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AMPK-sensed cellular energy state regulates the release of extracellular Fatty Acid Synthase
Biochemical and Biophysical Research Communications 378 (2009) 488–493
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
AMPK-sensed cellular energy state regulates the release of extracellular Fatty Acid Synthase Cristina Oliveras-Ferraros a,b,1, Alejandro Vazquez-Martin a,b,1, Jose Manuel Fernández-Real b,c, Javier A. Menendez a,b,* a
Catalan Institute of Oncology, Girona (ICO-Girona), Hospital de Girona ‘‘Dr. Josep Trueta”, Ctra. França s/n, E-17007 Girona, Catalonia, Spain Girona Biomedical Research Institute (IdIBGi), Girona, Catalonia, Spain c Department of Diabetes, Endocrinology and Nutrition, CIBEROBN Fisiopatología de la Obesidad y Nutrición CB06/03/010, Girona, Catalonia, Spain b
a r t i c l e
i n f o
Article history: Received 13 November 2008 Available online 24 November 2008
Keywords: Fatty Acid Synthase AMPK Cancer Hepatitis C Obesity Diabetes AICAR
a b s t r a c t Fatty Acid Synthase (FASN), a 250-kDa cytosolic multi-enzyme catalyzing eukaryotic de novo FA biogenesis, unexpectedly localizes in cancer cell culture supernatants and in the blood of cancer patients. High levels of ‘‘extracellular FASN” have recently been found in supernatants from Hepatitis C Virus-infected liver cells. The ultimate mechanism regulating FASN release, however, remained completely undefined. When the AMPK-activating drug AICAR was used to simulate an elevated AMP/ATP ratio in breast cancer cells, ELISA-based analyses revealed that extracellular FASN dramatically augmented in a dose- and timedependent manner. Immunoblotting procedures using a battery of anti-FASN antibodies further confirmed that, in response to AMPK activation, FASN protein is depleted from the cytosol to accumulate as different FASN isoforms in the extracellular milieu. siRNA-induced blockade of AMPK expression largely attenuated AICAR-promoted FASN release. FASN release might represent a previously unrecognized mechanism through which AMPK monitor and restores cellular energy state in response to increasing AMP/ATP ratios. Ó 2008 Elsevier Inc. All rights reserved.
Based on the intracellular localization of Fatty Acid Synthase (FASN)—the key multifunctional enzyme that plays a central role in the endogenous biogenesis of FAs [1,2]—two kinds of this lipogenic enzyme are classically recognized in eukaryotic cells: cytosolic (FASN I) and mitochondrial FASN (FASN II). While ‘‘cytosolic FASN” is the major responsible for the de novo biosynthesis of FAs, ‘‘mitochondrial FASN” provides the octanoyl precursor required for the essential lipoylation pathway [3]. Unexpectedly, recent studies have demonstrated that cultured cancer cells can excrete immunoreactive FASN into the extracellular space [4–7]. Importantly, significant elevations of ‘‘extracellular FASN” were also detected in the circulation of patients with breast, prostate, colon and ovarian cancer compared to healthy subjects. The fact that circulating FASN levels increase in parallel with different clinical stages supported the perception that the extracellular form of FASN should be considered a tumor marker capable to assess cancer virulence as its up-regulation is more pronounced in the more advanced stages of tumors [4,7]. Therefore, the current dogma in * Corresponding author. Address: Catalan Institute of Oncology, Girona (ICOGirona), Hospital de Girona ‘‘Dr. Josep Trueta”, Ctra. França s/n, E-17007 Girona, Catalonia, Spain. Fax: +34 972 217 344. E-mail addresses: [email protected], [email protected] (J.A. Menendez). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.067
the field states that the excess intracellular FASN—which increases during progression of tumor cells in the metastatic cascade—is excluded from the cytosol of tumor cells in a stage-related manner compared with healthy subjects. In the above mentioned scenario, the extracellular form of FASN should be considered a tumor marker capable to assess virulence as its up-regulation is expected to be more pronounced in the late (metastatic) stages of human malignancies. Interestingly, Yang et al. recently reported that, among 175 proteins identified from a cell culture supernatant fraction of human hepatocytes infected with the Hepatitis C virus (HCV), FASN was confirmed to be highly enriched [8]. Unfortunately, the ultimate mechanism regulating the release of FASN from the cytosolic compartment to the extracellular milieu in cancer and HCV-infected mammalian cells remains completely undefined. We here envisioned that the occurrence of ‘‘extracellular/circulating FASN” might represent a common feature in human diseases involving disorders in energy metabolism (e.g. lack of oxygen and/ or nutrients in rapidly growing carcinoma cells, intense energy utilization during a viral infection). Because AMP-activated protein kinase (AMPK) is a key ‘‘fuel gauge” maintaining the intracellular as well as body energy balance [9–12], we herein assessed whether changes in the phosphorylation (activation) status of AMPK might function as the master regulator of FASN release in response to
C. Oliveras-Ferraros et al. / Biochemical and Biophysical Research Communications 378 (2009) 488–493
cellular energy crisis. Using the AMPK-activating drug 5-amino-4imidazolecarboxamide riboside (AICAR [13,14]) to simulate an elevated AMP/ATP ratio in breast cancer cells and siRNA-induced specific knock-down of AMPK we likewise demonstrate that AMPK-sensed cellular energy state actively regulates the release of extracellular FASN in human breast cancer cells. Materials and methods Culture conditions. SKBR3 and MCF-7 breast cancer cells were obtained from the American Type Culture Collection and they where routinely grown in IMEM supplemented with 5% fetal bovine serum (FBS) and 2 mM L-Glutamine. Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. For the time-course studies, cells were plated in 100-mm tissue culture dishes and cultured in DMEM with 10% FBS until they reached 75–80% confluence. The cells were washed twice with serum-free DMEM, and incubated overnight in serum-free DMEM. Cells were then cultured for 48 h in 5 ml of 5% FBS-containing or serum-free DMEM in the presence or absence of 0.5 mM AICAR (Cell Signaling Technology, Inc.; Beverly, MA, USA) for 6, 24 and 48 h. After treatment, conditioned media were centrifuged to remove the cell debris and then used immediately or frozen at 80 oC until utilization. Cells were washed twice with cold-PBS and then lysed in buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerolphosphate, 1 mM Na3VO4, 1 lg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride) for 30 min on ice. The lysates were cleared by centrifugation in an Eppendorff tube (15 min at 14,000g, 4 oC). Protein content was determined against a standardized control using the Pierce Protein Assay Kit (Pierce, Rockford, IL, USA). Quantitative measurement of extracellular FASN. FASN concentrations were measured in duplicate by a sandwich enzyme immunoassay (FAS-detect ELISA, FASgen Inc., Baltimore, MA, USA) according to manufacturer’s instructions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/ml. Data are presented as means (columns) and 95% confidence intervals (bars) of three independent experiments carried out in duplicate. Transient transfection of small interference RNAs. The small interfering RNA (siRNAs) sequences used for targeted silencing of human a1/a2 AMPK isoforms and the negative control siRNA A were supplied by Santa Cruz Biotech (Santa Cruz, CA, USA). Transfections were performed as described in Santa Cruz technical bulletin. The day after the transfection, 0.5 mM AICAR was added to the medium and extracellular FASN was measured 48 h after the addition of AICAR. Immunoblotting. Fifteen microliter of either conditioned media (analyses of FASN expression in the extracellular milieu) or equal amounts of protein from whole cell lysates (i.e. 10 and 50 lg for the analyses of FASN and AMPK, respectively) were resuspended in 5X Laemli sample buffer (10 min at 70 oC), resolved by electrophoresis on 3–8% NuPAGE Tris–Acetate (FASN) or 10% SDS–PAGE (AMPK), and transferred onto nitrocellulose membranes. Testing for the phosphorylation status and total expression levels of AMPK was performed by immunoblotting procedures using the phosphoAMPKa Thr172 antibody (Clone 40H9) or total AMPK antibody (Cell Signalling Technology, Inc.; Beverly MA, USA). The primary antibodies for FASN immunoblotting were: The clone M3 (1:2000 dilution), a mouse anti-FASN monoclonal antibody (kindly provided by Dr. Ellen Pizer, Johns Hopkins University, MA, USA) diluted 1:5000; the clone 23 (1:250 dilution), a mouse anti-FASN monoclonal antibody obtained from BD Biosciences Pharmingen (San Diego, CA, USA); and the clone 3B3-1D6 (1:250 dilution), a mouse anti-FASN
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monoclonal antibody (M02) obtained from Abnova (Taiwan). Immunoreactive bands were visualized with ECL detection reagent (Pierce) following incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA) . Cell imaging. SKBR3 cells were seeded at 5000 cells/well in 96well clear bottom imaging tissue culture plates (Becton Dickinson Biosciences; San Jose, CA, USA) optimized for automated imaging applications. TritonÒ X-100 permeabilization and blocking, primary antibody staining (2.5 lg/ml of the anti-FASN mouse monoclonal antibody Clone 23), secondary antibody staining (Alexa FluorÒ 488 goat anti-mouse IgG; Invitrogen, Molecular Probes, Eugene, Oregon, USA) and counterstaining (Hoechst 33342; Invitrogen) were performed following BD Biosciences protocols. Images were captured on a BD PathwayTM 855 BioImager System with a 20 objective (NA 075 Olympus) according to the Recommended Assay Procedure and merged using BD AttovisionTM software. Statistical analyses. Two group comparisons were carried out by the Student’s t-test for paired and unpaired values. Comparisons of means of three or more groups were carried out by analysis of variance (ANOVA), and the existence of individual differences, in case of significant F values at ANOVA, was tested by Scheffé’s multiple contrasts. Results AICAR-induced activation of AMPK up-regulates extracellular expression of FASN We first examined the temporal accumulation of extracellular FASN in the serum-containing conditioned media of FASN-overexpressing SKBR3 cells [15,16] cultured in the absence or presence of the AMP analog AICAR. A slight but not statistically significant increase of extracellular FASN was observed as a function of SKBR3 cell growth (1.3-fold up-regulation after 48 h; Fig. 1A, top). Interestingly, a remarkable time-dependent accumulation of extracellular FASN occurred when SKBR3 cells were cultured in the presence of 0.5 mM AICAR. Thus, the concentration of extracellular FASN significantly increased by 3.3-fold as early as 6 h after AICAR treatment while a dramatic 6.9-fold up-regulation took place following 48 h exposure to AICAR (Fig. 1A, top). AICAR treatment likewise induced the phosphorylation of AMPK on Thr172 and this activation lasted for at least 24 h in SKBR3 cells (Fig. 1A, bottom). Serum starvation synergizes with AICAR to enhance FASN release We hypothesized that microenvironmental stresses such as serum starvation, which also enhances phosphorylation of AMPK [17,18], should further enhance the up-regulatory effects of AICAR on FASN release. Serum starvation significantly increased the basal level of FASN release (2.1-fold up-regulation after 48 h; Fig. 1B, top). Remarkably, a drastic potentiation of AICAR action on FASN release was observed upon serum starvation. Thus, extracellular FASN levels were increased by 4.9-fold as early as 6 h after AICAR treatment while a massive accumulation (74-fold increase) of the extracellular form of FASN was observed following 48 h treatment with AICAR in serum-starved SKBR3 cells (Fig. 1B, top). The potentiation of AICAR action by serum starvation on FASN release paralleled a synergistic increase in AMPK phosphorylation (Fig. 1B, bottom). siRNA-induced inhibition of AMPK signaling prevents AICAR-induced FASN release In order to determine if the effects of AICAR on FASN release were exclusively mediated by AMPK, we blocked this pathway
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Fig. 1. (A, B) AICAR-induced activation of AMPK up-regulates extracellular expression of FASN. (C) siRNA-induced knockdown of AMPK prevents AICAR-induced up-regulation of FASN release.
