QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.107.039479v1 73/1/215    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zu, L. Articles by Xu, G. Search for Related Content PubMed PubMed Citation Articles by Zu, L. Articles by Xu, G.

Salicylate Blocks Lipolytic Actions of Tumor Necrosis Factor- in Primary Rat Adipocytes

Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China (L.Z., H.J., J.H., C.X., S.P., M.L., G.X.); and Key Laboratory of Molecular Cardiovascular Sciences, the Ministry of Education of China, Beijing, China (L.Z., C.X., S.P., G.X.)

Received July 1, 2007; accepted October 2, 2007

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Increased systemic free fatty acids (FFA) impair insulin sensitivity. In obese and diabetic subjects, production of tumor necrosis factor- (TNF-), a proinflammatory cytokine, is elevated. TNF- has a variety of effects by inducing inflammation, decreasing glucose utilization, and stimulating adipocyte lipolysis to release FFA to plasma. High doses of nonsteroidal anti-inflammatory drug salicylates have long been recognized to lower blood FFA and glucose in humans, although the mechanisms are not fully understood. In this report, we show that sodium salicylate at therapeutic concentrations directly blocks TNF--stimulated lipolysis and therefore inhibits FFA release from primary rat adipocytes. To elucidate the cellular basis of this action, we show that salicylate suppresses TNF--induced extracellular signal-related kinase activation and intracellular cAMP elevation, two early events during the lipolysis response to TNF-. Furthermore, salicylate prevents the down-regulation of cyclic-nucleotide phosphodiesterase 3B, an enzyme responsible for cAMP hydrolysis. Perilipins coat intracellular lipid droplet surface by restricting lipase access to the triacylglycerol substrates. TNF- down-regulates perilipin but promotes its phosphorylation during lipolysis stimulation; these actions are efficiently reversed by salicylate. Salicylate slightly reduces basal but completely inhibits TNF--liberated lipase activity. In contrast, neither salicylate nor TNF- alters the protein levels of hormone-sensitive lipase and adipose triglyceride lipase. In addition, sodium salicylate restricts basal lipolysis simulated by a high concentration of glucose and significantly diminishes the high glucose-enhanced lipolysis response to TNF-. These results provide novel evidence that salicylate directly blocks TNF--mediated FFA efflux from adipocytes, hence reducing plasma FFA levels and increasing insulin sensitivity.

The nonsteroidal anti-inflammatory drugs sodium salicylate and acetylsalicylic acid (aspirin) are widely used to control pain, fever, and rheumatic arthritis. Aspirin is standard care for patients with diabetes with cardiovascular disease. In 1877, Ebstein found that high doses of sodium salicylate dramatically reduced glucosuria in patients with diabetes (Ebstein, 1877). Further early studies showed that high doses of salicylates also lowered blood glucose concentrations in rodents (Bizzi et al., 1965) and in diabetic humans (Reid et al., 1957; Carlson and Ostman, 1961). In contrast, conflicting results demonstrate that lower doses of aspirin (3 g/day for 3 days) may not improve glucose utilization in healthy (Newman and Brodows, 1983) and diabetic subjects (Bratusch-Marrain et al., 1985). Important discrepancies between these studies included lower salicylate dosages (<3 g/day) and therapeutic duration (a few days) in the more recent studies than in the earlier studies (6-9 g/day for 1-3 weeks). Although salicylate reduces TNF- production in rat macrophages (Vittimberga et al., 1999), administration of low-dose aspirin (325 mg/days) may result in a rebound increase in cytokine-induced synthesis of interleukin-1β and TNF- in human (Endres et al., 1996), which can be expected to impair insulin sensitivity. Most recently, several studies indicate that high doses of salicylates attenuate deleterious effects of lipids and therefore improve lipid-induced insulin resistance both in rodents (Kim et al., 2001; Yuan et al., 2001) and humans (Hundal et al., 2002; Möhlig et al., 2006).

Early studies indicated that salicylates lower serum FFA concentrations in healthy and diabetic subjects, which possibly contributes to their hypoglycemic effects. The FFA-lowering action may result from the suppression of FFA release from adipose tissue to plasma (Reid et al., 1957; Carlson and Ostman, 1961; Bizzi et al., 1965), because salicylates do not seem to affect FFA esterification and turnover. The FFA mobilization to plasma is governed by lipolytic reaction of adipocytes physiologically stimulated by catecholamines. Although the mechanism remains unidentified, salicylates can reduce catecholamine-stimulated lipolysis in isolated adipocytes (Stone et al., 1969; Schönhöfer et al., 1973).

In persons who are obese, elevated TNF- acts as a strong lipolytic stimulator (Hotamisligil et al., 1995). We (Ren et al., 2006) and others (Ryden et al., 2002) have indicated that activation of extracellular signal-related kinase (ERK) is a critical factor mediating TNF--induced lipolysis of adipocytes, whereas suppression of ERK signaling inhibits the lipolysis. Salicylates are able to inhibit ERK activation in neutrophils (Pillinger et al., 1998) and in TNF--stimulated fibroblasts (Schwenger et al., 1996). We hypothesize that salicylate may directly attenuate TNF--stimulated lipolysis response in adipocytes, which could be one of cellular bases for salicylate to reduce plasma FFA concentrations and thus improve insulin resistance.

