Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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ABSTRACT |
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Cytokines, such as tumor necrosis factor (TNF)
and interleukin-6, may contribute to the anorexia and cachexia of
infection, cancer, and AIDS. The present study tests the hypothesis
that endotoxin alters the expression of two key fat cell proteins, leptin and 3-adrenergic
receptor (
3-AR), through a
mechanism involving TNF-
. Increasing doses of
Escherichia coli endotoxin (lipopolysaccharide, LPS) resulted in dose-dependent elevations of
plasma leptin (maximal response ~7-fold, half-maximal effective dose
of ~16 µg/100 g body wt) and white fat leptin mRNA in C3/HeOUJ mice. LPS also produced a large decrease in adipose tissue
3-AR mRNA and a parallel
reduction in
-agonist-induced activation of adenylyl cyclase.
Changes in plasma leptin and
3-AR mRNA were preceded by an
approximately threefold increase in white fat TNF mRNA. TNF
administration resulted in changes similar to those seen with LPS. We
conclude that endotoxemia results in an induction of leptin mRNA and a
decrease in
3-AR mRNA in
adipose tissue, an effect that may be mediated by alterations in
TNF-
.
cytokine; tumor necrosis factor-; mice
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INTRODUCTION |
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ANOREXIA AND CACHEXIA, syndromes of depressed appetite, weight loss, and wasting, complicate many diseases, including cancer (33), AIDS (10), chronic infection, and other critical illnesses (3). Both decreased caloric intake and an increased metabolic rate have been observed in cachectic patients (8), but little is known regarding the specific mechanisms responsible for altered appetite and metabolism. Substantial animal and human data suggest a possible role of cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-1 (19); however, the cellular mechanisms by which these factors induce anorexia and cachexia are unknown.
Recent data indicate that cytokines alter the expression of several adipose tissue-specific genes (28). TNF decreases the activity of lipoprotein lipase (11), reduces expression of the glucose transporter GLUT-4 (29), and increases the rate of lipolysis (20). The effect of TNF on GLUT-4 is hypothesized to contribute to development of insulin resistance (15, 16). Related studies suggest a role for endogenous TNF in the regulation of fat cell size, with increases in fat cell lipid content leading to augmented TNF production, which in turn initiates responses to limit adipocyte size (14).
A variety of animal and human studies have suggested a possible
etiologic role for TNF- and other cytokines in cachexia (19, 34). We
hypothesize that TNF may contribute to wasting by altering adipocyte
gene expression. Two possible candidate genes are those coding for
synthesis of leptin and the
3-adrenoceptor
(
3-AR). Leptin is a fat cell
hormone that regulates both appetite and metabolic activity (for
review, see Refs. 2, 27). Recent data suggest that leptin is the
principal means by which information regarding the adequacy of
peripheral fat stores is communicated to the central nervous system,
with decreases in plasma leptin concentration playing a central role in
triggering adaptive responses to starvation (1). Moreover, Grunfeld et
al. (13) have demonstrated that lipopolysaccharide (LPS) and exogenous
TNF both prevent fasting-induced decreases in leptin. A second possible
site for cytokine action is the
3-AR, which is important in
transducing signals for sympathetically mediated lipolysis in white
adipose tissue and thermogenesis in brown adipose tissue (for review,
see Ref. 31). Little is known regarding possible effects of cytokines
on this receptor system; however, TNF is known to reduce
2-AR signal transduction in
select cell lines (5). The importance of the
3-AR system in energy homeostasis is highlighted by studies demonstrating weight gain after
targeted disruption of this receptor system (32) and weight loss with
administration of exogenous
3-AR agonists (36). The present
study examines the effect of LPS on these two adipocyte systems and
probes the potential role of TNF in mediating these actions.
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METHODS |
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Animal protocols.
All experimental procedures and protocols were approved by the
Institutional Animal Care and Use Committee and are accepted by the
American Association for Accreditation in Laboratory Animal Care.
Animals were housed at 25°C, five to a cage, with free access to
standard rodent diet and water. For all experiments, 8-wk-old C3H/HeOUJ
mice were used. In preliminary experiments, we found that high-dose LPS
produces marked anorexia, reducing food intake from 3.1 to 0 g/day.
