Activation of Protein Phosphatase 2A by Palmitate Inhibits AMP-activated Protein Kinase
2007; Elsevier BV; Volume: 282; Issue: 13 Linguagem: Inglês
10.1074/jbc.m608310200
ISSN1083-351X
AutoresYong Wu, Ping Song, Jian Xu, Miao Zhang, Ming-Hui Zou,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoElevated levels of free fatty acids contribute to cardiovascular diseases, but the mechanisms remain poorly understood. The present study was aimed to determine if free fatty acid inhibits the AMP-activated kinase (AMPK). Exposure of cultured bovine aortic endothelial cells (BAECs) to palmitate (0.4 mm) but not to palmitoleic or oleic acid (0.4 mm) for 40 h significantly reduced the Thr172 phosphorylation of AMPK-α without altering its protein expression or the phosphorylation of LKB1-Ser428, a major AMPK kinase in BAECs. Further, in LKB1-deficient cells, palmitate suppressed AMPK-Thr172 implying that the inhibitory effects of palmitate on AMPK might be independent of LKB1. In contrast, 2-bromopalmitate, a non-metabolizable analog of palmitate, did not alter the phosphorylation of AMPK and acetyl-CoA carboxylase. Further, palmitate significantly increased the activity of protein phosphatase (PP)2A. Inhibition of PP2A with either okadaic acid, a selective PP2A inhibitor, or PP2A small interference RNA abolished palmitate-induced inhibition on AMPK-Thr172 phosphorylation. Exposure of BAECs to C2-ceramide, a cell-permeable analog of ceramide, mimicked the effects of palmitate. Conversely, fumonisin B1, which selectively inhibits ceramide synthase and decreases de novo formation of ceramide, abolished the effects of palmitate on both PP2A and AMPK. Inhibition of AMPK in parallel with increased PP2A activity was founded in C57BL/6J mice fed with high fat diet (HFD) rich in palmitate but not in mice fed with HFD rich in oleate. Moreover, inhibition of PP2A with PP2A-specific siRNA but not scrambled siRNA reversed HFD-induced inhibition on the phosphorylation of AMPK-Thr172 and endothelial nitric-oxide synthase (eNOS)-Ser1177 in mice fed with high fat diets. Taken together, we conclude that palmitate inhibits the phosphorylation of both AMPK and endothelial nitric-oxide synthase in endothelial cells via ceramide-dependent PP2A activation. Elevated levels of free fatty acids contribute to cardiovascular diseases, but the mechanisms remain poorly understood. The present study was aimed to determine if free fatty acid inhibits the AMP-activated kinase (AMPK). Exposure of cultured bovine aortic endothelial cells (BAECs) to palmitate (0.4 mm) but not to palmitoleic or oleic acid (0.4 mm) for 40 h significantly reduced the Thr172 phosphorylation of AMPK-α without altering its protein expression or the phosphorylation of LKB1-Ser428, a major AMPK kinase in BAECs. Further, in LKB1-deficient cells, palmitate suppressed AMPK-Thr172 implying that the inhibitory effects of palmitate on AMPK might be independent of LKB1. In contrast, 2-bromopalmitate, a non-metabolizable analog of palmitate, did not alter the phosphorylation of AMPK and acetyl-CoA carboxylase. Further, palmitate significantly increased the activity of protein phosphatase (PP)2A. Inhibition of PP2A with either okadaic acid, a selective PP2A inhibitor, or PP2A small interference RNA abolished palmitate-induced inhibition on AMPK-Thr172 phosphorylation. Exposure of BAECs to C2-ceramide, a cell-permeable analog of ceramide, mimicked the effects of palmitate. Conversely, fumonisin B1, which selectively inhibits ceramide synthase and decreases de novo formation of ceramide, abolished the effects of palmitate on both PP2A and AMPK. Inhibition of AMPK in parallel with increased PP2A activity was founded in C57BL/6J mice fed with high fat diet (HFD) rich in palmitate but not in mice fed with HFD rich in oleate. Moreover, inhibition of PP2A with PP2A-specific siRNA but not scrambled siRNA reversed HFD-induced inhibition on the phosphorylation of AMPK-Thr172 and endothelial nitric-oxide synthase (eNOS)-Ser1177 in mice fed with high fat diets. Taken together, we conclude that palmitate inhibits the phosphorylation of both AMPK and endothelial nitric-oxide synthase in endothelial cells via ceramide-dependent PP2A activation. Withdrawal: Activation of protein phosphatase 2A by palmitate inhibits AMP-activated protein kinase.Journal of Biological ChemistryVol. 