Original Research—Sinonasal Disorders

Metformin Reduces TGF-b1–Induced Extracellular Matrix Production in Nasal Polyp–Derived Fibroblasts

Otolaryngology– Head and Neck Surgery 2014, Vol 150(1) 148–153 Ó American Academy of Otolaryngology—Head and Neck Surgery Foundation 2013 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0194599813513880 http://otojournal.org

Il-Ho Park, MD, PhD1*, Ji-Young Um2*, Sung-Moon Hong, MD1, Jung-Sun Cho, PhD2, Seung Hoon Lee, MD, PhD1, Sang Hag Lee, MD, PhD1, and Heung-Man Lee, MD, PhD1,2,3

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Abstract Background and Objects. Metformin is widely used to treat type 2 diabetes mellitus, and adenosine monophosphate– activated protein kinase (AMPK) is thought to be the target that mediates its effects. Recently, it has been demonstrated that metformin has antifibrotic effects beyond its antihyperglycemic action. The purposes of this study were to investigate the effect of metformin on TGF-b1–induced myofibroblast differentiation (a-smooth muscle actin [a-SMA]) and extracellular matrix (ECM) production and to determine the underlying mechanism of the action of metformin in nasal polyp–derived fibroblasts (NPDFs). Study Design. Basic research. Setting. The rhinology laboratory of Korea University Guro Hospital, Seoul, Korea. Methods. NPDFs from 7 patients were incubated with TGFb1 and treated with metformin or compound C, an inhibitor of AMPK. To determine the proliferation rate of nasal fibroblasts, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed. The expression levels of a-SMA and fibronectin were determined by reverse transcription–polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Phosphorylation of AMPK and phosphorylation of Smad2/3 were evaluated by Western blot analysis.

Keywords nasal polyposis, metformin, adenosine monophosphate– activated protein kinase, fibroblast, fibronectin Received June 12, 2013; revised October 22, 2013; accepted November 1, 2013.

N

asal polyps arise in the nose and paranasal sinuses and have the macroscopic appearance of a pedicle of inflammatory tissue arising from the mucosal surface and projecting into the lumen or cavity.1,2 The prevalence of nasal polyps is considered to be about 4%.3 The characteristic symptoms of nasal polyps are nasal obstruction, rhinorrhea, olfactory dysfunction, and headache. Although nasal polyps are benign, those symptoms can cause a considerable reduction in quality of life. Several different mechanisms have been proposed for the development of nasal polyps. However, most of these describe nasal polyp formation as an integrated process involving the mucosal epithelium, extracellular matrix (ECM), and inflammatory cells and their mediators. The myofibroblasts observed in nasal polyps are of the activated cell phenotype of fibroblasts and are thought to be the main source of ECM in nasal polyps.4-6 Metformin has been widely used for the treatment of type 2 diabetes mellitus since the 1950s, and adenosine monophosphate–activated protein kinase (AMPK) is thought to be the target that mediates its beneficial metabolic effects.7 AMPK is a heterotrimeric kinase complex composed of a

Results. In TGF-b1–induced NPDFs, metformin inhibited the expression of a-SMA and fibronectin, as confirmed by both RT-PCR and Western blot analysis. Metformin increased the phosphorylation of AMPK and the expression levels of aSMA and fibronectin. However, compound C reversed these effects. Metformin inhibited TGF-b1–induced phosphorylation of Smad2/3.

1 Department of Otorhinolaryngology–Head and Neck Surgery, Korea University College of Medicine, Seoul, Korea 2 Department of Biomedical Sciences, Korea University Graduate School, Seoul, Korea 3 Medical Devices Clinical Trial Center, Guro Hospital, Korea University, Seoul, Korea * These authors contributed equally to this work.

Conclusions. This study showed that metformin inhibits TGFb1–induced myofibroblast differentiation and ECM production in NPDFs via the Smad2/3 pathway. AMPK can be a therapeutic target for the prevention of ECM remodeling in nasal polyps.

