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Endocrinology Vol. 142, No. 12 5069-5075
Copyright © 2001 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Inhibition of Proteasome Activity Blocks the Ability of TNF{alpha} to Down-Regulate Gi Proteins and Stimulate Lipolysis

Leida M. Botion, Allan R. Brasier, Bing Tian, Vidya Udupi and Allan Green

Depto de Fisiologia e Biofísica–Instituto de Ciências Biológicas (L.M.B.), Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 31270-901; Department of Internal Medicine (A.R.B., B.T.), University of Texas Medical Branch, Galveston, Texas 77555; and the Bassett Research Institute (V.U., A.G.), Mary Imogene Bassett Hospital, Cooperstown, New York 13326

Address all correspondence and requests for reprints to: Allan Green, Ph.D., Basset Research Institute, Mary Imogene Bassett Hospital, One Atwell Road, Cooperstown, New York 13326.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prolonged treatment of rat adipocytes with TNF{alpha} increases lipolysis through a mechanism mediated, in part, by down-regulation of inhibitory G proteins (Gi). Separately, down-regulation of Gi by prolonged treatment with an A1-adenosine receptor agonist, N6-phenylisopropyl adenosine (PIA) increases lipolysis. To investigate the role of proteolysis in TNF{alpha} and PIA-mediated Gi down-regulation and stimulation of lipolysis, we used the protease inhibitors lactacystin (proteasome inhibitor) and calpeptin (calpain inhibitor). Rat adipocytes were preincubated for 1 h with lactacystin (10 µM) or calpeptin (50 µM), before 30-h treatment with either TNF{alpha} (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of {alpha}-subunits of Gi1 and Gi2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNF{alpha} and PIA stimulated lipolysis approximately 2-fold and caused Gi down-regulation. Although neither lactacystin nor calpeptin affected basal lipolysis, lactacystin completely inhibited both TNF{alpha} and PIA-stimulated lipolysis (the 50% inhibitory concentration was ~2 µM), whereas calpeptin had no effect. Similarly, lactacystin but not calpeptin blocked both PIA and TNF{alpha}-induced Gi down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNF{alpha} and PIA is secondary to Gi down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A NUMBER OF recent reports have suggested a role for adipose tissue-derived TNF{alpha} in the insulin resistance of obesity. Obese animals and humans express higher concentrations of TNF{alpha} mRNA and protein in their adipose tissue than do the lean, and addition of TNF{alpha} to various cell types induces insulin resistance (for review, see Ref. 1). In addition, several groups (including our own) have suggested that TNF{alpha} induces insulin resistance, at least partly, by increasing the rate of adipose tissue lipolysis. According to this hypothesis, the increased rate of lipolysis raises circulating FFA concentrations, which in turn antagonizes insulin action in muscle and liver (2, 3). The mechanism of the lipolytic effect of TNF{alpha} is only partly understood.

Previous studies from our laboratory have demonstrated that prolonged incubation of rat adipocytes in primary culture with TNF{alpha} increases the rate of basal, but not isoproterenol-stimulated, lipolysis (3). Similarly, TNF{alpha} has been shown to increase the rate of lipolysis in 3T3-L1 adipocytes (4, 5, 6). Recently, we reported that the mechanism of this lipolytic effect of TNF{alpha} in primary rat adipocytes involves down-regulation of the various isoforms of inhibitory G protein {alpha} (Gi{alpha}), especially Gi1{alpha}, without a change in the concentration of either isoform of Gs or G protein ß-subunits (7). The basal rate of lipolysis in isolated adipocytes is under tonic inhibition by endogenous adenosine that is spontaneously released by these cells (8, 9). Adenosine, through binding to A1-adenosine receptors and subsequent activation of Gi (10, 11), inhibits adenylyl cyclase (12, 13), decreases intracellular cAMP concentrations, and hence decreases the rate of lipolysis. Therefore, we have proposed that TNF{alpha} acts by blocking the action of endogenous adenosine and possibly other antilipolytic agonists. This hypothesis is supported by the finding that, when measured in the presence of adenosine deaminase, the rate of lipolysis was equal in control and TNF{alpha}-treated adipocytes (7). Furthermore, TNF{alpha}-treated adipocytes were resistant to the antilipolytic actions of both N6-phenylisopropyl adenosine (PIA), a nonhydrolyzable analog of adenosine that is a full agonist at the A1 adenosine receptor, and nicotinic acid. These findings strongly suggest that the lipolytic effect of prolonged treatment of adipocytes with TNF{alpha} is secondary to the Gi down-regulation (7).

