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Other Original Studies |
-Induced Apoptosis and Interleukin-1ß Release in Human Subcutaneous Adipocytes and Preadipocytes1
Division of Medical Sciences, University of Birmingham, Birmingham, United Kingdom B15 2TT
Address all correspondence and requests for reprints to: Dr. Margaret C. Eggo, The Medical School, University of Birmingham, Birmingham, United Kingdom B15 2TT. E-mail: m.c.eggo{at}.bham.ac.uk
Abstract
Tumor necrosis factor-
(TNF) can decrease adipose tissue mass,
but in obesity, adipose tissue hypertrophy persists despite increased
TNF expression. The hormonal milieu of obesity may antagonize the
adipostat effects of TNF. We examined the effects of insulin and the
synthetic glucocorticoid, dexamethasone (Dex), on TNF-induced
apoptosis and gene expression in human adipocytes and preadipocytes.
Using RT multiplex PCR, the expression of the proapoptotic genes
interleukin-1ß (IL-1ß)-converting enzyme (ICE) and TNF and the
antiapoptotic genes bcl-2, nuclear factor-
B (NFB),
and NFB inhibitory subunit, IB, were examined. The expression and
release of IL-1ß, a postulated downstream effector of ICE-mediated
apoptosis, were also determined. TNF increased the messenger
ribonucleic acid levels of ICE, TNF, IL-1ß, bcl-2,
and NFB in preadipocytes and adipocytes (P <
0.01). Dex inhibited TNF-induced messenger ribonucleic acid
expression of ICE, TNF, and IL-1ß (P < 0.01),
but not that of bcl-2 and NFB. TNF stimulated
IL-1ß release from preadipocytes and adipocytes up to 20-fold, but
the effect was abrogated by Dex. Apoptosis induced by TNF was
reduced to control levels (P < 0.01) by Dex.
Insulin had no significant effect on TNF-induced apoptosis and gene
expression. In obesity, glucocorticoids may reduce TNF actions in
adipose tissue by inhibiting TNF-induced apoptosis, IL-1ß release,
and TNF expression.
TUMOR NECROSIS factor- (TNF) could
reduce adipose tissue mass due to its ability to inhibit adipocyte
differentiation and lipogenesis (1), stimulate lipolysis
(2), and induce apoptosis (3). Circulating
levels and adipose tissue production of TNF are increased in obesity
(4), but despite this, adipose tissue hypertrophy
persists, suggesting that the hormonal milieu of obesity may modify the
adipostat effects of TNF and lead to TNF resistance
(5). Insulin and glucocorticoids are known to promote
adipogenesis through inducing adipocyte differentiation and increasing
lipogenesis. Insulin excess is associated with weight gain, and
elevated glucocorticoid levels, found in Cushing’s syndrome and in
experimental animal models, are associated with obesity. TNF
up-regulates the enzyme 11ß-hydroxysteroid dehydrogenase enzyme 1
that is responsible for the generation of cortisol from inactive
cortisone (6), an enzyme present in fat tissue and
elevated in central obesity (7). We hypothesized that in
obesity glucocorticoids and insulin antagonize the apoptotic effects of
TNF, leading to reduced control of adipose tissue mass.
In human preadipocytes and mature adipocytes, two TNF receptors (p55
and p80) exist. Long-term inhibition of insulin-stimulated glucose
transport and antiadipogenic effects are mediated through
down-regulation of the glucose transporter GLUT4 via the p55 TNF
receptor (8). TNF is known to induce apoptosis by
activating apoptosis executors, such as interleukin-1ß
(IL-1ß)-converting enzyme (ICE) and ICE-like cysteine proteases,
referred to as caspases. ICE cleaves the 33-kDa pro-IL-1ß into the
17.5-kDa, biologically active IL-1ß (9). Overexpression
of ICE causes apoptosis (10, 11), whereas mutations
involving the active site for catalytic activity (10) or
antisense treatment (12) eliminate the ability of ICE to
induce apoptosis. Treatment with an ICE-specific inhibitor
(13) or the product of crmA, a cowpox virus-
encoded cytokine response modifier gene that inhibits ICE and
ICE-related proteases (13, 14, 15), inhibits TNF-induced
apoptosis. IL-1ß, which is produced by macrophages and other
antigen-presenting cell types, plays a regulatory role in ICE-mediated
apoptosis (16). Pro-IL-1ß accumulates in the cell
cytosol (17), and mature IL-1ß is released during
apoptosis after cleavage by ICE (14, 16, 18). When
released, IL-1ß mediates TNF-induced apoptosis
(16).