Corresponding Author: Heung-Man Lee, MD, PhD, Department of Otorhinolaryngology–Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Guro-gu, Seoul 152-703, South Korea. Email: [email protected]

Park et al catalytic (a) subunit and 2 regulatory (b and g) subunits. AMPK is activated by energy stress when intracellular adenosine triphosphate (ATP) levels decline and intracellular AMP increases, as occurs during nutrient deprivation or hypoxia.8,9 For this reason, AMPK is called a ‘‘metabolic regulator.’’ Metformin has antifibrotic effects beyond its antihyperglycemic action. For example, metformin inhibited collagen synthesis in cardiac fibrosis and transforming growth factor (TGF)b–induced fibrogenic responses in hepatic stellate cells.10,11 Thus, we hypothesized that metformin may be a possible therapeutic agent for nasal polyps. The purposes of this study were to investigate the effects of metformin on TGF-b1–induced myofibroblast differentiation (a-smooth muscle actin [a-SMA]) and ECM production and to determine the underlying mechanism for metformin action in nasal polyp–derived fibroblasts (NPDFs).

Materials and Methods Reagents Metformin and compound C were purchased from Sigma (St Louis, Missouri). Metformin was dissolved in distilled water, and compound C was dissolved in dimethyl sulfoxide. The maximum final concentration of dimethyl sulfoxide was less than 0.1%.

Nasal Tissues and Fibroblast Culture Seven patients (4 men and 3 women; 42 6 6.3 years) with nasal polyps were recruited from the Department of Otorhinolaryngology, Korea University Medical Center, Korea. Informed consent was obtained from each patient, and the study was approved by the Korea University Medical Center Institutional Review Board (KUGGR2010-015). Nasal polyp tissues were obtained from the middle meatus at the beginning of the endoscopic surgical procedure. Histologic evaluation by pathologist showed that all nasal polyps were neutrophilic. All patients were nonsmokers and had not been treated with oral or topical corticosteroids or antibiotics for at least 4 weeks before surgery. NPDFs were isolated from surgical tissues by enzymatic digestion with collagenase (500 U/mL, Sigma), hyaluronidase (30 U/mL, Sigma), and DNase (10 U/mL, Sigma). After a 2-hour incubation in 5% CO2 at 37°C in a cultured plate, the cells were collected by centrifugation, washed twice, resuspended in Dulbecco’s modified Eagle’s medium containing 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 1% (v/v) 10,000 units/mL penicillin, and 10,000 mg/mL streptomycin (Invitrogen). Cells were allowed to attach the culture plate for 4 days. Nonadherent cells were removed by changing the medium. The purity of the obtained NPDFs was confirmed microscopically by observation of characteristic spindle-shaped cell morphology and by fluorescence-activated cell sorting. More than 95% of cultured NPDFs were positive for vimentin and Thy-1 (Santa Cruz Biotechnology, Santa Cruz, California), which are used as fibroblast markers, and were negative for E-cadherin (Santa Cruz Biotechnology), an

149 epithelial cell marker. Experimental cells were obtained from the fourth cell passage. Some of the cells were transferred to a liquid nitrogen freezer for storage and used for the additional experiments when needed.

MTT Assay To determine the proliferation rate of nasal fibroblasts, the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma) assay was performed. NPDFs were seeded into 96-well tissue culture plates with 4 3 105 cells/mL. After seeding, the media were replaced with various concentrations (0-40 mM) of metformin. After 72 hours, the cells were treated with MTT solution for 4 hours. Mitomycin C (Sigma, 10 mg/mL) was used as a positive control. The optical density of each well was measured at 570 nm using an automatic plate reader (F2000 Hitachi Ltd., Tokyo, Japan).

