37
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Spermidine Protects against Oxidative Stress in Inflammation Models Using Macrophages and Zebrafish

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Spermidine is a naturally occurring polyamine compound that has recently emerged with anti-aging properties and suppresses inflammation and oxidation. However, its mechanisms of action on anti-inflammatory and antioxidant effects have not been fully elucidated. In this study, the potential of spermidine for reducing pro-inflammatory and oxidative effects in lipopolysaccharide (LPS)-stimulated macrophages and zebrafish was explored. Our data indicate that spermidine significantly inhibited the production of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E 2 (PGE 2), and cytokines including tumor necrosis factor-α and interleukin-1β in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects of spermidine accompanied by a marked suppression in their regulatory gene expression at the transcription levels. Spermidine also attenuated the nuclear translocation of NF-κB p65 subunit and reduced LPS-induced intracellular accumulation of reactive oxygen species (ROS) in RAW 264.7 macrophages. Moreover, spermidine prevented the LPS-induced NO production and ROS accumulation in zebrafish larvae and was found to be associated with a diminished recruitment of neutrophils and macrophages. Although more work is needed to fully understand the critical role of spermidine on the inhibition of inflammation-associated migration of immune cells, our findings clearly demonstrate that spermidine may be a potential therapeutic intervention for the treatment of inflammatory and oxidative disorders.

          Related collections

          Most cited references37

          • Record: found
          • Abstract: found
          • Article: not found

          Antagonistic crosstalk between NF-κB and SIRT1 in the regulation of inflammation and metabolic disorders.

          Recent studies have indicated that the regulation of innate immunity and energy metabolism are connected together through an antagonistic crosstalk between NF-κB and SIRT1 signaling pathways. NF-κB signaling has a major role in innate immunity defense while SIRT1 regulates the oxidative respiration and cellular survival. However, NF-κB signaling can stimulate glycolytic energy flux during acute inflammation, whereas SIRT1 activation inhibits NF-κB signaling and enhances oxidative metabolism and the resolution of inflammation. SIRT1 inhibits NF-κB signaling directly by deacetylating the p65 subunit of NF-κB complex. SIRT1 stimulates oxidative energy production via the activation of AMPK, PPARα and PGC-1α and simultaneously, these factors inhibit NF-κB signaling and suppress inflammation. On the other hand, NF-κB signaling down-regulates SIRT1 activity through the expression of miR-34a, IFNγ, and reactive oxygen species. The inhibition of SIRT1 disrupts oxidative energy metabolism and stimulates the NF-κB-induced inflammatory responses present in many chronic metabolic and age-related diseases. We will examine the molecular mechanisms of the antagonistic signaling between NF-κB and SIRT1 and describe how this crosstalk controls inflammatory process and energy metabolism. In addition, we will discuss how disturbances in this signaling crosstalk induce the appearance of chronic inflammation in metabolic diseases. Copyright © 2013 Elsevier Inc. All rights reserved.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