We have also reported that prolonged treatment of rat adipocytes with certain agonists that inhibit lipolysis by inhibiting adenylyl cyclase can induce desensitization by mechanisms that include down-regulation of G proteins. Thus, PIA and PGE1, both of which acutely inhibit adenylyl cyclase, down-regulate Gi in adipocytes (14, 15, 16). In contrast, levels of the two forms of Gs{alpha} found in adipocytes are unaffected by these agents, demonstrating that the effect is specific for the Gi proteins. Consistent with our hypothesis that the lipolytic effect of TNF{alpha} is secondary to Gi down-regulation, prolonged incubation with PIA also results in an increase in the basal rate of lipolysis (7).

Little is known about the molecular mechanisms of agonist-promoted or TNF{alpha}-induced G protein down-regulation. Two broad mechanisms could be involved: a decrease in the rate of synthesis and/or an increase in the rate of degradation of Gi isoforms. Changes in the expression of both adipocyte Gi and Gs have been shown to occur in a number of animal models of pathophysiological states, such as chemically induced hypothyroidism (17), genetic obesity (18, 19), and insulin resistance (18, 19, 20, 21) and also in obese humans (22). Other studies suggest that at least in relation to Gi2, the down-regulation of this protein by {alpha}2A adrenergic receptors does not appear to be regulated by transcriptional events because the Gi2{alpha} mRNA levels were not changed by agonist exposure (23).

An alternative mechanism underlying G protein down-regulation may be an increase in the rate of G protein degradation. Intracellular proteolytic pathways are composed of lysosomal and nonlysosomal pathways in which intracellular proteases are directly responsible for the degradation of proteins. Nonlysosomal protease systems have been identified as important regulators of intracellular activities including programmed cell death, protein kinase abundance, and cell-cycle progression (24, 25, 26), and more recently, inducible proteolysis has also been shown to be important in hormonal control of gene expression (25). In viable cells, two prominent cytoplasmic protease systems have been identified. These include the calcium-activated neutral protease (calpain- calpastatin system), initially thought to be important in regulating turnover of protein kinases and key structural proteins in the cell, and the ubiquitin-proteasome pathway, mediating targeted turnover of misfolded and unstable proteins (24) as well as intact cellular proteins (25).

In the present report, we have used protease inhibitors to investigate the role of proteolysis in TNF{alpha}- and PIA-induced Gi down-regulation and to determine the relative contributions of the proteasome and calpain-mediated pathways. The results demonstrate that the proteasomal pathway is involved in both TNF{alpha}- and PIA-induced Gi down-regulation and stimulation of lipolysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats, approximately 45 d old, were purchased from Texas Animal Specialties (Houston, TX). Animals were on a 12-h light-dark cycle and fed Purina rat chow (Ralston Purina Co., St. Louis, MO). All animal protocols were approved by the Institutional Animal Care and Use Committee of the Mary Imogene Bassett Hospital.

Adipocyte isolation
Animals were killed by CO2 asphyxiation. Adipocytes were isolated under sterile conditions from epididymal fat pads by the method of Rodbell (27). 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 MgSO4, 0.5 mM KH2PO4, 20 mM HEPES (pH 7.4), plus 1% BSA.

Primary culture of adipocytes
After isolation, adipocytes were maintained in primary culture according to the method of Marshall et al. (28) in DMEM supplemented with 2% FBS, 20 mM HEPES (pH 7.4), 1% BSA and antibiotics. Isolated adipocytes were diluted (1:20 wt/vol), placed in sterile airtight polypropylene tubes with cells floating on top of the medium in a thin cell layer, and incubated for 1 h with lactacystin (10 µM) or calpeptin (50 µM), and then the cells were diluted 6-fold with media and TNF{alpha} or PIA were added at various concentrations. After 30 h of incubation, cells were washed three times with buffer described above.