TNF undermines its own killing powers by inducing the expression of
antiapoptotic mediators, such as bcl-2 (19) and
nuclear factor-B (NFB) (20). bcl-2
inhibits apoptosis in response to many different death-inducing
signals, such as overexpression of ICE (10), and has been
shown to activate transcription factor NFB through degradation of
the cytoplasmic inhibitor NFB inhibitory subunit (IB)
(21). NFB prevents TNF-induced apoptosis (22, 23), and knockout mice missing NFB die before birth,
apparently of massive apoptosis of liver cells (22). In
immune cells TNF expression is increased by lipopolysaccharide and
inhibited by glucocorticoids (24, 25). Similarly, in
adipose tissue, TNF release is stimulated by lipopolysaccharide
(26), but the effects of glucocorticoids and other
hormonal factors, such as insulin, on the expression of this cytokine
are unknown.
Glucocorticoids and insulin regulate apoptosis in a cell-specific
manner. Dexamethasone (Dex), a synthetic glucocorticoid, inhibits
TNF-induced cytotoxicity/apoptosis in TA1 preadipocytes
(27), mouse L-929 cells, human mammary carcinoma MCF-7
cells, and murine tumorigenic fibroblasts L-M cells
(28, 29, 30, 31), but in mouse lymphoma WEHI7.2 cells
(32), human leukemic 6TG1.1 T cells (33), and
human osteosarcoma U20S cells (34), Dex induces apoptosis.
Insulin has been shown to rescue many cells from apoptosis, but in
myeloma U266 cells, insulin increases apoptosis by activating caspase-3
(35).
To elucidate the mechanisms by which Dex and insulin may regulate
TNF effects, we examined DEX or insulin-mediated changes in
TNF-induced messenger ribonucleic acid (mRNA) expression of ICE,
TNF, bcl-2, NFB, IB, and IL-1ß. The release of
IL-1ß into the medium after TNF treatment and the effects of DEX
and insulin on this release were studied. We used both preadipocytes
and mature adipocytes from sc tissue for this study. Adipose tissue is
comprised of a heterogeneous population of preadipocytes and mature fat
cells with large lipid stores, both of which express receptors for
TNF.
Subjects and Methods
Human subjects
Subcutaneous adipose tissues were obtained from 16 patients undergoing either elective abdominal surgery (n = 5) or cosmetic abdominal liposuction (n = 11) in accordance with the guidelines of the local ethical committee. Twelve women (age, 27–64 yr; body weight, 65.2–82.0 kg) and four men (age, 35–72 yr; body weight, 76.1–86.7 kg) were fasted for at least 6 h preoperatively, and all underwent general anesthesia. None of the patients had diabetes or severe systemic illness, and none was taking medications known to influence adipose tissue mass, distribution, or metabolism. Variations in body weight, age, and sex did not influence the general conclusions reached, so the data were pooled.
Adipocyte culture
Adipose tissues were transferred to the laboratory within 1
h of removal and used immediately. Mature adipocytes (2 x
106 cells), prepared as described previously
(36), were cultured in suspension in 10 mL DMEM/Ham’s
F-12 with or without varying concentrations (0.3–10 nmol/L) of
recombinant human TNF (PeproTech, London, UK), Dex
(10-11–10-6
mol/L), and insulin
(10-10–10-6
mol/L) for the indicated periods of time (1–48 h). Cells were used for
total RNA extraction. Conditioned media were stored at -80 C and used
for analysis of IL-1ß release by enzyme-linked immunosorbent assay
(ELISA). To study adipocyte apoptosis, isolated mature adipocytes were
cultured using a modified ceiling culture method as previously
described (36). Adipocytes were then incubated for 24
h in serum-free DMEM/Ham’s F-12 with or without 6 nmol/L recombinant
human TNF in the presence or absence of
10-8 and
10-7 mol/L Dex and
10-7 mol/L insulin.
Preadipocyte culture
Preadipocytes that formed pellets after centrifugation at
250 x g (36) were resuspended in red
blood cell lysing buffer (154 mmol/L NH4Cl, 5.7
mmol/L K2HPO4, and 0.1
mmol/L ethylenediamine tetraacetate, pH 7.0) and incubated for 10 min.