            Introduction Macroautophagy (which we refer to as autophagy) is a cellular self-cannibalistic pathway in which parts of the cytosol or cytoplasmic organelles are enwrapped in double-membraned vesicles, autophagosomes, which then fuse with lysosomes (Klionsky, 2007). Autophagy plays a major role in the maintenance of cellular homeostasis, allows for the mobilization of energy reserves when external resources are limited, and is essential for the removal of damaged organelles and potentially toxic protein aggregates (Levine and Kroemer, 2008). At the organismal level, autophagy can mediate cytoprotection (for instance neuroprotection and cardioprotection in the context of ischemic preconditioning; Moreau et al., 2010) and delay the pathogenic manifestations of aging (Levine and Kroemer, 2009). Given the potential health and longevity-promoting effects of autophagy, pharmacological agents that stimulate autophagy at a low level of toxicity are urgently needed. Rapamycin and the so-called rapalogs are the most effective clinically used inducers of autophagy yet have severe immunosuppressive effects (Hartford and Ratain, 2007). Thus, alternative, nontoxic autophagy inducers (such as rilmenidine or carbamazepine) are being characterized for their pharmacological profile in suitable preclinical models (Hidvegi et al., 2010; Rose et al., 2010). Nontoxic compounds, such as resveratrol and spermidine, are also being evaluated for their potential to induce autophagy in vivo (Eisenberg et al., 2009; Morselli et al., 2010). Resveratrol is a natural polyphenol found in grapes, red wine, berries, knotweed, peanuts, and other plants. The interest in this molecule rose because it was suggested to mediate the cardioprotective effects of red wine (Baur and Sinclair, 2006). Resveratrol is also a potent inducer of autophagy (Scarlatti et al., 2008a,b), and this effect is mediated through the activation of sirtuin 1 (SIRT1), a NAD+-dependent deacetylase (Morselli et al., 2010). Resveratrol has been suggested to directly activate SIRT1 (Baur and Sinclair, 2006; Lagouge et al., 2006), although indirect effects may actually be preponderant (Beher et al., 2009; Pacholec et al., 2010). Spermidine is polyamine found in citrus fruit and soybean, which has recently been shown to increase the lifespan of yeast, nematodes, and flies in an autophagy-dependent fashion (Eisenberg et al., 2009). The transfection-enforced expression of SIRT1 is sufficient to stimulate autophagy in human cells (Lee et al., 2008). Starvation-induced autophagy (but not autophagy induced by rapamycin) requires SIRT1, both in vitro (in mammalian cells; Lee et al., 2008) and in vivo (in Caenorhabditis elegans; Morselli et al., 2010). Activated SIRT1 induces autophagy via its capacity to deacetylate acetyl lysine residues in other proteins (Lee et al., 2008). Conversely, knockdown of the acetyltransferase EP300 (Lee and Finkel, 2009), as well as inhibition of histone acetylases, potently induces autophagy (Eisenberg et al., 2009), indicating that protein deacetylation may play a general role in the initiation of the autophagic cascade. EP300 acetylates several autophagy-relevant proteins, including autophagy-related 5 (ATG5), ATG7, ATG12, and microtubule-associated protein 1 light chain 3 β (LC3; Lee and Finkel, 2009), whereas SIRT1 deacetylates ATG5, ATG7, LC3 (Lee et al., 2008), and the transcription factor forkhead box O3, which can stimulate the expression of proautophagic genes (Kume et al., 2010). As a result, protein (de)acetylation reactions influenced by sirtuins and other enzymes control autophagy at multiple levels, including the modification of autophagy core proteins and/or of transcriptional factors that control the expression of autophagic genes. Driven by these premises and incognita, we comparatively assessed the mechanisms of autophagy induction mediated by two distinct compounds that modulate protein acetylation, namely resveratrol and spermidine. We found that both agents induce autophagy through initially distinct yet convergent pathways that culminate in the acetylation and deacetylation of hundreds of proteins, with opposed patterns in distinct subcellular compartments. Based on this characterization, we demonstrated that these agents can stimulate autophagy in a synergistic fashion, both in vitro, in cultured human cells, and, in vivo, in mice. Results Sirtuin-dependent versus -independent autophagy induced by resveratrol and spermidine Spermidine and resveratrol were comparable in their autophagy stimulatory potency and induced hallmarks of autophagy with similar kinetics in human colon cancer HCT 116 cells. These signs included the redistribution of a GFP-LC3 chimera, which is usually diffuse, to cytoplasmic puncta and the lipidation of endogenous LC3, increasing its electrophoretic mobility (Fig. 1 and Fig. S1 A). In these conditions, neither spermidine nor resveratrol impaired oxidative phosphorylation (Fig. S1 B), ruling out that resveratrol might induce autophagy via mitochondriotoxicity (Dörrie et al., 2001). Knockdown of SIRT1 with a specific siRNA suppressed the proautophagic activity of resveratrol (Fig. 1, A and B) yet failed to affect spermidine-induced autophagy (Fig. 1 C). Similarly, the SIRT1 inhibitor EX527 (Peck et al., 2010) abolished autophagy induction by resveratrol but not by spermidine (Fig. 1, D–F). These results indicate that resveratrol and spermidine trigger autophagy through distinct mechanisms. Figure 1. SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A–C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1. (D–F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P 1.5-fold were included in statistical analyses. (B) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way. n = 560. Figure 6. Significant motifs among sites undergoing acetylation. (A) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Consensus acetylation motifs identified upon resveratrol (left) or spermidine (right) treatment are depicted using the MotifX algorithm (Schwartz and Gygi, 