Reverse Transcription-Polymerase Chain Reaction Total tissue RNA was extracted with NucleoSpin RNA II (Macherey-Nagel, Du¨ren, Germany) according to the manufacturer’s instructions. For real-time reverse transcription– polymerase chain reaction (RT-PCR), 200 ng to 1 mg of total RNAs was reverse transcribed to cDNA using ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) following the manufacturer’s protocols. Quantitative PCR was then carried out in a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) using 3 mL of cDNA template, 1 nmol of primers, and 12.5 mL of Power SYBR Green PCR Master Mix (Applied Biosystems), in a total volume of 25 mL reaction. The forward and reverse primers were as follows: GAPDH, 5#- GTGGATATTGTTGCCATCAATGACC-3# and 5#- GCCCCAGCCTTCTTCATGGTGGT-3#; a-SMA, 5#- GGTGCTGTCTCTCTATGCCTCTGGA-3# and 5#CCCATCAGGCAACTCGATACTCTTC-3#; fibronectin, 5#-ATTTGCTCCTGCACA TGC-3#, and 5#AGCCTGTACATCTAAAGGC. The cDNA were amplified with initial denaturation at 95°C for 10 minutes, followed PCR by 40 to 50 cycles of 95°C 15 for seconds, 58°C for 60 seconds, and finally 1 cycle of melting curve following cooling at 60°C for 60 seconds. To confirm amplification specificity, the PCR products from each primer pair were subjected to a melting curve analysis. Analysis of relative gene expression was done by evaluating q-RT-PCR data by the 2(-DDCt) method. Each experiment was repeated at least 3 times, and GAPDH was used as a housekeeping gene for internal control. The levels of gene expression were determined by normalizing relative to GAPDH expression.

Western Blot Analysis NPDFs were lysed using PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) and centrifuged for 30 minutes at 3000 rpm. NPDF lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore Inc, Billerica, Massachusetts). Membranes were blocked with 5% skim milk solution and were incubated with the following antibodies: a-SMA (Chemicon, Millipore Inc), fibronectin,

150

Otolaryngology–Head and Neck Surgery 150(1)

phosphorylated Smad2/3, pAMPK, and GAPDH antibodies (Santa Cruz Biotechnology). After incubation, the membranes were washed in Tris-buffered saline 0.1% Tween-20 buffer and then treated with peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G. Bands were visualized with HRP-conjugated secondary antibodies and an ECL system (Pierce, Rockford, Illinois).

Immunofluorescent Staining of a-SMA and Fibronectin Proteins NPDFs were fixed with 4% paraformaldehyde. NPDFs were permeabilized with 0.2% TritonX-100 in 1% bovine serum albumin (BSA) for 10 minutes, blocked with 3% BSA for 1 hour at room temperature, and incubated overnight at 4°C with rhodamine phalloidin (Invitrogen). Each sample was incubated with primary antibody and a-SMA or fibronectin. All stained nasal fibroblasts were captured and visualized using a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany). For nuclear counterstaining, Vectashield mounting medium with 4#,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, California) was used.

Statistical Analyses Results are shown as mean 6 standard deviation of the mean. The statistical significance of differences between groups was assessed by 1-way analysis of variance for factorial comparisons and by Tukey’s multiple comparison tests for multiple comparisons.

Results Effect of Metformin on Cytotoxicity in NPDFs To examine the effects of metformin on cell survival in NPDFs, an MTT assay was performed. A cell titration curve was generated from serial dilution of NPDFs and MTT reagent. The standard curve indicates a linear response between cell number and absorption at 570 nm. NPDFs were examined at concentrations ranging from 0 to 40 mM. Metformin did not affect cell survival until the concentration was 20 mM (Figure 1).