294Issue 27PreviewVOLUME 282 (2007) PAGES 9777–9788 Full-Text PDF Open AccessWithdrawal: Reactive nitrogen species is required for the activation of the AMP-activated protein kinase by statin in vivo.Journal of Biological ChemistryVol. 294Issue 27PreviewVOLUME 283 (2008) PAGES 20186–20197 Full-Text PDF Open Access Evidence accumulated over the past several years indicates that the AMP-activated protein kinase (AMPK) 2The abbreviations used are: AMPK, 5′-AMP activated-kinase; ACC, acetyl-CoA carboxylase; AICAR, 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AUC, area under the curve; BAEC, bovine aortic endothelial cell; BSA, bovine serum albumin; eNOS, endothelial nitric-oxide synthase; LKB1, Peutz-Jeghers syndrome kinase LKB1; OA, okadaic acid; ONOO–, peroxynitrite; VSMC, vascular smooth muscle cell; siRNA, short interference RNA; FFA, free fatty acid; EBM, endothelial basal medium; 2-BrP, 2-bromopalmitate; HFD, high fat diet; PP2C, protein phosphatase 2C. 2The abbreviations used are: AMPK, 5′-AMP activated-kinase; ACC, acetyl-CoA carboxylase; AICAR, 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AUC, area under the curve; BAEC, bovine aortic endothelial cell; BSA, bovine serum albumin; eNOS, endothelial nitric-oxide synthase; LKB1, Peutz-Jeghers syndrome kinase LKB1; OA, okadaic acid; ONOO–, peroxynitrite; VSMC, vascular smooth muscle cell; siRNA, short interference RNA; FFA, free fatty acid; EBM, endothelial basal medium; 2-BrP, 2-bromopalmitate; HFD, high fat diet; PP2C, protein phosphatase 2C. may be a therapeutic target for treating insulin resistance and type 2 diabetes (1Musi N. Curr. Med. Chem. 2006; 13: 583-589Crossref PubMed Scopus (53) Google Scholar). AMPK is a heterotrimeric protein formed by an α subunit, which contains the catalytic activity, and by the β and γ regulatory subunits important in maintaining stability of the heterotrimer complex (2Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). AMPK belongs to a family of energy-sensing enzymes functioning as a “fuel gauge” that monitors changes in the energy status of a cell (3Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1138) Google Scholar, 4Carling D. Biochimie (Paris). 2005; 87: 87-91Crossref PubMed Scopus (179) Google Scholar). When activated, AMPK shuts down anabolic pathways and promotes catabolism in response to an elevated AMP/ATP ratio by down-regulating the activity of several key enzymes of intermediary metabolism (4Carling D. Biochimie (Paris). 2005; 87: 87-91Crossref PubMed Scopus (179) Google Scholar). Two primary acute consequences of AMPK activation are 1) an increase in glucose uptake by induction of glucose 4 transporter microvesicle cytoplasm to membrane translocation and fusion and 2) an increase in fatty acid oxidation by phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis (5Taylor E.B. Ellingson W.J. Lamb J.D. Chesser D.G. Winder W.W. Am. J. Physiol. 2005; 288: E1055-E1061Crossref PubMed Scopus (50) Google Scholar). Therefore, the AMPK signal pathways are thought to play a central role in the regulation of cellular glucose and lipid homeostasis. The control of AMPK activity is complex, and the classic view is that AMPK is activated allosterically by an increase in the intracellular AMP/ATP ratios and/or by the phosphorylation of threonine 172 within the α subunit. Several protein kinases responsible for this phosphorylation have been identified. They include Peutz-Jeghers syndrome kinase LKB1 (LKB1) (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar), and the Ca2+/calmodulin-dependent protein kinase kinase (7Hurley R.L. Anderson K.A. Franzone J.M. Kemp B.E. Means A.R. Witters L.A. J. Biol. Chem. 2005; 280: 29060-29066Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar). Protein