2005). Among sites that were hyperacetylated in response to both agents, the K(F/Y) motif is significantly enriched when tested against the whole proteome (P 1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Among sites that were hypoacetylated in response to both agents, the KP motif is significantly enriched when tested against ABD (P 100 proteins that are part of the central network of autophagic regulators/executioners (Behrends et al., 2010). This suggests that both agents stimulate autophagy through a multipronged mechanism that involves a large number of (de)acetylation reactions. Although resveratrol can (directly or indirectly) activate SIRT1, a deacetylase (Baur and Sinclair, 2006; Lagouge et al., 2006; Beher et al., 2009; Pacholec et al., 2010), spermidine has been shown to inhibit acetylases (Erwin et al., 1984; Eisenberg et al., 2009). Based on this consideration, it appears paradoxical that neither of these two agents was able to provoke a general deacetylation state and that both of them actually stimulated a similar shift in the acetylation pattern, in which hundreds of proteins were deacetylated (more in the cytosol than in the nucleus), whereas several others were acetylated (more in the nucleus than in the cytosol). Cells harbor multiple deacetylases and acetylases (Hassig and Schreiber, 1997; Katan-Khaykovich and Struhl, 2002; Nakamura et al., 2010), and it appears plausible, yet remains to be proven, that inhibition of one (or a few) acetylase will activate compensatory reactions by other acetylases and/or impact the action of deacetylases so that the global cellular level of protein acetylation remains near to constant. As a significant trend, however, we observed that both resveratrol and spermidine stimulated the deacetylation of cytosolic proteins, such as ATG5 and LC3, and the acetylation of nuclear proteins, including multiple histones. It has been recently reported that lifespan extension by spermidine treatment (during conditions of chronological aging) is linked to deacetylation of nuclear histones and to an increase in the transcription of different autophagy-related genes (Eisenberg et al., 2009). Interestingly, autophagy was rapidly induced by both spermidine and resveratrol in cytoplasts prepared from proliferating human cells, and an extranuclear variant of SIRT1 was as efficient in inducing autophagy as the predominantly nuclear WT SIRT1. Collectively, these data suggest that protein deacetylation first stimulates autophagy predominantly through a cytosolic mechanism. These results not only illustrate the differences between quiescent and proliferating cells in terms of autophagy modulation but also suggest that after a fast and nuclear-independent autophagic response transcriptional reprogramming is required to maintain an increased basal autophagic activity (Kroemer et al., 2010), thus contributing to the previously reported lifespan extension. Although we have few mechanistic cues to understand the discrepancy in cytosolic versus nuclear (de)acetylation reactions induced by resveratrol and spermidine, it is tempting to explain the synergistic proautophagic action of both compounds by the network properties of acetylases and deacetylases. One tenth of the dose of spermidine or resveratrol, which optimally stimulates autophagy, has no major proautophagic effects, meaning that dose–response curves are rather steep (most likely caused by compensatory reactions that tend to maintain the homeostasis of the acetylproteome). However, the partial yet simultaneous activation of the deacetylase activity of SIRT1 by resveratrol and the concomitant inhibition of acetylases by spermidine can unbalance the acetylproteome, thereby synergistically stimulating autophagy. Resveratrol is a natural polyphenol contained in red wine and vegetables, whereas spermidine is a polyamine found in other healthy food, such as citrus fruit and soybean. When analyzed as individual compounds, neither polyphenols nor polyamines consumed with the normal diet may reach concentrations high enough to mediate pharmacological effects. Nonetheless, it is tempting to speculate that combinations of these agents—and perhaps that of other proautophagic dietary components—may affect the autophagic rheostat, as based on their distinct yet convergent mode of action. Materials and methods Chemical, cell line, and culture conditions Unless otherwise specified, chemicals were purchased from Sigma-Aldrich, culture media and supplements for cell culture were obtained from Invitrogen, and plasticware was purchased from Corning. Human colon carcinoma HCT 116 cells (gift from B. Volgelstein, Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD; Bunz et al., 1999) were cultured in McCoy’s 5A medium containing 10% fetal bovine serum, 100 mg/liter sodium pyruvate, 10-mM Hepes buffer, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (5% CO2 at 37°C). Cells were seeded in 6- and 12-well plates or in 10- and 15-cm dishes and grown for 24 h before treatment with 10-µM EX527 (Tocris Bioscience), 10- or 100-µM resveratrol, 10- or 100-µM spermidine, and 1-µM rapamycin or 1-nM bafilomycin A1 (Tocris Bioscience) for the time indicated in each experimental figure legend. Plasmids, transfection, and RNA interference in human cell cultures Cells were cultured in 12-well plates and transfected at 50% confluence with siRNAs targeting human SIRT1 (Ford et al., 2005), ATG5, or ATG7 (Thermo Fisher Scientific) or with an unrelated control siRNA by means of a transfection reagent (Oligofectamine; Invitrogen) following the manufacturer’s instructions. After 24 h, cells were transfected with a plasmid coding for a GFP-LC3 fusion (Kabeya et al., 2000). Transient plasmid transfections were performed with the Attractene reagent (QIAGEN) as suggested by the manufacturer, and unless otherwise indicated, cells were analyzed 24 h after transfection. Cells were transfected with a plasmid coding for RFP fused to LC3 (RFP-LC3; obtained from Invitrogen) in the presence of an empty vector (pcDNA3) or of