using siRNA directed against the two catalytic subunits of AMPK: a1 and a2. Interestingly, the specific ablation of endogenous AMPK by siRNA (75% reduction; Fig. 1C) was sufficient to slightly but significantly increase FASN release in SKBR3 cells. Moreover, AICAR-induced up-regulation of FASN release was completely prevented upon siRNA-induced knockdown of AMPK (Fig. 1C). AICAR-induced AMPK activation promotes the occurrence of extracellular FASN in various forms Earlier studies suggested that various extracellular immunoreactive forms of FASN can exist in the extracellular milieu of breast cancer cells and in breast cancer patients’ circulation [7]. We characterized extracellular FASN molecules recognized by monoclonal antibodies in immunoblotting analyses of conditioned medium from SKBR3 cells grown in the absence or presence of AICAR. Twenty-four and 48 h after AICAR treatment, the monoclonal anti-FASN antibody M3 recognized one major FASN form that corresponded to the its high molecular mass (260 kDa) (Supplemental 1A). The M3 anti-FASN antibody further recognized a minor intermediate form of FASN (150 kDa) that accumulated significantly in the extracellular milieu of SKBR3 following 48 h exposure to AICAR. A similar pattern of extracellular immunoreactive FASN forms was observed when using the monoclonal anti-FASN anti-
body Clone 23 (Supplemental 1B). Thus, this anti-FASN antibody recognized the accumulation of one major FASN form (260 kDa) and an intermediate size FASN form (150 kDa) in the extracellular milieu of AICAR-treated SKBR3 cells. Moreover, a third minor size FASN form (<25 kDa) could be detected after 48 h treatment with AICAR. In contrast to the M3 and Clone 23 anti-FASN antibodies, only the major large molecular mass FASN form (260 kDa) was found when using the monoclonal 3B31-1D6 anti-FASN antibody (Supplemental 1C). Immunoblotting analyses of extracellular FASN of conditioned media obtained from serum-starved SKBR3 cells revealed a similar but significantly weaker pattern of the extracellular FASN forms observed after AICAR treatment. AICARinduced up-regulation of extracellular FASN was not restricted to the FASN-overexpressing SKBR3 breast cancer model as ELISAbased and Western blotting analyses confirmed that this phenomenon similarly occurs in moderately-FASN expressing MCF-7 breast cancer cells (Supplemental 1D). Differential excretion of extracellular immunoreactive FASN forms from the cytosol to the extracellular space To evaluate whether, upon cellular release, the high molecular FASN form (260 kDa) could be degraded into smaller fragments or whether cancer cells can generate smaller fragments of FASN
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that are actively and specifically excluded from their cytosols upon microenvironmental conditions leading to AMPK activation, we took two different experimental approaches: first, we monitored the expression status of FASN in individual SKBR3 cells using an automated confocal platform capable to capture high content imaging of SKBR3 cell cultures growing in individual wells. Images clearly revealed that, upon AICAR-induced AMPK activation, a significant decrease in the cytoplasmic accumulation of FASN protein occurs in a high percentage of individual SKBR3 cells (Fig. 2A). We then monitored the temporal accumulation of each FASN form both in the extracellular milieu and in cell lysates of SKBR3 cells growing in the absence or presence of AICAR. AICAR treatment significantly downregulated—in a time-dependent manner—the high molecular mass FASN form (260 kDa) in the cytosolic compartment of SKBR3 cells (Fig. 2B). This decrease in the intracellular content of the 260 kDa-FASN band paralleled the appearance of this in the extracellular milieu of SKBR3 cells. Interestingly, AICAR-induced activation of AMPK up-regulated—in a time-dependent manner—the cytosolic accumulation of the intermediate size FASN form (150 kDa). The appearance of the 150 kDa-FASN band in
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cytosolic compartment was already detectable 24 h after AICAR treatment while the accumulation of the intermediate size FASN form was not detectable in the extracellular milieu until 24 h later. Discussion Earlier studies suggested that extracellular expression of the lipogenic enzyme FASN may be a time-dependent and hormonalindependent process that solely occurs in human breast cancer cells [4–7]. However, no direct evidences for the ultimate molecular mechanism(s) regulating FASN release were provided. Our current findings reveal for the first time that, upon sensing energy crisis in an AMPK-dependent manner, cancer cells actively exclude FASN molecules from their cytosolic pools into the extracellular milieu (Fig. 2C). AMPK is a key cellular fuel gauge that is exquisitely sensitive to changes in the levels of the low-energy indicator AMP [9–12]. On binding of AMP, AMPK phosphorylates several downstream targets, including key anabolic enzymes, inhibiting their activity and this way switching off major energy-consuming anabolic processes including DNA synthesis and lipid production. We
Fig. 2. (A, B) AICAR treatment promotes the release of several extracellular FASN forms. (C) Model of relationship between AMPK-sensed cellular energy status and release of extracellular FASN in cancer cells.