Cells were centrifuged at 150 x g for 5 min and
resuspended in DMEM/Ham’s F-12 medium supplemented with 15% bovine
FCS (First Link UK Ltd., Brierley Hill, UK). Cells (2.5 x
104/cm2) were cultured for
72 h in six-well plates to reach confluence and were incubated
(1–24 h) in serum-free DMEM/Ham’s F-12 medium with or without varying
concentrations of recombinant human TNF (0.3–10 nmol/L), Dex
(10-11–10-7
mol/L), and insulin
(10-10–10-6
mol/L). Cells were used for total RNA extraction. Conditioned media
were stored at -80 C and used for measurement of IL-1ß by ELISA. For
apoptosis studies, preadipocytes were cultured on 20 x 20-mm
coverslips in six-well plates and treated with or without 6 nmol/L
TNF in the presence or absence of Dex
(10-8–10-7
mol/L) and insulin (10-7
mol/L).
RNA extraction
Total RNA was extracted from cultured preadipocytes and adipocytes using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. RNA was treated with RQ-1 deoxyribonuclease (Promega Corp., Madison, WI) and quantified by absorbance at 260 nm in a spectrophotometer (Ultrospec III, Pharmacia LKB, Piscataway, NJ). The integrity of the RNA was verified by ethidium bromide staining of ribosomal RNA (rRNA) bands on a 1% agarose gel.
Analysis of TNF-induced gene expression by RT-multiplex PCR
(RT-MPCR)
RT-MPCR amplifies multiple genes in a single PCR reaction.
Therefore, variations in RNA quality, initial quantitation errors, and
random tube to tube variations in RT-PCR reactions can be compensated.
Additionally, the expression of the gene of interest can be normalized
against the amplified internal control. The mRNA expression of ICE,
TNF, NFB, bcl-2, IB, and the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed by
RT-MPCR (Maxim Biotech, Inc., San Francisco, CA) according to the
manufacturer’s protocol with modifications. Two micrograms of total
RNA were reverse transcribed for 1 h at 42 C in 25 µL buffer
[50 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 10 mmol/L
MgCl2, and 10 mmol/L dithiothreitol] containing
50 U AMV reverse transcriptase, 1 µg random hexamer, 1 mmol/L
deoxy-NTP, and 40 U RNasin ribonuclease inhibitor. All reagents were
supplied by Promega Corp. complementary DNA (cDNA) was
denatured for 5 min at 94 C, and 2.5 µL cDNA from each sample were
used for subsequent PCR amplification in a final volume of 25 µL
buffer containing 2.5 U Taq DNA polymerase (Promega Corp.), and supplied sense and antisense primers (Maxim Biotech, Inc., USA). To determine the linearity of amplification
for quantitation, PCR was performed for 26–40 cycles, and
amplification of all genes was linear between 26–32 cycles. For
further PCR, reactions were performed at 96 C for 1 min and 56 C for 4
min for 2 cycles and at 94 C for 1 min and 56 C for 2.5 min for 29
cycles. The amplified fragments correspond to 921 bp (GAPDH), 658 bp
(ICE), 535 bp (TNF), 409 bp (the p65 subunit of NFB), 235 bp
(bcl-2), and 158 bp (the L-factor subunit of IB). PCR was
negative when reactions were performed without AMV reverse
transcriptase during RT reactions, indicating the absence of DNA
contamination in RNA samples.
To provide an additional control for each RT-MPCR, a parallel 18S rRNA RT-PCR using the same cDNA, PCR conditions, and 18S primers was performed using the QuantumRNA 18S kit, which contains both 18S rRNA primers and competimers (AMS Biotechnology Europe Ltd., Abingdon, UK). 18S rRNA competimers are modified at their 3'-ends to block extension by DNA polymerase. By mixing 18S primers with increasing concentrations of 18S competimer, the overall PCR amplification efficiency of 18S cDNA was reduced without the primers becoming limiting due to abundance of 18S rRNA. In this study, a 3:7 ratio of 18S primers to competimers was chosen, and PCR was performed at 56 C for 16–36 cycles to determine the linear range of amplification. The linear range was 18–26 cycles, and 23 cycles were chosen for further PCR to offset any variations in RT-MPCR. The 488-bp 18S PCR product was separated on 2% agarose gels stained with ethidium bromide.
Analyses of IL-1ß mRNA by RT-MPCR
RT-MPCR was performed to analyze IL-1ß mRNA using 18S rRNA as an internal standard. QuantumRNA 18S primers and competimers at a ratio of 2:8 were added to the PCR reaction with two IL-1ß primers that were designed using the Primer3 software available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html. The primer sites are located at nucleotides 591–610, and 955–974 of human IL-1ß complete cDNA (GenBank accession no. M15330). The primer sequences are: upstream, 5'-AATGACAAAATACCTGTGGC-3'; and downstream, 5'-AAACCTTTCTGTTCCCTTTC-3'. RT was performed using the method described above. PCR was carried out at 94, 55, and 70 C, 1 min each, for 29 cycles (within the linear range of amplification). The amplified IL-1ß (384-bp) and 18S (488-bp) cDNAs were separated on 2% agarose gels.