In this report, we show that sodium salicylate at therapeutic concentrations directly inhibits adipocyte lipolysis response to TNF-. Salicylate attenuates TNF--induced ERK activation, cellular cAMP elevation, and the down-regulation of cyclic-nucleotide phosphodiesterase 3B (PDE3B), thus restricting lipolysis. Furthermore, salicylate corrects TNF--dysregulated protein levels and phosphorylation state of perilipins on adipocyte lipid droplet surface, inhibits TNF--liberated lipase activity, but does not alter the protein expression of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This study provides novel evidence that salicylate directly antagonizes TNF--stimulated FFA efflux from adipocytes to plasma, thus lowering systemic FFA levels and increasing insulin sensitivity.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Recombinant rat TNF- was purchased from Pepro-Tech EC (London, UK). Sodium salicylate was from Beijing Chemical Reagents Co. (Beijing, China). Phenol red-free Dulbecco's modified Eagle's medium (DMEM) containing glucose (5 mM) and protein kinase A (PKA) inhibitor H89 were from Sigma Chemical (St. Louis, MO). Enzyme materials used for enzymatic assays were products of Totobo Co. (Tokyo, Japan). Antibodies against ERK-1, phospho-ERK1/2, PDE3B, anti-Gi1, actin, and horseradish peroxidase (HRP)-conjugated second antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against rat perilipin and rat HSL (He et al., 2006) were generous gifts from Dr. Londos at the U.S. National Institutes of Health (Bethesda, MD). Antibody against phosphorylated PKA substrate (RRXS/T motif) was from Cell Signaling Technology (Danvers, MA). Rat anti-ATGL antibody was from Cayman Chemical (Ann Arbor, MI). Nitrocellulose blot membrane, prestained protein molecular weight marker, and ultrasensitive enhanced chemiluminescence (ECL) detection reagents were from Applygen Technologies Inc. (Beijing, China).

Isolation and Culture of Primary Rat Adipocytes. Adipocytes were isolated from epididymal fat pads of Sprague-Dawley rats (150-180 g) according to our laboratory method (He et al., 2006; Jiang et al., 2007). The fat pads were minced and digested in 5 ml of Krebs-Ringer solution containing 0.75 mg/ml type I collagenase, 200 nM adenosine, 25 mM HEPES, pH 7.4, and 1% defatted bovine serum albumin. After incubation for 40 min at 37°C in a water bath with shaking at 100 cycles/min, cells were filtered through a nylon mesh and washed 3 times with warmed DMEM containing 200 nM adenosine. Adipocytes floating on the top of the tube were packed by centrifuging at 200g for 3 min. Every 25 µl of packed adipocytes was resuspended in 500 µl of phenol red- and serum-free DMEM containing 2% defatted bovine serum albumin and preincubated at 37°C for 1 h before treatments (He et al., 2006). Next, adipocytes were incubated in the presence or absence of the tested agents, followed by the assays described below.

Fatty Acid Assay. The concentration of FFA in the culture medium was determined by colorimetric assay as described previously (Itaya, 1977) with some modifications. In brief, 50 µl of culture medium was mixed with 120 µl of isooctane and 80 µl of cupric acetate-pyridine. The mixture was vortexed and centrifuged for 10 min at 12,000g at room temperature. The upper organic phase (80 µl) was transferred to a clean tube. One hundred eighty microliters of the color development reagent consisting of diphenylcarbazone and diphenylcarbazide in methanol was then added to the tube. The mixture was vortexed for 5 s, and the color of the reaction was developed immediately. The absorbance of the color reaction at 540 nm was spectrophotometrically measured in a 96-well plate.

Glycerol Assay. Glycerol content released in culture medium of adipocytes served as an index of lipolysis and was determined at the absorption at 490 nm (He et al., 2006; Ren et al., 2006), by use of a colorimetric assay (GPO Trinder reaction) kit from Applygen Technologies Inc. Lipolysis data were expressed as micromoles of glycerol or FFA per milliliter of packed cell volume of adipocytes.

Western Blot. Adipocytes were packed and lysed in sample buffer containing 62 mM Tris-HCl, pH 6.8, 5% SDS, 0.1 mM sodium orthovanadate, and 50 mM sodium fluoride (He et al., 2006). After centrifugation at 12,000g for 10 min at 4°C, the lysate was transferred to a new tube and heated at 95°C for 5 min. Protein content in the extracts was determined by use of a bicinchoninc acid protein assay kit from Applygen Technologies Inc. Equal amounts of proteins were loaded and separated by 10% SDS-polyacrylamide gel electrophoresis, then transferred to a nitrocellulose membrane. The membranes were blocked for 1 h in 5% nonfat milk in 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, and 0.05% Tween 20) (Xu et al., 2005, 2006), then incubated with primary antibodies overnight at 4°C, followed by incubation for 1 h with HRP-conjugated secondary antibodies. The blots were developed by use of an enhanced chemiluminescence (ECL) detection kit. If required, the antibodies bound to membranes were removed by a commercial stripping solution from Applygen Technologies Inc. Blots were then reprobed with use of other antibodies and developed as described above. Densitometric analysis of protein bands involved use of NIH Image software (http://rsb.info.nih.gov/nih-image/).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Sodium salicylate inhibition of TNF--stimulated lipolysis in rat adipocytes is concentration-dependent. Packed primary rat adipocytes (25 µl) were resuspended and pretreated at 37°C for 1 h with 0.5, 1.0, or 5.0 mM sodium salicylate (NaSal) and then incubated for 24 h in the presence of 50 ng/ml TNF-, sodium salicylate, or both. Glycerol (A) and FFA (B) released in the culture media were lipolysis indexes. The data are expressed as micromoles per milliliter packed cell volume (PCV) of adipocytes. Results are means ± S.E.M. of three separate experiments performed in triplicate. *, p < 0.01 versus control; , p < 0.05; , p < 0.01 versus TNF-.