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now present the notion that the AMPK-energy-sensing-system not only might prevent lipid production by phosphorylating and inactivating Acetyl-CoA Carboxylase (ACACA) [19], the first committed enzyme in the endogenous biogenesis of FAs, but further by depleting FASN from the cytosol. This notion is based on the following findings: first, cancer cells treated with 5-aminoimidazole-4-carboxamide (AICA) riboside (a cell-permeable nucleoside that, on cell entry is phosphorylated by adenosine kinase to AICA ribotide (ZMP), thus mimicking the effects of AMP in activating AMPK) dramatically up-regulate the expression of extracellular FASN. Second, specific inhibition of the AMPK enzyme by the RNA interference technique largely prevented AICAR-induced elevation of extracellular FASN. Using gel-filtration chromatography Wang et al. examined the protein profiles of extracellular FASN in cell lysates and conditioned media of cultured breast cancer cells as well as in serum from breast cancer patients [7]. These authors observed that gelfiltration profiles of FASN varied among various samples. While the serum-free-conditioned media and cell lysate contained one major FASN form that corresponded to its high molecular mass (260 kDa), a minor intermediate size FASN form was further detected in the cell lysate. Interestingly, this smaller form of FASN was the sole FASN form detected in serum from breast cancer patients. Wang et al. suggested that the high molecular mass FASN protein could be degraded into smaller FASN fragments due to proteolytic action [7]. In this scenario, the intermediate size of cancerassociated FASN in patients’ circulation was suggested to represent a degraded product from large size FASN originated during blood drawing or sample processing. Our current findings do not support this elucidation. The high molecular FASN form (260 kDa), which accumulates in the extracellular milieu of cancer cells in parallel to its depletion in the cytosolic compartment, is not degraded into smaller fragments upon release. Pharmacological mimicking of low-energy status in cancer cells rather triggers a generation of the intermediate size FASN form (150 kDa) which, in turns, appears later on in the conditioned medium. This finding may suggest that circulating FASN in cancer patients does not constitute a degradation phenomenon of its high molecular form into smaller fragments. Alternatively, cancer cells could generate smaller fragments of FASN that are actively and specifically excluded from their cytosols upon AMPK-activating microenvironmental conditions. Forthcoming studies should clarify both the cause and the function, if any, of the intermediate size FASN form. As cancers grow, regions of the tumor may extend beyond the capacity of its vasculature to provide nutrients. Paradoxically, this cellular energy crisis induced by biophysical constraints continues to occur and is further enhanced in advanced disease (i.e. invasive and metastatic stages) as excessive amounts of pro-angiogenic factors are produced that result in disrupted chaotic vasculature [20– 23]. HCV is a slow-growing virus and signs of disease in the liver do not usually show up until 10–20 years after initial exposure. Therefore, HCV must maintain beneficial host cell functions for an extended period. However, as the infection proceeds, intense energy utilization could potentially induce cellular stress responses that could be deleterious to the viral infection [24]. In both scenarios (i.e. metastatic cancer and virus infection), AMP/ATP ratios rise, AMPK becomes phosphorylated, and P-AMPK will then attempt to conserve cellular energy through its well known ability to promote catabolism while inhibiting anabolism by phosphorylating several downstream targets. AMPK-induced phosphorylation of its prototypical target ACACA will decrease ACACA Vmax and its affinity for the allosteric activator citrate, thus shutting down this energy-consuming pathway [10,19]. In addition to this acute effect on the phosphorylation/activation status of ACACA, activated AMPK chronically decreases the expression of the transcriptional regulator SREBP-1c, thus suppressing the synthesis of ACACA, FASN
and other enzymes that regulate lipid synthesis [19]. An enhanced release of FASN may provide an alternative and rapid mechanism to prevent further energy consumption and to circumvent the AMPK-induced inhibition of mTOR-dependent protein synthesis. If the extracellular form of FASN can further signal the occurrence of energy stress to adjacent and/or distal target cells certainly merits further investigation. In summary, we provide evidence to suggest that FASN release from cancer cells is an active and controlled process that takes place by AMPK-sensed stimuli that increase the cellular AMP/ATP ratio (i.e. energy crisis at the cellular level). It will be interesting to measure the extracellular FASN level not only in HCV patients but also in individuals suffering AMPK-related disorders in which insulin resistance is prominent. If it correlates with the natural history of disease processes such as steatosis, hepatocarcinogenesis, obesity and type 2 diabetes, then the extracellular FASN levels may become a useful diagnostic and/or prognostic universal marker in chronic metabolic diseases. Acknowledgments J.A.M. is the recipient of a Basic, Clinical and Translational Research Award (BCTR0600894) from the Susan G. Komen Breast Cancer Foundation (TX, USA). This work was supported in part by Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria—FIS, Spain, Grants CP05-00090, PI06-0778 and RD06-0020-0028 to J.A.M.). J.A.M. was also supported by a Grant from the Fundación Científica de la Asociación Española Contra el Cáncer (Spain). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.11.067. References [1] F.B. Hillgartner, L.M. Salati, A.G. Goodridge, Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis, Physiol. Rev. 75 (1995) 47–76. [2] C.F. Semenkovich, Regulation of fatty acid synthase (FAS), Prog. Lipid Res. 36 (1995) 43–53. [3] A. Witkowski, A.K. Joshi, S. Smith, Coupling of the de novo fatty acid biosynthesis and lipoylation pathways in mammalian mitochondria, J. Biol. Chem. 282 (2007) 14178–14185. [4] Y. Wang, F.P. Kuhajda, J.N. Li, E.S. Pizer, W.F. Han, L.J. Sokoll, D.W. Chan, Fatty acid synthase (FAS) expression in human breast cancer cell culture supernatants and in breast cancer patients, Cancer Lett. 167 (2001) 99– 104. [5] Y. Wang, F.P. Kuhajda, L.J. Sokoll, D.W. Chan, Two-site ELISA for the quantitative determination of fatty acid synthase, Clin. Chim. Acta 304 (2001) 107–115. [6] Y.Y. Wang, F.P. Kuhajda, P. Cheng, W.Y. Chee, T. Li, K.J. Helzlsouer, L.J. Sokoll, D.W. Chan, A new model ELISA, Based on two monoclonal antibodies, for quantification of fatty acid synthase, J. Immunoassay Immunochem. 