Images of UV-illuminated agarose gels were captured using UVP ImageStore 5000 (UV Products Ltd., San Gabriel, CA). The density of cDNA bands were analyzed using Gelbase/Gelblot software (UV Products, Ltd.). Relative changes in the mRNA levels of apoptosis-related genes were assessed by comparing the density ratios of the gene of interest against both GAPDH and 18S. Changes in IL-1ß mRNA expression were assessed by comparing the density ratios of IL-1ß against 18S cDNA.
Quantitation of IL-1ß release with ELISA
IL-1ß release was measured using a Quantikine immunoassay kit that detects active IL-1ß (the mature cleaved form) in cell culture supernatants (R\|[amp ]\|D Systems Europe Ltd., Abingdon, UK). Briefly, recombinant human IL-1ß standards and cell culture media were added to the microtiter plate that was precoated with a monoclonal antibody specific for human IL-1ß. Bound IL-1ß was detected with a horseradish peroxidase-conjugated polyclonal antibody using hydrogen peroxide and chromogen as substrates. Chemiluminescence, developed after the addition of sulfuric acid, was measured at 450 nmol/L and for correction at 540 nmol/L using a Labsystems bichromatic multiskan microplate reader (Labsystems, Farnborough, UK).
Apoptosis detection by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-fluorescein nick-end labeling and propidium iodide staining (TUNEL)
Apoptosis was detected by TUNEL and propidium iodide (PI) staining of nuclear DNA using methods previously described (36). Apoptotic features shown by both PI and FITC fluorescence of cell nuclei were identified, and apoptotic indexes were obtained by counting 250 cells in at least 4 fields of view.
Statistics
All experiments in the study were performed using adipose tissue
from at least three patients (n 
3). Data from representative
preparations are shown. One-way ANOVA and paired Student’s
t test were used for data analysis in the study. Data are
the mean ± SEM. P < 0.05
was considered significant.
Results
TNF increased the mRNA expression of ICE, TNF, NFB, and
bcl-2 in human preadipocytes and adipocytes
After RT-MPCR and parallel 18S rRNA RT-PCR, regulation of the
expression of apoptosis-related genes by TNF was analyzed by
comparing their density ratios against those of the housekeeping gene
GAPDH and 18S rRNA. The data are shown in Fig. 1A
. TNF treatment (8 h) induced
dose-dependent increases in the expression of ICE, TNF, NFB, and
bcl-2 in human preadipocytes (Fig. 1A; P <
0.01 at 1 nmol/L or more), whereas it had no significant effect on the
expression of IB. A concentration of 6 nmol/L (
100 ng/mL) TNF
was chosen for subsequent experiments, because significant effects were
found at this concentration. The use of GAPDH as an internal control
for RT-MPCR in the study was validated by plotting the density ratios
of each gene against GAPDH and 18S rRNA independently, and this
revealed closely comparable changes (variation <10%) in the
expression of each gene.
|
shows the time course of the effects of TNF treatment on
preadipocytes. ICE mRNA was increased 2.5-fold (P <
0.01), with a maximum increase at 8 h. TNF mRNA was increased
up to 3-fold at 1 h (P < 0.01), followed by a
gradual decline to the control level at 16 h. There were gradual
increases in NFB and bcl-2 mRNA, rising to 2-fold at
8 h (P < 0.01).
In mature human adipocytes, dose-dependent increases in the gene
expression of ICE, TNF, NFB, and bcl-2 were also
observed after treatment with TNF (Fig. 2A). At 6 nmol/L, TNF increased the
expression of ICE 3-fold (P < 0.01), that of TNF
2.5-fold (P < 0.01), that of NFB 2.1-fold
(P < 0.01), and that of bcl-2 2.7-fold
(P < 0.01). Figure 2B shows that these increases were
observed after 24-h treatment. They coincide with the general decline
in these mRNAs in control (nontreated) samples during 2- to 24-h
incubation (data not shown).