Assay of Adipose Lipase Activity. After the treatments, adipocytes were washed twice with warmed PBS buffer and packed by centrifugation. The 50 µl of packed adipocytes was lysed in 120 µl of buffer containing 50 mM Tris-HCl, pH 7.4, and 1 mM EDTA. After being vortexed vigorously, the lysate was centrifuged at 12,000g for 15 min at 4°C. The infranatant phase below the fat cake fraction was transferred to a new tube, then centrifuged at 12,000g for 5 min at 4°C. The supernatant was used for the determination of cellular lipase activity against emulsified triolein substrate (Peled and Krenz, 1981). The mixture was incubated for 30 min at 37°C, when the lipases hydrolyze emulsified triolein to produce glycerol. The release of glycerol from triolein hydrolysis represented the activity of adipose lipase and was assayed as described above.

Cell Viability Assays. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay were performed according to the manufacturer instructions of two commercial kits available from Applygen Technologies Inc. The MTT assay was based on the reduction of tetrazolium salt to colored formazan product by mitochondrial enzymes present only in living cells. Cell viability rates by MTT assay were directly proportional to the absorbance values of optical density measured at 570 nm in a 96-well plate and were presented as percentage of the control values. LDH activity was measured based on the conversion of lactate to pyruvate in the presence of LDH with parallel reduction of NAD⁺, and the further reaction of pyruvate with 2,4-dinitrophenylhydrazine to form a colored hydrazone product that has a high optical density in the wavelength range of 400 to 500 nm. The absorbance at 440 nm was determined and used for calculating LDH activity. The cell viability index of LDH was presented as a percentage of LDH leakage in medium compared with total LDH activity.

Statistical Analysis. Data are expressed as means ± S.E.M. One-way analysis of variance Tukey's test involved use of GraphPad Prism version 4.0. p < 0.05 was considered significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Sodium Salicylate Blocked TNF--Induced Lipolysis of Primary Adipocytes. Triacylglycerol lipolysis results in the release of FFA and glycerol from fat cells. To determine the effect of sodium salicylate on TNF--induced lipolysis, primary rat adipocytes were pretreated at 37°C for 1 h with 0.5, 1.0, or 5.0 mM sodium salicylate, then incubated for 24 h with 50 ng/ml TNF-, sodium salicylate, or both. The levels of glycerol and FFA released in the culture media were determined as indices of lipolysis. TNF- promoted the levels of glycerol (Fig. 1A) and FFA (Fig. 1B) released in the culture media, which indicated that TNF- stimulated remarkable lipolysis. The TNF--mediated lipolytic action was readily inhibited by sodium salicylate at 0.5 mM (p < 0.05) and completely blocked at 5 mM (p < 0.01), so the antilipolytic effect of sodium salicylate was concentration-dependent.

Next, adipocytes were pretreated with 5 mM sodium salicylate for 1 h, then incubated for 6, 12, or 24 h with 50 ng/ml TNF-, 5 mM sodium salicylate, or both. TNF--stimulated releases of glycerol (Fig. 2A) and FFA (Fig. 2B) were increased at 6 h after treatment and further enhanced at 12 or 24 h. The lipolytic action of TNF- was completely suppressed by the addition of 5 mM sodium salicylate in the media. The basal releases of FFA and glycerol from unstimulated adipocytes were also slightly inhibited by sodium salicylate. Partial oxidation and/or re-esterification of fatty acids within adipocytes may account for the fact that the ratio of fatty acid to glycerol was less than the theoretical 3:1 proportion.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Salicylate inhibition of TNF--stimulated lipolysis is time-dependent. Rat adipocytes were pretreated at 37°C for 1 h with 5 mM sodium salicylate (NaSal) and then stimulated for 6, 12, or 24 h with 50 ng/ml TNF-, sodium salicylate, or both. Glycerol (A) and FFA (B) released in the culture media were lipolysis indexes. The data are means ± S.E.M. of three separate experiments performed in triplicate.

View this table: [in this window] [in a new window]   TABLE 1 Sodium salicylate reduces lipolysis in adipocytes pre-stimulated with TNF- Primary adipocytes were pre-stimulated with TNF- (25 and 50 ng/ml) for 24 h, and then the incubation media were freshly replaced. The cells were incubated for another 24 h in the presence of TNF- or/and 5 mM sodium salicylate (NaSal). Glycerol released in the culture media was then assayed as an index of lipolysis. Data are means ± S.E.M. of three individual experiments each assayed in triplicate.

Evaluation of adipocyte viability by LDH assay indicated no significant changes of LDH leakage into the culture media from adipocytes incubated for 24 h in the presence of sodium salicylate, TNF-, or both (Table 2). Furthermore, the MTT assay revealed that no obvious cytotoxicity of adipocytes was observed under same conditions (Table 2). These data suggest that the tested agents did not affect adipocyte viability.

View this table: [in this window] [in a new window]   TABLE 2 Effects of TNF- and sodium salicylate on cell viability of adipocytes Primary rat adipocytes were pretreated at 37°C for 1 h with 5 mM sodium salicylate (NaSal), then incubated for 24 h in the presence of 50 ng/ml TNF-, sodium salicylate, or both. The MTT assay and LDH leakage assay were performed as described under Materials and Methods. Cell viability data by MTT assay were represented as percentage of the control values. LDH leakage data were expressed as percentage of LDH activity in the culture media compared with total LDH activity present both in the culture media and in whole adipocyte extracts. Results are means ± S.E.M. of three individual experiments performed in triplicate.