phosphorylation signal transduction systems are balanced and regulated delicately by both phosphatase and kinase. Since AMPK is activated by (a) protein kinase(s) at the threonine 172 residue, one can easily assume that AMPK can be regulated negatively by (a) serine/threonine phosphatase(s). To date, a wide range of physiological stressors, pharmacological agents, and hormones associated with increase in the intracellular AMP/ATP ratios have been demonstrated to activate AMPK (8Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2321) Google Scholar). AMPK is also thought to be regulated by glycogen (9Polekhina G. Gupta A. Michell B.J. van Denderen B. Murthy S. Feil S.C. Jennings I.G. Campbell D.J. Witters L.A. Parker M.W. Kemp B.E. Stapleton D. Curr. Biol. 2003; 13: 867-871Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), which is the major cellular storage form of carbohydrates and thus, an additional indicator of cellular energy status. Lipids are the other major energy source for cellular metabolism. Recent studies (10Clark H. Carling D. Saggerson D. Eur. J. Biochem. 2004; 271: 2215-2224Crossref PubMed Scopus (84) Google Scholar, 11Suchankova G. Tekle M. Saha A.K. Ruderman N.B. Clarke S.D. Gettys T.W. Biochem. Biophys. Res. Commun. 2005; 326: 851-858Crossref PubMed Scopus (107) Google Scholar) in heart and liver have revealed that AMPK may be sensitive to the “lipid status” of a cell, and activation may be influenced by intracellular fatty acid availability independent of the cellular AMP levels. Indeed, a reduced fat oxidative capacity has been reported in type 2 diabetic patients (12Kelley D.E. Simoneau J.A. J. Clin. Invest. 1994; 94: 2349-2356Crossref PubMed Scopus (410) Google Scholar). Also, high fat diet feeding significantly decreases phospho-AMPK in the liver and muscles of rodents (13Muse E.D. Obici S. Bhanot S. Monia B.P. McKay R.A. Rajala M.W. Scherer P.E. Rossetti L. J. Clin. Invest. 2004; 114: 232-239Crossref PubMed Scopus (327) Google Scholar, 14Wilkes J.J. 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In an attempt to understand the mechanism underlying which chronically increased FFA inhibits AMPK, we examined the effects of the saturated fatty acid palmitate, which makes up 30–40% of plasma FFA, in cultured endothelial cells and in mice. Here we report that palmitate inhibited AMPK via ceramide-dependent PP2A activation in vivo. Materials—Bovine aortic endothelial cells (BAECs) and cell culture media were obtained from Clonetics Inc. (Walkersville, MD). BAECs were maintained in EBM with 2% serum and growth factors. HeLa-S3 and A549 cells were obtained from ATCC (Manassas, VA). Dulbecco's modified Eagle's medium/Ham's F-12 medium was purchased from Mediatech, Inc. (Herndon, VA). 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) was purchased from Toronto Research Chemicals, Inc. (Toronto, Canada). FFA-free bovine serum albumin (BSA), palmitic acid, oleic acid, palmitoleic acid, okadaic acid, C2-ceramide, fumonisin B1, and EDTA were obtained from Sigma. Peroxynitrite (ONOO–) was from Calbiochem (La Jolla, CA). 2-Bromopalmitate was from Aldrich Chemical Co. (Milwaukee, WI). The antibodies against phosphor-ACC (Ser79), phosphor-AMPK (Thr172), AMPK, phosphor-LKB1 (Ser428), LKB1, and phosphor-eNOS (Ser1177) were purchased from Cell Signaling Inc. (Beverly, MA). The antibodies against ACC were obtained from Alpha Diagnostic International, Inc. (San Antonio, TX). The antibody against PP2A was obtained from Upstate Biotechnology (Lake Placid, NY). Other chemicals and organic solvents, if not indicated, were obtained from Sigma with the highest grade. Other assay kits or antibodies, if not mentioned here, are indicated under “Experimental Procedures.” Cell Culture and Treatments—BAECs were grown in EBM supplemented with 2% fetal bovine serum. HeLa-S3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% serum. A549 cells were grown in Ham's F-12 medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). Rat vascular smooth muscle cells (VSMCs) were