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-induced ICE and TNF mRNA expression
To determine the effects of Dex and insulin on TNF-induced
apoptosis-related gene expression, preadipocytes were treated with 6
nmol/L TNF for 8 h in the presence/absence of varying
concentrations of Dex and insulin. Figure 3A shows the dose-dependent inhibitory
effect of Dex on TNF-induced gene expression. Dex
(10-7 mol/L) significantly
reduced TNF-induced ICE mRNA by 56% (P < 0.01) and
TNF mRNA by 54% (P < 0.01). The reductions in
TNF-induced expression of NFB (by 16%) and bcl-2 (by
14%) were not statistically significant. Insulin treatment
(10-10–10-6
mol/L) had no significant effect on TNF- induced gene expression
(data not shown).
|
shows the effects of Dex and insulin on TNF-induced gene
expression in mature human adipocytes. Dex treatment
(10-7 mol/L for 24 h)
decreased TNF-induced mRNA expression of ICE by 29%
(P < 0.05) and TNF by 36% (P <
0.01), but had no significant effect on NFB and bcl-2
expression. Insulin alone
(10-7 mol/L for 24 h)
had no significant effect on TNF-induced gene expression. Combined
treatment with Dex (10-7
mol/L) and insulin (10-7
mol/L), however, synergistically inhibited TNF induced mRNA
expression of ICE by 52% (P < 0.01), TNF by 57%
(P < 0.01), NFB by 37% (P <
0.01), and bcl-2 by 12% (P > 0.05).
Similarly, in preadipocytes the combination of Dex and insulin
synergistically inhibited TNF-induced expression of ICE by 67%
(P < 0.01), TNF by 69% (P <
0.01), NFB by 35% (P < 0.01), and bcl-2
by 29% (P < 0.01).
Dex inhibited TNF-induced IL-1ß mRNA and protein release in
human preadipocytes and mature adipocytes
The expression and regulation of IL-1ß by TNF, Dex, and
insulin in human adipose cells were investigated. In human
preadipocytes, treatment with 6 nmol/L recombinant human TNF
increased IL-1ß mRNA expression 5.3-fold (P < 0.01)
at 1 h and 4.2-fold (P < 0.01) at 8 h.
Preincubation with 10-7
mol/L Dex for 8 h reduced TNF-induced IL-1ß mRNA to almost
control levels at both time points. Coincubation with TNF and Dex
(10-7 mol/L) inhibited
TNF-induced IL-1ß mRNA by 93% at 1 h (P <
0.01) and 83% at 8 h (P < 0.01; Fig. 4). Coincubation with
10-7 mol/L insulin
inhibited TNF-induced IL-1ß expression by 11%, but the inhibition
was not significant (data not shown).
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for 24 h (Fig. 5). Preincubation with
10-7 mol/L Dex (12 h) and
cotreatment with Dex and TNF reduced TNF-induced IL-1ß release
to control levels. Treatment with Dex alone
(10-7 mol/L) reduced
IL-1ß release to 60.6% of the control level. Treatment with insulin
(10-7 mol/L) produced no
effect on IL-1ß release. Combined treatment with insulin
(10-7 mol/L) and Dex
(10-7 mol/L) had no
further effect on TNF-induced IL-1ß release than Dex alone.
|
(6 nmol/L for 2–24 h) had no significant
effect on IL-1ß mRNA expression (Fig. 6). Coincubation with 6 nmol/L TNF and
10-7 mol/L Dex for 24
h reduced IL-1ß mRNA to 30% of that in TNF (alone)-treated
samples (P < 0.01). Coincubation with
10-7 mol/L insulin
decreased IL-1ß mRNA by 27% (P < 0.01).
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increased IL-1ß release from mature adipocytes 1.5-fold after
8 h (25.5 vs. 37.2 pg/mL; P < 0.01),
5.2-fold after 8–24 h (9.2 vs. 48 pg/mL; P
< 0.001), and 20-fold after 24–48 h of treatment (3.4 vs.
68.1 pg/mL; P < 0.001) as shown in Fig. 7. Treatment with Dex
(10-10–10-6
mol/L) decreased TNF-induced IL-1ß release in a dose-dependent
manner (data not shown). At
10-7 mol/L, it completely
abrogated TNF- induced IL-1ß release (P <
0.001; Fig. 7). In contrast, insulin
(10-7 mol/L) had no effect
on IL-1ß release. Combined treatment with insulin and Dex had no
further effect on TNF-induced IL-1ß release than Dex alone.