Salicylate Inhibited TNF--Stimulated ERK Phosphorylation. Activation of ERK may be one of the major mechanisms by which TNF- induces adipocyte lipolysis (Ryden et al., 2002; Ren et al., 2006). We next examined whether sodium salicylate affected TNF--induced activation of ERK1/2. Primary rat adipocytes were incubated for 0.5, 6, and 24 h with 50 ng/ml TNF-, 5 mM sodium salicylate, or both. Proteins extracted from adipocytes underwent immunoblotting. Neither TNF- nor sodium salicylate altered the level of total ERK-1 proteins, but TNF- induced a notable phosphorylation of ERK1/2 at 30 min after treatment. ERK1/2 activation was sustained at high levels 6 and 24 h after TNF- stimulation (Fig. 3); these alterations paralleled the elevated lipolysis of adipocytes (Fig. 2). Sodium salicylate at 5 mM slightly attenuated basal level of the ERK1/2 phosphorylation in unstimulated adipocytes but completely eliminated the promoted ERK phosphorylation in TNF--stimulated adipocytes (Fig. 3). These effects were concomitant with the inhibitory action of sodium salicylate on basal- and TNF--stimulated lipolysis (Fig. 2). In addition, we also examined the phosphorylation of two other mitogen-activated protein kinases, p38 and JNK (c-Jun-NH₂-terminal kinase); phosphorylation of JNK was rarely detected, but that of p38 was not affected by TNF- or sodium salicylate (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 3. Inhibition of ERK activation by salicylate during TNF--stimulated lipolysis. Adipocytes were preincubated for 1 h with 5 mM sodium salicylate (NaSal), then incubated for 0.5, 6, or 24 h with 50 ng/ml TNF- in the presence or absence of 5 mM sodium salicylate. The culture media were collected and used for glycerol assay (data not shown). The adipocytes were lysed and the equivalent amounts of proteins underwent immunoblotting with use of primary antibodies against phosphorylated-ERK1/2 (p-ERK). After incubation with HRP-conjugated secondary antibodies, the blots were developed by use of an enhanced chemiluminescence detection kit. To detect total ERK-1, the blots were stripped and reprobed with primary anti-ERK-1 antibodies (ERK). The phosphorylated-ERK1/2 levels were quantitated densitometrically. The data of three separate experiments are expressed as percentage of control. *, p < 0.05; **, p < 0.01 versus control; , p < 0.01 versus TNF-.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Sodium salicylate reverses TNF--down-regulated PDE3B. Adipocytes were pretreated for 1 h with 5 mM sodium salicylate (NaSal) and then incubated for 24 h with or without 50 ng/ml TNF-. A, the cells were lysed and equivalent amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were immunoblotted with anti-PDE3B antibodies and peroxidase-conjugated secondary antibodies. After the development of the PDE3B bands, the blots were reprobed with use of anti-actin antibodies. B, the bands were quantitated densitometrically. The PDE3B/actin ratios are the mean ± S.E.M. of three separate experiments. *, p < 0.05 versus control; , p < 0.05 versus TNF-.

View this table: [in this window] [in a new window]   TABLE 3 Effects of TNF- and sodium salicylate on intracellular cAMP levels. Primary rat adipocytes were pretreated at 37°C for 1 h with 5 mM sodium salicylate (NaSal), then incubated for 24 h in the presence of 50 ng/ml TNF-, sodium salicylate, or both. The culture media were collected and assayed for glycerol as an index of lipolysis. Intracellular cAMP content was determined by use of a commercial 125I radioimmunoassay kit. Results (means ± S.E.M.) are representative of three separate experiments each performed in sextuple.