Inhibitory Effect of Metformin on mRNA Expression of a-SMA and Fibronectin in TGF-b1–Induced NPDFs It is already well known that TGF-b1 is a potent stimulator of differentiation of fibroblasts into myofibroblasts. NPDFs were treated with TGF-b1 for 24 hours and analyzed for a-SMA, a marker of myofibroblast differentiation, and expression of ECM such as fibronectin. Expression levels of a-SMA and fibronectin mRNA were determined by real-time RT-PCR. Both a-SMA and fibronectin mRNA levels were increased after TGF-b1 stimulation. Relative mRNA expression levels were measured and quantitated. To investigate the effects of metformin on myofibroblast differentiation and the production of fibronectin in TGFb1–induced NPDFs, NPDFs were treated with TGF-b1 and metformin for 24 hours. mRNA levels of a-SMA and fibronectin were significantly decreased. We also observed

Figure 1. Cytotoxicity test using an MTT assay of metformin. MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide.

that compound C, an AMPK inhibitor, reversed the inhibitory effect of metformin when cells were pretreated with it 1 hour before the treatment of metformin (Figure 2). Protein expression levels were demonstrated by Western blot and immunofluorescence staining (Figure 3). a-SMA and fibronectin levels increased in TGF-b1–induced NPDFs, and metformin significantly reduced the expression of these proteins. Taken together, these results indicate that metformin inhibits TGF-b1–induced myofibroblast differentiation and fibronectin in NPDFs.

Increased Phosphorylation of AMPK by Metformin To investigate whether metformin promotes AMPK activation in NPDFs, we confirmed the phosphorylation of AMPK. Phosphorylated AMPK expression levels were demonstrated by Western blot. Stimulation with TGF-b1 for 24 hours increased the level of phosphorylated AMPK in NPDFs. Metformin inhibited the increased expression of phosphorylated AMPK in TGF-b1–induced NPDFs. Compound C again reversed the inhibitory effect of metformin (Figure 4).

Inhibitory Pathway of Smad2/3 by Metformin in TGF-b1–Induced NPDFs Smad2/3 is one of the main transcription factors of the TGF-b signaling pathway. We determined if the effects of metformin on NPDFs are related to the Smad2/3 pathway. The expression level of phosphorylated Smad2/3 was demonstrated by Western blot. TGF-b1 increased the level of phosphorylated AMPK in NPDFs. Metformin inhibited the increase in the expression of phosphorylated AMPK in TGF-b1–induced NPDFs. Compound C reversed the inhibitory effect of metformin (Figure 5). These data suggest that metformin inhibits a-SMA and fibronectin expression via the Smad2/3 pathway (Figure 6).

Discussion The present study showed that metformin suppresses myofibroblast differentiation and ECM production in TGF-b1– stimulated NPDFs and that the effect of metformin is

Park et al

151

Figure 2. Effects of metformin with or without compound C on a-SMA (A) and fibronectin (B) mRNAs in TGF-b1–induced nasal fibroblasts. Values are mean 6 SD of independent samples. a-SMA, (a-smooth muscle actin; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TGF, transforming growth factor. *P \.05 versus control. yP \.05 versus TGF-b1 only.

Figure 3. Effects of metformin with or without compound C on the expression of a-SMA and fibronectin protein levels in TGF-b1– induced nasal fibroblasts. (A) Western blotting. (B) Immunofluorescence staining (red, a-SMA; green, fibronectin). Scale bar = 100 mm.

Figure 4. Effects of metformin with or without compound C on phosphorylation of adenosine monophosphate–activated protein kinase (p-AMPK) in TGF-b1–induced nasal fibroblasts. The expression of p-AMPK and AMPK was determined by Western blotting (representative of independent experiments).

mediated by the activation of AMPK. The data also showed that pretreatment of NPDFs with metformin decreased the level of pSmad2/3 induced by TGF-b1 stimulation. Taken together, these results suggest that