different constructs for the overexpression of GFP-tagged WT SIRT1 or a SIRT1 variant mutated in the nuclear localization signal, which mostly localizes in the nucleus (Tanno et al., 2007). For fluorescence microscopy determinations, cells cultured on coverslips were fixed in paraformaldehyde (4% wt/vol) for 15 min at RT, washed three times in PBS, and mounted with mounting medium (Vectashield; Vector Laboratories). Fluorescence microscopy Confocal fluorescent images were captured using a confocal fluorescence microscope (TCS SP2; Leica). For experiments with HCT 116 cells, an Apochromat 63× 1.3 NA immersion objective was used, whereas for the analysis of GFP-LC3 mice tissue sections, an Apochromat 40× 1.15 NA immersion objective was used. All the acquisitions were made at RT with fixed cells/tissue slides. Images were acquired with a camera (DFC 350 FX 1.8.0; Leica) using LAS AF software (Leica) and processed with Photoshop (CS2; Adobe) software. Specifically, picture processing involved cropping of representative areas and linear adjustments of contrast and brightness and was performed using Photoshop (with equal adjustment parameters for all pictures); no explicit γ correction was used. Nonconfocal microscopy of yeast strains carrying the EGFP-tagged Atg8 protein was performed with a microscope (Axioskop; Carl Zeiss, Inc.) using a Plan Neofluar objective lens (Carl Zeiss, Inc.) with a 63× magnification and 1.25 NA in oil at RT. Images were taken with a camera (SPOT 9.0 Monochrome 6; Diagnostic Instruments, Inc.), acquired using the Metamorph software (6.2r4; Universal Imaging Corp.), and processed with IrfanView (version 3.97) and Photoshop (CS2) software. Specifically, picture processing involved coloring and cropping of representative areas and was performed with IrfanView. In addition, linear adjustments of contrast and brightness were applied with Photoshop (using equal adjustment parameters for all pictures); no explicit γ correction was used. Nonconfocal microscopy of C. elegans was performed with a microscope (AxioImager Z2; Carl Zeiss, Inc.) using a Plan Neofluar 40× objective with a 0.75 NA and a 63× Plan Neofluar objective with an NA of 1.25 in oil at RT. Images were taken with a camera (AxioCam MRc5; Carl Zeiss, Inc.) with Axiovision software (Carl Zeiss, Inc.) without further processing. The different fluorophores used in this work were GFP and RFP for HCT 116 cells, EGFP for yeast experiments, DsRed for C. elegans analyses, and GFP for mice tissue sections. Nuclei were counterstained by Hoechst (Invitrogen). Immersion oil (Immersol; Carl Zeiss, Inc.) was used for all microscopy analyses. Immunoblotting, immunoprecipitation, and phosphokinase array For immunoblotting, cells were collected, washed with cold PBS, and lysed as previously described (Criollo et al., 2007). 25 µg of protein was then separated on 4–12% Bis-Tris acrylamide (Invitrogen) or 12% Tris-glycine SDS-PAGE precast gels (Bio-Rad) and electrotransferred to Immobilon membranes (Millipore) according to standard procedures. Unspecific binding sites were saturated by incubating membranes for 1 h in 0.05% Tween 20 (vol/vol in TBS) supplemented with 5% nonfat powdered milk (wt/vol in TBS) followed by overnight incubation with primary antibodies specific for acetylated lysine, phosphoacetyl-CoA carboxylase α (Ser79), acetyl-CoA carboxylase α, phospho-AKT1 (Thr308), AKT1, LC3, phosphomitogen-activated protein kinase 8 (Tyr183/Thr185), phospho-PRKAA1 (Thr172), PRKAA1, cyclin-dependent kinase inhibitor 1B, phosphoprotein tyrosine kinase 2 β (Tyr402), phosphoribosomal protein S6 kinase (Thr421/Ser424), ribosomal protein S6 kinase, stress-induced phosphoprotein 1 (Cell Signaling Technology), ATG7 (Sigma-Aldrich), phosphocyclin-dependent kinase inhibitor 1B (Abcam), TP53 (DO7), p62, SIRT1 (Santa Cruz Biotechnology), and peptidylprolyl isomerase A (cyclophilin A; Enzo Life Sciences, Inc.). Revelation was performed with appropriate HRP-labeled secondary antibodies (SouthernBiotech) plus a chemoluminescent substrate (SuperSignal West Pico; Thermo Fisher Scientific). An antiglyceraldehyde-3 phosphate dehydrogenase antibody (Millipore) was used to control the equal loading of lanes. For immunoprecipitation, extracts from 8 × 106 cells were lysed, and 500 µg of protein was precleaned for 1 h with 15 µl protein G–Sepharose 4 Fast Flow (GE Healthcare) followed by incubation for 2 h in the presence of an antiacetylated lysine (Cell Signaling Technology) antibody. Subsequent immunoblotting was performed by means of TrueBlot-HRP (eBioscience) secondary antibodies. For phosphokinase array analyses, the Proteome Profiler kit (R&D Systems) was used according to the manufacturer’s instructions. Yeast aging and autophagy measurements For chronological aging experiments, WT BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) S. cerevisiae or the respective sir2 deletion mutant (Δsir2) from the European Saccharomyces Cerevisiae Archive for Functional Analysis were inoculated from fresh overnight cultures to an absorbance of 0.1 (∼106 cells/ml) and grown at 28°C on Synthetic Complete 2% glucose medium. Aliquots were taken to perform survival plating at the indicated time points (Fig. 2 D, line graph) as previously described (Herker et al., 2004). Representative aging experiments are shown with at least three independent samples. Spermidine was added to stationary cultures at day 1 of the aging experiments (Eisenberg et al., 2009). Dihydroethidium staining was performed as previously described (Büttner et al., 2007), and the superoxide-driven conversion to ethidium was quantified either on a fluorescence plate reader (GeniosPro; TECAN) or on a cytofluorometer (FACSAria; BD) followed by first-line statistical analysis by means of the FACSDiva software (BD). Autophagy was monitored by vacuolar localization of Atg8p using fluorescence microscopy or by immunoblotting of cells ectopically expressing an EGFP-Atg8 chimera (Eisenberg et al., 2009) with anti-GFP (Sigma-Aldrich) and antiglyceraldehyde-3 phosphate dehydrogenase