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-induced apoptosis of human preadipocytes and
adipocytes
After 24-h treatment with 6 nmol/L TNF with
or without treatment with Dex
(10-8 and
10-7 mol/L) and insulin
(10-7 mol/L), apoptosis of
preadipocytes and mature adipocytes was assayed by TUNEL and PI
staining of nuclear DNA (36). Abnormal nuclear changes
characteristic of apoptosis, including clumping of chromatin beneath
the nuclear envelope, convolution of the nuclear outline, and
disintegration of the cell nucleus, were observed. After 24 h in
serum-free conditions, 10.2% of preadipocytes were undergoing
apoptosis (Fig. 8A). Treatment with Dex
alone had no significant effect. Treatment with TNF (6 nmol/L)
increased apoptosis almost 2-fold to 18.6%, but preincubation with
10-7 mol/L Dex for 8
h before treatment with TNF completely blocked TNF-induced
apoptosis (mean ± SEM, 10.7 ± 2.1%;
P < 0.01 vs. TNF-treated). Cotreatment
with Dex (10-7 and
10-8 mol/L) also
effectively inhibited TNF-induced apoptosis (P <
0.01 vs. TNF-treated). Insulin alone
(10-7 mol/L) and in
combination with TNF had no significant effect on apoptotic indexes.
Combined treatment with insulin and Dex had no additional effect in
preventing TNF-induced apoptosis than Dex alone.
|
). Treatment with Dex alone during serum
deprivation did not significantly affect the apoptotic index.
Treatment with TNF increased apoptosis almost 2-fold to 38.6 ±
2.5% (P < 0.01). Coincubation with
10-7 mol/L Dex
significantly decreased apoptosis to 25.4 ± 1.7%
(P < 0.01 vs. TNF-treated) and to
31.5 ± 2.6% (P < 0.01 vs.
TNF-treated) with 10-8
mol/L Dex. Insulin alone
(10-7 mol/L) and in
combination with TNF had no significant effect on apoptotic indexes.
Combined treatment with insulin and Dex had no further effect in
preventing TNF-induced apoptosis than Dex alone. Discussion
Consistent with the general belief that TNF induces both pro-
and antiapoptotic mediators, TNF treatment of human sc preadipocytes
and mature adipocytes led to dose-dependent increases in the gene
expression of ICE, TNF, NFB, and bcl-2. In
preadipocytes, the increase in TNF mRNA was rapid (maximum at 1
h), whereas the increases in ICE, NFB, and bcl-2 mRNAs
occurred later (1–8 h). Rapid induction of endogenous TNF mRNA and
subsequent protein translation by exogenous TNF have been reported
in human myosarcoma (KYM-S) cells, murine tumorigenic fibroblasts (L-M)
(37), and human ZR-75–1 breast carcinoma cells
(38) and have been associated with acquired resistance to
TNF-induced cytotoxicity in these cell lines. We confirmed by
immunostaining (data not shown) that adipocytes produce detectable
levels of TNF. The local concentrations of TNF in adipose
interstitial fluid are unknown, but could conceivably reach the
concentrations used in this study. This and the findings that exogenous
TNF induces apoptosis in both human preadipocytes and mature
adipocytes (3) questions the association of overexpression
with resistance in human adipose cells. Locally produced TNF may
potentiate its adipostat effects by autocrine/paracrine effects on its
own production.
Dex treatment inhibited TNF-induced mRNA expression of ICE and
TNF in both sc preadipocytes and adipocytes. Insulin in combination
with DEX produced a synergistic inhibition of TNF-induced mRNA
expression of ICE, TNF, NFB, and bcl-2. The selective
inhibitory effects of Dex may offset the balance between TNF-induced
proapoptotic and antiapoptotic mediators and thereby modify the
apoptotic effects of TNF. The mRNA expression of IB in
preadipocytes and adipocytes was not affected by treatment with TNF,
Dex, and/or insulin, but the expression of this gene was low in all
conditions.
In mature sc adipocytes, TNF-mediated increases in mRNA expression
were observed only after 24-h treatment compared with 1–8 h for the
preadipocytes. The differences in the time course of TNF-mediated
changes in the two cell populations may relate to the culture
conditions. Preadipocytes were cultured in serum-containing medium for
72 h (to reach confluence) before TNF treatment, whereas mature
adipocytes were treated immediately after isolation. The mRNAs of
ICE, TNF, NFB, and bcl-2 in control (nontreated)
adipocytes decreased with incubation time (2–24 h), suggesting that
the isolation procedure itself may have increased the expression of
these genes and overshadowed the early effects of TNF, such as the
rapid induction of endogenous TNF mRNA seen in preadipocytes.
