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Originally published In Press as doi:10.1074/jbc.M703701200 on July 16, 2007

J. Biol. Chem., Vol. 282, Issue 37, 26793-26801, September 14, 2007
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Nicotine-induced Activation of AMP-activated Protein Kinase Inhibits Fatty Acid Synthase in 3T3L1 Adipocytes

A ROLE FOR OXIDANT STRESS*

Zhibo An{ddagger}1, Hong Wang§1, Ping Song§1, Miao Zhang§, Xuemei Geng{ddagger}, and Ming-Hui Zou{ddagger}§2

From the {ddagger}Vascular Biology Laboratory, Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, Tennessee 37922 and the §Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104

Received for publication, May 4, 2007 , and in revised form, July 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies suggest that the AMP-activated protein kinase (AMPK) acts as a major energy sensor and regulator in adipose tissues. The objective of this study was to investigate the role of AMPK in nicotine-induced lipogenesis and lipolysis in 3T3L1 adipocytes. Exposure of 3T3L1 adipocytes to smoking-related concentrations of nicotine increased lipolysis and inhibited fatty acid synthase (FAS) activity in a time- and dose-dependent manner. The effects of nicotine on FAS activity were accompanied by phosphorylation of both AMPK (Thr172) and acetyl-CoA carboxylase (ACC; Ser79). Nicotine-induced AMPK phosphorylation appeared to be mediated by reactive oxygen species based on the finding that nicotine significantly increased superoxide anions and 3-nitrotyrosine-positive proteins, exogenous peroxynitrite (ONOO–) mimicked the effects of nicotine on AMPK, and N-acetylcysteine (NAC) abolished nicotine-enhanced AMPK phosphorylation. Inhibition of AMPK using either pharmacologic (insulin, compound C) or genetic means (overexpression of dominant negative AMPK; AMPK-DN) abolished FAS inhibition induced by nicotine or ONOO–. Conversely, activation of AMPK by pharmacologic (nicotine, ONOO–, metformin, and AICAR) or genetic (overexpression of constitutively active AMPK) means inhibited FAS activity. Notably, AMPK activation increased threonine phosphorylation of FAS, and this effect was blocked by adenovirus encoding dominant negative AMPK. Finally, AMPK-dependent FAS phosphorylation was confirmed by 32P incorporation into FAS in adipocytes. Taken together, our results strongly suggest that nicotine, via ONOO– activates AMPK, resulting in enhanced threonine phosphorylation and consequent inhibition of FAS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
AMP-activated protein kinase (AMPK)3 is a highly conserved eukaryotic protein that acts as a cellular energy sensor (1–4). AMPK is a heterotrimer consisting of {alpha}-, beta-, and {gamma}-subunits, each of which has at least two isoforms (1–4). AMPK is activated by stresses such as physical exercise, hypoxia, and nutrient depletion that result in a rise in the cellular AMP/ATP ratios (1–4). AMPK activity is also regulated by hormones (1–4) or oxidant stress (5) via an AMP-independent pathway. Activation of AMPK requires phosphorylation at Thr172, which resides in the activation loop of the {alpha}1- and {alpha}2-subunits (6). Several upstream kinases (AMPKKs) are capable of phosphorylating AMPK at Thr172. Included among these are LKB1 (7–9), calcium calmodulin-dependent kinase kinase (CaMKK) (10–13), and transforming growth factor-beta-activated kinase-1 (TAK-1) (14, 15). AMPK has been implicated in the regulation of several aspects of intermediary metabolism, including glucose transport, gluconeogenesis, glycogenolysis, lipolysis, and sterol synthesis (1–4).

Cigarette smoking is associated with weight loss, and cessation of smoking often leads to weight gain (16). Nicotine, which is readily absorbed from cigarette smoke, is elevated in the plasma of habitual smokers throughout the course of a day (17, 18). Nicotine has a direct effect on the release of cytokines and free fatty acids from adipocytes (20), which express nicotinic acetylcholine receptors (19). Human studies (21, 24) indicate that systemic nicotine infusion stimulates lipolysis in human adipose tissue via activation of local beta-adrenergic and nicotinic acetylcholine receptors (22, 23). However, the mechanisms by which nicotine regulates adipocyte function and contributes to weight loss in smokers remain unknown.

Accumulation of fat depends on the balance between fat synthesis (i.e. lipogenesis) plus re-esterification and breakdown (i.e. lipolysis and fatty acid oxidation). Fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) are the two major enzymes regulating these processes (25, 26). Evidence suggests that AMPK plays an important role in fatty acid metabolism, in part, through its ability to regulate ACC activity (27, 28). For example, activation of AMPK in liver, muscle, and heart results in phosphorylation and inhibition of ACC, which catalyzes the conversion of acetyl-CoA to malonyl-CoA. This is the rate-limiting step of fatty acid biosynthesis. The malonyl-CoA inhibits carnitine palmitoyltransferase I, which transports fatty acids into mitochondria for oxidation. Thus the simple inhibition of ACC prevents a futile cycle by promoting synthesis and blocking oxidation (29).