Therefore, to avoid confounding effects of varying food intake on
leptin expression, all animals were fasted after LPS (or saline)
administration. To determine the effect of increasing doses of LPS on
the expression of leptin and
3-AR mRNA, animals
(n = 5/group) received intraperitoneal
endotoxin at one of the following doses: 0, 0.5, 5.0, 50, or 500 mg/100 g. For this dose-response study, animals were killed 16 h after LPS
administration. After the animals were killed, blood was obtained for
measurement of leptin concentration, and epididymal white fat was
removed and rapidly frozen in liquid nitrogen for quantification of
3-AR, leptin, and TNF mRNA
concentrations and adenylyl cyclase responses to
-agonist
stimulation.
RT-PCR.
For RT-PCR of TNF- mRNA, 1 µg of total RNA was reverse transcribed
with Moloney murine leukemia virus RT (GIBCO BRL, Gaithersburg, MD) for
30 min at 37°C in a solution containing 500 nM oligo(dT), 0.2 mM of
each dNTP, 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol.
RNA template was digested with RNase H. cDNA was denatured for 5 min at
94°C and submitted to 30 cycles of amplification in a solution
containing 0.5 U of Taq polymerase
(Perkin Elmer) and 1 mM sense and antisense oligonucleotides. For TNF,
the amplified fragment corresponds to nucleotides 946-1738 of the
mouse genomic sequence (23). This sequence spans an intron (intron 2);
we are thus able to determine that the amplified fragment does not
arise from genomic contamination. The sense and antisense primers are
1) GCC CAG ACC CTC ACA CTC AG and
2) CTT GGG GCA GGG GCT CTT GA. To
determine the linearity of amplification for quantitation, PCR was
performed for 25-30 cycles and found to be linear over this range.
For further PCR reactions, 27 cycles were chosen. The PCR cloning of
the leptin and
3-AR RNase
protection assay (RPA) probes is described below.
Mouse leptin and 3-AR
cRNA probe synthesis.
Fragments of the leptin and
3-AR genes were amplified by
using RT-PCR with specific primers containing
EcoR
I/BamH I primer linkers. These cDNA
fragments were cloned into the transcription vector, pGEM-4Z. The
leptin PCR product is a 331-bp fragment that spans the intron and
corresponds to 92-2,155 bp of the mouse genomic sequence (37). The
3-AR PCR product used for probe
is a 257-bp fragment corresponding to 1,090-1,347 bp of the mouse
3-AR sequence (18). Both probes
were sequenced in their entirety before use. Mouse white fat cDNA was
used as template for the PCR reactions (GIBCO BRL). Radiolabeled cRNA
probes for use in the RPA were synthesized from cloned cDNA fragments
using [
-32P]CTP
(NEN) and T7 polymerase (Ambion, Austin, TX) according to their
published protocol.
RNA quantification by RPA.
RNA was extracted from fat tissue by the method of Chomczynski using
RNAzol (Tel-test, Friendswood, TX) and quantitated
spectrophotometrically at 260/280 nm. RPAs were performed as previously
described (21). In brief, 20 µg of total RNA were coprecipitated with
~105 counts/min of the cRNA probe and then dissolved in 30 µl of
hybridization buffer [80% formamide, 40 mM PIPES (pH 6.4), 400 mM NaCl, and 1 mM EDTA]. The solution was then heated for 5 min
at 85°C, followed by an overnight incubation at 55°C. RNase
digestion was accomplished by using 300 µl of RNase solution
[RNase A (40 µg/ml, Sigma) and RNase T1 (800 U/ml, GIBCO BRL)
in 300 mM sodium acetate and 5 mM EDTA] at 30°C for 60 min
and was terminated by addition of 10 ml of 20% SDS and 2.5 ml of
proteinase K (15 mg/ml) for 30 min at 37°C. After phenol-chloroform
extraction, RNA was precipitated with 20 µg of tRNA as a carrier.