Sodium Salicylate Inhibited TNF--Induced Phosphorylation and down-Regulation of Perilipin. Perilipin coats the surface of intracellular lipid droplets (Xu et al., 2006) and is a major PKA substrate in adipocytes. Down-regulation or phosphorylation of perilipins facilitates lipase-catalyzed hydrolysis of triacylglycerols (Sztalryd et al., 2003; He et al., 2006; Ren et al., 2006). TNF--elevated cellular cAMP, which can result in PKA activation, might result in perilipin phosphorylation. First, we determined whether the PKA might manipulate the phosphorylation of perilipins in TNF--stimulated adipocytes. After the glycerol assays, the adipocyte extracts underwent immunoblotting with use of the specific antibody against a phospho-PKA motif, and then the blots were reprobed with the anti-perilipin antibody. Stimulation with TNF- for 6 h significantly increased perilipin phosphorylation (Fig. 5A) and elevated early lipolysis by 30.3% (Fig. 5B), which can be suppressed by a PKA inhibitor, H89, indicating that the PKA activation accounted for those effects. Next, we evaluated the protein level and phosphorylation of perilipin in the adipocytes treated with TNF- and/or sodium salicylate. TNF- did not alter perilipin levels but greatly promoted its phosphorylation by 6 h (Fig. 5, A, C, and D). Perilipins were significantly down-regulated by 24 h, but the ratio of phosphorylated perilipins to total species remained at a high level (Fig. 5, C and D), which reveals that the long-term lipolytic action of TNF- is related to multiple dysregulation of perilipin proteins. Coincubation with sodium salicylate prevented TNF--mediated phosphorylation and down-regulation of perilipin proteins (Fig. 5, C and D), therefore attenuating the lipolytic action of TNF-.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Effects of sodium salicylate on perilipin phosphorylation and down-regulation stimulated by TNF-. After glycerol assays, adipocyte extracts underwent immunoblotting analysis. Phosphorylated perilipin (P-peri) was detected by use of an antibody against a specific motif (RRXS/T) of the PKA phosphosubstrate. Next, to assess total perilipin proteins, the blots were stripped and reprobed with rabbit anti-perilipin antibodies. The patterns of the protein bands recognized by the specific antibodies separately against PKA phosphosubstrate or perilipin are completely overlapped on the developed X-ray film. In final steps, the blots were stripped again and reprobed with anti-actin antibodies. A, immunoblot. Adipocytes were pretreated for 1 h with a PKA inhibitor H89 and then incubated for 6 h with 50 ng/ml TNF-. B, lipolysis assay in parallel to the tests of A. C, immunoblots. Adipocytes were pretreated for 1 h with 5 mM sodium salicylate (NaSal) and then incubated for 6 or 24 h with 50 ng/ml TNF-, sodium salicylate, or both. D, densitometric analysis of protein bands shown in C. The ratios of phosphorylated and total perilipins (P-Peri/Peri) are the mean ± S.E.M. of three experiments. Peri, total perilipin; P-Peri, phosphorylated perilipin; NaSal, sodium salicylate; *, p < 0.05 versus control; , p < 0.05 versus TNF-.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Effects of sodium salicylate and TNF- on activity and protein level of adipose lipases. A, lipase activity. After incubation for 24 h, primary adipocytes were collected, washed, and lysed. The supernatant was used for the determination of cellular lipase activity. Results are expressed as percentage of the control values and are means ± S.E.M. of three individual experiments performed in triplicate. *, p < 0.05 versus control; , p < 0.05 versus TNF-. B, immunoblotting. Adipocytes were pretreated for 1 h with 5 mM sodium salicylate and then incubated with 50 ng/ml TNF- for 6 or 24 h. Adipocyte extracts underwent immunoblotting with use of the antibodies against HSL or ATGL.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Sodium salicylate restricts the lipolytic actions of TNF- enhanced by a high concentration of glucose. The culture media were supplied with normal- (5 mM) or high-concentration (25 mM) of glucose. Adipocytes were preincubated with 5 mM sodium salicylate for 1 h, then incubated for 24 h with or without 50 ng/ml TNF-. Glycerol released in the culture media was determined as an index of lipolysis. The data are means ± S.E.M. of three experiments performed in triplicate. **, p < 0.01 compared with no 25 mM glucose; , p < 0.05 versus 25 mM glucose alone; , p < 0.01 versus TNF- plus 25 mM glucose.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The mechanisms by which TNF- stimulates adipocyte lipolysis are multifactorial. ERK activation is a major and early signal in the regulation of TNF--stimulated lipolysis (Ryden et al., 2002; Ren et al., 2006). Our data indicate that TNF- promotes significant phosphorylation of ERK1/2 in primary adipocytes at 30 min after stimulation, and the activation remains high at 6 and 24 h. Under the same conditions, phosphorylation of the two other MAPKs, p38 and JNK, was undetectable or unchanged, which is consistent with our previous observation (Ren et al., 2006). In parallel to the ERK activation mediated by TNF-, the release of FFA and glycerol from adipocytes was increased at 6 h. Salicylates can inhibit ERK activation in neutrophils (Pillinger et al., 1998) and in TNF--stimulated fibroblasts (Schwenger et al., 1996); they may therefore limit lipolysis response to ERK activation in adipose cells. Consistent with this hypothesis, sodium salicylate attenuated TNF--activated ERK phosphorylation in adipocytes, thereby abrogating the lipolytic action of TNF-. Salicylate also slightly inhibited basal ERK phosphorylation in unstimulated adipocytes, which may account for its inhibitory effect on basal lipolysis.

Alterations in protein expression are also involved in the regulation of the chronic lipolytic response to TNF-. Our study confirms that PDE3B, responsible for cellular cAMP hydrolysis, can be down-regulated by TNF- (Rahn Landström et al., 2000), an effect associated with promoted adipocyte cAMP levels. When the TNF--mediated down-regulation of PDE3B is prevented by sodium salicylate, the elevation of cellular cAMP falls and the lipolytic action of TNF- is consequently eliminated. Other investigators have indicated that TNF- down-regulates inhibitory G_i-proteins, leading to a withdrawal of endogenous inhibition in adenylyl cyclase, hence elevating cAMP concentrations and stimulating lipolysis in adipocytes (Gasic et al., 1999). We observed that salicylate prevented TNF--mediated decrease of G_i1 isoforms (data now shown), which might also account for the antilipolytic effects of salicylate.

