveness of the preadipocytes from a CBG-deficient individual compared with control preadipocytes. We studied GR number, because the
GR is a significant regulator of glucocorticoid action in many tissues
and because glucocorticoids downregulate their own receptor. We also
compared their 11-HSD activity, as local interconversion of cortisol
and cortisone is known to have significant effects on glucocorticoid
responsiveness at a tissue level, and again glucocorticoids regulate
(increase) type 1 11
-HSD activity (6). There were no
differences in either GR number or characteristics or in 11
-HSD
activity between CBG-negative individuals and control subjects. The
enhanced differentiation occurred only in the presence of
rosiglitazone, suggesting that the effect requires the presence of
PPAR
-mediated mechanisms. This is not surprising, as glucocorticoids induce CAATT-enhancing binding protein (C/EBP)-
, which in turn induces PPAR
and C/EPB
. PPAR
and C/EBP
act synergistically to promote adipocyte differentiation (21).
It is important to consider whether the differences in the CBG-negative
preadipocyte's replication and differentiation could be due to effects
other than CBG's effects on cortisol action. CBG also binds
17-hydroxyprogesterone, but as no progesterone binding has been
shown in human adipose tissue (5, 28), it is unlikely that
the differences we have demonstrated are secondary to direct changes in
progesterone responsiveness.
Genetic abnormalities resulting in a reduced amount of CBG protein or cortisol-binding activity are rare (26), but hyperinsulinemia is associated with reduced CBG concentrations (10). In in vitro studies of HepG2 cells, insulin reduced CBG secretion in a dose-dependent manner, suggesting a causal relationship (7). If, as our study suggests, lower CBG concentrations influence adipose tissue development, this may be another aspect of the pathophysiology of the metabolic syndrome. This may be important in the treatment of type 2 diabetes mellitus when insulin-sensitizing agents are compared with agents that increase insulin concentrations. Furthermore, efforts to increase CBG concentrations toward normal may attenuate some aspects of the metabolic syndrome.
In summary, we have shown that genetic CBG deficiency is associated with enhanced proliferation and differentiation of human preadipocytes. We were unable to elicit mechanisms by which CBG deficiency influences preadipocyte metabolism. The effect of CBG deficiency on preadipocyte function may be due to altered circulating cortisol kinetics or an effect on adipocyte development/programming early in life. We propose that the increased proliferation and enhanced differentiation of CBG-negative preadipocytes may promote adipose tissue deposition and explain the obesity seen in individuals with genetic CBG deficiency. Furthermore, these observations may be relevant to obesity occurring with suppressed CBG concentrations associated with hyperinsulinemia.
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ACKNOWLEDGEMENTS |
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The rosiglitazone was provided by Glaxo SmithKline. J. B. Prins is a Wellcome Senior Research Fellow in Medical Science. D. J. Torpy is a recipient of a Sylvia and Charles Viertel Clinical Investigatorship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Prins, Dept. of Diabetes and Endocrinology, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Queensland 4102, Australia (E-mail : jprins{at}soms.uq.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 28, 2003;10.1152/ajpendo.00262.2002
Received 13 June 2002; accepted in final form 13 January 2003.
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