After resuspension in 4 ml of loading buffer [80% formamide, 20 mM EDTA (pH 8.0), 0.1% bromophenol blue, and 0.1% xylene
cyanole], the samples were separated by electrophoresis on a
denaturing 8 M urea-6% acrylamide gel, followed by drying and exposure
to XAR-5 (Kodak) film for 12-48 h at 70°C. Protected
fragments were quantified after exposure of PhosphoImager screens by
image-analysis software (Imagequant, Molecular Dynamics, Sunnyvale,
CA). Because it has been shown that LPS and cytokines increase levels
of mRNA for actin and cyclophylin (13), two mRNAs commonly used for
normalizing data, equal amounts of total RNA were coprecipitated in our
RPA protocol.
Measurement of plasma leptin. Mouse plasma leptin concentrations were measured by radioimmunoassay (Linco Research, St. Charles, MO). Samples were incubated overnight with mouse leptin antibody, after which 125I-labeled leptin was added and allowed to equilibrate for 18 h. Precipitated antibody was separated by centrifugation and counted in a Minaxi 5000 gamma counter, and leptin concentrations were derived by interpolation from a standard curve. The sensitivity of this assay is 0.2 ng/ml, and intra-assay variability is <3%.
Adenylyl cyclase activity.
Frozen adipose tissue was homogenized in 2 ml of cold buffer [0.1
M HEPES (pH 7.4), 25 mg/ml aprotinin, and 25 mg/ml leupeptin] in
ice using a Tissuemizer high-speed cutting blade (Tekmar, Cincinnati, OH). The homogenate was used immediately for protein determination and
adenylyl cyclase assays. Adenylyl cyclase activity was determined as
previously described with minor modifications (4). Briefly, adipocyte
membranes (60 mg) were incubated for 10 min at 30°C in 100 ml of
buffer [50 mM HEPES (pH 8.0), 50 mM NaCl, 0.4 mM EGTA, 0.25 mg/ml
BSA, 5 mM MgCl2, 1 mM
[-32]ATP
(0.1-0.3 mCi/mmol), 1 mM cAMP, 7 mM creatine phosphate, and 50 m/ml creatine kinase]. Adenylyl cyclase activity was determined in the basal (unstimulated) state and in response to GTP (10 mM), GTP
plus isoproterenol (100 mM), GTP plus the
3-AR-selective agonist
CL-316243 (1 nM-100 mM), forskolin (10 mM),
NaF-AlCl3 (10 mM NaF + 100 mM
AlCl3), and
MnCl2 (20 mM). Preliminary
experiments confirmed the linearity of adenylyl cyclase activity at the
protein concentrations and incubation times used. cAMP was isolated by sequential column chromatography through Dowex and alumina (24), with
column recoveries of 75-90%. Protein was assayed with the Pierce
Chemical (Rockford, IL) bicinchoninic acid (BCA) protein assay reagent,
consisting of BCA and copper sulfate solutions (3). BSA was used as a
standard (26).
Data analysis. ANOVA techniques were used to evaluate effects of both varying doses of LPS and exposure times on plasma leptin and fat cell mRNA levels. Dunnett's test was used for pairwise comparisons. Nonlinear curve fit analysis was used to establish dose-response relationships. Effects of TNF on leptin and fat cell mRNA were evaluated by use of independent t-tests, with Bonferonni correction for multiple comparisons. RNA data are expressed as counts and are expressed as means ± SE. Effects of LPS on basal and stimulated adenyl cyclase activity were analyzed by paired, two-tailed Student's t-tests. Dose-response curves of CL-316243-stimulated adenyl cyclase activity in LPS and control animals were determined by use of nonlinear curve-fit analysis and were compared by Friedman nonparametric repeated measures test, with Dunn's multiple posttest used for comparisons between groups. Data were considered to be significantly different at P < 0.05.
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RESULTS |
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Escherichia coli endotoxin produced
dose-dependent increases in plasma leptin concentrations (Fig.
1), with a half-maximal effective dose of
~16 µg/100 g. At the highest LPS dose (500 µg/100 g), plasma
leptin concentrations were approximately seven times higher than in
saline-treated controls (13.9 ± 0.6 vs. 2.0 ± 0.06 ng/ml). LPS
had similar effects on white fat leptin mRNA (Fig. 2A). In
contrast to the observed effect on leptin, LPS administration reduced
white fat 3-AR mRNA (Fig.
2B). Adenyl cyclase responses of
adipose tissue membranes from animals treated with LPS demonstrated reduced responses to both nonselective and
3-selective agonists (Fig.