During lipolysis, catecholamine or TNF- elevates cellular cAMP to activate PKA. PKA phosphorylates downstream HSL and perilipin, which cooperatively confer a full lipolytic reaction (Sztalryd et al., 2003). Perilipins are localized at intracellular lipid droplet surface, by functioning as a barrier to restrict lipase access to the triacylglycerol core stored within the lipid droplets (Sztalryd et al., 2003). When perilipin is down-regulated (Ren et al., 2006) or its phosphorylation state is up-regulated (Sztalryd et al., 2003; He et al., 2006), its barrier function could be impaired, thus leading to increased lipolysis (Sztalryd et al., 2003). By using two separate antibodies against phosphorylated PKA substrate and perilipin, we confirmed that the activation of PKA is at least partly responsible for TNF--induced perilipin phosphorylation and early (6 h) lipolytic elevation, because these effects can be blunted by the PKA inhibitor H89. Furthermore, we observed a two-stage alteration of perilipin protein levels in response to TNF- and sodium salicylate. First, the phosphorylation of perilipin was accelerated but total perilipin level unchanged on 6-h stimulation with TNF-, when glycerol release started to increase. The addition of sodium salicylate suppressed perilipin phosphorylation and therefore inhibited lipolysis stimulated by TNF-. Then, 24-h stimulation with TNF- caused a significant decrease in total perilipin levels, but the strength of perilipin phosphorylation remained high. Clearly, salicylate efficiently prevents TNF--mediated early phosphorylation or subsequent loss of perilipin. Perilipin phosphorylation is essential in governing the full lipolysis response to various stimulators (Sztalryd et al., 2003; He et al., 2006). In addition, we (Ren et al., 2006) and other investigators (Souza et al., 1998) have previously demonstrated the lipolytic action of TNF- associated with perilipin down-regulation, whose effects can be restored by the thiazolidinedione antidiabetic agent BRL 49653 (Souza et al., 1998) or the classic antihyperglycemic drug metformin (Ren et al., 2006). Obviously, the amelioration of TNF--dysregulated perilipin and its phosphorylation state is an important basis for the antilipolytic effects of salicylate.

HSL and ATGL are two major lipases for adipose triacylglycerol hydrolysis. With 24-h stimulation, TNF- causes a moderate increase in total lipase activity in adipocytes. Sodium salicylate slightly reduces basal lipase activity but completely inhibits TNF--liberated lipase activity. We were surprised to find that TNF- (as well as sodium salicylate) did not alter the protein levels of HSL and ATGL in primary rat adipocytes. TNF- is reported to down-regulate HSL and ATGL mRNA (Kralisch et al., 2005) and HSL protein (Souza et al., 1998) in differentiated 3T3-L1 adipocytes. However, Green et al. (1994) did not find TNF- to affect HSL protein expression during the lipolytic process in a study also involving primary rat adipocytes. These discrepancies in lipase expression may be due to the different responses of TNF- in fibroblast-derived differentiating 3T3-L1 adipocytes and primary rat adipocytes. HSL is phosphorylated on PKA activation and translocated from the cytosol to the lipid droplet surface to initiate lipolysis in adipocytes; this translocation requires fully phosphorylatable perilipin (Sztalryd et al., 2003). In contrast, ATGL is not a target for PKA phosphorylation and lacks the translocation reaction during lipolytic stimulation (Zimmermann et al., 2004). Whether sodium salicylate and TNF- influence HSL phosphorylation remains to be further clarified.

A high concentration of glucose is also an effective lipolytic stimulator for adipocytes, as observed in the present and prior studies (Green et al., 2004; Ren et al., 2006). TNF--mediated lipolysis can be further enhanced by a high glucose supplied in the adipocyte culture; the salicylate restricts the basal lipolysis simulated alone by a high concentration of glucose at 25 mM and also diminishes the high glucose-enhanced lipolysis response to TNF-. This antilipolytic characteristic of salicylate could be particularly beneficial in limiting FFA efflux from adipose tissue and reducing systemic FFA concentrations under high glucose conditions.

High doses of salicylates have long been recognized to concurrently lower blood FFA and glucose levels in healthy and diabetic subjects (Reid et al., 1957; Carlson and Ostman, 1961; Bizzi et al., 1964, 1965). Although salicylates can also inhibit catecholamine-stimulated lipolysis (Stone et al., 1969; Schönhöfer et al., 1973), they remain effective in lowering plasma FFA level in adrenalectomized or hypothyroid animals (Bizzi et al., 1965), which implies that the FFA-lowering effects probably relate more to the antilipolytic actions against lipolysis mediated by neither catecholamines nor thyroxine. Obesity is considered a chronic inflammatory state. TNF- production is greatly increased in adipose and nonadipose tissues of obese and diabetic persons (Hotamisligil et al., 1995). TNF- stimulates lipolysis to liberate FFA flux from adipocytes and promotes plasma FFA concentrations, hence impairing insulin sensitivity. Thus, direct suppression of TNF--mediated lipolysis by salicylates provides a novel explanation for salicylates lowering plasma FFA levels.

Recent studies suggest that salicylate can reverse obesity- and diet-induced insulin resistance by inactivating IB kinase β (IKKβ) (Kim et al., 2001; Yuan et al., 2001; Hundal et al., 2002). However, another study has indicated that conditional abrogation of IKKβ fails to prevent obesity-induced insulin resistance in vivo (Rohl et al., 2004), which argues against a basis of the salicylate to improve insulin resistance via inhibiting IKKβ. It is noteworthy that in the former studies, the amelioration of insulin sensitivity by salicylates via inactivating IKKβ is virtually accompanied by a 50% decrease of plasma FFA levels in obese and diabetic animals (Yuan et al., 2001) and humans (Hundal et al., 2002). Therefore, it may be rational to speculate that the FFA-lowering effects via limiting FFA efflux from adipocytes could be more critical than the IKKβ-inhibition to contribute to the hypoglycemic actions of the salicylates.

In conclusion, we have presented novel evidence showing that salicylate at therapeutic concentrations directly antagonizes adipocyte lipolysis response to TNF- through multiple mechanisms. This antilipolytic action definitely restricts FFA mobilization from adipocytes to plasma, which could be a cellular basis for the roles of salicylates in reducing systemic FFA levels and improving insulin sensitivity. Future studies are necessary to determine whether the antilipolytic effect of salicylates contributes to their hypoglycemic actions in vivo.

	Footnotes

 
This work was supported by grants 30670779 and 30370535 from the National Natural Science Foundation of China and grant 5072030 from the Beijing Natural Science Foundation. This work was supported by the Program for New Century Excellent Talents in the University, of the Education Ministry of China (NECT-04-0023), and by the Major State Basic Research Development Program of China (2006CB503903).