23 (2002) 279–292. [7] Y.Y. Wang, F.P. Kuhajda, J. Li, T.T. Finch, P. Cheng, C. Koh, T. Li, L.J. Sokoll, D.W. Chan, Fatty acid synthase as a tumor marker: its extracellular expression in human breast cancer, J. Exp. Ther. Oncol. 4 (2004) 101–110. [8] W. Yang, B.L. Hood, S.L. Chadwick, S. Liu, S.C. Watkins, G. Luo, T.P. Conrads, T. Wang, Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production, Hepatology 48 (2008) 1396–1403. [9] H. Koh, J. Chung, AMPK links energy status to cell structure and mitosis, Biochem. Biophys. Res. Commun. 362 (2007) 789–792. [10] F.P. Kuhajda, AMP-activated protein kinase and human cancer: cancer metabolism revisited, Int. J. Obes. (Lond.) 32 (Suppl. 4) (2008) S36–S41. [11] T. Williams, J.E. Brenman, LKB1 and AMPK in cell polarity and division, Trends Cell Biol. 18 (2008) 193–198. [12] J.E. Brenman, AMPK/LKB1 signaling in epithelial cell polarity and cell division, Cell Cycle 6 (2007) 2755–2759. [13] J.M. Corton, J.G. Gillespie, S.A. Hawley, D.G. Hardie, 5-Aminoimidazole-4carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?, Eur J. Biochem. 229 (1995) 558–565.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
AMPK-sensed cellular energy state regulates the release of extracellular Fatty Acid Synthase Cristina Oliveras-Ferraros a,b,1, Alejandro Vazquez-Martin a,b,1, Jose Manuel Fernández-Real b,c, Javier A. Menendez a,b,* a
Catalan Institute of Oncology, Girona (ICO-Girona), Hospital de Girona ‘‘Dr. Josep Trueta”, Ctra. França s/n, E-17007 Girona, Catalonia, Spain Girona Biomedical Research Institute (IdIBGi), Girona, Catalonia, Spain c Department of Diabetes, Endocrinology and Nutrition, CIBEROBN Fisiopatología de la Obesidad y Nutrición CB06/03/010, Girona, Catalonia, Spain b
a r t i c l e
i n f o
Article history: Received 13 November 2008 Available online 24 November 2008
Keywords: Fatty Acid Synthase AMPK Cancer Hepatitis C Obesity Diabetes AICAR
a b s t r a c t Fatty Acid Synthase (FASN), a 250-kDa cytosolic multi-enzyme catalyzing eukaryotic de novo FA biogenesis, unexpectedly localizes in cancer cell culture supernatants and in the blood of cancer patients. High levels of ‘‘extracellular FASN” have recently been found in supernatants from Hepatitis C Virus-infected liver cells. The ultimate mechanism regulating FASN release, however, remained completely undefined. When the AMPK-activating drug AICAR was used to simulate an elevated AMP/ATP ratio in breast cancer cells, ELISA-based analyses revealed that extracellular FASN dramatically augmented in a dose- and timedependent manner. Immunoblotting procedures using a battery of anti-FASN antibodies further confirmed that, in response to AMPK activation, FASN protein is depleted from the cytosol to accumulate as different FASN isoforms in the extracellular milieu. siRNA-induced blockade of AMPK expression largely attenuated AICAR-promoted FASN release. FASN release might represent a previously unrecognized mechanism through which AMPK monitor and restores cellular energy state in response to increasing AMP/ATP ratios. Ó 2008 Elsevier Inc. All rights reserved.
Based on the intracellular localization of Fatty Acid Synthase (FASN)—the key multifunctional enzyme that plays a central role in the endogenous biogenesis of FAs [1,2]—two kinds of this lipogenic enzyme are classically recognized in eukaryotic cells: cytosolic (FASN I) and mitochondrial FASN (FASN II). While ‘‘cytosolic FASN” is the major responsible for the de novo biosynthesis of FAs, ‘‘mitochondrial FASN” provides the octanoyl precursor required for the essential lipoylation pathway [3]. Unexpectedly, recent studies have demonstrated that cultured cancer cells can excrete immunoreactive FASN into the extracellular space [4–7]. Importantly, significant elevations of ‘‘extracellular FASN” were also detected in the circulation of patients with breast, prostate, colon and ovarian cancer compared to healthy subjects. The fact that circulating FASN levels increase in parallel with different clinical stages supported the perception that the extracellular form of FASN should be considered a tumor marker capable to assess cancer virulence as its up-regulation is more pronounced in the more advanced stages of tumors [4,7]. Therefore, the current dogma in * Corresponding author. Address: Catalan Institute of Oncology, Girona (ICOGirona), Hospital de Girona ‘‘Dr. Josep Trueta”, Ctra. França s/n, E-17007 Girona, Catalonia, Spain. Fax: +34 972 217 344. E-mail addresses: [email protected], [email protected] (J.A. Menendez). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.067
the field states that the excess intracellular FASN—which increases during progression of tumor cells in the metastatic cascade—is excluded from the cytosol of tumor cells in a stage-related manner compared with healthy subjects. In the above mentioned scenario, the extracellular form of FASN should be considered a tumor marker capable to assess virulence as its up-regulation is expected to be more pronounced in the late (metastatic) stages of human malignancies. Interestingly, Yang et al. recently reported that, among 175 proteins identified from a cell culture supernatant fraction of human hepatocytes infected with the Hepatitis C virus (HCV), FASN was confirmed to be highly enriched [8]. Unfortunately, the ultimate mechanism regulating the release of FASN from the cytosolic compartment to the extracellular milieu in cancer and HCV-infected mammalian cells remains completely undefined. We here envisioned that the occurrence of ‘‘extracellular/circulating FASN” might represent a common feature in human diseases involving disorders in energy metabolism (e.g. lack of oxygen and/ or nutrients in rapidly growing carcinoma cells, intense energy utilization during a viral infection). Because AMP-activated protein kinase (AMPK) is a key ‘‘fuel gauge” maintaining the intracellular as well as body energy balance [9–12], we herein assessed whether changes in the phosphorylation (activation) status of AMPK might function as the master regulator of FASN release in response to