cultured from rat thoracic aortas as described previously (17Jiang B. Xu S. Hou X. Pimentel D.R. Cohen R.A. J. Biol. Chem. 2004; 279: 20363-20368Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Cultured cells were used between passages 5 and 10. When 70% confluent, the cells were washed with serum-free medium and then maintained in Dulbecco's modified Eagle's medium/F-12 with 0.1% fetal calf serum for 24 h. All cells were incubated in a humidified atmosphere of 5% CO2/95% air at 37 °C. When the cells were 60–70% confluent, the maintenance medium was removed and cells were treated with fatty acids (palmitic acid, oleic acid, or palmitoleic acid, 0.4–0.8 mm) for 1–40 h. Fatty acids were added to the culture medium as a fatty acid-BSA complex. Briefly, a stock solution of fatty acids was directly prepared in 95% ethanol and kept at 4 °C. The fatty acids were added to the cell cultures coupled to fatty acid-free bovine serum albumin (BSA) in the ratio of 2 mol of fatty acid to 1 mol of albumin. These complexes were constituted in stopper-covered flasks by adding the appropriate volume of the ethanolic FFAs solution to the albumin previously dissolved in culture medium containing no fetal bovine serum (here termed SF-EBM). These solutions were gently stirred and sterilized by filtration through 0.2-μm filters. Final concentrations of ethanol in the culture medium were kept to <0.1%. The concentrations of endotoxins in BSA were very low, as assessed by the supplier (3 enzyme units/mg BSA versus ∼30–60 enzyme units/mg of BSA for standard albumin preparations). Fatty acid-BSA complexes were added to culture dishes at a final concentration of 0.4 mm fatty acid. Controls were incubated with equal concentrations of FFA-free albumin as present in fatty acid-treated cells. In some experiments, BAECs were incubated in the presence or absence of either 2-bromopalmitate (2-BrP, 0.4 mm), C2-ceramide (15 μm), fumonisin B1 (15 μm), AICAR (2 mm), or okadaic acid (OA, 2 nm), and peroxynitrite (ONOO–, 50 μm). C2-ceramide and fumonisin B1 were first dissolved in prewarmed 37 °C Me2SO (Sigma) at a concentration of 15 mm and were simply added to the cell culture media resulting in a 1000-fold dilution. The final Me2SO concentration is 0.1%. For control experiments, BAECs were exposed to solvent alone (0.1% Me2SO). Cell Viability and Apoptosis—To exclude the potential contribution of cell death to the effects of FFA on AMPK phosphorylation in our experimental conditions, we first verified cell viability after 40 h of culture in the presence of either 0.4 mm palmitic or oleic acid. Cells were rinsed with phosphate-buffered saline, trypsinized, washed with medium, centrifuged, and resuspended in phosphate-buffered saline. Next, cells were mixed with the same volume of 0.25% trypan blue and transferred to a slide for 3 min. A total of 300 cells was microscopically counted using a hemocytometer to determine the dead cell (stained blue) rate. The experiments were performed in triplicate. Compared with the control (cells not exposed to FFA), no significant differences were detected for cell viability after exposing the cells for 40 h to the treatment conditions. Apoptosis was measured using the Cell Death Detection ELISA (Roche Diagnostics, Mannheim, Germany) to detect cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) according to the manufacturer's protocol. Data were normalized for comparison by total protein concentration (Bio-Rad). Animals—Male C57BL/6J mice were purchased from the Jackson Laboratory. The animals were maintained in a temperature-controlled room (22 °C) on a 12-h light-dark cycle. The study was approved by the Institutional Animal Care and Use Committee at University of Oklahoma Health Sciences Center. One week after arrival, mice were divided into three groups and were fed with normal chow, diet rich in palmitic acid (palmitate-HFD, high palmitate, 16:0 = 50% of total fatty acids) or diet rich in oleic acid (oleate-HFD, high oleic, 18:1 = 80% of total fatty acids, Research Diets, New