antibodies. For biochemical quantifications of the autophagic flux, AP activity was assayed according to published methods (Noda et al., 1995). In brief, WT or Δsir2 BY4741 cells were transformed and selected for stable insertion of a pTN9 HindIII fragment encoding for the cytosolic Pho8ΔN60 protein. AP activity was then assessed in 1.5 µg of crude protein extracts by measuring the conversion of α-naphtyl phosphate to naphthalene using a GeniosPro fluorescence plate reader with excitation and emission wavelengths at 340 nm and 485 nm, respectively (Noda et al., 1995). To correct for intrinsic AP activity, WT or Δsir2 yeast cells lacking the pTN9 HindIII fragment were simultaneously assayed, and these values were used for background subtraction, giving the vacuolar (autophagic) AP activity. C. elegans strains, genetics, and pharmacology We followed standard procedures for C. elegans strain maintenance. Nematode-rearing temperature was kept at 20°C. The following strains were used in this study: N2, WT Bristol isolate, and VC199, sir-2.1(ok434)IV. The VC199 strain was provided by the C. elegans Gene Knockout Project at the Oklahoma Medical Research Foundation, which is part of the International C. elegans Gene Knockout Consortium and the Caenorhabditis Genetics Center and is funded by the National Institutes of Health National Center for Research Resources. The construction of the p lgg-1 DsRed::LGG-1 reporter plasmid has been described previously (Samara et al., 2008). Spermidine was dissolved in sterilized water to a stock solution concentration of 100 mM. Escherichia coli (OP50) bacteria on seeded nematode growth medium (NGM) plates were killed by UV irradiation for 10 min (0.5 J) using a UV cross-linker (Bio-Link BLX-E365; Vilber Lourmat). A range of spermidine concentrations was prepared by dilutions in 100 µl of sterilized water and applied to the top of the agar medium (7-ml NGM plates). Plates were then gently swirled to allow the drug to spread to the entire NGM surface. Identical drug-free water solutions were used for the control plates. Plates were then allowed to dry overnight. The procedure was repeated each time worms were transferred to fresh plates (every 2–3 d during the first 2 wk and every week thereafter). Worms were incubated at 20°C. C. elegans autophagy measurements Images from transgenic embryos expressing a DsRed::LGG-1 fusion protein were acquired using a 540 ± 15–nm band-pass excitation filter and a 575-nm long-pass emission filter. Experiments were performed at 20°C, with photography exposure time kept identical for each embryo. Emission intensity was measured on grayscale images with a pixel depth of 8 bits (256 shades of gray). We calculated the mean and maximum pixel intensity for each embryo in these images using the ImageJ software (National Institutes of Health). For each transgenic line, we processed ≥25 images over at least three independent trials. C. elegans lifespan analysis Lifespan assays were performed at 20°C. Synchronous animal populations were generated by hypochlorite treatment of gravid adults to obtain tightly synchronized embryos that were allowed to develop into adulthood under appropriate conditions. Progeny were grown at 20°C through the L4 larval stage and then transferred to fresh plates in groups of 10–20 worms per plate for a total of 100–150 individuals per experiment. The day of egg harvest was set as t = 0. Animals were transferred to fresh plates every 2–4 d and were examined every day for touch-provoked movement and pharyngeal pumping until death. Worms that died (because of internally hatched eggs, extruded gonads, or desiccation upon crawling on the plate edge) were censored and incorporated as such into the dataset. Each survival assay was repeated at least three times. SILAC cell culture, sample processing, and analysis HCT 116 cells were cultured for 2 wk in three different SILAC media (Invitrogen) containing either (1) light isotopes ofl-arginine and l-lysine (Arg0/Lys0), (2) l-arginine–13C6 HCl (Euroisotop) and l-lysine 2HCl 4,4,5,5-D4 (Arg6/Lys4; Euroisotop), or (3) l-arginine–13C6 15N4 HCl and l-lysine 13C6 15N4 HCl (Arg10/Lys8; Invitrogen) and complemented as previously described (Blagoev and Mann, 2006). Cells were treated for 2 h with 100 µM spermidine (Arg10/Lys8) or 100 µM resveratrol (Arg6/Lys4) and then lysed and subdivided in nuclear, mitochondrial, and cytosolic fractions as previously described (Gurbuxani et al., 2003). The cytosolic fraction was precipitated using ice-cold (−20°C) acetone (4 vol of the sample extract), vortexed, and placed for 2 h at −20°C. The resulting solution was centrifuged for 10 min at 16,000 g, and the supernatant was removed. The pellet was subsequently washed twice with ice-cold 4:1 acetone/water. The final pellet was dried using a concentrator (SpeedVac; Thermo Fisher Scientific) for 10–15 min. All fractions were dissolved in denaturant 6:2-M urea/thiourea (both obtained from Merck), and Benzonase (Merck) was added to the nuclear fraction. All steps were performed at RT to avoid carbamoylation of amines. Reduction of cysteines was performed with 5-mM DTT (Sigma-Aldrich) for 30 min followed by alkylation with 11-mM iodoacetamide for 20 min in the dark. The proteins were digested with 1:100 protease LysC (Wako Chemicals USA, Inc.) for 3.5 h, diluted four times with 50-mM ammonium bicarbonate, and then digested with 1:100 trypsin (Promega) overnight. The nuclear fraction mixture was centrifuged at 10,000 g for 10 min, and the supernatant was filtered through a 0.45-µm MillexHV filter (Millipore). From each fraction, ∼100 µg of digested protein was collected for isoelectric focusing, a step required for subsequent normalization. The acetyl lysine peptide enrichment was performed as previously described (Choudhary et al., 2009). After peptide enrichment and isoelectric focusing, samples were subjected to MS analysis, and data were processed as described in the next paragraph. Preparation of cytoplasts Trypsinized cells previously transfected with GFP-LC3 cDNA