Lipolysis assay
Lipolysis was measured by following the rate of glycerol release, as previously described (29). After washing, cells were incubated at 37 C for 30 min, and glycerol released was assayed using a kit from Sigma (St. Louis, MO).

Northern blot analysis of G proteins
Following 24-h incubation of rat adipocytes with TNF{alpha}, total cellular RNA was prepared from adipocytes using Tri-Reagent (Sigma). Five to ten micrograms total RNA were separated on formaldehyde-agarose gel electrophoresis (1%) and transferred to nylon membranes following the directions contained in the Northern blotting kit (Ambion, Inc., Austin TX). Plasmids containing the cDNA for rat Gi1{alpha}, Gi2{alpha}, and Gi3{alpha} (30) were provided by Dr. Graeme Milligan (University of Glasgow, Scotland, UK). The cDNA probes were labeled with [{alpha}32P]CTP by Prime-a-Gene labeling system (Promega Corp., Madison, WI) using the manufacturer’s directions. After hybridization, membranes were washed and exposed to phosphor screen for 1–4 h. The Northern blots were analyzed using Storm (Molecular Dynamics, Inc., Sunnyvale, CA). All blots were examined for homogeneity in RNA loading by monitoring 18S rRNA.

Isolation of adipocyte membranes
Following incubation for 30 h, adipocytes were washed three times in the buffer used for adipocyte isolation with no glucose and only 1% BSA followed by one wash in homogenizing buffer (250 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulphonyl fluoride, 20 mM HEPES, pH 7.4). The cells were then homogenized by vigorous mixing in 16 x 100-mm glass test tubes on a vortex mixer. The homogenate was centrifuged for 5 min at 1,000x g, and the supernatant was centrifuged for 30 min at 16,000 x 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 preparations was determined by the Bradford method (31) using a kit from Bio-Rad Laboratories, Inc., (Hercules, CA) and bovine {gamma}-globulin as a standard.

Quantification of G proteins by Western blotting
The {alpha}-subunits of Gi1 and Gi2 were quantified using antiserum SG1, which was raised against a synthetic peptide (KENLKDCGLF) corresponding to the C-terminal 10 amino acids of transducin (which is absent from adipocytes), as described in our previous reports (7, 15, 16). This antiserum recognizes the {alpha}-subunit of Gi1 and Gi2 equally, because in the C-terminal region these two molecules are identical and differ from transducin in only one amino acid. Adipocyte crude membrane fractions were diluted to equal protein concentrations, further diluted with an equal volume of 2x concentrated Laemmli sample buffer (32) (70 mM Tris, 10% glycerol, 2% SDS, 10% mercaptoethanol) and heated for 5 min at 95 C. The membranes were resolved on SDS-PAGE (12% acrylamide, 0.06% bisacrylamide, run at 35 mA constant current) and then electrophoretically transferred to nitrocellulose. The gels were 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 and 500 mM NaCl (pH 7.5) and then washed once for 15 min in TBS containing 0.2% IGEPAL (Sigma). Following further washes with TBS, the nitrocellulose membranes were incubated overnight with primary antiserum (in 1% dried milk/TBS diluted 1:2000 for chemiluminescent detection). The membranes were washed again several times with TBS-IGEPAL and incubated with secondary antibody for 1 h (goat antirabbit IgG in 1% dried milk/TBS diluted 1:3000). 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 were quantified using an UltraScan laser densitometer (Amersham Pharmacia Biotech).

26S Proteasome assay
Ten micrograms cytoplasmic protein were added to assay buffer [20 mM Tris-HCl (pH 8.0), 1 mM ATP, and 2 mM MgCl2] in the presence of the synthetic fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (Suc-Leu-Leu-Val-Tyr-AMC; final concentration, 60 µM) (33) in a final volume of 1 ml. The tubes were incubated at 30 C for 30 min, after which the reaction was terminated by the addition of 1 ml cold ethanol and the lysate was spun at 12,000 x g for 10 min at 4 C. Proteolytic hydrolysis of the peptidyl-7-amino bond liberates the highly fluorescent 7-amino-4-methylcoumarin (AMC). Fluorescence was measured in a fluorometer at 440-nm emission (Iex380). For each assay, a standard curve was measured with known dilution of 7-amino- methylcoumarin.