Brunswick, NJ) for 3 months. Food intake and body weight were measured once a week. The intraperitoneal glucose tolerance test was performed after 12 weeks in 6 mice from each dietary group. Briefly, mice fasted for 12 h and blood was drawn from the tail vein at 0, 5, 15, 30, 60, and 120 min after intraperitoneal injection of glucose (2 g/kg body weight). The blood glucose was assayed using a blood glucose meter (LifeScan, Inc.). The trapezoid rule was used to determine the area under the curve (AUC) for glucose concentrations in each animal. Finally, after 12 weeks, blood samples were taken from the intraorbital, retrobulbar plexus from non-fasted, anesthetized mice to measure basal plasma levels of glucose and insulin. Insulin was determined using a Mouse Insulin Elisa Kit (Linco Research, St. Charles, MO). The mice were sacrificed, and thoracic aortas were immediately isolated and snap frozen in liquid nitrogen. The manipulation times were reduced to the minimum between aorta isolation and storage in liquid nitrogen. To avoid additional phosphorylation/dephosphorylation, Krebs-Ringer bicarbonate solution, which was used to rinse isolated aortas, was insufflated with 95% O2 and 5% CO2, and the mixtures were placed on ice to prevent tissue hypoxia. Mouse aortas were subsequently homogenated for determination of AMPK, ACC, and eNOS. In some experiments, the adventitia was removed from the isolated aortas. Aortas were then cut open along the ordinate axis, and the endothelium was removed by gentle rubbing of the intima with curved forceps. Immunohistochemistry—Ceramide formation was determined using immunohistochemistry. After incubation with media containing palmitic, palmitoleic, or oleic acid, the cells were fixed with 100% methanol for 3 min at –20 °C and then blocked with 1% normal goat serum in phosphate-buffered saline. Ceramide was detected using a specific anti-ceramide antibody (Alexis, Carlsbad, CA; clone MID 15B4, 1:200 dilutions in blocking buffer) and assayed using an Alexa 488-labeled anti-mouse antibody (Molecular Probes, 1:200 dilution in blocking buffer). Cells were examined under a fluorescence microscope (Olympus IX71) and pictures were taken for analysis. siRNA Construction and Infection—Double-stranded RNA with sequence 5′-CCAUUCUUCGAGGAAAUCAtt-3′ was designed to target the open reading frame of the bovine PP2A catalytic subunit Cα (GenBank™ accession number M16968) and purchased from Ambion (Austin, TX). A scrambled sequence served as control. Transient infection of siRNA was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were plated in a 6-well plate at 2.0 × 105 cells/well in 1.5 ml of EBM supplemented with 10% serum without antibiotics. BAECs were transfected with either scrambled or PP2A-specific siRNA oligonucleotides at a final concentration of 80 nm. No cell toxicity was observed at the concentrations of siRNA used. The PP2A expression was verified in Western blots. We found the maximal inhibition of PP2A was achieved after 2 days infection, and cells were treated on day 3 post infection. For mice, Double-stranded RNA, with sequence 5′-GCCUCUUGUCAUCAACAGCtt-3′, was designed to target the open reading frame of the mouse PP2A catalytic subunit Cα (GenBank™ accession number NM019411) and was purchased from Ambion. 50 μg of siRNAs was mixed with in vivo-jetPEI™ (Qbiogene, Carlsbad, CA) at an N/P ratio of 5 at room temperature for 15 min. For intravenous administration, 200 μl of the mixture containing the indicated amounts of siRNA was injected retro-orbitally. After 48 h, the mice were anesthetized and sacrificed. Aortas were isolated for biochemical assays. Assay of Phosphatase Activities—PP2A activity was measured by using threonine phosphopeptide (KRpTIRR) as the substrate with the PP2A Immunoprecipitation Phosphatase Assay Kit (Upstate Biotechnology). The cells were lysed in lysis buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 1 mm EGTA, 10% glycerol, 1.5 mm magnesium chloride, 1% Triton X-100, 1 μg of leupeptin/ml, 50 units of aprotinin/ml, 1 mm phenylmethylsulfonyl fluoride). Clarified supernatants were incubated with anti-PP2A antibody and protein A-agarose for 2 h at 4 °C. As for mice, PP2A immunoprecipitates in total aorta were prepared as described previously (18Brewis N. Ohst K. Fields K. Rapacciuolo A. Chou D. Bloor C. Dillmann W. Rockman H. Walter G. Am. J. Physiol. 