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J Biol Chem, Vol. 274, Issue 10, 6770-6775, March 5, 1999
From the Prolonged treatment (12-24 h) of
adipocytes with tumor necrosis factor TNF TNF The basal rate of lipolysis in isolated adipocytes is normally
inhibited by endogenous adenosine that is spontaneously released from
these cells (11, 12). Adenosine, through binding to A1 adenosine receptors and subsequent activation of Gi,
inhibits adenylyl cyclase, decreases intracellular cyclic AMP
concentrations, and hence decreases the rate of lipolysis. These
observations led us to hypothesize that TNF Animals--
Male Sprague-Dawley rats were used for experiments.
Animals approximately 45 days old were purchased from Texas Animal
Specialties (Houston, TX). They were maintained on a 12-h light-dark
cycle and fed Purina rat chow (Ralston Purina, St. Louis, MO) and tap water ad libitum.
Adipocyte Isolation--
Animals were killed by CO2
asphyxiation. Adipocytes were isolated from epididymal fat pads by the
collagenase digestion method (13). Digestion was carried out at
37 °C with constant shaking (140 cycles/min) for 45 min. Cells were
filtered through nylon mesh (1 mm) and washed three times with buffer
containing 137 mM NaCl, 5 mM KCl, 4.2 mM NaHCO3, 1.3 mM
CaCl2, 0.5 mM MgCl2, 0.5 mM KH2PO4, 0.5 mM
MgSO4, 20 mM HEPES (pH 7.4), plus 1% bovine serum albumin.
Lipolysis Assay--
Adipocytes were suspended at a 5% final
concentration (w/v) in the above described buffer supplemented with 5 mM glucose. In some experiments, adenosine deaminase (10 µg/ml) was included in the incubation medium to prevent accumulation
of endogenously produced adenosine, which inhibits lipolysis. Cells
were incubated at 37 °C in a final volume of 0.5 ml for 30 min with
constant shaking. Preliminary experiments (not shown) established that the rate of lipolysis is constant for at least 45 min under these conditions. Four minutes before the incubation was ended shaking was
stopped to allow cells to float. Infranatant was transferred to another
set of tubes and heated at 70 °C for 10 min to inactivate any
enzymes released by the cells. Glycerol was assayed enzymatically by
the method of McGowan et al. (14) using a kit from Sigma.
Primary Culture of Adipocytes--
After isolation, adipocytes
were maintained in primary culture according to the method of Marshall
et al. (15). Briefly, after isolation and digestion under
sterile conditions, the cells were washed three times with Dulbecco's
modified Eagle's medium supplemented with 2% fetal bovine serum, 20 mM HEPES (pH 7.4), 1% bovine serum albumin, and
antibiotics. After that, adipocytes were resuspended in the same
medium, supplemented with 1 µg/ml adenosine deaminase, and incubated
at 37 °C for up to 4 days at a final concentration of 1 g of
cells/120 ml of medium.
Isolation of Adipocyte Membranes--
Following incubation for
various times, adipocytes were washed three times in the buffer used
for adipocyte isolation with no glucose and only 1% bovine serum
albumin followed by one wash in homogenizing buffer (250 mM
sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 20 mM HEPES, pH 7.4). The cells were then
homogenized by vigorous mixing in 16 × 100-mm glass test tubes on
a vortex mixer. The homogenate was centrifuged for 5 min at 1,000 × g, and the supernatant was centrifuged for 30 min at
16,000 × g. The pellet was suspended in 154 mM NaCl, 10 mM MgCl2, 50 mM HEPES, pH 7.6, and frozen at Antisera--
Each antiserum was raised against a synthetic
decapeptide corresponding to a peptide sequence in the antigen of
interest as described before (16).