metformin modulates the TGF-b1–induced phenotype change and ECM production in NPDFs. Although the cause of nasal polyps and the pathophysiological mechanisms are still poorly understood, there is accumulating evidence supporting the idea that the differentiation of fibroblasts into myofibroblasts plays a key role.12 Myofibroblasts expressing a-SMA produce large amounts of ECM components. Accumulation of ECM is an important process in the characteristic structural modification of nasal polyps.13 In nasal polyps, growth factors including TGF-b1 stimulate fibroblast proliferation and myofibroblast differentiation.14 TGF-b has been reported to be involved in the pathogenesis of nasal polyposis and chronic rhinosinusitis.15 Increased production of TGF-b induces fibroblast activation and differentiation, leading to stromal fibrosis. Among the 3 isoforms of TGF-b, TGF-b1 is the isoform most relevant to the pathogenesis of nasal polyps.16 In our previous study, we showed that TGF-b1 significantly increases the expression of a-SMA as well as the production of collagen in NPDFs.6 The activation of AMPK provides a unified explanation for its pleiotropic beneficial effects.7 Initially, AMPK was

152

Figure 5. Effects of metformin with or without compound C on phosphorylation of Smad2/3 (pSmad2/3) protein in TGF-b1– induced nasal fibroblasts. Expression of pSmad2/3 and Smad2/3 was determined by Western blotting (representative of independent experiments). TGF, transforming growth factor.

Figure 6. Hypothetical schema of the action of metformin in TGFb1–induced nasal polyp–derived fibroblasts. AMPK, adenosine monophosphate-activated protein kinase; TGF, transforming growth factor.

characterized as a protein activated by nutrient and bioenergetic stress that raises intracellular AMP and lowers ATP. Recent studies have revealed novel functions of AMPK, including the regulation of a cell cycle check point, the decision to enter autophagy or apoptosis, and cell fate decisions in development.17-20 AMPK is highly sensitive to incremental changes in AMP and to elevations in the AMP/ ATP ratio.21 AMPK exists as a heterotrimer with a catalytic a-subunit and regulatory b- and g-subunits. Stimulation of AMPK requires phosphorylation of a critical threonine residue (Thr-172) in the activation loop of the a-subunit. In cells, AMPK activation slows metabolic reactions that consume ATP and stimulates reactions that produce ATP, thereby restoring the AMP/ATP ratio and the normal cellular energy state. The results of the present study showed that AMPK inhibits TGF-b1–induced myofibroblast differentiation in NPDFs, and thus we conclude that changes in the AMP/ATP ratio may influence the development of nasal polyps.

Otolaryngology–Head and Neck Surgery 150(1) Smad2/3 is one of the main transcriptional factors of the TGF-b signaling pathway. In fibroblasts, the regulation of myofibroblast differentiation by TGF-b1 is mediated by phosphorylation of Smad2/3, which subsequently complexes with Smad4 and translocates to the nucleus, where the dimer binds to the promoter region of the a-SMA gene. We examined whether phosphorylation of Smad2/3 by TGF-b1 is inhibited by metformin and confirmed that pretreatment of NPDFs with metformin decreased the level of pSmad2/3. The results of this study resemble those of an earlier study that reported that metformin suppresses the phosphorylation of Smad3 in response to TGF-b1 and in turn inhibits the nuclear translocation and transcriptional activity of Smad3 in adult mouse cardiac fibroblasts.10 However, another study showed that AMPK exerts antifibrotic effects via the regulation of p300 without altering TGF-b1–induced Smad in hepatic stellate cells.11 Therefore, we can assume that the effect of AMPK on fibroblasts is different according to the tissue in which they are located. This is the first study concerning the effects of metformin and AMPK on NPDFs. This study examined the inhibitory role of metformin on TGF-b1–induced myofibroblast differentiation and ECM production in NPDFs via the Smad2/3 pathway. The frequent recurrence of nasal polyps after endoscopic sinus surgery requires additional therapeutic modalities. The results of the present study suggest metformin and AMPK may be possible candidates for the inhibition of nasal polyp formation. However, further testing including in vivo studies will be required to verify this finding. Author Contributions Il-Ho Park, design, drafting the article, analysis and interpretation of data, revision; Ji-Young Um, design, drafting the article, acquisition of data; Sung-Moon Hong, acquisition of data, drafting the article interpretation of data; Jung-Sun Cho, acquisition of data, analysis of data, revising; Seung Hoon Lee, conception and design, revising; Sang Hag Lee, conception and design, revising; Heung-Man Lee, conception and design, revising.

