Tumor Necrosis Factor alpha  Stimulates Lipolysis in Adipocytes by Decreasing Gi Protein Concentrations*

Slavisa GasicDagger , Bing Tian§, and Allan GreenDagger

From the Dagger  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

Prolonged treatment (12-24 h) of adipocytes with tumor necrosis factor alpha  (TNFalpha ) stimulates lipolysis. We have investigated the hypothesis that TNFalpha stimulates lipolysis by blocking the action of endogenous adenosine. Adipocytes were incubated for 48 h with TNFalpha , 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 TNFalpha -treated cells, but with adenosine deaminase lipolysis increased in the controls to approximately that in the TNFalpha -treated cells. This suggests that TNFalpha 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 alpha -subunits of all three Gi subtypes was detected by Western blotting without a change in Gs proteins or beta -subunits. Gi2alpha was about 50% of control, whereas Gi1alpha and Gi3alpha 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 TNFalpha stimulates lipolysis by blunting endogenous inhibition of lipolysis. The mechanism appears to be a Gi protein down-regulation.

    INTRODUCTION
Top
Abstract
Introduction
References

TNFalpha 1 is a multifunctional cytokine important in many pathological and physiological states (1). A series of recent reports has demonstrated that adipose tissue expresses TNFalpha mRNA and protein. Furthermore, adipose tissue from obese animals and humans expresses considerably more TNFalpha than does tissue from their lean counterparts (2, 3). This excess expression of TNFalpha 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, TNFalpha is not measurable in the circulation of obese subjects and is therefore considered an autocrine or paracrine regulator of adipose tissue metabolism.

TNFalpha has several effects on adipocytes that may be related to the development of type 2 diabetes in obese subjects. It has been reported that TNFalpha 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, TNFalpha 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 TNFalpha also stimulates lipolysis both in rat adipocytes maintained in primary culture (7) and in 3T3-L1 adipocytes (8-10). We found that TNFalpha -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 TNFalpha over the time course of our studies. This suggests that the major effect of TNFalpha is to increase the basal rate of adipocyte lipolysis and activate existing hormone-sensitive lipase.

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 TNFalpha stimulates adipocyte lipolysis indirectly by blocking the action of endogenous adenosine. Here, we report that TNFalpha 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 Gialpha isoforms, especially Gi1alpha .

    MATERIALS AND METHODS

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 -70 °C. Protein concentration of the membrane suspensions was determined by the Bradford method using a kit from Bio-Rad and bovine gamma -globulin as a standard.

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).

    RESULTS

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 TNFalpha stimulates lipolysis indirectly by preventing adenosine accumulation or by abolishing the antilipolytic action of adenosine. To test this hypothesis, we measured the basal and TNFalpha -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 TNFalpha (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 TNFalpha -treated cells. Adenosine deaminase increased the rate of lipolysis in control cells to approximately the same rate as in TNFalpha -treated cells. However, in the TNFalpha -treated cells, adenosine deaminase had little or no additional stimulatory effect.


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Fig. 1.   Effect of adenosine deaminase on the lipolytic action of TNFalpha . Adipocytes were incubated for 2 days in primary culture without or with TNFalpha . 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 TNFalpha -treated cells. ADA, adenosine deaminase.

The above finding suggests that TNFalpha 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 TNFalpha 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 TNFalpha -treated cells as compared with controls. Furthermore, maximal inhibition of lipolysis was markedly blunted in the TNFalpha -treated cells, such that PIA inhibited lipolysis in TNFalpha -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 TNFalpha -treated cells relative to control cells.


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Fig. 2.   Effect of TNFalpha on the ability of PIA to inhibit lipolysis. Adipocytes were incubated for 2 days without or with TNFalpha (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 TNFalpha (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").


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Fig. 3.   Effect of TNFalpha on the ability of nicotinic acid to inhibit lipolysis. Adipocytes were incubated for 2 days without or with TNFalpha (30 ng/ml). The cells were washed and incubated with adenosine deaminase (10 µg/ml) and various concentrations of nicotinic acid 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.

Together, these findings indicate that TNFalpha 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 alpha -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 TNFalpha . The blot shown in A was probed with an antiserum (SG1) that binds to the alpha -subunits of Gi1 and Gi2. Although these two alpha -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 alpha -subunit of Gi3. As can be seen from these blots, the membranes from TNFalpha -treated adipocytes contained markedly less of each Gialpha subtype than did the membranes from control cells. Quantification of Western blots from three separate experiments is shown in Fig. 4C. This revealed that TNFalpha decreased the concentration of Gi1alpha by an average of approximately 80%, Gi2alpha by approximately 50%, and Gi3alpha by approximately 60% compared with controls, that is each Gi subtype was markedly down-regulated after incubation of adipocytes with TNFalpha .


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Fig. 4.   TNFalpha decreases cellular concentrations of Gialpha -subtypes. Adipocytes were incubated for 2 days with (+) or without (-) TNFalpha as indicated. The cells were then homogenized, and crude plasma membranes were isolated and analyzed on Western blots. A, 50 µg of membrane protein were loaded per lane, and the blot was probed with antiserum SG1 to label Gi1alpha and Gi2alpha as indicated. B, 150 µg of protein were loaded per lane, and the blot was probed with antiserum I3B to label Gi3alpha . C, results of densitometry. Bands from TNFalpha -treated cells were compared with their respective bands from control cells. Data are mean ± S.E. from three separate experiments.