Quantification of G-proteins by Western Blotting--
Adipocyte
crude membrane fractions prepared as described previously (17) were
diluted to equal protein concentrations, further diluted with an equal
volume of 2× concentrated Laemmli sample buffer (18) (70 mM Tris, 10% glycerol, 2% SDS, 10% mercaptoethanol), and
heated for 5 min at 95 °C. The membranes were resolved on SDS-polyacrylamide gel electrophoresis (12.5% acrylamide, 0.06% bisacrylamide, run at a 35 mA constant current) and then
electrophoretically transferred to nitrocellulose. The gels were
typically loaded with 50 µg of protein/lane. The nitrocellulose
membranes were blocked for 2 h with 5% dried skim milk in
Tris-buffered saline (TBS, consisting of 20 mM Tris-HCl,
500 mM NaCl, pH 7.5) and then washed once for 15 min in TBS
containing 0.2% Nonidet P-40 detergent (Sigma). Following further
washes with TBS, the nitrocellulose membranes were incubated overnight
with primary antiserum (in 1% dried milk/TBS diluted 1:200 for
colorimetric detection or 1:2000-5000 for chemiluminescent detection).
The membranes were washed again several times with TBS-Nonidet P-40 and
incubated with secondary antibody for 1 h (goat anti-rabbit IgG
coupled to alkaline phosphatase in 1% dried milk/TBS diluted 1:3000
for colorimetric detection or goat anti-rabbit IgG coupled to
horseradish peroxidase in 1% dried milk/TBS diluted 1:3000 for
chemiluminescent detection). After several more washes, membranes were
incubated with chemiluminescent detection reagents according to
directions given by Amersham Pharmacia Biotech, and membranes were
exposed to photographic films with repeated exposures as needed. Blots and films were quantified using an Amersham Pharmacia Biotech UltraScan
laser densitometer.
Lactate Dehydrogenase Assay--
Media from adipocytes incubated
in primary culture were assayed for lactate dehydrogenase activity by
measuring the rate of decrease in A340 in the
presence of pyruvate and NADH (19).
Lipolysis in isolated adipocytes is under tonic inhibition because
of endogenously produced adenosine, which acts through binding to
A1 adenosine receptors (20). Therefore, the basal rate of
lipolysis in adipocytes is determined, at least partly, by the action
of endogenous adenosine. We hypothesized that TNF The above finding suggests that TNF
Tumor Necrosis Factor 
Stimulates Lipolysis in Adipocytes by
Decreasing Gi Protein Concentrations*
,¶
Bassett Research Institute, The Mary Imogene
Bassett Hospital, Cooperstown, New York 13326 and the
§ Department of Pharmacology, University of Texas Medical
Branch, Galveston, Texas 77555
ABSTRACT
Top
Abstract
Introduction
References
(TNF) stimulates
lipolysis. We have investigated the hypothesis that TNF stimulates
lipolysis by blocking the action of endogenous adenosine. Adipocytes
were incubated for 48 h with TNF, and lipolysis was measured in
the absence or presence of adenosine deaminase. Without adenosine
deaminase, the rate of glycerol release was 2-3-fold higher in the
TNF-treated cells, but with adenosine deaminase lipolysis increased
in the controls to approximately that in the TNF-treated cells. This
suggests that TNF blocks adenosine release or prevents its
antilipolytic effect. Both N6-phenylisopropyl
adenosine and nicotinic acid were less potent and efficacious
inhibitors of lipolysis in treated cells. A decrease in the
concentration of -subunits of all three Gi subtypes was detected by Western blotting without a change in Gs
proteins or 
-subunits. Gi2 was about 50% of control,
whereas Gi1 and Gi3 were about 20 and
40% of control values, respectively. The time course of Gi
down-regulation correlated with the stimulation of lipolysis.
Furthermore, down-regulation of Gi by an alternative approach (prolonged incubation with
N6-phenylisopropyl adenosine) stimulated
lipolysis. These findings indicate that TNF stimulates lipolysis by
blunting endogenous inhibition of lipolysis. The mechanism appears to
be a Gi protein down-regulation.