ABBREVIATIONS: FFA, free fatty acids; TNF-, tumor necrosis factor-; ERK, extracellular signal-related kinase; PDE3B, phosphodiesterase 3B; HSL, hormone-sensitive lipase; ATGL, adipose triglyceride lipase; DMEM, Dulbecco's modified Eagle's medium; PKA, cAMP-dependent protein kinase; HRP, horseradish peroxidase; PCV, packed cell volume; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; JNK, c-Jun-NH₂-terminal kinase; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; IKKβ, IB kinase β.

Address correspondence to: Dr. Guoheng Xu, Department of Physiology and Pathophysiology, Peking (Beijing) University Health Science Center, 38 Xueyuan Road, Beijing 100083, China. E-mail: xug{at}hsc.pku.edu.cn

	References

Top Abstract Materials and Methods Results Discussion References

 
Bergman RN and Ader M (2000) Free fatty acids and pathogenesis of type 2 diabetes mellitus. Trends Endocrinol Metab 11: 351-356.[CrossRef][Medline]

Bizzi A, Codegoni AM, and Garattini S (1964) Salicylate, a powerful inhibitor of free fatty acid release. Nature 204: 1205.[Medline]

Bizzi A, Garattini S, and Veneroni E (1965) The action of salicylate in reducing plasma free fatty acids and its pharmacological consequences. Br J Pharmacol 25: 187-196.[Medline]

Bratusch-Marrain PR, Vierhapper H, Komjati M, and Waldhausl WK (1985) Acetylsalicylic acid impairs insulin-mediated glucose utilization and reduces insulin clearance in healthy and non-insulin-dependent diabetic man. Diabetologia 28: 671-676.[CrossRef][Medline]

Carlson LA and Ostman J (1961) Effect of salicylates on plasma free fatty acid in normal and diabetic subjects. Metabolism 10: 781-787.[Medline]

Ebstein W (1877) Zur therapie des diabetes mellitus, insbesondere uber die anwendung des salicylsauren natron bei demselben. Berliner Klinische Wochenschrift 24: 337-340.

Endres S, Whitaker RE, Ghorbani R, Meydani SN, and Dinarello CA (1996) Oral aspirin and ibuprofen increase cytokine-induced synthesis of IL-1 beta and of tumour necrosis factor-alpha ex vivo. Immunology 87: 264-270.[CrossRef][Medline]

Gasic S, Tian B, and Green A (1999) Tumor necrosis factor stimulates lipolysis in adipocytes by decreasing G_i protein concentrations. J Biol Chem 274: 6770-6775.[Abstract/Free Full Text]

Green A, Dobias SB, Walters DJ, and Brasier AR (1994) Tumor necrosis factor increases the rate of lipolysis in primary cultures of adipocytes without altering levels of hormone-sensitive lipase. Endocrinology 134: 2581-2588.[Abstract/Free Full Text]

Green A, Rumberger JM, Stuart CA, and Ruhoff MS (2004) Stimulation of lipolysis by tumor necrosis factor-alpha in 3T3-L1 adipocytes is glucose dependent: implications for long-term regulation of lipolysis. Diabetes 53: 74-81.[Abstract/Free Full Text]

He J, Jiang H, Tansey JT, Tang C, Pu S, and Xu G (2006) Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes. Biochim Biophys Acta 1761: 247-255.[Medline]

Hotamisligil GS, Arner P, Caro JF, Atkinson RL, and Spiegelman BM (1995) Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 95: 2409-2415.[Medline]

Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, and Shulman GI (2002) Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest 109: 1321-1326.[CrossRef][Medline]

Insel PA (1996) Analgesic-antipyretic and antiinflammatory agents and drugs employed in the treatment of gout, in Goodman & Gilman's The Pharmacological Basis of Therapeutics (Harman JG and Limbird LE eds) pp 617-657, McGraw-Hill, New York.

Itaya K (1977) A more sensitive and stable colorimetric determination of free fatty acids in blood. J Lipid Res 18: 663-665.[Abstract]

Jiang H, He J, Pu S, Tang C, and Xu G (2007) Heat shock protein 70 is translocated to lipid droplets in rat adipocytes upon heat stimulation. Biochim Biophys Acta 1771: 66-74.[Medline]

Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, et al. (2001) Prevention of fat-induced insulin resistance by salicylate. J Clin Invest 108: 437-446.[CrossRef][Medline]

Kiortsis DN, Mavridis AK, Vasakos S, Nikas SN, and Drosos AA (2005) Effects of infliximab treatment on insulin resistance in patients with rheumatoid arthritis and ankylosing spondylitis. Ann Rheum Dis 64: 765-766.[Abstract/Free Full Text]

Kralisch S, Klein J, Lossner U, Bluher M, Paschke R, Stumvoll M, and Fasshauer M (2005) Isoproterenol, TNFalpha, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes. Mol Cell Endocrinol 240: 43-49.[CrossRef][Medline]

Möhlig M, Freudenberg M, Bobbert T, Ristow M, Rochlitz H, Weickert MO, Pfeiffer AF, and Spranger J (2006) Acetylsalicylic acid improves lipid-induced insulin resistance in healthy men. J Clin Endocrinol Metab 91: 964-967.[Abstract/Free Full Text]

Newman WP and Brodows RG (1983) Aspirin causes tissue insensitivity to insulin in normal man. J Clin Endocrinol Metab 57: 1102-1106.[Abstract/Free Full Text]