To determine whether TNFalpha causes a general decrease in the concentration of G-proteins, we also measured relative concentrations of Gsalpha , which couples stimulatory receptors to adenylyl cyclase. Adipocytes were incubated in primary culture with or without TNFalpha ; 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 Gsalpha including the 43- and 47-kDa isoforms expressed in adipocytes (16). As demonstrated in Fig. 5A, treatment with TNFalpha did not alter the concentration of either isoform of Gsalpha . Similarly, TNFalpha did not alter the cellular concentration of G-protein beta -subunits as demonstrated by Western blotting with antiserum BN2 (Fig. 5B).


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Fig. 5.   Effect of TNFalpha on adipocyte Gsalpha and G-protein beta -subunits. Adipocytes were treated without (-) or with (+) TNFalpha , and plasma membranes were isolated as described in the legend to Fig. 4. Western blots of membranes (50 µg/lane) were probed with antiserum to Gsalpha (A) or beta -subunits (B). The 47- and 43-kDa isoforms of Gsalpha and the 36-kDa beta -subunit band are indicated by arrows. The blots are representative of three separate experiments.

To further investigate the relationship between Gi down-regulation and stimulation of lipolysis, we determined the time course of the loss of Gi1alpha and Gi2alpha (Fig. 6). TNFalpha 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 TNFalpha -induced activation of lipolysis that we have published previously (7).


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Fig. 6.   Time course of TNFalpha -induced Gi down-regulation. A, adipocytes were incubated with TNFalpha (100 ng/ml) for the times indicated and then homogenized, and Gi1alpha and Gi2alpha were analyzed on Western blots with SG1 antibody as described in the legend to Fig. 4. B, densitometric analysis of blots from three similar experiments. Closed circles, Gi1alpha ; open circles, Gi2alpha .

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 TNFalpha 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 TNFalpha (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 TNFalpha or PIA is secondary to the down-regulation of Gi.


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Fig. 7.   Prolonged treatment of adipocytes with PIA stimulates lipolysis. A, adipocytes were incubated with no additions, TNFalpha (100 ng/ml), or PIA (300 nM) for 48 h and then washed, and the rate of lipolysis was measured. B, a time course for the effect of PIA at a concentration of 100 nM. C, a dose-response curve for the PIA effect using a 24-h treatment time.

PIA acutely inhibits lipolysis, but as demonstrated above prolonged treatment with PIA down-regulates Gi and stimulates lipolysis. Because prolonged treatment of adipocytes with TNFalpha has similar effects to prolonged treatment with PIA (i.e. Gi down-regulation and activation of lipolysis), we hypothesized that TNFalpha may have an acute inhibitory effect of lipolysis. However, we were unable to determine any such effect of TNFalpha (data not shown).

Because TNFalpha 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 TNFalpha -treated adipocytes (data not shown), indicating that TNFalpha did not affect cell viability.

    DISCUSSION

In a previous report, we demonstrated that prolonged incubation of adipocytes with TNFalpha 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 TNFalpha stimulates lipolysis in rat adipocytes. TNFalpha 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 TNFalpha 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 TNFalpha -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 TNFalpha -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 TNFalpha caused a pronounced inhibition of the ability of both PIA and nicotinic acid to inhibit lipolysis, we investigated the effect of TNFalpha on concentrations of Gi. These experiments demonstrated that TNFalpha caused a marked down-regulation of all three Gialpha 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 TNFalpha also can result in Gi down-regulation in a manner similar to that induced by agonists, that is TNFalpha down-regulated Gi2alpha levels less than either Gi1alpha or Gi3alpha , 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 beta -subunits from adipocytes (16, 17). By contrast, TNFalpha did not affect cellular concentrations of beta -subunits. This, together with the observation that TNFalpha has no acute inhibitory effect on lipolysis, suggests that the mechanism by which TNFalpha down-regulates Gi is different from that by which PIA and other inhibitory agonists down-regulate Gi.

Clearly, TNFalpha down-regulates Gialpha -subunits in adipocytes and stimulates lipolysis. The findings suggest that this Gi down-regulation explains the lipolytic effect of TNFalpha . 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 TNFalpha 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 TNFalpha . 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 TNFalpha . Finally, the time course of both TNFalpha -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 TNFalpha .

Stimulatory G-proteins (Gs) were not affected by treatment of adipocytes with TNFalpha . Similarly, in a recent study of beta -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 TNFalpha induced an increase in Gi proteins in rat cardiomyocytes and that increased adenylyl cyclase activity was a consequence (30). This suggests that TNFalpha has tissue-specific effects on G-protein concentrations.

The exact signaling mechanism for the effect of TNFalpha is not known. TNFalpha has two receptor subtypes widely expressed on all cells, including adipocytes (31). Most TNFalpha effects are mediated through activation of a p55 receptor, which is trimerized upon TNFalpha 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 TNFalpha 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.

TNFalpha has been reported to induce insulin resistance of adipocytes (5). Furthermore, adipose tissue from obese animals and humans contains more TNFalpha mRNA and protein than adipose tissue from lean animals and humans (2). This increased expression of TNFalpha 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). TNFalpha -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, TNFalpha -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 TNFalpha 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 TNFalpha 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 TNFalpha 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 TNFalpha 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 TNFalpha 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 TNFalpha 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: TNFalpha , tumor necrosis factor-alpha ; TBS, Tris-buffered saline; PIA, N6-phenylisopropyl adenosine.

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Abstract
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