INTRODUCTION
Top
Abstract
Introduction
References
1 is a
multifunctional cytokine important in many pathological and
physiological states (1). A series of recent reports has demonstrated
that adipose tissue expresses TNF mRNA and protein. Furthermore,
adipose tissue from obese animals and humans expresses considerably
more TNF than does tissue from their lean counterparts (2, 3). This
excess expression of TNF in adipose tissue may form a link between
obesity and development of insulin resistance, which often leads to
type 2 diabetes in obese subjects (4). However, TNF is not
measurable in the circulation of obese subjects and is therefore
considered an autocrine or paracrine regulator of adipose tissue metabolism.
has several effects on adipocytes that may be related to the
development of type 2 diabetes in obese subjects. It has been reported
that TNF induces insulin resistance possibly by inducing serine
phosphorylation of insulin receptor substrate-1 and converting it to an
inhibitor of the insulin receptor tyrosine kinase (5). In addition,
TNF causes a net depletion of the adipose tissue triglyceride.
Initially, this was thought to be largely because of a decrease in the
activity of lipoprotein lipase (6). However, we and others have
reported that TNF also stimulates lipolysis both in rat adipocytes
maintained in primary culture (7) and in 3T3-L1 adipocytes (8-10). We
found that TNF-induced stimulation of lipolysis in primary
adipocytes is chronic in nature, taking approximately 6-12 h before a
measurable effect is observed (7). Furthermore, neither the rate of
isoproterenol-stimulated lipolysis nor the concentration of
hormone-sensitive lipase (the rate-limiting enzyme for lipolysis) was
affected by TNF over the time course of our studies. This suggests
that the major effect of TNF is to increase the basal rate of
adipocyte lipolysis and activate existing hormone-sensitive lipase.
stimulates adipocyte
lipolysis indirectly by blocking the action of endogenous adenosine.
Here, we report that TNF disrupts the ability of an A1
adenosine receptor agonist and of nicotinic acid to inhibit lipolysis.
Furthermore, the mechanism of this disruption appears to be specific
down-regulation of Gi isoforms, especially
Gi1.
MATERIALS AND METHODS
70 °C. Protein
concentration of the membrane suspensions was determined by the
Bradford method using a kit from Bio-Rad and bovine 
-globulin as a standard.
RESULTS
stimulates
lipolysis indirectly by preventing adenosine accumulation or by
abolishing the antilipolytic action of adenosine. To test this
hypothesis, we measured the basal and TNF-stimulated rate of
lipolysis in the absence and presence of adenosine deaminase. Adenosine
deaminase hydrolyzes adenosine into inosine and hence abolishes
adenosine-induced inhibition of lipolysis. Adipocytes were incubated in
primary culture for 48 h without or with TNF (100 ng/ml). After
incubation, the cells were washed three times, and the rate of
lipolysis (glycerol release) was measured in the presence or absence of
adenosine deaminase (10 µg/ml) (Fig.
1). As in our previous report (7), the
rate of lipolysis was markedly higher in the TNF-treated cells.
Adenosine deaminase increased the rate of lipolysis in control cells to
approximately the same rate as in TNF-treated cells. However, in the
TNF-treated cells, adenosine deaminase had little or no additional
stimulatory effect.
View larger version (17K):
[in a new window]
Fig. 1.
Effect of adenosine deaminase on the
lipolytic action of TNF . Adipocytes were
incubated for 2 days in primary culture without or with TNF. The
cells were then washed and incubated for 1 h in the absence or
presence of adenosine deaminase (10 µg/ml) as indicated. Lipolysis
was measured as glycerol release. Data are mean ± S.E. of three
separate experiments. White bars represent control cells;
black bars represent TNF-treated cells. ADA,
adenosine deaminase.
stimulates lipolysis either by
disrupting the pathway by which adenosine inhibits lipolysis or by
inhibiting adenosine release from the cells. To distinguish between
these alternatives, a full dose-response curve for lipolysis inhibition
with PIA, a nonhydrolyzable analog of adenosine that is a full agonist
at A1 adenosine receptors, was obtained (Fig. 2). Adipocytes were incubated with TNF
for 48 h as before, washed, and incubated with adenosine deaminase
to prevent accumulation of endogenous adenosine plus various
concentrations of PIA. Glycerol release was measured after 30 min.