Ofei F, Hurel S, Newkirk J, Sopwith M, and Taylor R (1996) Effects of an engineered human anti-TNF-alpha antibody (CDP571) on insulin sensitivity and glycemic control in patients with NIDDM. Diabetes 45: 881-885.[Abstract]

Peled N and Krenz MC (1981) A new assay of microbial lipases with emulsified trioleoyl glycerol. Anal Biochem 112: 219-222.[CrossRef][Medline]

Pillinger MH, Capodici C, Rosenthal P, Kheterpal N, Hanft S, Philips MR, and Weissmann G (1998) Modes of action of aspirin-like drugs: salicylates inhibit erk activation and integrin-dependent neutrophil adhesion. Proc Natl Acad Sci U S A 95: 14540-14545.[Abstract/Free Full Text]

Rahn Landström T, Mei J, Karlsson M, Manganiello V, and Degerman E (2000) Down-regulation of cyclic-nucleotide phosphodiesterase 3B in 3T3-L1 adipocytes induced by tumour necrosis factor alpha and cAMP. Biochem J 346: 337-343.[CrossRef][Medline]

Reid J, Macdougall AI, and Andrews MM (1957) Aspirin and diabetes mellitus. Br Med J 2: 1071-1074.[Free Full Text]

Ren T, He J, Jiang H, Zu L, Pu S, Guo X, and Xu G (2006) Metformin reduces lipolysis in primary rat adipocytes stimulated by tumor necrosis factor-alpha or isoproterenol. J Mol Endocrinol 37: 175-183.[Abstract/Free Full Text]

Rohl M, Pasparakis M, Baudler S, Baumgartl J, Gautam D, Huth M, De Lorenzi R, Krone W, Rajewsky K, and Bruning JC (2004) Conditional disruption of IkappaB kinase 2 fails to prevent obesity-induced insulin resistance. J Clin Invest 113: 474-481.[CrossRef][Medline]

Ryden M, Dicker A, van Harmelen V, Hauner H, Brunnberg M, Perbeck L, Lonnqvist F, and Arner P (2002) Mapping of early signaling events in tumor necrosis factor--mediated lipolysis in human fat cells. J Biol Chem 277: 1085-1091.[Abstract/Free Full Text]

Schönhöfer PS, Sohn J, Peters HD, and Dinnendahl V (1973) Effects of sodium salicylate and acetylsalicylic acid on the lipolytic system of fat cells. Biochem Pharmacol 22: 629-637.[CrossRef][Medline]

Schwenger P, Skolnik EY, and Vilcek J (1996) Inhibition of tumor necrosis factor-induced p42/p44 mitogen-activated protein kinase activation by sodium salicylate. J Biol Chem 271: 8089-8094.[Abstract/Free Full Text]

Souza SC, Yamamoto MT, Franciosa MD, Lien P, and Greenberg AS (1998) BRL 49653 blocks the lipolytic actions of tumor necrosis factor-alpha: a potential new insulin-sensitizing mechanism for thiazolidinediones. Diabetes 47: 691-695.[Abstract]

Stone DB, Brown JD, and Steele AA (1969) Effect of sodium salicylate on induced lipolysis in isolated fat cells of the rat. Metabolism 18: 620-624.[CrossRef][Medline]

Sztalryd C, Xu G, Dorward H, Tansey JT, Contreras JA, Kimmel AR, and Londos C (2003) Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. J Cell Biol 161: 1093-1103.[Abstract/Free Full Text]

Uysal KT, Wiesbrock SM, Marino MW, and Hotamisligil GS (1997) Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function. Nature 389: 610-614, 1997.[CrossRef][Medline]

Vittimberga FJ Jr, McDade TP, Perugini RA, and Callery MP (1999) Sodium salicylate inhibits macrophage TNF-alpha production and alters MAPK activation. J Surg Res 84: 143-149.[CrossRef][Medline]

Xu G, Sztalryd C, and Londos C (2006) Degradation of perilipin is mediated through ubiquitination-proteasome pathway. Biochim Biophys Acta 1761: 83-90.[Medline]

Xu G, Sztalryd C, Lu X, Tansey JT, Gan JW, Dorward H, Kimmel AR, and Londos C (2005) Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway. J Biol Chem 280: 42841-42847.[Abstract/Free Full Text]

Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, and Shoelson SE (2001). Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293: 1673-1677.[Abstract/Free Full Text]

Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, et al. (2004) Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306: 1383-1386.[Abstract/Free Full Text]

This article has been cited by other articles:

				L. Zu, J. He, H. Jiang, C. Xu, S. Pu, and G. Xu Bacterial Endotoxin Stimulates Adipose Lipolysis via Toll-Like Receptor 4 and Extracellular Signal-regulated Kinase Pathway J. Biol. Chem., February 27, 2009; 284(9): 5915 - 5926. [Abstract] [Full Text] [PDF]

				T. Zhang, J. He, C. Xu, L. Zu, H. Jiang, S. Pu, X. Guo, and G. Xu Mechanisms of metformin inhibiting lipolytic response to isoproterenol in primary rat adipocytes J. Mol. Endocrinol., January 1, 2009; 42(1): 57 - 66. [Abstract] [Full Text] [PDF]

				N. Anderson and J. Borlak Molecular Mechanisms and Therapeutic Targets in Steatosis and Steatohepatitis Pharmacol. Rev., September 1, 2008; 60(3): 311 - 357. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.107.039479v1 73/1/215    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zu, L. Articles by Xu, G. Search for Related Content PubMed PubMed Citation Articles by Zu, L. Articles by Xu, G.