Whether FAS is regulated post-translationally remains unclear (30–33). FAS is a multifunctional enzyme that catalyzes the synthesis of long-chain fatty acids, primarily palmitate, using acetyl-CoA and malonyl-CoA as substrates and NADPH as a reducing equivalent (31–33). FAS is a homodimer of 250-kDa subunits (33). The FAS subunit is organized into discrete domains, which comprise different activities. The N- to C-terminal arrangement of FAS activities is as follows: beta-ketoacyl synthase, acetyl-CoA transacylase, malonyl-CoA transacylase, dehydratase, enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase (33). Under normal conditions, FAS is expressed mostly in the lipogenic tissues (i.e. liver and adipose tissue). Unlike ACC, FAS is not subject to allosteric regulation, and there is no evidence to suggest that FAS is post-translationally modified (29–31). Rather, FAS is regulated at the transcriptional level (30–33). Whether or not AMPK alters FAS activity remains unknown. The present study was designed to explore the molecular mechanisms by which nicotine activates AMPK and to determine whether AMPK regulates FAS activity in adipocytes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's medium/Ham's F-12 (50:50, v/v) with L-glutamine and 15 mM HEPES (DMEM/F-12) and phosphate-free DMEM/F-12 were purchased from Mediatech (Herndon, VA). Phenol red-free DMEM/F-12 was purchased from Caisson Laboratories (Rexburg, ID). F-12K medium was purchased from American Type Culture Collection (ATCC, Manassas, VA). Compound C was purchased from Calbiochem, and 2', 7'-dichlorodihydrofluorerscein diacetate (DCFH-DA) was from Molecular Probes (Eugene, OR). Peroxynitrite (ONOO–) was obtained from Calbiochem. Antiphospho-AMPK{alpha} (Thr172) and anti-AMPK{alpha} antibodies were from BioSource (Camarillo, CA). Anti-phospho-ACC (Ser79) antibody was from Cell Signaling (Beverly, MA). Anti-LKB1 and anti-3-nitrotyrosine antibodies were from Upstate (Charlottesville, VA). Anti-beta-actin antibody was from Abcam (Cambridge, MA). BCA Protein Assay Kit was from Pierce. Dexamethasone, 3-isobutyl-1-methylxanthine, N-acetylcysteine (NAC), fatty acid-free bovine serum albumin, free glycerol reagent, glycerol standard solution, insulin, NADPH, acetyl-CoA, and malonyl-CoA were purchased from Sigma. [32P]Orthophosphate was purchased from GE Healthcare. RIPA lysis buffer supplemented with protease inhibitor mixture were from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against FAS was from Novus Biologicals, Inc. (Littleton, CO) and BD Biosciences. All other reagents were obtained from Fisher Scientific.

Cell Culture—3T3L1 fibroblasts were obtained from ATCC and were cultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (10,000 units/ml and 10,000 µg/ml, respectively) at 37 °C in 95% air, 5% CO2. Two days after reaching confluence, cultures were incubated in DMEM/F-12 containing 10% FBS, 500 µM 3-isobutyl-1-methylxanthine, 10 µg/ml insulin, and 250 nM dexamethasone for 72 h to induce differentiation of 3T3L1 fibroblasts into adipocytes. Cultures were then given fresh DMEM/F-12 with 10% FBS, and media were replaced every 2 days there-after until more than 85% of the cells contained lipid droplets. 3T3L1 adipocytes were used for experimentation 7 to 10 days after the induction of differentiation. Adipocytes were incubated in phenol red-free DMEM/F-12 supplemented with 2% fatty acid-free bovine serum albumin for 2 h prior to experimentation.

LKB1-deficient A549 cells were obtained from ATCC. Cells were grown to 70% confluence in F-12K medium containing 10% FBS and 1% penicillin/streptomycin prior to experimentation.

Adenoviral Infection—The 3T3L1 cells were infected (multiplicity of infection = 50) with a replication-defective adenoviral vector expressing green fluorescence protein (Ad-GFP), dominant-negative mutants for AMPK (Ad-AMPK-DN), a constitutively active mutant of AMPK (Ad-AMPK-CA), wild type of LKB1 (LKB1-WT), and the kinase-dead LKB1 (LKB1-KD), as described previously (5, 34, 35) with modification. Differentiated 3T3L1 cells were incubated in DMEM/F-12 with 1% fetal bovine serum overnight. The cells were then washed and incubated in fresh DMEM with 0.1% fetal bovine serum for an additional 12 h prior to experimentation. Under these conditions, infection efficiency was typically >80% as determined by Ad-GFP expression.

Treatment of 3T3L1 Cells with Peroxynitrite—The concentrations of ONOO– were determined spectrophotometrically in 0.1 M NaOH ({epsilon}302 = 1670 M–1 s–1). Confluent 3T3L1 adipocytes grown in 6-well plates were rinsed twice with phosphate-buffered saline buffer (pH 7.4) and overlaid with 950 µlof100 mM HEPES buffer (pH 7.4). Next, 50 µl of concentrated ONOO– in 0.1 mM NaOH was quickly but evenly added to the plates, which were rapidly rotated on orbital shakers at room temperature. There was no pH shift during treatment with ONOO–. Equal volumes of 0.1 mM NaOH or decomposed ONOO– (decomposed in 1 mol/liter Tris buffer (pH 7.4) and kept for 5 min or overnight) were employed as controls.