2000; 279: H1307-H1318Crossref PubMed Google Scholar). The washed immunoprecipitates were resuspended in p-nitrophenyl phosphate Ser/Thr assay buffer, provided by the kit, and incubated for 2 h at 4°C. After washing the beads three times, the diluted phosphopeptide (750 μm) and Ser/Thr assay buffer were added, and the mixture was incubated for 10 min at 30 °C followed by addition of the Malachite Green Phosphate Detection Solution. PP2A activity in the reactive system was determined by measuring the absorbance at 650 nm and comparing absorbance values of samples to negative controls containing no enzyme. The activity of PP2C in cell culture with p-nitrophenyl phosphate as substrate was essentially assayed as previously described (19Takai A. Mieskes G. Biochem. J. 1991; 275: 233-239Crossref PubMed Scopus (159) Google Scholar). The initial rate of liberation of p-nitrophenol was determined spectrophotometrically at 405 nm. Western Blot Analysis—BAEC, HeLa-S3, A549, and VSMC cells were washed twice with cold phosphate-buffered saline and lysed in cold radioimmune precipitation assay buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and a mixture of protease inhibitors (Roche Applied Science). The protein concentrations were determined with a bicinchoninic acid protein assay system (Pierce). Proteins were subjected to Western blots. The antibody bindings were detected by using ECL-Plus, as described previously (20Davis B.J. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; 55: 496-505Crossref PubMed Scopus (359) Google Scholar). Quantification of Western Blot—The intensity (area × density) of the individual bands on Western blots was measured by densitometry (model GS-700, Imaging Densitometer, Bio-Rad). The background was subtracted from the calculated area. Expression of Data and Statistics—Data are expressed as mean ± S.E. Intergroup comparisons were performed by Student's paired t test. p < 0.05 was considered significant. Effects of Palmitate on Phosphorylation of AMPK and Its Downstream Enzyme, ACC-Ser79—Previous studies had demonstrated that palmitate activates AMPK in rats heart (10Clark H. Carling D. Saggerson D. Eur. J. Biochem. 2004; 271: 2215-2224Crossref PubMed Scopus (84) Google Scholar) and skeletal muscle cells (21Fediuc S. Gaidhu M.P. Ceddia R.B. J. Lipid Res. 2006; 47: 412-420Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) after acute treatment. To investigate whether or not palmitate activated AMPK in endothelial cells, BAECs were treated with various concentrations of palmitate for 1–40 h. AMPK activation was monitored in Western blots by staining with a specific antibody against phosphorylated Thr172 of AMPK, which is essential for its activity (22Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar). AICAR is a compound taken up by the cells and phosphorylated to the monophosphate form ZMP, which can accumulate in the cell, mimicking the effect of AMP on AMPK phosphorylation and activation (4Carling D. Biochimie (Paris). 2005; 87: 87-91Crossref PubMed Scopus (179) Google Scholar, 23Ruderman N.B. Saha A.K. Vavvas D. Witters L.A. Am. J. Physiol. 1999; 276: E1-E18Crossref PubMed Google Scholar). Thus, AICAR (2 mm, 2 h) was used as a positive control for AMPK activation. Because ACC is a substrate for AMPK (23Ruderman N.B. Saha A.K. Vavvas D. Witters L.A. Am. J. Physiol. 