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cellular energy crisis. Using the AMPK-activating drug 5-amino-4imidazolecarboxamide riboside (AICAR [13,14]) to simulate an elevated AMP/ATP ratio in breast cancer cells and siRNA-induced specific knock-down of AMPK we likewise demonstrate that AMPK-sensed cellular energy state actively regulates the release of extracellular FASN in human breast cancer cells. Materials and methods Culture conditions. SKBR3 and MCF-7 breast cancer cells were obtained from the American Type Culture Collection and they where routinely grown in IMEM supplemented with 5% fetal bovine serum (FBS) and 2 mM L-Glutamine. Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. For the time-course studies, cells were plated in 100-mm tissue culture dishes and cultured in DMEM with 10% FBS until they reached 75–80% confluence. The cells were washed twice with serum-free DMEM, and incubated overnight in serum-free DMEM. Cells were then cultured for 48 h in 5 ml of 5% FBS-containing or serum-free DMEM in the presence or absence of 0.5 mM AICAR (Cell Signaling Technology, Inc.; Beverly, MA, USA) for 6, 24 and 48 h. After treatment, conditioned media were centrifuged to remove the cell debris and then used immediately or frozen at 80 oC until utilization. Cells were washed twice with cold-PBS and then lysed in buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerolphosphate, 1 mM Na3VO4, 1 lg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride) for 30 min on ice. The lysates were cleared by centrifugation in an Eppendorff tube (15 min at 14,000g, 4 oC). Protein content was determined against a standardized control using the Pierce Protein Assay Kit (Pierce, Rockford, IL, USA). Quantitative measurement of extracellular FASN. FASN concentrations were measured in duplicate by a sandwich enzyme immunoassay (FAS-detect ELISA, FASgen Inc., Baltimore, MA, USA) according to manufacturer’s instructions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/ml. Data are presented as means (columns) and 95% confidence intervals (bars) of three independent experiments carried out in duplicate. Transient transfection of small interference RNAs. The small interfering RNA (siRNAs) sequences used for targeted silencing of human a1/a2 AMPK isoforms and the negative control siRNA A were supplied by Santa Cruz Biotech (Santa Cruz, CA, USA). Transfections were performed as described in Santa Cruz technical bulletin. The day after the transfection, 0.5 mM AICAR was added to the medium and extracellular FASN was measured 48 h after the addition of AICAR. Immunoblotting. Fifteen microliter of either conditioned media (analyses of FASN expression in the extracellular milieu) or equal amounts of protein from whole cell lysates (i.e. 10 and 50 lg for the analyses of FASN and AMPK, respectively) were resuspended in 5X Laemli sample buffer (10 min at 70 oC), resolved by electrophoresis on 3–8% NuPAGE Tris–Acetate (FASN) or 10% SDS–PAGE (AMPK), and transferred onto nitrocellulose membranes. Testing for the phosphorylation status and total expression levels of AMPK was performed by immunoblotting procedures using the phosphoAMPKa Thr172 antibody (Clone 40H9) or total AMPK antibody (Cell Signalling Technology, Inc.; Beverly MA, USA). The primary antibodies for FASN immunoblotting were: The clone M3 (1:2000 dilution), a mouse anti-FASN monoclonal antibody (kindly provided by Dr. Ellen Pizer, Johns Hopkins University, MA, USA) diluted 1:5000; the clone 23 (1:250 dilution), a mouse anti-FASN monoclonal antibody obtained from BD Biosciences Pharmingen (San Diego, CA, USA); and the clone 3B3-1D6 (1:250 dilution), a mouse anti-FASN
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monoclonal antibody (M02) obtained from Abnova (Taiwan). Immunoreactive bands were visualized with ECL detection reagent (Pierce) following incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA) . Cell imaging. SKBR3 cells were seeded at 5000 cells/well in 96well clear bottom imaging tissue culture plates (Becton Dickinson Biosciences; San Jose, CA, USA) optimized for automated imaging applications. TritonÒ X-100 permeabilization and blocking, primary antibody staining (2.5 lg/ml of the anti-FASN mouse monoclonal antibody Clone 23), secondary antibody staining (Alexa FluorÒ 488 goat anti-mouse IgG; Invitrogen, Molecular Probes, Eugene, Oregon, USA) and counterstaining (Hoechst 33342; Invitrogen) were performed following BD Biosciences protocols. Images were captured on a BD PathwayTM 855 BioImager System with a 20 objective (NA 075 Olympus) according to the Recommended Assay Procedure and merged using BD AttovisionTM software. Statistical analyses. Two group comparisons were carried out by the Student’s t-test for paired and unpaired values. Comparisons of means of three or more groups were carried out by analysis of variance (ANOVA), and the existence of individual differences, in case of significant F values at ANOVA, was tested by Scheffé’s multiple contrasts. Results AICAR-induced activation of AMPK up-regulates extracellular expression of FASN We first examined the temporal accumulation of extracellular FASN in the serum-containing conditioned media of FASN-overexpressing SKBR3 cells [15,16] cultured in the absence or presence of the AMP analog AICAR. A slight but not statistically significant increase of extracellular FASN was observed as a function of SKBR3 cell growth (1.3-fold up-regulation after 48 h; Fig. 1A, top). Interestingly, a remarkable time-dependent accumulation of extracellular FASN occurred when SKBR3 cells were cultured in the presence of 0.5 mM AICAR. Thus, the concentration of extracellular FASN significantly increased by 3.3-fold as early as 6 h after AICAR treatment while a dramatic 6.9-fold up-regulation took place following 48 h exposure to AICAR (Fig. 1A, top). AICAR treatment likewise induced the phosphorylation of AMPK on Thr172 and this activation lasted for at least 24 h in SKBR3 cells (Fig. 1A, bottom). Serum starvation synergizes with AICAR to enhance FASN release We hypothesized that microenvironmental stresses such as serum starvation, which also enhances phosphorylation of AMPK [17,18], should further enhance the up-regulatory effects of AICAR on FASN release. Serum starvation significantly increased the basal level of FASN release (2.1-fold up-regulation after 48 h; Fig. 1B, top). Remarkably, a drastic potentiation of AICAR action on FASN release was observed upon serum starvation. Thus, extracellular FASN levels were increased by 4.9-fold as early as 6 h after AICAR treatment while a massive accumulation (74-fold increase) of the extracellular form of FASN was observed following 48 h treatment with AICAR in serum-starved SKBR3 cells (Fig. 1B, top). The potentiation of AICAR action by serum starvation on FASN release paralleled a synergistic increase in AMPK phosphorylation (Fig. 1B, bottom). siRNA-induced inhibition of AMPK signaling prevents AICAR-induced FASN release In order to determine if the effects of AICAR on FASN release were exclusively mediated by AMPK, we blocked this pathway
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Fig. 1. (A, B) AICAR-induced activation of AMPK up-regulates extracellular expression of FASN. (C) siRNA-induced knockdown of AMPK prevents AICAR-induced up-regulation of FASN release.