Immunoprecipitation and Western Blot Analysis—Immunoprecipitation and Western blots were performed as described elsewhere (5, 34, 35). Band intensity was quantified using the FluorChem Imaging System from Alpha Innotech (San Leandro, CA).

Assay of Intracellular Reactive Oxygen Species (ROS)—ROS were measured using the dichlorofluorescein (DCF) assay. In this assay, DCFH-DA, a cell permeable nonfluorescent precursor, is hydrolyzed by nonspecific esterases within cells to release DCF, which is readily oxidized by intracellular H2O2. The assay was performed using a modified version of the method previously described (34, 35). Briefly, differentiated 3T3L1 adipocytes were preincubated with phenol red-free DMEM/F-12 containing 40 µM DCFH-DA at 37 °C for 30 min protected from light. DCF fluorescence was then read at excitation and emission wavelengths of 485 and 545 nm, respectively. All experimental DCF fluorescence values were corrected for background.

Lipolysis Assay—3T3L1 adipocytes grown in 6-well plates were incubated in phenol red-free DMEM/F-12 containing 2% bovine serum albumin for 2 h. Cultures were then rinsed twice with the same media and incubated with the indicated treatment suspended in 500 µl of DMEM/F-12 containing 2% bovine serum albumin. All inhibitors were added 30 min before treatment. Lipolysis was assayed in duplicate or triplicate by measuring glycerol release into the incubation medium with Free Glycerol Reagent.

Assay of FAS Activity—FAS activity was determined in duplicate by measuring the malonyl-CoA- and acetyl-CoA-dependent oxidation of NADPH according to the method of Nepokroeff (36). After treatment, cells were immediately washed with 2 ml of ice-cold phosphate-buffered saline buffer and collected, using a rubber spatula, in homogenization buffer (250 mM sucrose, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 100 µM phenylmethylsulfonyl fluoride). The cell lysates were sonicated five times with 10-s pulses in an Ultrasonic Dismemberator with 80% output (Model 500, Fisher Scientific). The lysates were clarified by centrifugation at 13,000 x g for 60 min at 4 °C. The resulting supernatant was assayed for protein concentration and subsequently, for FAS activity. Reactions were carried out at 37 °C using a SpectraMax® Plus384 Absorbance Microplate Reader System equipped with a temperature controller (Molecular Devices Ltd., Sunnyvale, CA). For each assay, both reference (blank) and sample wells were measured simultaneously with absorbance at 340 nm. The reaction was linear in the ranges employed. Final concentrations of the assay mixture (per ml) were potassium phosphate buffer, 60 mmol; acetyl-CoA, 75 µmol; malonyl-CoA, 100 nmol; and NADPH, 100 µmol. The change in concentrations of NADPH during oxidation was calculated using the following equation: {Delta}C ={Delta}A/E, where {Delta}C = change in concentrations of NADPH, {Delta}A = change in absorbance, and E = extinction coefficient of NADPH ({epsilon}340 nm = 6.22 mM–1 cm–1). FAS activity was expressed as nanomole of NADPH oxidized/min/mg of protein.

Site-directed Mutagenesis of Human LKB1 and Plasmid DNA Transfection—Plasmids encoding wild type LKB1 (LKB1-WT) and kinase-dead LKB1 mutants (LKB1-KD; Asp194 mutated to Ala) were prepared as previously reported (37). For adenovirus construction, the LKB1 open reading frame for wild type (WT) point mutations were released from the plasmid DNA of the WT-LKB1 pCI-neo, and D194A/pCI-neo vectors, and then cloned into the EcoRI/NotI sites of transfer vector pCR259 in the Transpose-Ad adenoviral vector system (Q-Biogen, Canada, catalog number AES3000). The resulting vectors, WT-LKB1/pCR259 and D194A/pCR259, were transformed to Transpose-Ad 294 Escherichia coli cells for homolog-based recombination with the adenovirus genome. Positive clone selection, characterization, and large-scale adenovirus amplification were carried out according to the manufacturer's instructions.

Detection of 32P Incorporation into FAS in Intact Adipocytes—Confluent 3T3L1 adipocytes were starved in phosphate-free DMEM/F-12 with 0.1% fetal bovine serum overnight, then pretreated with 10 µM Compound C (Comp C) or Me2SO as vehicle for 30 min. The cells were treated with AICAR (2 mM) or metformin (2 mM) for 30 min. Then the cells were labeled with [32P]orthophosphate (0.3 mCi/ml) for 4 h at 37 °C. The cells were washed with ice-cold phosphate-buffered saline and immediately lysed on ice with RIPA lysis buffer supplemented with protease inhibitor mixture (Santa Cruz Biotechnology, Santa Cruz, CA) for 20 min. Cell lysates were centrifuged 18 min at 14,000 x g, and supernatants were immunoprecipitated with the specific anti-FAS antibody (Novus Biologicals, Littleton, CO). Immunoprecipitates were separated by using 5% SDS-PAGE, transferred to nitrocellulose membranes, and radiolabeled proteins were visualized by autoradiography, and FAS levels were detected by Western blotting with anti-FAS antibody.