were incubated in 3 ml of complete medium supplemented with 7.5 mg/ml cytochalasin B for 45 min at 37°C. This cell suspension was layered onto a discontinuous Ficoll density gradient (3 ml of 55%, 1 ml of 90%, and 3 ml of 100% Ficoll; GE Healthcare) in complete medium containing 7.5 mg/ml cytochalasin B. Gradients were prepared in ultracentrifuge tubes and preequilibrated at 37°C in a CO2 incubator overnight. Gradients containing cell suspensions were centrifugated in a prewarmed rotor (SW41; Beckman Coulter) at 100,000 g for 20 min at 32°C. The cytoplast-enriched fraction was collected from the interface between 55 and 90% Ficoll layers, washed in complete RPMI 1640 medium, and incubated for 4 h at 37°C before resveratrol, spermidine, or rapamycin treatment. Mouse experiments and tissue processing C57BL/6 mice obtained from Charles River Laboratory were bred and maintained according to both the Federation for Laboratory Animal Science Associations and the Animal Experimental Ethics Committee guidelines. They were housed in a temperature-controlled environment with 12-h light/dark cycles and received food and water ad libitum. Mice were injected intraperitoneally with 5 or 50 mg/kg spermidine and with 2.5 or 25 mg/kg 3 h before anesthetization and killing. Mice tissues were immediately frozen in liquid nitrogen after extraction and homogenized in a 20-mM Tris buffer, pH 7.4, containing 150-mM NaCl, 1% Triton X-100, 10-mM EDTA, and protease inhibitor cocktail (Complete; Roche). Tissue extracts were then centrifuged at 12,000 g at 4°C, and supernatants were collected. Protein concentration in the supernatants was evaluated by the bicinchoninic acid technique (BCA protein assay kit; Thermo Fisher Scientific). Quantitative analysis of GFP-LC3 dots in mice tissue sections To avoid postmortem autophagy induction, dead mice were immediately perfused with 4% paraformaldehyde (wt/vol in PBS, pH 7.4). Tissues were then harvested and further fixed with the same solution for ≥4 h followed by treatment with 15% sucrose (wt/vol in PBS) for 4 h and with 30% sucrose (wt/vol in PBS) overnight. Tissue samples were embedded in Tissue-Tek OCT compound (Sakura) and stored at −70°C. 5-µm-thick tissue sections were generated with a cryostat (CM3050 S; Leica), air dried for 1 h, washed in PBS for 5 min, dried at RT for 30 min, and mounted with Vectashield antifading medium. In each organ, the number of GFP-LC3 dots was counted in five independent visual fields from at least five mice using a confocal fluorescence microscope (TCS SP2). SILAC sample processing and analysis Upon digestion, peptides were concentrated and desalted on SepPak C18 (Waters) purification cartridges and eluted using a highly organic buffer (80% acetonitrile and 0.5% acetic acid). Eluates were lyophilized in a vacuum centrifuge, dissolved in immunoprecipitation buffer (50-mM MOPS, pH 7.2, 10-mM sodium phosphate, and 50-mM sodium chloride), and mixed with anti–acetyl lysine antibodies conjugated to agarose beads. The mixture was left for 12 h at 4°C on a rotation wheel. The flow-through (containing nonbound peptides) was removed, and beads were washed four times with the immunoprecipitation buffer and twice with deionized water. Bead-bound peptides were eluted with 0.1% trifluoroacetic acid. The same enrichment procedure was performed again on the flow-through containing the unbound peptides from the first enrichment. Six eluates and three separately collected digested protein samples were desalted using SepPak C18 cartridges, and 5% of each acetyl lysine–enriched eluate was used for MS analysis. The rest of the samples were subsequently separated into 12 fractions by isoelectric focusing using the 3100 OFFGEL Fractionator (Agilent Technologies) as previously described (Hubner et al., 2008). Gel strips and amfolyte buffer were purchased from GE Healthcare. Focusing was performed for 20 kV/h at a maximum current of 50 µA and maximum power of 200 mW. 10 µl of 10% trifluoroacetic acid was added to each fraction and STAGE (stop and go extraction) tipped as previously described (Rappsilber et al., 2007) before MS analysis, which was performed on either an LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific) connected to a 1200 Series Nanoflow HPLC system (Agilent Technologies) or an LTQ Orbitrap Velos spectrometer (Thermo Fisher Scientific; Olsen et al., 2009) connected to an 1100 Series Nanoflow HPLC system (Agilent Technologies) using a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated by reversed phase chromatography using an in-house–made fused silica emitter (75-µm internal diameter) packed with 3-µm reversed phase material (Reprosil-Pur C18-AQ; Dr. Maisch GmbH). Peptides were loaded in 98% solvent A (0.5% acetic acid) followed by a 100-min linear gradient to 50% solvent B (80% acetonitrile and 0.5% acetic acid). Survey full-scan MS spectra (mass to charge [m/z] range of 300–200, with a resolution of 60,000 at an m/z of 400) were acquired followed by fragmentation of the 10 (in the case of using the LTQ Orbitrap XL) or the 20 (in the case of using the LTQ Orbitrap Velos) most intense multiply charged ions. Ions selected for MS/MS were placed on a dynamic exclusion list for 45 s. Real-time internal lock mass recalibration was used during data acquisition (Olsen et al., 2005). For unfractionated acetyl lysine–enriched eluates, an additional MS analysis was performed on the LTQ Orbitrap Velos using higher energy collision dissociation fragmentation (normalized collision energy of 40) of the 10 most intense ions from each MS spectrum creating MS/MS spectra at a resolution of 7,500. All samples used for protein normalization were analyzed on a mass spectrometer (LTQ FT ULTRA; Thermo Finnigan) in which the five most intense ions from each precursor scan were selected for fragmentation in the LTQ. In this case, no real-time lock mass recalibration was used. Reverse-phase chromatography settings were the same as described for the analysis performed on the Orbitrap spectrometers. SILAC data processing Raw files