There was a pronounced rightward shift in the dose-response curve for
lipolysis inhibition by PIA in TNF-treated cells as compared with
controls. Furthermore, maximal inhibition of lipolysis was markedly
blunted in the TNF-treated cells, such that PIA inhibited lipolysis
in TNF-treated adipocytes by only about 40% as compared with almost
70% inhibition in control cells, that is PIA was both less efficacious
and less potent as an inhibitor of lipolysis in TNF-treated cells
relative to control cells.
View larger version (18K):
[in a new window]
Fig. 2.
Effect of TNF on the
ability of PIA to inhibit lipolysis. Adipocytes were incubated for
2 days without or with TNF (30 ng/ml). The cells were washed and
incubated with adenosine deaminase (10 µg/ml) and various
concentrations of PIA as indicated. Glycerol release was measured after
30 min. Data are mean ± S.E. of three separate experiments.
Open circles, controls; closed circles,
TNF-treated cells.
To determine whether the loss of inhibition of lipolysis is generalized
or specific to the A1 adenosine receptor, we investigated the effect of another inhibitor of lipolysis, nicotinic acid. The
antilipolytic action of nicotinic acid was also markedly blunted after
a 2-day treatment with TNF (Fig. 3)
with almost complete loss of the inhibitory effect. Nicotinic acid
inhibits lipolysis by binding to a receptor that is distinct from the
A1 adenosine receptor but is coupled to inhibition of
adenylyl cyclase by pertussis toxin-sensitive G-proteins (see
"Discussion").
|
Together, these findings indicate that TNF disrupts a component of
the signal transduction pathway by which both adenosine and nicotinic
acid inhibit lipolysis. G-proteins are a crucial link between receptor
occupation and second messenger activation, and their concentrations in
rat adipocytes can be regulated (21). Receptors that decrease
intracellular cyclic AMP concentrations and inhibit lipolysis act
through a group of G-proteins collectively termed Gi.
Therefore, we measured the relative concentrations of the -subunits
of all three Gi subtypes expressed in rat adipocytes. Fig.
4 shows Western blots of plasma membrane
preparations isolated from adipocytes incubated for 2 days without or
with TNF. The blot shown in A was probed with an
antiserum (SG1) that binds to the -subunits of Gi1 and
Gi2. Although these two -subunits are almost identical
in molecular mass, it is possible to resolve them by using a very low
concentration of bisacrylamide in the gels, as we have reported before
(16). The blot in B was probed with an antibody (I3B) that
is specific for the -subunit of Gi3. As can be seen from
these blots, the membranes from TNF-treated adipocytes contained
markedly less of each Gi subtype than did the membranes
from control cells. Quantification of Western blots from three separate
experiments is shown in Fig. 4C. This revealed that TNF
decreased the concentration of Gi1 by an average of approximately 80%, Gi2 by approximately 50%, and
Gi3 by approximately 60% compared with controls, that
is each Gi subtype was markedly down-regulated after
incubation of adipocytes with TNF.
|
To determine whether TNF causes a general decrease in the
concentration of G-proteins, we also measured relative concentrations of Gs, which couples stimulatory receptors to adenylyl
cyclase. Adipocytes were incubated in primary culture with or without
TNF; plasma membrane preparations were isolated and analyzed on
Western blots as before but probed with CS1 antibody, which binds to
all known splice variants of Gs including the 43- and
47-kDa isoforms expressed in adipocytes (16). As demonstrated in Fig.