Assays of Intracellular Triglyceride Contents—Confluent 3T3L1 adipocytes were cultured in DMEM/F-12 supplemented with 10% FBS. Differentiated 3T3L1 adipocytes were exposed to either nicotine (1 µM) or AICAR (0.5 mM) for 96 h with a daily change of media. 3T3 L1 cells were lysed in phosphate-buffered saline (pH 7.4) by sonication. Intracellular triglyceride contents in lysates were measured enzymatically using Infinity Triglyceride reagents from Thermo Scientific (Melbourne, Australia) according to the manufacturer's instructions.

Statistical Analysis—Results are expressed as the mean ± S.E. Comparisons of group means were performed using oneway ANOVA test. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Nicotine Increases the Phosphorylation of AMPK, ACC, and LKB1—Because phosphorylation of AMPK at Thr172 is correlated with increased kinase activity and ACC is a downstream target of AMPK (6), we examined the effects of nicotine on the levels of Thr172-phosphorylated AMPK (AMPK-P) and Ser79-phosphorylated ACC (ACC-P) in adipocytes. Differentiated 3T3L1 adipocytes were exposed to nicotine ranging from 6 nM to 60 µM for 1 h. As shown in Fig. 1, A and B, concentrations from 6 nM to 600 nM resulted in a dose-dependent increase in AMPK-P. Low concentrations of nicotine (6 nM) were sufficient to elicit significant increases in AMPK-P. Significant increases in the phosphorylation of AMPK were also observed at concentrations of 60 and 600 nM, respectively (Fig. 1A). However, increasing nicotine concentrations to 600 nM did not result in any additional increase in AMPK-P (Fig. 1A).

The effects of nicotine on AMPK-P and ACC-P were also time-dependent (Fig. 1, C and D). AMPK-P was increased as early as 10 min after exposure of adipocytes to smoking-relevant concentrations of nicotine (100 nM). Prolonged exposure of adipocytes to nicotine was associated with additional increases in the levels of both AMPK-P and ACC-P, which reached a maximum at 2 h after exposure.

LKB1 Was Recently Identified as One of the Major AMPK Kinases—Our laboratory has demonstrated that the phosphorylation of LKB1 at Ser428 is required for AMPK activation (37). Thus, we examined whether nicotine alters Ser428 phosphorylation of LKB1 in adipocytes. As depicted in Fig. 2, A and B, nicotine dose-dependently increased the LKB1 Ser428 phosphorylation in adipocytes.


Figure 1
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  		FIGURE 1.
Nicotine increases phosphorylation of AMPK at Thr172 and ACC at Ser79 in 3T3L1 adipocytes.3T3L1 cells were treated with various concentrations of nicotine for the indicated periods of time, and extracts were analyzed for levels of phosphorylated AMPK (Thr172) and ACC (Ser79) by Western blotting. The blotsshown are representative of at least three blots obtained from three independent assays. A and B, nicotine dose-dependently increases phosphorylated AMPK (Thr172) as well as phosphorylated ACC (Ser79), a down-stream enzyme of AMPK. C and D, nicotine increases the phosphorylation of both AMPK (Thr172) and ACC(Ser79) in a time-dependent manner (n = 3, {clubsuit}, p < 0.05, nicotine versus control, Student t test).

 


Figure 2
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  		FIGURE 2.
Activation of AMPK by nicotine is LKB1-dependent. A and B, nicotine increases the phosphorylation of LKB1 at Ser428 in a dose-dependent manner. C and D, overexpression of kinase-dead LKB1 abolishes nicotine-enhanced AMPK activation in adipocytes. Differentiated 3T3L1 adipocytes were infected with adeno-viruses encoding LKB1 wild type (LKB1-WT) or the kinase-dead LKB1 mutants (LKB1-KD) prior to nicotine exposure (100 nM, 1 h). AMPK activation was assayed by the Thr172 phosphorylation of AMPK in adipocytes (n = 4, {clubsuit}, p < 0.05, LacZ versus LKB1 WT; {dagger}, p < 0.05, LKB1 WT versus LKB1-KD; one-way ANOVA).

 
Nicotine-induced AMPK Phosphorylation Is LKB1-dependent— To examine the role of LKB1 in nicotine-induced AMPK activation, adipocytes were infected with adenoviruses encoding with LacZ, LKB1-WT, or LKB1-KD. As expected, nicotine did not alter the levels of AMPK-P in adipocytes infected with adenoviruses encoding LacZ. In contrast, nicotine significantly increased the levels of AMPK-P in adipocytes overexpressing LKB1 WT but not in adipocytes overexpressing LKB1-KD (Fig. 2, C and D). Taken together, these results suggest that LKB1 is required for nicotine-enhanced AMPK activation in adipocytes.

Nicotine Increases Oxidative Stress—Work from our laboratory (5, 34–37) as well as from another laboratory (38) has demonstrated that AMPK is activated by oxidants such as ONOO– or H2O2 and that nicotine increases oxidant stress in brain, liver, and vascular cells. To determine whether nicotine increases oxidant stress in adipocytes, the formation of ROS was measured in nicotine-exposed 3T3L1 adipocytes. As shown in Fig. 3A, exposure of adipocytes to concentrations of nicotine ranging from 60 nM to 6 µM significantly increased the generation of ROS. In parallel, nicotine significantly increased the levels of 3-nitrotyrosine (3-NT)-positive proteins (Fig. 3B), a footprint of reactive nitrogen species such as ONOO–. This result implies that nicotine increased the formation of ONOO–, a potent oxidant formed by superoxide and nitric oxide.