Calpain activity assay
Calpain activity was measured in intact adipocytes using the peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC as previously described (34, 35). Adipocytes were washed and plated in 500 µl (10% suspension) per well in 24-well plates and 200 µl assay buffer (115 mM NaCl, 1 mM KH2PO4, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 25 mM HEPES buffer, pH 7.4) was added to all wells. Suc-Leu-Leu-Val-Tyr-AMC substrate (62.5 µM) was added to each well at the start of the assay, and cellular fluorescence was quantified at 5-min intervals starting immediately after the addition of substrate, with a Cytofluor Series fluorometer (Applied Biosystems, Foster City, CA) with 360 nm excitation and 460 nm emission filters.

Lactate dehydrogenase (LDH) assay
Media from adipocyte incubations were assayed for LDH by measuring the rate of decrease in A340 in the presence of pyruvate and reduced nicotinamide adenine dinucleotide, as described by Vassault (36).

Statistical analysis
All experiments were repeated at least three times on different days. Statistical analysis was, as appropriate, by paired t test or by one-way ANOVA followed by the Tukey test for pair-wise comparisons of treatment groups with the use of SigmaStat 2.0 (Jandel, San Rafael, CA). P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have previously demonstrated that long-term exposure of adipocytes to TNF{alpha} increases the basal rate of lipolysis (3) by a mechanism that involves down-regulation of Gi{alpha} isoforms, especially Gi1{alpha} (7). We hypothesized that the decrease in Gi levels could result from a decrease in Gi expression or an increase in Gi degradation or both. To test these hypotheses, adipocytes were first incubated in primary culture for 24 h in the presence or absence of TNF{alpha} and steady-state Gi1{alpha} and Gi2{alpha} mRNA levels in control and treated cells were determined by Northern blot analysis relative to 18S RNA (Fig. 1Go). TNF{alpha} did not detectably alter the abundance of either Gi1{alpha} or Gi2{alpha} mRNA. This finding suggests that the mechanism of TNF{alpha}-induced down-regulation is an increase in the rate of G protein degradation, rather than an inhibitory effect on G protein gene expression.



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  		Figure 1. Northern blots for Gi {alpha} isoforms in TNF{alpha}-treated cells. Adipocytes were incubated without or with TNF{alpha} (50 ng/ml) for 24 h. RNA was isolated, analyzed on Northern blots, and probed with cDNAs for Gi1{alpha} or Gi2{alpha}, as indicated. The lower panels show 18S RNA.

 
We next investigated the participation of cytoplasmic proteolytic pathways in TNF{alpha}-induced changes in lipolysis and Gi levels. Calpain and the ubiquitin/proteasome systems are important in signaling and in degradation of specific intracellular proteins for which specific cell-permeant inhibitors are available. Calpeptin (Z-Leu-norleucinal), a competitive inhibitor of the m- and µ calpain catalytic subunits, has no detectable inhibitory activity for the 26S proteasome (37). Conversely lactacystin, an irreversible inhibitor of the ß- catalytic activity of the 26S proteasome (38), has no effect on basal or TNF{alpha}-inducible calpain activity (37). To determine whether either of these pathways is involved in the effect of TNF{alpha} on lipolysis, adipocytes were pretreated for 1 h with 10 µM lactacystin or 50 µM calpeptin. TNF{alpha} (50 ng/ml) was added and the cells were incubated in primary culture for a further 30 h. After incubation, the cells were washed three times, and the rate of basal lipolysis (glycerol release) was measured after a further 30-min incubation. After TNF{alpha} stimulation, the rate of basal lipolysis increased approximately 2-fold (Fig. 2Go), consistent with our previous reports (3, 7). Neither lactacystin nor calpeptin treatment alone had any effect on lipolysis when compared with the control group. However, lactacystin completely blocked the lipolytic effect of TNF{alpha} whereas calpeptin was without effect. In combination, no additional effect of calpeptin was seen over that of lactacystin (data not shown). These observations are consistent with a predominant role of the proteasome pathway mediating TNF{alpha}-stimulated lipolysis.