5A, treatment with TNF did
not alter the concentration of either isoform of Gs.
Similarly, TNF did not alter the cellular concentration of G-protein
-subunits as demonstrated by Western blotting with antiserum BN2
(Fig. 5B).
|
To further investigate the relationship between Gi
down-regulation and stimulation of lipolysis, we determined the time
course of the loss of Gi1 and Gi2 (Fig.
6). TNF caused a measurable decrease
in concentration of both G-proteins within 12 h and a maximal
decrease by approximately 24 h. This time course is in good
agreement with that for TNF-induced activation of lipolysis that we
have published previously (7).
|
We have previously reported that prolonged treatment of adipocytes with
PIA (or other agonists that inhibit adenylyl cyclase) down-regulates
Gi in adipocytes (16, 17). This agonist-induced down-regulation results in heterologous desensitization of lipolysis to
other agonists that act through the inhibition of adenylyl cyclase
(17). However, we have not previously investigated the effect of
prolonged treatment of adipocytes with PIA on basal rates of lipolysis.
Therefore, as an independent assessment of whether Gi
down-regulation is sufficient to stimulate lipolysis, we incubated
adipocytes with TNF or PIA (300 nM) for 24 h,
washed the cells, and measured the rate of lipolysis over 30 min as
before. We have previously reported that this concentration of PIA is maximal for Gi down-regulation (16). Interestingly,
prolonged incubation with PIA was equally effective at stimulating
lipolysis as was TNF (Fig.
7A). Further experiments
demonstrated that the time course for the stimulatory effect of PIA on
lipolysis (Fig. 7B) corresponds to that for PIA-induced
Gi down-regulation (16). Similarly, the dose-response
relationship for the chronic lipolytic effect of PIA (Fig.
7C) is essentially identical to the dose-response relationship for Gi down-regulation (16). Together, these
findings strongly suggest that the lipolytic effect of prolonged
treatment of adipocytes with either TNF or PIA is secondary to the
down-regulation of Gi.
|
PIA acutely inhibits lipolysis, but as demonstrated above prolonged
treatment with PIA down-regulates Gi and stimulates
lipolysis. Because prolonged treatment of adipocytes with TNF has
similar effects to prolonged treatment with PIA (i.e.
Gi down-regulation and activation of lipolysis), we
hypothesized that TNF may have an acute inhibitory effect of
lipolysis. However, we were unable to determine any such effect of
TNF (data not shown).
Because TNF has a cytotoxic effect on some cell types, we determined
the degree of adipocyte lysis by measuring lactate dehydrogenase activity in the media. There was no significant difference in lactate
dehydrogenase activity released into the incubation media between
control and TNF-treated adipocytes (data not shown), indicating that
TNF did not affect cell viability.
| |
DISCUSSION |
|---|
In a previous report, we demonstrated that prolonged incubation of
adipocytes with TNF increased the basal but not
isoproterenol-stimulated rate of lipolysis and that the concentration
of hormone-sensitive lipase was unaffected (7). We now have
investigated the mechanism by which TNF stimulates lipolysis in rat
adipocytes. TNF induces a 2-3-fold increase in the rate of release
of glycerol from rat adipocytes incubated for at least 12 h in
primary culture (7).
In isolated adipocytes, the cyclic AMP concentration and hence the rate
of lipolysis are very low because of the presence of adenosine, which
is spontaneously released by these cells (11). After removal of
endogenous adenosine, adenylyl cyclase activity increases, and
lipolysis proceeds at almost maximal rates (22), that is lipolysis is
tonically inhibited by endogenous adenosine in the basal state.
Therefore, we hypothesized that TNF may stimulate lipolysis by
releasing this endogenous inhibition. This hypothesis was supported by
the finding that, when measured in the presence of adenosine deaminase,
the rate of lipolysis was equal in control and TNF-treated
adipocytes. Results of lipolysis inhibition experiments using the
adenosine analog, PIA, and nicotinic acid suggest that this is because
of a decrease in the ability of the cells to respond to inhibitors of lipolysis.