were processed with MaxQuant v.1.0.13.13 (Cox and Mann, 2008) into centroided data and submitted to database searching with Mascot v.2.2 (Matrix-Science). Preprocessing by MaxQuant was performed to determine charge states, miscleavages, and SILAC states and to filter the MS/MS spectra, keeping the six most intense peaks within a 100-D bin. Cysteine carbamidomethyl was chosen as a fixed modification, whereas N-terminal acetylation and methionine oxidation were chosen as variable modifications. Furthermore, acetylation of light, medium, and heavy isotope lysines (Lys0/Lys4/Lys8) was chosen as a variable modification. Processed MS/MS spectra were searched against a concatenated target decoy database of forward and reversed sequences from the International Protein Index database (152,616 sequences; FASTA file created 5/6/2008). For the search, trypsin/P + DP was chosen for the in silico protein digestion allowing four miscleavages. The mass tolerance for the MS spectra acquired in the Orbitrap was set to 7 ppm, whereas the MS/MS tolerance was set to 0.6 D for the collision-induced dissociation MS/MS spectra from the LTQ and to 0.04 D for the higher energy collision dissociation MS/MS spectra. Upon peptide search, protein and peptide identification was performed given an estimated maximal false discovery rate of 1% at both the protein and peptide level. For false discovery rate calculation, posterior error probabilities were calculated based on peptides of at least six amino acids having a Mascot score of ≥10. For protein quantification, only unmodified peptides, peptides modified by N-terminal acetylation, and methionine oxidation were calculated. If a counterpart to a given lysine-acetylated peptide was identified, this counterpeptide was also excluded by protein quantitation. According to the protein group assignment performed by MaxQuant, both razor and unique peptides are used for protein quantification. A minimum of two ratio counts was required for protein quantification. For quantification of lysine-acetylated sites, the least modified peptides were used. The ratios for the sites were normalized by the corresponding protein ratios to account for eventual changes in protein abundance. In case a protein ratio was not determined, normalization was based on a logarithm transformation algorithm as previously described (Cox and Mann, 2008). Cell respiration and mitochondrial substrate oxidations Cell respiration and mitochondrial substrate oxidation were polarographically measured at 37°C in 250 µl of a buffer containing 0.3-M mannitol, 10-mM KCl, 5-mM MgCl2, 1 mg/ml BSA, and 10-mM KH2PO4, pH 7.4 (Rustin et al., 1994). Respiration was measured on intact cells (final concentration of ∼106/ml), which were subsequently permeabilized by 0.01% digitonin to study mitochondrial substrate oxidation. 10-mM malate plus10-mM glutamate oxidation was measured in the presence of 200-µM ADP. 10-mM succinate oxidation by digitonin-permeabilized cells was measured in the presence of 2-µM rotenone and 200-µM ADP. Sequential addition of 2-µM oligomycin, a specific inhibitor of the mitochondrial ATPase, and 2-µM carbonyl cyanide m-chlorophenyl hydrazone, a potent mitochondrial uncoupler, allowed for the determination of the respiratory control value associated with succinate oxidation. Functional analysis of proteins regulated by deacetylation or acetylation To decipher the functional context of the proteins associated with the drug-specific regulation of proteins by deacetylation and acetylation, GO term (Ashburner et al., 2000) enrichment was performed using the Cytoscape (Shannon et al., 2003) plugin BiNGO (Biological Networks Gene Ontology tool; Maere et al., 2005) and PANTHER (Protein Analysis Through Evolutionary Relationships) classification system. For the enrichment analysis, proteins regulated by ≥1.5-fold were included, and p-values were calculated by Fisher’s exact test after the Benjamini–Hochberg adjustment for multiple testing (Benjamini and Hochberg, 1995). A significance level of 0.05 (corresponding to the maximal false discovery rate) and a minimum of five proteins in at least one of the subsets of each given significant GO term were set as thresholds. Statistical analysis Statistical analyses were performed using the Prism software package (GraphPad Software, Inc.), the Office 2003 Excel software package (Microsoft), and the statistical environment R (R Development Core Team). In cell culture experiments, values were compared using unpaired Student’s t tests. For multiple comparisons, we used the one-factor analysis of variance corrected by posthoc Bonferroni test. C. elegans survival curves were created using the product-limit method of Kaplan and Meier. The log-rank (Mantel–Cox) test was used to evaluate differences between survival and determine p-values. Online supplemental material Fig. S1 describes the kinetics of autophagy induction after 100-µM resveratrol and spermidine treatment and shows measurements of cell respiration and mitochondrial substrate oxidation in the absence (control) or presence of 100-µM resveratrol or spermidine. Fig. S2 shows the measurement of SILAC fraction purity and analysis of the acetylation status of autophagy essential proteins after spermidine and/or resveratrol treatment. Fig. S3 schematically shows the biological processes associated with the differentially acetylated proteins in response to resveratrol and spermidine treatment by means of GO enrichment. Table S1 shows all the detected acetyl lysine–containing motifs in the different subcellular compartments after spermidine or resveratrol treatment and their relative enrichment in treated samples as compared with the corresponding control. Table S2 shows the list of proteins belonging to the human autophagy network interacting with the proteins detected in SILAC analysis. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201008167/DC1.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Reprogramming mitochondrial metabolism in macrophages as an anti-inflammatory signal.