3), whereas basal adenylyl cyclase activity was not
altered (85 ± 9.5 and 66 ± 15.0 pmol
cAMP · mg
protein
1 · 10 min
1). There was no
effect of LPS on cyclase activation by G protein activators (GTP and
NaF) or by direct activators of adenylyl cyclase (forskolin and Mn).
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Studies examining effects of LPS on adipocyte TNF mRNA expression
demonstrated increased expression of this cytokine [26.6 ± 1.1 vs. 88.4 ± 8.1 (arbitrary units × 103),
n = 5, P < 0.01]. When TNF was
administered to mice, leptin and
3-AR leptin mRNA expression
changed in a manner similar to that seen with LPS (Fig.
4), and plasma leptin concentrations increased
~10-fold (1.63 ± 0.1 vs. 16.8 ± 0.6, n = 4, P < 0.01). To further probe the
relationship between TNF and adipocyte gene transcription, the time
course of LPS-induced alterations in fat cell TNF,
3-AR mRNA, and plasma leptin
was determined (Fig. 5). TNF induction was observed 2 h
post-LPS and appeared to be maximal at this time (Fig.
5A).
3-AR mRNA was decreased at 4 h,
with the maximal decrease seen at 8 and 16 h (Fig.
5B). Plasma leptin concentrations
were increased in two of five animals at 8 h and in five of five
animals at 16 h (Fig. 5C).
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DISCUSSION |
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The present study demonstrates that endotoxin administration results in
dramatic changes in the expression of TNF, leptin, and
3-AR in white fat. LPS
increases leptin production in a dose-dependent manner and, at higher
doses, produces plasma levels significantly above those seen in
untreated mice. LPS also reduces adipose tissue
3-AR mRNA levels and results in
reduced
3-AR agonist-induced adenylyl cyclase activation. Changes in fat cell leptin and
3-AR mRNA concentrations are
preceded by increases in adipose tissue TNF mRNA. Moreover,
administration of exogenous TNF is sufficient to produce the changes in
leptin and
3-AR mRNA produced
by endotoxin. These data suggest that LPS alters the synthesis of these
fat cell proteins by stimulation of
TNF.
Other investigators have shown that endotoxin increases leptin
production. Grunfeld et al. (13) demonstrated anorexia and increased
leptin mRNA in hamsters treated with endotoxin, IL-1, and TNF. Using
Western blot analysis, they demonstrated that leptin levels in fasted,
LPS-treated animals are similar to those seen in control fed animals.
The authors hypothesized that cytokines block the normal inhibition of
leptin synthesis that occurs with fasting. In contrast, we observed
marked elevations of plasma leptin concentrations in LPS-treated mice.
Levels in our animals were far greater than those seen in fed animals.
These data suggest pathological overexpression of leptin rather than
simply interference with the signaling mechanism responsible for
fasting-induced inhibition of leptin synthesis. Kirchgessner et al.
(17) present data both in vivo (mouse model) and in vitro (3T3-L1
adipocytes in culture) to suggest a mechanism by which TNF- acts
directly on adipocytes to release preformed pools of leptin, since
TNF-
stimulation is insensitive to cyclohexamide, a protein
synthesis inhibitor, while being significantly inhibited by the
secretion inhibitor brefeldin A. Although this mechanism may be
important in the rapid regulation of leptin release, this does not
explain the significant rise in the steady-state concentration of
leptin mRNA that we and other investigators have demonstrated with LPS
and TNF-
. Thus both transcriptional and posttranslational mechanisms
may be important in the leptin reponse to TNF-
.
Endotoxin and TNF have not previously been reported to affect
3-AR mRNA levels or
3-AR function. Our results
indicate a dramatic reduction in mRNA levels, reflecting either a
reduction in mRNA synthesis or an accelerated breakdown of this
message. Accompanying the reduced
3-AR mRNA expression was a
reduced adenyl cyclase response to
3-AR agonist stimulation.
Observation of normal responses to direct activators of G proteins and
adenyl cyclase suggests a specific effect on
3-ARs and not their signal transduction pathways. Although the magnitude of the reduction in
adenyl cyclase response was smaller than the decrease in mRNA concentration, cyclase studies were performed 16 h after LPS
administration. Because there is a significant lag between changes in
3-AR mRNA expression and
changes in
3-AR
cyclase-mediated activity (7), inadequate time may have elapsed to
manifest the full consequences of reduced receptor synthesis.