Nicotine-activated AMPK Is Oxidative Stress-dependent—Although nicotine clearly increased oxidant stress in adipocytes, it was unclear if oxidant stress was responsible for nicotine-enhanced AMPK activation. To this end, we examined the effect of NAC, a thiol-containing antioxidant, on nicotine-enhanced AMPK phosphorylation. As depicted in Fig. 3C, NAC did not alter the basal levels of AMPK-P in adipocytes. However, it significantly suppressed nicotine-enhanced AMPK phosphorylation.

Peroxynitrite Increases AMPK Phosphorylation—Because nicotine increased 3-NT, a footprint of reactive nitrogen such as ONOO–, we next determined if ONOO– could mimic the effects of nicotine on AMPK in adipocytes. As shown in Fig. 3D, exposure of adipocytes to chemically synthesized ONOO– dose-dependently increased both AMPK-P and ACC-P in 3T3L1 adipocytes.

Nicotine-stimulated Lipolysis Is Independent of AMPK—It has been reported that nicotine stimulates lipolysis in humans and rats (8, 9). Thus we examined the effect of nicotine on lipolysis in adipocytes. As depicted in Fig. 4A, exposure of adipocytes to isoproterenol (ISO, 10 µM) stimulated glycerol release in adipocytes by 4–5-fold. Similar to ISO, exposure of 3T3L1 adipocytes to nicotine for 1 h significantly increased glycerol release in a dose-dependent manner (Fig. 4A). Glycerol release stimulated by 600 nM nicotine was linear up to 2 h after treatment, and was significantly different from control at each time point (Fig. 4B).


Figure 3
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  		FIGURE 3.
Nicotine-induced AMPK activation is mediated by reactive oxygen species. A, nicotine increases ROS production in adipocytes after 1 h of exposure, as measured by the DCF assays (n = 6, *, p < 0.05). B, nicotine increases levels of 3-nitrotyrosine-positive proteins in adipocytes. C, NAC suppresses nicotine-enhanced AMPK phosphorylation in adipocytes (n = 3; control versus nicotine, {clubsuit}, p < 0.05; nicotine versus nicotine + NAC; {dagger}, p < 0.05). D, adipocytes treated with ONOO– exhibit dose-dependent increases in phosphorylation of AMPK (Thr172) and ACC (Ser79). All data shown are expressed as mean ± S.E. (n = 4, {clubsuit}, p < 0.05, versus vehicle, one-way ANOVA). WB, Western blot.

 


Figure 4
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  		FIGURE 4.
Nicotine-enhanced lipolysis is independent of AMPK. A, nicotine dose-dependently increases glycerol release in adipocytes (n = 3; control versus nicotine or ISO, *, p < 0.05). B, nicotine-induced glycerol release is time-dependent (n = 3). C, adipocytes were stimulated with nicotine (100 nM) for 1 h in the absence or presence of compound C (10 µM), a potent AMPK inhibitor (n = 3). Glycerol release induced by nicotine was unaffected by AMPK inhibition. All data shown are expressed as mean ± S.E.

 
To test the hypothesis that AMPK contributes to nicotine-induced lipolysis, cultures were preincubated with compound C, a potent inhibitor of AMPK. The presence of compound C did not alter nicotine-enhanced glycerol release (Fig. 4C). The same was true for ISO-enhanced glycerol release. Thus, AMPK did not appear to be required for either ISO- or nicotine-stimulated lipolysis.

Nicotine Inhibits FAS Activity without Altering FAS Expression— We next determined if nicotine altered FAS activity in adipocytes. As shown in Fig. 5A, exposure of adipocytes to nicotine (1 and 100 nM) suppressed FAS activity in a dose-dependent manner (Fig. 5A). A smoking-related concentration of nicotine (100 nM) significantly inhibited FAS activity by 30%. The inhibitory effects of nicotine on FAS activity were also time-dependent (Fig. 5B). The inhibitory effects of nicotine reached a maximum at 60 min and remained stable for 120 min (Fig. 5B). This time course of FAS inhibition was consistent with the time course of nicotine-enhanced AMPK activation (Fig. 1, C and D). However, nicotine did not alter the levels of FAS expression (Fig. 1C).

Nicotine Reduces the Levels Triglyceride in 3T3L1 Adipocytes— We next determined if AMPK activation by nicotine altered the levels of triglycerides in adipocytes. Differentiated 3T3L1 adipocytes were exposed to nicotine (10–6 M) or AICAR (5 x 10–4 M) for 96 h. As shown in Fig. 5D, both nicotine and AICAR significantly decreased the levels of triglyceride in differentiated 3T3L adipocytes.

Peroxynitrite Inhibits Basal FAS Activity—The finding that endogenous ONOO– was involved in nicotine-enhanced AMPK activation led us to test whether chemically synthesized ONOO– could reproduce the effects of nicotine on FAS and AMPK. ONOO– (50 to 100 µM) significantly suppressed FAS activity within 5 min of treatment (Fig. 6A). However, ONOO– did not alter FAS protein levels (Fig. 6B), implying that the effect of ONOO– was not because of decreased expression of FAS. These FAS inhibitory effects of ONOO– were not associated with alterations in Ser473 phosphorylation of Akt or Akt activity (data not shown).