1999; 276: E1-E18Crossref PubMed Google Scholar), the determination of ACC phosphorylation also served as an indicator of AMPK activity. As shown in Fig. 1, AICAR elicited a 2.8- to 3.3-fold increase in both AMPK and ACC-Ser79, respectively. As depicted in Fig. 1A, exposure to low concentrations of palmitate (0.05–0.2 mm) for 40 h had no effect on the basal levels of both AMPK-Thr172 and ACC-Ser79 in BAECs. However, exposure of BAECs to palmitate at high concentrations (0.4 and 0.6 mm) for 40h significantly suppressed the levels of both AMPK-Thr172 and ACC-Ser79 (Fig. 1, A and B). We next examined the time dependence of palmitate on both AMPK-Thr172 and ACC-Ser79. Interestingly, acute exposure of BAECs to palmitate for 1 h caused a transient increase in both AMPK-Thr172 and ACC-Ser79 phosphorylation. The levels of AMPK and ACC were reduced to the basal levels at 5-h incubation. Prolonged incubation of BAECs with palmitate (20–40 h) caused a significant reduction of both AMPK-Thr172 and ACC-Ser79 (Fig. 1, C and D). In addition, palmitate did not affect the total content of both AMPK and ACC (Fig. 1), suggesting that palmitate-suppressed phosphorylation of both AMPK and ACC was not due to altered expression of AMPK and ACC. As depicted in Fig. 1 (E and F), 0.4 mm palmitate also reduced both basal and AICAR-stimulated AMPK and ACC phosphorylation at 40 h. Because 0.4 mm palmitate caused the greatest extent of inhibition on the phosphorylation of AMPK and ACC at 40 h, we exposed BAECs to 0.4 mm palmitate in the rest of the study. Inhibition of AMPK by Palmitate Is LKB1-independent—Two recent studies (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 24Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar) had identified that LKB1 acts as one of the AMPK upstream kinases, AMPK kinase. We then determined whether or not palmitate-induced AMPK inhibition was due to its inhibition on an AMPK kinase such as LKB1. We determined if palmitate altered the phosphorylation of LKB1 at Ser428, which is essential for ONOO–-induced AMPK activation (25Xie Z. Dong Y. Zhang M. Cui M.Z. Cohen R.A. Riek U. Neumann D. Schlattner U. Zou M.H. J. Biol. Chem. 2006; 281: 6366-6375Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). As depicted in Fig. 2A, ONOO– significantly increased the levels of LKB1-Ser428 phosphorylation compared with its control. In contrast, palmitate did not alter the levels of LKB1-Ser428. We next determined if palmitate inhibited AMPK in either HeLa-S3 or A549, two LKB1-deficient cells. Similar to that seen in BAECs, palmitate (0.4 mm) inhibited the Thr172 phosphorylation of AMPK in both HeLa-S3 and A549 by 60 and 70%, respectively (Fig. 2, C and E). Because palmitate inhibited AMPK Thr172 phosphorylation in both BAEC and LKB1-depleted cells, these results suggest that palmitate-induced reduction of AMPK-Thr172 might be independent of its upstream kinase, LKB1. Inhibition of AMPK by Palmitate Requires Palmitate Activation but Is β-Oxidation-independent—The inhibitory effects of saturated FFA on AMPK phosphorylation could either be due to a direct action or due to products generated by its metabolism. To distinguish between direct and indirect effects of FFA we determined the effects of a non-metabolizable analog of palmitate, 2-BrP, on AMPK and ACC phosphorylation. As shown in Fig. 3, 2-BrP, unlike palmitate, did not suppress the phosphorylation of both AMPK and ACC, suggesting that the effect of palmitate was dependent on its metabolism in endothelial cells. AMPK, as defined by its name, is regulated by the ratios of AMP to ATP. Increased oxidation of palmitate might inhibit AMPK by increasing ATP via enhanced mitochondrial oxidation of palmitate. To this end, BAECs were preincubated with 2-BrP (0.4 mm) 2 h prior to the addition of palmitate. 2-BrP inhibits long-chain fatty acid β-oxidation and consequent ATP production by irreversibly binding to carnitine-palmitoyltransferase 1 (26Chase J.F. Tubbs P.K. Biochem. J. 1972; 129: 55-65Crossref PubMed Scopus (118) Google Scholar). As shown in Fig. 3 (A and B), 2-BrP did not alter palmitate-induced inhibition on both AMPK and ACC phosphorylation. These results suggest that increased oxidation of FFA was unlikely a contributor to palmitate-induced AMPK inhibition. Inhibition of AMPK by Palmitate Is Independent of Apoptosis— There is evidence that palmitate
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