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  		Figure 2. The lipolytic effect of TNF{alpha} is blocked by a proteasome inhibitor. Adipocytes were incubated for 1 h with 10 µM lactacystin (proteasome inhibitor) or 50 µM calpeptin (calpain inhibitor), then TNF{alpha} (50 ng/ml) was added and the cells incubated for 30 h. Cells were washed and glycerol release was measured after 30 min. *, P < 0.05 vs. other groups.

 
To determine the effective concentration of protease inhibitors on lipolysis, we incubated cells for 30 h with TNF{alpha} plus various concentrations of lactacystin or calpeptin. After incubation the cells were washed and glycerol release was determined as before. As can be seen in Fig. 3Go, the lipolytic effect of TNF{alpha} was blocked by lactacystin in a dose-dependent manner, with a 50% inhibitory concentration of approximately 2 µM and saturation at 5 µM, but calpeptin had no effect at concentrations up to 100 µM. It should be pointed out that these were the concentrations present during the 1-h preincubation and that the cells were subsequently diluted 6-fold for the 30-h treatment with TNF{alpha} (see Materials and Methods). However, this does not affect the conclusion that lactacystin blocks the effect of TNF{alpha} but calpeptin does not.



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  		Figure 3. Dose-response curve for the effect of protease inhibitors on lipolysis. Adipocytes were incubated with several concentrations of lactacystin or calpeptin for 1 h, TNF{alpha} (50 ng/ml) was then added, and the cells incubated for 30 h. Cells were then washed and glycerol release was measured over 30 min. The half-maximally effective concentration of lactacystin was approximately 2 µM.

 
Because prolonged treatment of adipocytes with PIA has similar effects on lipolysis to prolonged treatment with TNF{alpha} (7), we also investigated the effect of the protease inhibitors on the chronic lipolytic effect of PIA. Adipocytes were preincubated for 1 h with or without lactacystin or calpeptin as before, and then PIA (300 nM) was added and the cells were incubated for 30 h. After incubation, the cells were washed and the rate of glycerol release was measured. As can be seen in Fig. 4Go, adipocytes incubated in the presence of PIA for 30 h showed an approximately 2-fold increase in lipolytic rates, as we have previously reported (7). The presence of lactacystin in the incubation medium decreased PIA-stimulated glycerol release, but calpeptin was without effect. The results suggest participation of the proteasome in the chronic lipolytic effect of PIA.



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  		Figure 4. PIA-stimulated lipolysis is blocked by lactacystin. Adipocytes were incubated for 1 h with lactacystin (Lacta; 10 µM) or calpeptin (Calp; 50 µM), PIA (300 nM) was then added, and the cells incubated for 30 h. Cells were then washed and glycerol release was measured over 30 min. *, P < 0.05; **, P < 0.01 vs. other groups.

 
To assess the effect of protease inhibitors on Gi levels, adipocytes were treated with lactacystin or calpeptin for 1 h, and then TNF{alpha} (50 ng/ml) was added and the cells were incubated for 30 h. Cells were then washed, and crude plasma membranes were prepared and analyzed on Western blots with antibodies that bind to the {alpha}-subunits of Gi1 and Gi2. Consistent with previous results (7), prolonged incubation of adipocytes with TNF{alpha} decreased the cellular concentrations of both Gi1{alpha} and Gi2{alpha}, as shown in Fig. 5Go. In the presence of lactacystin, the ability of TNF{alpha} to decrease Gi1{alpha} and Gi2{alpha} concentrations is completely blocked, in good agreement with that for TNF{alpha}-induced activation of lipolysis. Conversely, treatment with calpeptin did not alter the cellular concentration of either isoform of Gi{alpha}. These findings suggest a contribution of the proteasome in TNF{alpha}-induced Gi down-regulation.



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  		Figure 5. TNF{alpha} decreases cellular concentrations of Gi1{alpha} and Gi2{alpha}. After 30 h of incubation, adipocytes were homogenized, and crude membranes were isolated and analyzed on Western blots (50 µg membrane protein per lane). The blots were probed with antiserum SG1 to label Gi1{alpha} and Gi2{alpha}. The insets above the bars are representative examples of Gi subtypes. P < 0.05 vs. other groups.