The decreased ability of endogenous adenosine and of exogenous PIA to
inhibit lipolysis could be because of a loss of adenosine receptors.
However, this is unlikely because the TNF-treated cells also did not
respond to nicotinic acid. Although nicotinic acid receptors are not
well characterized, inhibition of lipolysis by nicotinic acid is
pertussis toxin-sensitive (23, 24), and furthermore nicotinic acid
inhibition of adenylyl cyclase is GTP-dependent (25).
Therefore nicotinic acid inhibits adipocyte lipolysis through a
G-protein-coupled receptor, although the identity of this receptor is unknown.
Because TNF caused a pronounced inhibition of the ability of both
PIA and nicotinic acid to inhibit lipolysis, we investigated the effect
of TNF on concentrations of Gi. These experiments demonstrated that TNF caused a marked down-regulation of all three
Gi proteins, which most likely explains the inability of both PIA and nicotinic acid to inhibit lipolysis in these cells.
We have previously demonstrated that agonist-induced down-regulation of
Gi results in decreased sensitivity to all agents that act
through the same pathway forming a mechanism for heterologous desensitization (17, 26). We now report that TNF also can result in
Gi down-regulation in a manner similar to that induced by
agonists, that is TNF down-regulated Gi2 levels less
than either Gi1 or Gi3, as we have
reported for agonist-induced Gi down-regulation (16, 17).
However, the inhibitory agonists also cause a marked (approximately
50%) loss of G-protein -subunits from adipocytes (16, 17). By
contrast, TNF did not affect cellular concentrations of
-subunits. This, together with the observation that TNF has no
acute inhibitory effect on lipolysis, suggests that the mechanism by
which TNF down-regulates Gi is different from that by
which PIA and other inhibitory agonists down-regulate
Gi.
Clearly, TNF down-regulates Gi-subunits in adipocytes
and stimulates lipolysis. The findings suggest that this Gi
down-regulation explains the lipolytic effect of TNF. However, the
cellular concentration of G-proteins generally exceeds that of
receptors by approximately an order of magnitude (17). This raises the
question of whether Gi down-regulation by itself can
account for TNF stimulation of lipolysis. Several lines of evidence
support the conclusion that loss of Gi is indeed sufficient
for stimulation of lipolysis. First, we have demonstrated that
down-regulation of Gi by another agent (PIA) that
presumably works through a different mechanism stimulates lipolysis
equally to TNF. Second, treatment of adipocytes with pertussis
toxin, which ADP ribosylates and inactivates the various isoforms of
Gi, results in marked stimulation of lipolysis (27). Third,
prolonged treatment with growth hormone has been reported to stimulate
adipocyte lipolysis, and growth hormone has also been reported to
decrease Gi expression in adipocytes (28), suggesting that
the lipolytic effect of growth hormone may have a similar mechanism to
that of TNF. Finally, the time course of both TNF-induced and
PIA-induced Gi down-regulation corresponds closely to the
time course for stimulation of lipolysis. We conclude that the
concentration of Gi proteins in adipocytes may be a major
regulatory end point used by several hormones and cytokines to
influence cyclic AMP levels on a long term basis. Thus, Gi
down-regulation or inactivation can stimulate lipolysis and account for
the lipolytic effect of TNF.
Stimulatory G-proteins (Gs) were not affected by treatment
of adipocytes with TNF. Similarly, in a recent study of
-adrenergic receptor down-regulation following isoproterenol
infusion in vivo, we did not detect concomitant
down-regulation of adipocyte Gs (29). Therefore, it appears
that the concentration of Gs in adipocytes is not regulated
in the same manner as are concentrations of Gi. One study
found that TNF induced an increase in Gi proteins in rat
cardiomyocytes and that increased adenylyl cyclase activity was a
consequence (30). This suggests that TNF has tissue-specific effects
on G-protein concentrations.