              Mitochondria are master regulators of metabolism. Mitochondria generate ATP by oxidative phosphorylation using pyruvate (derived from glucose and glycolysis) and fatty acids (FAs), both of which are oxidized in the Krebs cycle, as fuel sources. Mitochondria are also an important source of reactive oxygen species (ROS), creating oxidative stress in various contexts, including in the response to bacterial infection. Recently, complex changes in mitochondrial metabolism have been characterized in mouse macrophages in response to varying stimuli in vitro. In LPS and IFN-γ-activated macrophages (M1 macrophages), there is decreased respiration and a broken Krebs cycle, leading to accumulation of succinate and citrate, which act as signals to alter immune function. In IL-4-activated macrophages (M2 macrophages), the Krebs cycle and oxidative phosphorylation are intact and fatty acid oxidation (FAO) is also utilized. These metabolic alterations in response to the nature of the stimulus are proving to be determinants of the effector functions of M1 and M2 macrophages. Furthermore, reprogramming of macrophages from M1 to M2 can be achieved by targeting metabolic events. Here, we describe the role that metabolism plays in macrophage function in infection and immunity, and propose that reprogramming with metabolic inhibitors might be a novel therapeutic approach for the treatment of inflammatory diseases.
                Bookmark

                Author and article information

                Journal
                Biomol Ther (Seoul)
                Biomol Ther (Seoul)
                Biomol Ther (Seoul)
                ksp
                Biomolecules & Therapeutics
                The Korean Society of Applied Pharmacology
                1976-9148
                2005-4483
                March 2018
                06 April 2017
                : 26
                : 2
                : 146-156
                Affiliations
                [1 ]Anti-Aging Research Center and Department of Biochemistry, Dongeui University College of Korean Medicine, Busan 47227, Republic of Korea
                [2 ]Departments of Parasitology and Genetics, Kosin University College of Medicine, Busan 49267, Republic of Korea
                [3 ]Natural products Research Team, Marine Biodiversity Institute of Korea, Seocheon 33662, Republic of Korea
                [4 ]Department of Pharmacy, College of Pharmacy, Inje University, Gimhae 50834, Republic of Korea
                [5 ]Department of Microbiology, College of Medicine, Inje University, Busan 47392, Republic of Korea
                [6 ]Department of Chemistry, College of Natural Sciences, Pusan National University, Busan 46241, Republic of Korea
                [7 ]Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 46241, Republic of Korea
                [8 ]Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea
                [9 ]Department of Molecular Biology, College of Natural Sciences & Human Ecology, Dongeui University, Busan 47340, Republic of Korea
                Author notes
                [†]

                The first two authors contributed equally to this work.

                [* ]Corresponding Author: E-mail: choiyh@ 123456deu.ac.kr , Tel: +82-51-850-7413, Fax: +82-51-853-4036
                Article
                bt-26-146
                10.4062/biomolther.2016.272
                5839493
                28365977
                ba475eea-f754-4e2c-9fe3-3871d9207a6b
                Copyright © 2018 The Korean Society of Applied Pharmacology

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 11 December 2016
                : 25 December 2016
                : 18 January 2017
                Categories
                Original Article

                spermidine,macrophages,zebrafish,anti-inflammation,anti-oxidant

                Comments

                Comment on this article