TNF (6 nmol/L) increased IL-1ß mRNA expression 5-fold in human sc
preadipocytes, but had no significant effect on mature adipocytes. Dex
decreased TNF-induced IL-ß mRNA expression to almost control
levels in preadipocytes and to 70% of the control levels in mature
adipocytes. Dex has been shown to decrease IL-1ß mRNA expression in
the human promonocytic cell line U-937 by inhibiting IL-1ß gene
transcription and decreasing the stability of IL-1ß mRNA
(39). Furthermore, Dex is known to down-modulate (via
glucocorticoid receptor protein) the activity of the activator
protein-1 complex, a transcription factor that regulates the expression
of various cytokines, including TNF and IL-1ß (40, 41). The inhibition of IL-1ß and TNF mRNAs by Dex in the
present study may be mediated by similar mechanisms.
Dex also abrogated TNF-induced IL-1ß protein release from human sc
preadipocytes and adipocytes. The decrease in IL-1ß release may
result from decreased IL-1ß mRNA expression in treated cells.
However, in mature adipocytes the discrepancy between the effects of
Dex on IL-1ß mRNA (no effect at 8 h and only 30% reduction at
24 h) and the effects on IL-1ß release (40% decrease in total
IL-1ß release after 8 h and 91% decrease after 8–24 h)
suggests that decreased IL-1ß release may also result from inhibition
of ICE activation. The time-dependent (8–48 h) decrease in IL-1ß
release from control mature adipocytes may correlate with their
recovery from the isolation procedure. TNF treatment increased
IL-1ß protein release from both preadipocytes (5 fold) and adipocytes
(1.5- to 20-fold), indicating ICE activation in these cells. Dex
inhibits IL-1ß release from murine peritoneal macrophages
(42) and decreases circulating IL-1ß levels in mice
(43). The inhibition of IL-1ß release from both adipose
cells and macrophages may thus reflect a general effect of
glucocorticoids on production of this cytokine.
IL-1ß plays a pivotal immunological role in host defense against
infections. It also stimulates lipolysis and inhibits lipogenesis by
inhibiting the expression of fatty acid transport protein and fatty
acid translocase in adipose tissue (44). In this study we
have shown mRNA expression of IL-1ß and the release of assayable
protein. The finding that IL-1ß is released by human sc adipose cells
and that it is regulated by TNF may have significant in
vivo implications. Firstly, increased TNF production in
obesity, infection, or malignancy may act on adipose tissue to increase
the expression and release of IL-1ß, which, in turn, regulates lipid
metabolism and synergizes with other effects of TNF. Secondly,
adipose tissue constitutes up to 10–30% of the total body weight and
produces a range of cytokines, including TNF (4, 5);
IL-6 (45); complement factors D, B, and C3
(46); and macrophage colony-stimulating factor
(47). The finding that this large depot also produces and
releases IL-1ß in response to TNF suggests a possible
immunological role for adipose tissue. Conceivably, elevated TNF
during infection may stimulate IL-1ß release from adipose tissue,
potentiating the host immune response. These effects of TNF and
IL-1ß may be modulated by glucocorticoids in obesity or
therapeutically administered for combating inflammatory diseases.
Whether glucocorticoids exert the same antagonistic effects on
TNF-induced apoptosis and interleukin-1ß release in adipocytes
from visceral fat and other fat depots remains to be determined.
The outcome of TNF activation, i.e. cell death or cell
survival, depends on the balance of pro- and antiapoptotic mediators.
Dex selectively inhibited TNF-induced gene expression of ICE,
TNF, IL-1ß, and IL-1ß release in human sc adipocytes and
preadipocytes. Combined treatment with Dex and insulin synergistically
inhibited TNF-induced gene expression, although insulin alone had no
significant effect. Dex treatment decreased TNF-induced apoptosis to
control levels, but in apoptosis assays, there was no synergism with
insulin. This may be because insulin in combination with Dex inhibited
not only the proapoptotic genes, ICE and TNF, which were induced by
TNF, but also the antiapoptotic genes, NFB and Bcl-2. Consistent
with the lack of synergy of insulin with Dex on apoptosis are the data
showing no synergistic effect on IL-1ß release, which supports the
role of this cytokine in mediating the effects of TNF. ICE is only
one member of the large caspase family. The effects of Dex on other
TNF-induced apoptosis mediators, such as other ICE-related
proteases, may be involved in its inhibition of apoptosis.