Figure 5
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  		FIGURE 5.
Nicotine induces dose- and time-dependent inhibition of FAS activity in 3T3L1 adipocytes. A, dose-dependent effects of nicotine on FAS activity (n = 8, {clubsuit}, p < 0.05, paired Student's t test). B, time-dependent effects of nicotine on FAS activity. The results represent two sets of three individual assays. C, expression of FAS in adipocytes exposed to nicotine (100 nM). The blot is a representative of three blots obtained from three individual experiments. D, nicotine reduces the levels of triglyceride in 3T3L1 adipocytes. Differentiated 3T3L1 adipocytes were exposed to either nicotine (1 µM) or AICAR (0.5 mM) for 96 h (n = 4, one-way ANOVA with Dunnett's multiple comparison test).

 


Figure 6
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  		FIGURE 6.
Peroxynitrite (ONOO–) inhibits FAS activity. 3T3L1 cells were treated for 10 min with ONOO– (0–100 µM) or NaOH vehicle, and FAS activity and protein levels were measured. A, ONOO– inhibits FAS activity in adipocytes (n = 7, control versus ONOO–, {clubsuit}, p < 0.05). B, ONOO– does not alter FAS protein levels in adipocytes. The blot is representative of five blots obtained from five individual assays. C, Thr172 phosphorylation of AMPK is inhibited by insulin but is increased by ONOO–. The blot is representative of five blots obtained from five individual assays. D, ONOO– inhibitsinsulin-enhanced FAS activity(n=7, controlversusONOO–, {clubsuit},p <0.05;n=7, insulinversusinsulin plus ONOO–, {dagger}, p < 0.05). All data shown are expressed as mean ± S.E. from a one-way ANOVA test.

 
Insulin-stimulated FAS Activity Is Blocked by Peroxynitrite and Is Associated with AMPK Inhibition— We next determined if AMPK was involved in ONOO–-induced FAS inhibition in adipocytes. Because ONOO– activated AMPK but inhibited FAS, inhibition of AMPK would be predicted to increase FAS activity. As depicted in Fig. 6C, insulin (10 nM, 10 min) attenuated the phosphorylation of both AMPK and ACC without altering AMPK expression. Conversely, insulin (10 nM) significantly increased FAS activity without altering FAS levels (Fig. 6D). In addition, ONOO–, at a concentration that activated AMPK (Fig. 6C), attenuated insulin-stimulated FAS activity (Fig. 6D).

Activation of AMPK by Pharmacologic Means Inhibits FAS Activity—AMPK activators were tested for their ability to mimic the effects of ONOO– or nicotine on FAS. As expected, exposure of adipocytes to the AMPK activators AICAR or metformin significantly increased the phosphorylation of both AMPK and ACC (Fig. 7A). Neither AICAR nor metformin altered the expression of FAS in adipocytes up to 4 h after treatment (Fig. 7B). However, AICAR- and metformin-induced AMPK activation significantly inhibited basal FAS activity as well as insulin-stimulated FAS activity (Fig. 7C).

AMPK Negatively Regulates FAS in Adipocytes—Although our results showed that ONOO–, metformin, and AICAR activated AMPK but inhibited FAS, it remained possible that the effects of these reagents on FAS were unrelated to their activation on AMPK. To exclude this possibility, we examined the effect of overexpression of AMPK-CA on FAS activity. Overexpression of AMPK-CA in adipocytes increased AMPK activity by 2-fold.4 As shown in Fig. 7D, overexpression of AMPK-CA but not GFP significantly suppressed FAS activity.

We also examined whether pharmacologic and genetic inhibition of AMPK abolished the inhibitory effects of AMPK activators and ONOO– on FAS activity. Compound C abolished ONOO–-induced FAS inhibition (Fig. 8A). In the same way, overexpression of Ad-AMPK-DN, but not GFP, significantly attenuated ONOO–-induced FAS inhibition (Fig. 8B). Similar results were obtained under conditions of AICAR- and metformin-induced FAS inhibition (Fig. 8C). Taken together, these results strongly suggest that AMPK inhibits FAS in adipocytes.


Figure 7
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  		FIGURE 7.
Pharmacologic activation of AMPK inhibits FAS in adipocytes. A, exposure of adipocytes to the AMPK activators, AICAR or metformin, increases the phosphorylation of AMPK (Thr172). The blot is represent-ative of three blots obtained from three individual assays. B, neither AICAR nor metformin alters the FAS protein levels. The blot is representative of five blots obtained from five individual assays. C, inhibition of FAS activity by AICAR and metformin (n = 7; control versus metformin or AICAR, {clubsuit}, p < 0.05; control versus insulin; {dagger}, p < 0.05; insulin versus insulin + AICAR and insulin versus insulin + metformin, {ddagger}, p < 0.05). D, overexpression of constitutively active AMPK (AMPK-CA), but not GFP, suppresses FAS activity in adipocytes (n = 5; control versus AMPK-CA, {clubsuit}, p < 0.05). All data shown are expressed as mean ± S.E. from a two-way ANOVA test.