using siRNA directed against the two catalytic subunits of AMPK: a1 and a2. Interestingly, the specific ablation of endogenous AMPK by siRNA (75% reduction; Fig. 1C) was sufficient to slightly but significantly increase FASN release in SKBR3 cells. Moreover, AICAR-induced up-regulation of FASN release was completely prevented upon siRNA-induced knockdown of AMPK (Fig. 1C). AICAR-induced AMPK activation promotes the occurrence of extracellular FASN in various forms Earlier studies suggested that various extracellular immunoreactive forms of FASN can exist in the extracellular milieu of breast cancer cells and in breast cancer patients’ circulation [7]. We characterized extracellular FASN molecules recognized by monoclonal antibodies in immunoblotting analyses of conditioned medium from SKBR3 cells grown in the absence or presence of AICAR. Twenty-four and 48 h after AICAR treatment, the monoclonal anti-FASN antibody M3 recognized one major FASN form that corresponded to the its high molecular mass (260 kDa) (Supplemental 1A). The M3 anti-FASN antibody further recognized a minor intermediate form of FASN (150 kDa) that accumulated significantly in the extracellular milieu of SKBR3 following 48 h exposure to AICAR. A similar pattern of extracellular immunoreactive FASN forms was observed when using the monoclonal anti-FASN anti-
body Clone 23 (Supplemental 1B). Thus, this anti-FASN antibody recognized the accumulation of one major FASN form (260 kDa) and an intermediate size FASN form (150 kDa) in the extracellular milieu of AICAR-treated SKBR3 cells. Moreover, a third minor size FASN form (<25 kDa) could be detected after 48 h treatment with AICAR. In contrast to the M3 and Clone 23 anti-FASN antibodies, only the major large molecular mass FASN form (260 kDa) was found when using the monoclonal 3B31-1D6 anti-FASN antibody (Supplemental 1C). Immunoblotting analyses of extracellular FASN of conditioned media obtained from serum-starved SKBR3 cells revealed a similar but significantly weaker pattern of the extracellular FASN forms observed after AICAR treatment. AICARinduced up-regulation of extracellular FASN was not restricted to the FASN-overexpressing SKBR3 breast cancer model as ELISAbased and Western blotting analyses confirmed that this phenomenon similarly occurs in moderately-FASN expressing MCF-7 breast cancer cells (Supplemental 1D). Differential excretion of extracellular immunoreactive FASN forms from the cytosol to the extracellular space To evaluate whether, upon cellular release, the high molecular FASN form (260 kDa) could be degraded into smaller fragments or whether cancer cells can generate smaller fragments of FASN
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that are actively and specifically excluded from their cytosols upon microenvironmental conditions leading to AMPK activation, we took two different experimental approaches: first, we monitored the expression status of FASN in individual SKBR3 cells using an automated confocal platform capable to capture high content imaging of SKBR3 cell cultures growing in individual wells. Images clearly revealed that, upon AICAR-induced AMPK activation, a significant decrease in the cytoplasmic accumulation of FASN protein occurs in a high percentage of individual SKBR3 cells (Fig. 2A). We then monitored the temporal accumulation of each FASN form both in the extracellular milieu and in cell lysates of SKBR3 cells growing in the absence or presence of AICAR. AICAR treatment significantly downregulated—in a time-dependent manner—the high molecular mass FASN form (260 kDa) in the cytosolic compartment of SKBR3 cells (Fig. 2B). This decrease in the intracellular content of the 260 kDa-FASN band paralleled the appearance of this in the extracellular milieu of SKBR3 cells. Interestingly, AICAR-induced activation of AMPK up-regulated—in a time-dependent manner—the cytosolic accumulation of the intermediate size FASN form (150 kDa). The appearance of the 150 kDa-FASN band in
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cytosolic compartment was already detectable 24 h after AICAR treatment while the accumulation of the intermediate size FASN form was not detectable in the extracellular milieu until 24 h later. Discussion Earlier studies suggested that extracellular expression of the lipogenic enzyme FASN may be a time-dependent and hormonalindependent process that solely occurs in human breast cancer cells [4–7]. However, no direct evidences for the ultimate molecular mechanism(s) regulating FASN release were provided. Our current findings reveal for the first time that, upon sensing energy crisis in an AMPK-dependent manner, cancer cells actively exclude FASN molecules from their cytosolic pools into the extracellular milieu (Fig. 2C). AMPK is a key cellular fuel gauge that is exquisitely sensitive to changes in the levels of the low-energy indicator AMP [9–12]. On binding of AMP, AMPK phosphorylates several downstream targets, including key anabolic enzymes, inhibiting their activity and this way switching off major energy-consuming anabolic processes including DNA synthesis and lipid production. We
Fig. 2. (A, B) AICAR treatment promotes the release of several extracellular FASN forms. (C) Model of relationship between AMPK-sensed cellular energy status and release of extracellular FASN in cancer cells.