 
To determine whether protease inhibitors prevented the PIA-mediated Gi down-regulation, we treated adipocytes with lactacystin and calpeptin as before, and then PIA was added and the cells were incubated for 30 h. Crude plasma membranes were isolated and analyzed on Western blots. The results in Fig. 6Go show a decrease in Gi1{alpha} and Gi2{alpha} levels in PIA-treated cells, compared with control cells, as we have reported before (7, 15, 16). Interestingly, incubation of adipocytes with lactacystin was equally effective at blocking PIA-induced Gi1{alpha} and Gi2{alpha} down-regulation as it was in preventing TNF{alpha}-induced down-regulation. Again, calpeptin had no effect on Gi levels. These findings suggest that both PIA-induced and TNF{alpha}-induced Gi down-regulation involves the proteasome.



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  		Figure 6. PIA decreases cellular concentrations of Gi1{alpha} and Gi2{alpha}. After 30 h of incubation, adipocytes were homogenized, and crude membranes were isolated and analyzed on Western blots (50 µg membrane protein per lane). The blots were probed with antiserum SG1 to label Gi1{alpha} and Gi2{alpha}. The insets above the bars are representative examples of Gi subtypes. P < 0.05 vs. other groups.

 
To investigate further the role of the proteasome in Gi down-regulation, the effect of TNF{alpha} and PIA on proteasome activity was measured in the presence or absence of lactacystin. Hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC was used to measure proteasome activity in whole-cell lysates, as described in Materials and Methods. We have previously shown that hydrolysis of the fluorogenic substrate is dependent on proteasome activity because no proteolysis is seen after depletion of proteasome from cellular lysates by ultracentrifugation (37). As shown in Fig. 7aGo, in TNF{alpha}-treated cells, Suc-Leu-Leu-Val-Tyr-AMC hydrolysis was 2-fold higher than that observed in control cells. In cells pretreated with the potent irreversible proteasome inhibitor lactacystin, lower proteasome activity was seen, indicating that the cellular proteasome activity was inhibited. This effect was consistent for either control or TNF{alpha}-treated cells. Similarly, an increase in proteasome activity was seen in adipocytes treated with PIA, and lactacystin inhibited proteasome activity, both in control and PIA-treated cells (Fig. 7BGo). Therefore, it appears that both TNF{alpha} and PIA can cause an increase in proteasome activity in adipocytes.



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  		Figure 7. Proteasome activity in adipocytes treated with TNF{alpha} or PIA. Adipocytes were treated with TNF{alpha} (50 ng/ml) or PIA (300 nM) and protease inhibitors, as described in Figs. 1Go and 3Go. The proteasome activity was assayed in cellular extracts using Suc-Leu-Leu-Val-Tyr-7-AMC as a substrate. The inset shows a standard curve with known dilutions of AMC. *, P < 0.05 TNF{alpha} vs. other groups; **, P < 0.01 vs. other groups.

 
To determine whether an increase in calpain-like protease activity may be contributing to decreased Gi concentrations in adipocytes, the effects of TNF{alpha} and PIA on calpain activity was examined using the fluorescent peptide substrate, in the presence or absence of calpeptin. Because the measurements of dynamic changes in intracellular calpain activity in broken cells is hampered owing to the presence of endogenous calpastatin inhibitor that rapidly associates with active calpains following cell disruption (37), we monitored the hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC fluorometrically in intact adipocytes (37). As can be seen in Fig. 8AGo, TNF{alpha} and calpeptin alone or combined produced a decrease in calpain activity in relation to control cells. Similar results were obtained in PIA-treated adipocytes as shown in Fig. 8BGo.



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  		Figure 8. Effect of TNF{alpha} or PIA on calpain activity in intact adipocytes. Adipocytes were incubated for 1 h with lactacystin (10 µM) or calpeptin (50 µM), then TNF{alpha} (50 ng/ml) or PIA (300 nM) was added and the cells incubated for 30 h. Cells were then washed and loaded with calpain substrate at time 0. Accumulation of calpain hydrolytic products was measured at 5-min intervals in a microplate fluorometer.