 


Figure 8
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  		FIGURE 8.
AMPK-dependent fatty acid synthase inhibition in adipocytes. A, compound C, a potent inhibitor of AMPK, abolishes ONOO–-induced FAS inhibition (n = 4; control versus ONOO–, {clubsuit}, p < 0.05, ONOO– versus ONOO– + compound C; {dagger}, p < 0.05, two-way ANOVA). B, overexpression of adenoviral, dominant-negative AMPK {alpha} (AD-AMPK-DN) abolishes ONOO–-induced FAS inhibition (n = 4; control versus ONOO–, {clubsuit}, p < 0.05; ONOO– versus ONOO– + AMPK-DN, {dagger}, p < 0.05, two-way ANOVA test). The adenovirus vector encoding GFP (Ad-GFP) was employed as a negative control. C, the AMPK activators, AICAR or metformin, inhibit FAS activity. AMPK-DN reversed the inhibitory effects of both AICAR and metformin on FAS activity (n = 4; control versus activator-treated, {clubsuit}, p < 0.05; activator treated versus activator-treated + AMPK-D, {dagger}, p < 0.05, one-way ANOVA test). D, AMPK-dependent threonine phosphorylation of FAS. FAS immunoprecipitates were analyzed for phosphorylated threonine by Western blotting. Reciprocal co-immunoprecipitation/Western blot (IP/WB) experiments were performed to confirm the results. AICAR increased the threonine phosphorylation of FAS, which was abolished by overexpression of AMPK-DN. The blot is representative of at least three blots obtained from three individual assays. All data shown are expressed as mean ± S.E.

 
AMPK-dependent Phosphorylation of FAS in Adipocytes—It is generally accepted that FAS is regulated mainly by controlling rates of transcription, and there is no evidence to suggest that FAS is subject to allosteric regulation and posttranslational modification. Because ONOO– inhibited FAS within minutes and none of the AMPK activators (i.e. ONOO–, metformin, and AICAR) altered the levels of FAS protein in adipocytes, we hypothesized that AMPK might inhibit FAS by increasing the phosphorylation of FAS. To this end, FAS was immunoprecipitated from adipocytes treated with or without AICAR, and immunoprecipitates were subjected to Western blot analysis of phosphorylated Ser, phosphorylated Tyr, and phosphorylated Thr. As shown in Fig. 8D, AICAR treatment increased levels of Thr-phosphorylated FAS, and this effect was blocked by overexpression of AMPK-DN, but not GFP. Reciprocal experiments in which phospho-Thr immunoprecipitates were subjected to Western blot analysis of FAS yielded identical results (Fig. 8D). No Tyr(P) or Ser(P) was detected in FAS immunoprecipitates from control or AICAR-treated adipocytes (data not shown). These data are consistent with the hypothesis that AMPK inhibits FAS activity by increasing Thr phosphorylation of FAS.

Fatty Acid Synthase Is Phosphorylated by AMPK in Vivo—In vivo phosphorylation was analyzed in 3T3L1 adipocytes by labeling with [32P]orthophosphate. As depicted in Fig. 9, A and B, AMPK activator AICAR dramatically enhanced 32P incorporation into FAS by 8-fold (n = 3, p < 0.001). Similarly, metformin, another AMPK activator increased 32P incorporation into FAS (FAS phosphorylation) by 5-fold (n = 3, p < 0.001). Interestingly, pretreatment with compound C, a potent AMPK inhibitor, disrupted either AICAR or metformin-enhanced FAS phosphorylation in adipocytes. Taken together, these data suggest that AMPK activation increased FAS phosphorylation, as evidenced by an AMPK-dependent 32P incorporation into FAS.


Figure 9
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  		FIGURE 9.
AMPK-dependent phosphorylation of fatty acid synthase in intact adipocytes. Differentiated 3T3L1 adipocytes were starved in phosphor-free DMEM/F-12 with 0.1% fetal bovine serum overnight. After pretreatment with 10 µM compound C (Comp C) or Me2SO (Vehicle) for 30 min, the cells were stimulated with either 2 mM AICAR or 2 mM metformin for 30 min in the presence of [32P]orthophosphate (1 mCi) for 4 h at 37 °C. A, AMPK activators significantly increase [32P]orthophosphate incorporation into FAS, which are suppressed by AMPK inhibitor, compound C. The blot is a representative of three independent experiments. B, [32P]orthophosphate incorporation of FAS. Densitometry analysis of FAS phosphorylation caused by AMPK activators, metformin and AICAR (n = 3; {clubsuit}, p < 0.001 AICAR, metformin versus vehicle; Comp C/Met versus Met; {dagger}, p < 0.01 Comp C/AICAR versus AICAR, one-way ANOVA test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
AMPK activity is stimulated by an increase in the intracellular AMP/ATP ratios, which results from stresses such as exercise, hypoxia, oxidant stress, and glucose deprivation (1–4). Here we demonstrate that nicotine, a major component of cigarette liquid phases, activates AMPK in an LKB1-dependent fashion in adipocytes. Nicotine concentrations of 10 to 1000 nM triggered AMPK activation. These concentrations of nicotine are relevant to smoking because nicotine concentrations are ~90–100 nM over the first 6–8 h of the day in regular smokers (17, 18). In addition, the average peak plasma levels of nicotine in smokeless tobacco users have been reported to range from ~22 to 30 ng/ml (equal to 136 to 185 nM) (17, 18). In the present study, the level of nicotine causing AMPK activation was 100 nM (16.2 ng/ml), which is well within the range of plasma nicotine levels found in cigarette smokers and in users of smokeless tobacco.