The exact signaling mechanism for the effect of TNF is not known.
TNF has two receptor subtypes widely expressed on all cells,
including adipocytes (31). Most TNF effects are mediated through
activation of a p55 receptor, which is trimerized upon TNF binding
and activates membrane neutral sphingomyelinase. As a consequence,
ceramide is released and activates various intracellular substrates
like ceramide-activated protein kinase and a specific phosphatase (32,
33). Cell-permeable analogs of ceramide have been able to mimic many
TNF actions (34). We incubated adipocytes in primary cultures with
one such analog, C2-ceramide. Neither the rate of lipolysis
nor cellular concentrations of Gi were affected by this
compound (data not shown), indicating that the ceramide second
messenger pathway is probably not involved.
TNF has been reported to induce insulin resistance of adipocytes
(5). Furthermore, adipose tissue from obese animals and humans contains
more TNF mRNA and protein than adipose tissue from lean animals
and humans (2). This increased expression of TNF is believed to be
central to the insulin resistance of obesity and may be key to the
relationship between obesity and the development of type 2 diabetes
(2). TNF-induced insulin resistance has been reported to be caused
by phosphorylation of insulin receptor substrate-1, which in turn
causes inhibition of the tyrosine kinase activity of the insulin
receptor (5). However, TNF-induced insulin receptor substrate-1
phosphorylation is quite slow to develop, and another group has been
unable to confirm the finding (35).
An alternative explanation for the mechanism by which increased adipose
tissue TNF may lead to insulin resistance is that the insulin
resistance is secondary to stimulation of adipocyte lipolysis. Several
arguments can be made to support this hypothesis. First, circulating
free fatty acid concentrations are known to be elevated in obese
subjects and in patients with type 2 diabetes (36-38). Second, free
fatty acids are known to induce insulin resistance in vivo
(39, 40). In addition, free fatty acids stimulate hepatic
gluconeogenesis (41), providing another mechanism by which increased
adipocyte lipolysis may lead to insulin resistance. Recent studies have
demonstrated that insulin inhibits hepatic glucose production primarily
through indirect mechanisms (42) most likely by decreasing adipose
tissue lipolysis and hence decreasing circulating free fatty acids.
Therefore, increased rates of adipose tissue lipolysis would be
expected to induce insulin resistance of both muscle and liver, and
this may provide a mechanism by which TNF induces insulin resistance
in vivo. Furthermore, in a recent study Souza et
al. (43) reported that a thiazolidinedione (BRL 49653) blocked the
lipolytic effect of TNF in 3T3-L1 adipocytes. They proposed that
this is an important mechanism by which thiazolidinediones improve
insulin resistance. Further work will be needed to address this question.
In conclusion, our findings demonstrate that the lipolytic effect of
TNF in primary cultures of adipocytes can be attributed mainly to a
decrease in the concentration of Gi proteins. Further work
will be required to determine the mechanism by which TNF decreases
cellular concentrations of Gi.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Dr. Dianne DeCamp
(University of Texas Southwestern Medical Center, Dallas, TX) for
generously providing us with recombinant human TNF and to Professor
Graeme Milligan (University of Glasgow, Glasgow, Scotland) for the
antisera used in these studies.
| |
FOOTNOTES |
|---|
* This work was supported in part by a grant from the American Diabetes Association.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.
¶ To whom correspondence should be addressed: Basset Research Inst., the Mary Imogene Bassett Hospital, One Atwell Rd., Cooperstown, NY 13326. Tel.: 607-547-3048; Fax: 607-547-3061; E-mail: allan.green{at}bassett.org.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
TNF, tumor
necrosis factor-;
TBS, Tris-buffered saline;
PIA, N6-phenylisopropyl adenosine.
| |
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