 
To evaluate possible effects on cell viability, we measured the effect of all the agents used on adipocyte lysis by measuring lactate dehydrogenase activity in the media. There was no significant difference in LDH activity released into incubation media between control and TNF{alpha}, PIA, or protease inhibitor-treated adipocytes (data not shown), indicating that the treatments did not affect cell viability. Additionally, we have found that the lipolytic effect of TNF{alpha} is reversible (3), further supporting the conclusion that cell viability is unaffected. Similarly, we have demonstrated that PIA-induced Gi down-regulation is reversible when PIA is removed from the cells, with concentrations returning to control levels within 2–3 d (16).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have reported previously that prolonged incubation of adipocytes with either TNF{alpha} (3) or PIA (7) increases the rate of lipolysis by a mechanism that involves down-regulation of Gi (7). We now have investigated further the mechanism by which TNF{alpha} and PIA down-regulate Gi and stimulate lipolysis in adipocytes. Our results show that lactacystin, a specific proteasome inhibitor, blocks the ability of both TNF{alpha} and PIA to chronically increase the rate of lipolysis. Conversely calpeptin (a selective calpain inhibitor) had no effect, at concentrations as high as 100 µM. Similar effects were obtained in adipocytes incubated with PIA for 30 h, in which there was observed a reduction in the rate of lipolysis in presence of lactacystin and no effect with calpeptin. The specificity of these protease inhibitors for proteasome and calpain activities was directly measured in vitro by Han et al. (37). In a variety of mammalian cells, lactacystin blocks degradation of both long-lived and short-lived proteins similarly, by inhibiting nonlysosomal pathway for protein degradation in cells and did not reduce cell viability (39), as shown in the present study, or protein synthesis for at least several hours (39). These differential effects of the inhibitors (lactacystin and calpeptin) on lipolysis are consistent with a predominant role of the proteasome pathway mediating the chronic lipolytic effect of both TNF{alpha} and PIA.

Corroborating previous results, prolonged incubation of adipocytes with TNF{alpha} or PIA produce down-regulation of Gi1{alpha} and Gi2{alpha}, and the mechanism does not appear to be secondary to a decrease in mRNA concentrations (Fig. 1Go). G proteins do not seem to be regulated at the transcriptional level. In particular, the promoter regions of all three Gi{alpha} subunits, up to 500 bp upstream of the ATG codon are guanine + cytosine-rich, with several guanine-cytosine boxes present in all three genes (35). This suggests that Gi{alpha} subunits can be considered housekeeping genes, making it unlikely that they are regulated by changes in gene expression. Consistent with this, we have found that after both agonist-induced and TNF{alpha}-induced down-regulation of Gi, mRNA concentrations do not change, despite an evident loss of protein. Also relevant to our studies is a recent report (23) indicating that the down-regulation of Gi2{alpha} observed in transfected Chinese hamster ovary cells expressing the human {alpha}2AR after 24 h of exposure of the cells to epinephrine was not accompanied by a decrease in Gi2{alpha} mRNA. Together these findings strongly suggest that the mechanism for Gi down-regulation is an increase in the rate of proteolysis mediated by the proteasome pathway, rather than decreased transcription or translation.

As additional evidence for proteasome-mediated, calpain-independent pathway for Gi down-regulation, we analyzed the effect of TNF{alpha} or PIA on both protease activities. We observe here that long-term treatment with either TNF{alpha} or PIA significantly changes proteasome activity in adipocytes. The proteasome is a multiprotein complex of approximately 14 nonidentical polypeptides responsible for energy-dependent selective hydrolysis of cytoplasmic structural and regulatory proteins. Recently, it has been appreciated that proteasome activity can be regulated by inflammatory hormones that can induce the synthesis of two inducible ß-catalytic forms of the proteasome, low molecular mass proteins-2 and -7 (LMP-2 and 7) that are encoded within the major histocompatibility class II locus. Following their synthesis, LMP-2 and -7 are incorporated into the proteasome at the expense of other constitutive subunits to alter its substrate specificity (40). In particular, interferons, particularly interferon-{gamma}, and TNF induce coordinate expression of LMP-2 and -7 in epithelial cells to influence antigen processing and cell surface-associated major histocompatibility class I expression (41, 42). Here we observe that TNF and PIA induce proteasome activity in adipocytes and that changes in proteasome activity is linked to changes in lipolytic rate through relief of tonic Gi-mediated inhibition. It will be of interest to determine in future studies whether this is mediated through increased expression of LMP subunits.