Disclosures Competing interests: None. Sponsorships: None. Funding source: This study was funded by the Bumsuk Academic Scholarship Foundation.

References ¨ nerci 1. Seethala RR, Pant H. Pathology of nasal polyps. In: O TM, Ferguson BJ, eds. Nasal Polyposis. New York, NY: Springer; 2010:17-26. 2. Thomas M, Yawn B, Price D, et al. EPOS primary care guidelines: European position paper on the primary care diagnosis and management of rhinosinusitis and nasal polyps 2007—a summary. Prim Care Respir J. 2008;17:79-89. 3. Hedman J, Kaprio J, Poussa T, et al. Prevalence of asthma, aspirin intolerance, nasal polyposis and chronic obstructive

Park et al

4.

5.

6.

7.

8. 9.

10.

11.

12.

pulmonary disease in a population-based study. Int J Epidemiol. 1999;28:717-722. Nakagawa T, Yamane H, Nakai Y, Shigeta T, Takashima T, Takeda Z. Comparative assessment of cell proliferation and accumulation of extracellular matrix in nasal polyps. Acta Otolaryngol Suppl. 1998;538:205-208. Wang QP, Escudier E, Roudot-Thoraval F, et al. Myofibroblast accumulation induced by transforming growth factor-b is involved in the pathogenesis of nasal polyps. Laryngoscope. 1997;107:926-931. Park IH, Park SJ, Cho JS, et al. Role of reactive oxygen species in transforming growth factor beta1-induced alpha smoothmuscle actin and collagen production in nasal polyp-derived fibroblasts. Int Arch Allergy Immunol. 2012;159:278-286. Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001; 108:1167-1174. Hardie DG. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol. 2007;8:774-785. Kemp B, Stapleton D, Campbell D, et al. AMP-activated protein kinase, super metabolic regulator. Biochem Soc Trans. 2003;31:162-168. Xiao H, Ma X, Feng W, et al. Metformin attenuates cardiac fibrosis by inhibiting the TGFb1–Smad3 signalling pathway. Cardiovasc Res. 2010;87:504-513. Lim JY, Oh MA, Kim WH, et al. AMP-activated protein kinase inhibits TGF-b-induced fibrogenic responses of hepatic stellate cells by targeting transcriptional coactivator p300. J Cell Physiol. 2012;227:1081-1089. Norlander T, Westrin KM, Fukami M, et al. Experimentally induced polyps in the sinus mucosa. Laryngoscope. 1996;106: 196-203.

153 13. Kakoi H, Hiraide F. A histological study of formation and growth of nasal polyps. Acta Otolaryngol. 1987;103:137-144. 14. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007;87:245-313. 15. Watelet JB, Claeys C, Perez-Novo C, et al. Transforming growth factor 1 in nasal remodeling: differences between chronic rhinosinusitis and nasal polyposis. Am J Rhinol. 2004;18:267-272. 16. Serpero L, Petecchia L, Sabatini F, et al. The effect of transforming growth factor (TGF)-beta1 and (TGF)-beta2 on nasal polyp fibroblast activities involved upper airway remodeling: modulation by fluticasone propionate. Immunol Lett. 2006;105:61. 17. Jones RG, Plas DR, Kubek S, et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell. 2005;18:283-293. 18. Shaw RJ, Kosmatka M, Bardeesy N, et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A. 2004;101:3329-3335. 19. Liang J, Shao SH, Xu ZX, et al. The energy sensing LKB1– AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis. Nat Cell Biol. 2007;9:218-224. 20. Narbonne P, Roy R. Inhibition of germline proliferation during C. elegans dauer development requires PTEN, LKB1 and AMPK signalling. Development. 2006;133:611-619. 21. Kahn BB, Alquier T, Carling D, et al. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 2005;1:15-25.