Glucocorticoids and TNF exert opposing effects on food intake and on
adipose differentiation and lipogenesis. Both are produced in adipose
tissue and may thus regulate adipose tissue mass in an
autocrine/paracrine as well as a neuroendocrine manner. Our findings
that Dex inhibited TNF-induced apoptosis, IL-1ß release, and
TNF expression in adipose tissue provide the first direct in
vitro evidence that glucocorticoids inhibit TNF actions in
adipose tissue and delineate a possible mechanism by which TNF
resistance occurs in obesity.
Acknowledgments
We thank all the operative surgeons and theater staff at the University Hospitals Trust, and Mr. P. L. Levick at the Priory Hospital in Birmingham for their assistance.
Footnotes
1 This work was supported by the University of Birmingham, the
British Diabetic Association, and Eli Lilly & Co. UK. 
2 Current address: Jean Mayer U.S. Department of Agriculture Human
Nutrition Research Center on Aging, Tufts University, Boston,
Massachusetts 02111-1524.
Received October 3, 2000.
Revised January 12, 2001.
Accepted March 16, 2001.
References
prevents the differentiation of human adipocyte precursor
cells and causes delipidation of newly developed fat cells. J Clin
Endocrinol Metab. 76:742–747.[Abstract]
induces apoptosis of human adipose cells. Diabetes. 46:1939–1944.[Abstract]
in human obesity and insulin resistance. J
Clin Invest. 95:2409–2415.
. Cell. 73:625–627.[CrossRef][Medline]
and interleukin 1ß enhance
the cortison/cortisol shuttle. J Exp Med. 186:189–198.B
activation in primary hippocampal neurons. J Biol Chem. 274:8531–8538.B during monocyte differentiation is associated
with activation of HIV-gene expression. Res Virol. 142:233–238.[CrossRef][Medline]
B through
the degradation of the cytoplasmic inhibitor IB. J Biol
Chem. 273:23946–23951.B in preventing TNF--induced cell death. Science. 274:782–784.B. Science. 274:784–787. release from human
adipose tissue in vitro. J Endocrinol. 163:33–38.[Abstract]
priming increases glucocorticoid inhibition of
TNF- induced cytotoxicity/apoptosis. J Clin Invest. 98:1409–1416.[Medline]
cytotoxicity. J Endocrinol Invest. 16:591–599.[Medline]
B activity in human leukemic T cells. Endocrinology. 139:3813–3821.This article has been cited by other articles:
 |
|
 |
|
  A. R. Schwartz, S. P. Patil, A. M. Laffan, V. Polotsky, H. Schneider, and P. L. Smith Obesity and Obstructive Sleep Apnea: Pathogenic Mechanisms and Therapeutic Approaches Proceedings of the ATS, February 15, 2008; 5(2): 185 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wu, T. Pew, M. Zou, D. Pang, and S. D. Conzen Glucocorticoid Receptor-induced MAPK Phosphatase-1 (MPK-1) Expression Inhibits Paclitaxel-associated MAPK Activation and Contributes to Breast Cancer Cell Survival J. Biol. Chem., February 11, 2005; 280(6): 4117 - 4124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Skurk, V. van Harmelen, and H. Hauner Angiotensin II Stimulates the Release of Interleukin-6 and Interleukin-8 From Cultured Human Adipocytes by Activation of NF-{kappa}B Arterioscler. Thromb. Vasc. Biol., July 1, 2004; 24(7): 1199 - 1203. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. N. Fain, S. W. Bahouth, and A. K. Madan Haptoglobin release by human adipose tissue in primary culture J. Lipid Res., March 1, 2004; 45(3): 536 - 542. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Y. Um, H.-S. Chung, M.-Y. Song, H.-D. Shin, and H.-M. Kim Association of Interleukin-1{beta} Gene Polymorphism with Body Mass Index in Women Clin. Chem., March 1, 2004; 50(3): 647 - 650. [Full Text] [PDF]
|
|
|
|
 |
|
 I. Herr, E. Ucur, K. Herzer, S. Okouoyo, R. Ridder, P. H. Krammer, M. von Knebel Doeberitz, and K.-M. Debatin Glucocorticoid Cotreatment Induces Apoptosis Resistance toward Cancer Therapy in Carcinomas Cancer Res., June 15, 2003; 63(12): 3112 - 3120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Friedberg, E. Zoumakis, N. Hiroi, T. Bader, G. P. Chrousos, and Z.'e. Hochberg Modulation of 11{beta}-Hydroxysteroid Dehydrogenase Type 1 in Mature Human Subcutaneous Adipocytes by Hypothalamic Messengers J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 385 - 393. [Abstract] [Full Text] [PDF]
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