In contrast to their effects on proteasome activity, we observe that both TNF{alpha} and PIA decrease calpain proteolytic activity in treated adipocytes. This observation is surprising because TNF is a rapid and potent inducer of calpain activity in hepatocytes through a mechanism involving membrane to cytoplasmic redistribution of the 80-kDa catalytic subunit of m-calpain (37). In HepG2 cells, increases in the cytoplasmic abundance of m-calpain are detectable between 2 and 10 min after TNF treatment, but time points longer than 15 min were not examined (37). Therefore, these two observations may be reconciled by either the consequence of cell type-specific effects of TNF or may be owing to the chronicity of TNF stimulation in the adipocyte model (required for lipolysis). Further investigation will be required to resolve this issue.

The mechanism of G-protein activation in response to an agonist involves dissociation of the {alpha}-subunit from ß{gamma}. This event could unmask {epsilon}-amino groups of lysine residues on the {alpha}-subunit. {epsilon}-Amino groups have been identified as the site of coupling of ubiquitin to proteins (43). There is evidence that certain G-protein {alpha}-subunits dissociate from the plasma membrane, become more loosely associated (44), or dissociate from multimeric to monomeric forms (45) in response to agonists. These changes could make these proteins a better substrate for ubiquitination. Alternatively, disassociated G-protein subunits may be accessible to cytoplasmic serine-threonine kinases that could phosphorylate Gi and allow recognition by specific ubiquitin ligases. In the well-studied example of inhibitory nuclear factor {kappa}B (I{kappa}B) proteolysis, signal inducible phosphorylation in its NH2 regulatory domain is required for recognition by the ubiquitin ligase, E3RSI{kappa}B (E3RSI{kappa}B does not bind the nonphosphorylated regulatory domain [46 ]). Finally, TNF- or PIA-inducible proteasome activity may have sufficiently different substrate recognition properties that Gi is more efficiently recognized. The requirement for ubiquitination, phosphorylation, or both will require further investigation. It has been demonstrated that the platelet-derived growth factor receptor is ubiquitinated in response to binding of its agonist (47). This finding suggests a role for ubiquitin in agonist-induced down- regulation of platelet-derived growth factor receptor. Finally, it has been reported that transducin (a G protein involved in phototransduction and highly homologous to the Gis) is a substrate for ubiquitin-dependent proteolysis (48).

As mentioned above, G proteins may become more loosely associated with the plasma membrane on activation. Therefore, a possibility that must be considered is that the G-protein {alpha}-subunits may be present in the cytoplasm after TNF{alpha} treatment, rather than totally lost from the cell. Our attempts to perform Western blots on whole-cell lysates were unsuccessful because of the high background produced by the many proteins present in such extracts (data not shown). However, we believe that this is an unlikely explanation for our findings. First, the time course of our experiments is quite prolonged. It is unlikely that G protein {alpha}-subunits would remain undegraded in the cytoplasm for prolonged periods. And second, it is difficult to envisage a mechanism by which a proteasome inhibitor would prevent translocation of an intact, active G protein from the plasma membrane to some other intracellular compartment. Even if this were the case, the conclusion would not be very different because the G protein would still be in a location in which it was functionally inactive (i.e. not in the plasma membrane). However, it is possible that further experiments would reveal G protein degradation products within the cell.

In summary, we describe a central role for Gi down- regulation in control of lipolysis in response to chronic exposure to both TNF{alpha} and PIA. Our findings suggest that proteasome-mediated proteolysis is involved either directly or indirectly in the mechanism of Gi down-regulation and that targeting this pathway may be one method for control of insulin resistance syndromes in humans.


   Acknowledgments
 
We are grateful to Bob Burns and Greg Waldron for help with the figures.


   Footnotes
 
This work was partially supported by the Stephen C. Clark fund. L.M.B. was the recipient of a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Federal University of Minas Gerais (Brazil) fellowship.

Abbreviations: AMC, 7-Amino-4-methylcoumarin; LDH, lactate dehydrogenase; I{kappa}B, inhibitory nuclear factor {kappa}B; LMP, low molecular mass proteins; PIA, N6-phenylisopropyl adenosine; TBS, Tris-buffered saline.

Received May 11, 2001.

Accepted for publication August 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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