The observed changes in leptin and
3-AR mRNA after LPS
administration were preceded by augmented adipose tissue TNF mRNA production. Adipose tissue TNF mRNA was maximally increased at 2 h,
whereas changes in
3-AR mRNA
were not seen until 4 h (maximal at 8 h), and plasma leptin levels did
not increase until 8-16 h after LPS. This temporal sequence
suggests that TNF plays a critical intermediary role in LPS-induced
changes in leptin and
3-AR.
This hypothesis is supported by the observation that exogenous TNF
produced changes in adipocyte gene expression identical to those seen
with LPS. Although the observed temporal sequences are consistent with
adipose tissue TNF being principally responsible for affecting leptin
and
3-AR synthesis, we cannot
exclude the participation of other cytokines in modulating adipocyte
mRNA synthesis, especially in light of reports demonstrating leptin stimulation by exogenous IL-1 (25). It is also possible that circulating cytokines, rather than locally produced TNF, are
responsible for the observed effects of LPS. Systemic cytokines could
act directly on the adipocyte, or indirectly, via diverse effects on
hormonal secretion and autonomic nervous system activity (30). Finally,
we do not know the identity of the cell type responsible for TNF
production in adipose tissue (adipocyte vs. stromal cell vs. resident
macrophage).
The clinical relevance of the observed changes in fat cell protein synthesis is unknown at this time, but we speculate that the rise in plasma leptin levels with LPS and TNF administration may contribute to sepsis-induced weight loss. Pathological production of TNF occurs in many animal models of cachexia and in some patients with cancer cachexia. Moreover, administration of exogenous TNF causes weight loss; however, little is known about the mechanisms by which TNF alters appetite and metabolism. Given the central role that leptin plays in regulating both appetite and metabolic rate, it is attractive to postulate that TNF-induced hyperleptinemia suppresses appetite and prevents the usual compensatory responses to starvation. However, recent data from LPS-treated, leptin-deficient mice suggest that leptin is not essential for LPS-induced appetite suppression (6). TNF-induced hyperleptinemia may still contribute to weight loss in some cachectic states by preventing the normal adaptive responses to starvation. Available data suggest that a fall in plasma leptin concentration is the trigger responsible for neuroendocrine changes that reduce metabolic rate and terminate nonessential functions, such as reproduction, during starvation (1, 9). Cytokine-mediated increases in leptin would appear to interfere with this signaling and prevent initiation of adaptive responses. Leptin is not elevated in many wasting states (i.e., AIDS; Refs. 12, 35), and additional studies are required to determine whether hyperleptinemia contributes to weight loss in states with elevated cytokines.
The physiological relevance of reduced
3-AR expression in LPS-treated
animals is unclear.
3-ARs are
important in sympathetically mediated lipolysis and thermogenesis.
3-AR knockout mice are prone to
excess weight gain (22), presumably as a result of reduced energy
expenditure and impaired fat utilization. LPS administration causes a
marked increase in sympathetic nervous system activity. Increased
sympathetic traffic to adipose tissue augments lipolysis and
nonshivering thermogenesis. Whether the reductions in
3-AR expression and
agonist-induced adenylyl cyclase production that we observed act to
attenuate sympathetic effects in fat is not known.
The present study examines only short-term effects of LPS
administration; it is not known whether long-standing infections are
characterized by chronically elevated leptin levels. Equally important,
it is not known whether cytokines increase leptin levels in humans.
Finally, although leptin and
3-AR play an important role in
metabolic regulation, the contribution of cytokine-mediated changes in
the expression of these fat cell proteins to metabolic abnormalities in
sepsis and other states with elevated cytokines remains unknown.
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ACKNOWLEDGEMENTS |
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D. Brown is a Foundation for Anesthesia Education and Research/Marion Roussel Anesthesiology Research Fellow.
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FOOTNOTES |
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Address for reprint requests: D. E. Berkowitz, Dept. of Anesthesiology and Critical Care Medicine, Tower 711, The Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287-8711.
Received 27 August 1997; accepted in final form 4 February 1998.
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