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now present the notion that the AMPK-energy-sensing-system not only might prevent lipid production by phosphorylating and inactivating Acetyl-CoA Carboxylase (ACACA) [19], the first committed enzyme in the endogenous biogenesis of FAs, but further by depleting FASN from the cytosol. This notion is based on the following findings: first, cancer cells treated with 5-aminoimidazole-4-carboxamide (AICA) riboside (a cell-permeable nucleoside that, on cell entry is phosphorylated by adenosine kinase to AICA ribotide (ZMP), thus mimicking the effects of AMP in activating AMPK) dramatically up-regulate the expression of extracellular FASN. Second, specific inhibition of the AMPK enzyme by the RNA interference technique largely prevented AICAR-induced elevation of extracellular FASN. Using gel-filtration chromatography Wang et al. examined the protein profiles of extracellular FASN in cell lysates and conditioned media of cultured breast cancer cells as well as in serum from breast cancer patients [7]. These authors observed that gelfiltration profiles of FASN varied among various samples. While the serum-free-conditioned media and cell lysate contained one major FASN form that corresponded to its high molecular mass (260 kDa), a minor intermediate size FASN form was further detected in the cell lysate. Interestingly, this smaller form of FASN was the sole FASN form detected in serum from breast cancer patients. Wang et al. suggested that the high molecular mass FASN protein could be degraded into smaller FASN fragments due to proteolytic action [7]. In this scenario, the intermediate size of cancerassociated FASN in patients’ circulation was suggested to represent a degraded product from large size FASN originated during blood drawing or sample processing. Our current findings do not support this elucidation. The high molecular FASN form (260 kDa), which accumulates in the extracellular milieu of cancer cells in parallel to its depletion in the cytosolic compartment, is not degraded into smaller fragments upon release. Pharmacological mimicking of low-energy status in cancer cells rather triggers a generation of the intermediate size FASN form (150 kDa) which, in turns, appears later on in the conditioned medium. This finding may suggest that circulating FASN in cancer patients does not constitute a degradation phenomenon of its high molecular form into smaller fragments. Alternatively, cancer cells could generate smaller fragments of FASN that are actively and specifically excluded from their cytosols upon AMPK-activating microenvironmental conditions. Forthcoming studies should clarify both the cause and the function, if any, of the intermediate size FASN form. As cancers grow, regions of the tumor may extend beyond the capacity of its vasculature to provide nutrients. Paradoxically, this cellular energy crisis induced by biophysical constraints continues to occur and is further enhanced in advanced disease (i.e. invasive and metastatic stages) as excessive amounts of pro-angiogenic factors are produced that result in disrupted chaotic vasculature [20– 23]. HCV is a slow-growing virus and signs of disease in the liver do not usually show up until 10–20 years after initial exposure. Therefore, HCV must maintain beneficial host cell functions for an extended period. However, as the infection proceeds, intense energy utilization could potentially induce cellular stress responses that could be deleterious to the viral infection [24]. In both scenarios (i.e. metastatic cancer and virus infection), AMP/ATP ratios rise, AMPK becomes phosphorylated, and P-AMPK will then attempt to conserve cellular energy through its well known ability to promote catabolism while inhibiting anabolism by phosphorylating several downstream targets. AMPK-induced phosphorylation of its prototypical target ACACA will decrease ACACA Vmax and its affinity for the allosteric activator citrate, thus shutting down this energy-consuming pathway [10,19]. In addition to this acute effect on the phosphorylation/activation status of ACACA, activated AMPK chronically decreases the expression of the transcriptional regulator SREBP-1c, thus suppressing the synthesis of ACACA, FASN
and other enzymes that regulate lipid synthesis [19]. An enhanced release of FASN may provide an alternative and rapid mechanism to prevent further energy consumption and to circumvent the AMPK-induced inhibition of mTOR-dependent protein synthesis. If the extracellular form of FASN can further signal the occurrence of energy stress to adjacent and/or distal target cells certainly merits further investigation. In summary, we provide evidence to suggest that FASN release from cancer cells is an active and controlled process that takes place by AMPK-sensed stimuli that increase the cellular AMP/ATP ratio (i.e. energy crisis at the cellular level). It will be interesting to measure the extracellular FASN level not only in HCV patients but also in individuals suffering AMPK-related disorders in which insulin resistance is prominent. If it correlates with the natural history of disease processes such as steatosis, hepatocarcinogenesis, obesity and type 2 diabetes, then the extracellular FASN levels may become a useful diagnostic and/or prognostic universal marker in chronic metabolic diseases. Acknowledgments J.A.M. is the recipient of a Basic, Clinical and Translational Research Award (BCTR0600894) from the Susan G. Komen Breast Cancer Foundation (TX, USA). This work was supported in part by Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria—FIS, Spain, Grants CP05-00090, PI06-0778 and RD06-0020-0028 to J.A.M.). J.A.M. was also supported by a Grant from the Fundación Científica de la Asociación Española Contra el Cáncer (Spain). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.11.067. References [1] F.B. Hillgartner, L.M. Salati, A.G. Goodridge, Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis, Physiol. Rev. 75 (1995) 47–76. [2] C.F. Semenkovich, Regulation of fatty acid synthase (FAS), Prog. Lipid Res. 36 (1995) 43–53. [3] A. Witkowski, A.K. Joshi, S. Smith, Coupling of the de novo fatty acid biosynthesis and lipoylation pathways in mammalian mitochondria, J. Biol. Chem. 282 (2007) 14178–14185. [4] Y. Wang, F.P. Kuhajda, J.N. Li, E.S. Pizer, W.F. Han, L.J. Sokoll, D.W. Chan, Fatty acid synthase (FAS) expression in human breast cancer cell culture supernatants and in breast cancer patients, Cancer Lett. 167 (2001) 99– 104. [5] Y. Wang, F.P. Kuhajda, L.J. Sokoll, D.W. Chan, Two-site ELISA for the quantitative determination of fatty acid synthase, Clin. Chim. Acta 304 (2001) 107–115. [6] Y.Y. Wang, F.P. Kuhajda, P. Cheng, W.Y. Chee, T. Li, K.J. Helzlsouer, L.J. Sokoll, D.W. Chan, A new model ELISA, Based on two monoclonal antibodies, for quantification of fatty acid synthase, J. Immunoassay Immunochem. 23 (2002) 279–292. [7] Y.Y. Wang, F.P. Kuhajda, J. Li, T.T. Finch, P. Cheng, C. Koh, T. Li, L.J. Sokoll, D.W. Chan, Fatty acid synthase as a tumor marker: its extracellular expression in human breast cancer, J. Exp. Ther. Oncol. 4 (2004) 101–110. [8] W. Yang, B.L. Hood, S.L. Chadwick, S. Liu, S.C. Watkins, G. Luo, T.P. Conrads, T. Wang, Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production, Hepatology 48 (2008) 1396–1403. [9] H. Koh, J. Chung, AMPK links energy status to cell structure and mitosis, Biochem. Biophys. Res. Commun. 362 (2007) 789–792. [10] F.P. Kuhajda, AMP-activated protein kinase and human cancer: cancer metabolism revisited, Int. J. Obes. (Lond.) 32 (Suppl. 4) (2008) S36–S41. [11] T. Williams, J.E. Brenman, LKB1 and AMPK in cell polarity and division, Trends Cell Biol. 18 (2008) 193–198. [12] J.E. Brenman, AMPK/LKB1 signaling in epithelial cell polarity and cell division, Cell Cycle 6 (2007) 2755–2759. [13] J.M. Corton, J.G. Gillespie, S.A. Hawley, D.G. Hardie, 5-Aminoimidazole-4carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?, Eur J. Biochem. 229 (1995) 558–565.
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