Nicotine also significantly increased the production of ROS as detected by DCF, and increased the levels of 3-nitrotyrosine-positive proteins. These results are consistent with numerous other reports linking nicotine with ROS. For example, there is evidence (39–41) that (±)-nicotine and its enantiomers generate Formula, H2O2, and ONOO–. Nicotine infusion has been shown to impair endothelium-dependent relaxation, presumably via O2. release (42). In addition, superoxide dismutase can reverse endothelial dysfunction (21, 44), supporting a role for oxidant stress in smoking-related pathologies (43). In active smokers, plasma proteins such as fibrinogen, transferrin, plasminogen, and ceruloplasmin have been shown to possess nitrated tyrosine residues (45). Chronic smoking induces intra-platelet 3-NT formation, which is associated with increased platelet aggregation and reduced platelet levels of glutathione and ascorbate (45–47). A more recent study revealed that mitochondrial Mn-superoxide dismutase undergoes tyrosine nitration in mouse hearts following exposure to cigarette smoke (48). 3-NT has also been observed in plasma isolated from smokers or following infusion with nicotine (49).

Interestingly, ONOO–, like nicotine, was able to induce phosphorylation of both AMPK at Thr172 and LKB1 at Ser428. This is consistent with previous work by our laboratory, which showed that ONOO– induces activation of AMPK and LKB1 in endothelial cells and rat vascular smooth muscle cells. Importantly, the ability of NAC, a thiol-containing antioxidant, to abolish nicotine-enhanced AMPK activation suggests that oxidants, like ONOO–, mainly contribute to the activation of AMPK by nicotine.

The anti-lipogenic effects of nicotine are attributed, in part, to its ability to negatively regulate ACC and FAS via AMPK resulting in inhibition of lipogenesis and fat oxidation. AMPK activation by nicotine or AICAR reduces the levels of triglycerides in differentiated 3T3L1 adipocytes. In addition, nicotine increases lipolysis (increased glycerol levels, Fig. 4). Nicotine might activate AMPK, which causes a metabolic effect for nicotine that could be linked to weight loss.

However, the effects of AMPK on FAS are much less clear. To date, there is no evidence to suggest that FAS is post-translationally modified (30, 31). Rather, FAS is thought regulated at the transcriptional level (30, 31). In the present study, we have found that, like ACC, FAS inhibited by nicotine-induced AMPK activation. Nicotine (or ONOO–) inhibited FAS activity in cultured adipocytes within 10 min of treatment, without altering FAS protein levels. These inhibitory effects could be reversed by treatments that blocked AMPK activity (i.e. chemical inhibition and overexpression of dominant negative mutants). Conversely, treatments that activated AMPK (i.e. ONOO–, AMPK activators, and overexpression of constitutively active AMPK) diminished basal and insulin-enhanced FAS activity. Notably, FAS inhibition was accompanied by increased threonine phosphorylation and [32P]orthophosphate incorporation into FAS, which was sensitive to AMPK inhibition. Unpublished data from our laboratory have revealed that incubation of recombinant AMPK {alpha}1beta1{gamma}1or {alpha}2beta1{gamma}1/LKB1 with FAS, which had been immunoprecipitated from adipocytes, did not increase [32P]orthophosphate incorporation into FAS in vitro kinase assays.4 Therefore, it seems probable that AMPK does not directly phosphorylate FAS, but activates an unknown intermediary kinase(s). Whatever the case, our data clearly demonstrate that FAS is subject to AMPK-regulated phosphorylation and strongly suggest that AMPK-induced FAS phosphorylation inhibits lipogenesis. The finer molecular details of FAS inhibition by AMPK will require additional experimentation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL079584, HL080499, and HL074399 and a research award from the Research Management Group, Philip Morris International, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this article. Back

2 To whom correspondence should be addressed: BSEB 325, 941, Stanton L. Young Blvd., Oklahoma City, OK 73104. Tel.: 405-271-3974; Fax: 405-271-3973; E-mail: ming-hui-zou{at}ouhsc.edu.

3 The abbreviations used are: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; AMPK-DN, adenovirus encoding dominant negative AMPK; AMPK-CA, adenovirus encoding constitutively active AMPK; FAS, fatty acid synthase; NAC, N-acetylcysteine; 3-NT, 3-nitrotyrosine; ONOO–, peroxynitrite; DCFH-DA, 2', 7'-dichlorodihydrofluorescein diacetate; ISO, isoproterenol; DMEM, Dulbecco's modified Eagle's medium; ROS, reactive oxygen species; WT, wild type; ANOVA, analysis of variance; FBS, fetal bovine serum; AD-GFP, adenoviral vector expressing green fluorescence protein. Back

4 Z. An and M.-H. Zou, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Yunzhou Dong for technical support for LKB1 adenoviruses.



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