Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrol-induced secretome effects

BBAPAP-39352; No. of pages: 12; 4C: 4 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap

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Johan Renes a,⁎, Anja Rosenow a, Nadia Roumans a, Jean-Paul Noben b, Edwin C.M. Mariman a

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Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrol-induced secretome effects

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Article history: Received 30 September 2013 Received in revised form 25 April 2014 Accepted 28 April 2014 Available online xxxx

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Keywords: Human adipocytes Calorie restriction Adipokines Metabolic syndrome 2-DE LC–MS/MS

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NUTRIM School for Nutrition, Toxicology and Metabolism, Department of Human Biology, Maastricht University, Maastricht, The Netherlands Biomedical Research Institute, Hasselt University, and School of Life Sciences, Transnationale Universiteit Limburg, Diepenbeek, Belgium

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Obesity is characterized by dysfunctional white adipose tissue (WAT) that ultimately may lead to metabolic diseases. Calorie restriction (CR) reduces the risk for age and obesity-associated complications. The impact of CR on obesity has been examined with human intervention studies, which showed alterations in circulating adipokines. However, a direct effect of CR on the human adipocyte secretome remains elusive. Therefore, the effect of a 96 h low glucose CR on the secretion profile of in vitro cultured mature human SGBS adipocytes was investigated by using proteomics technology. Low-glucose CR decreased the adipocyte triglyceride contents and resulted in an altered secretion profile. Changes in the secretome indicated an improved inflammatory phenotype. In addition, several adipocyte-secreted proteins related to insulin resistance showed a reversed expression after low-glucose CR. Furthermore, 6 novel CR-regulated adipocyte-secreted proteins were identified. Since resveratrol (RSV) mimics CR we compared results from this study with data from our previous RSV study on the SGBS adipocyte secretome. The CR and RSV adipocyte secretomes partly differed from each other, although both treatment strategies lead to secretome changes indicating a less inflammatory phenotype. Furthermore, both treatments induced SIRT1 expression and resulted in a reversed expression of detrimental adipokines associated with metabolic complications. © 2014 Published by Elsevier B.V.

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1. Introduction

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White adipose tissue (WAT) stores the surplus of the body's energy as triglycerides (TG) and regulates energy metabolism by endocrine/ paracrine secretion of signaling molecules, known as adipokines. The pattern of secreted adipokines from WAT cells includes extracellular matrix proteins, hormones, neurotrophins, cytokines as well as proteins involved in angiogenesis and lipid and glucose metabolism [1–3]. Excess energy storage results in elevated fat mass, which is characterized by morphological, histological and functional changes of the WAT, including the alteration of secreted adipokines. These changes lead to dysfunction of the WAT, which is associated with obesity-related disorders such as type 2 diabetes mellitus, coronary heart disease and cancers [1,4,5]. Bariatric surgery, drugs, exercise and/or calorie restriction (CR) are common methods to treat obesity and its complications. Especially CR reduces the risk for age- and obesity-associated disease and may lead

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Abbreviations: CR, calorie restriction; ECM, extracellular matrix; FFA, free fatty acids; RSV, resveratrol; SGBS, Simpson–Golabi–Behmel syndrome; TG, triglycerides; WAT, white adipose tissue ⁎ Corresponding author at: Department of Human Biology, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel.: +31 43 388 1633; fax: +31 43 36 70976. E-mail address: [email protected] (J. Renes).

to improved life quality and longer lifespan [6,7]. In yeast, these effects are mediated by the silent information regulator 2 (SIR2) [8]. Beneficial effects of CR in mammals have been attributed to Sirtuin 1 (SIRT1), the mammalian homologue of SIR2 [9]. Within mouse 3T3-L1 adipocytes the lipolysis of TG and the release of free fatty acid (FFA) are regulated by SIRT1 [9]. The effects of CR on obesity and its associated complications have been mainly investigated with human intervention studies. Varady et al. [10] demonstrated an improved circulating adipokine profile together with a decreased adipocyte size already by a weight loss of 5% in severely obese women. Beneficially altered adipokine profiles may be attributed to the decreased adipocyte size since adipokine expression and adipocyte size are correlated [11]. Yet, the molecular mechanisms of CR-mediated effects on adipokine expression and release are still poorly understood [12]. Simpson–Golabi–Behmel syndrome (SGBS) fat cells provide an ideal model to investigate the human adipocyte secretome [3]. As such, we analyzed the effect of low-glucose CR on mature SGBS adipocytes to investigate the relation between reduction of the TG content and changes in the adipokine secretion pattern. The differences between the secretome of fully differentiated calorie restricted and non-calorie restricted SGBS adipocytes were analyzed by two-dimensional gel electrophoresis (2-DE) and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS).

http://dx.doi.org/10.1016/j.bbapap.2014.04.023 1570-9639/© 2014 Published by Elsevier B.V.

Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

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2. Experimental section

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2.1. Materials

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Modified phenol red free DMEM/F12 medium without glucose, 0.5% trypsin–EDTA and 10,000 u/ml penicillin/streptomycin were obtained from Life Technologies (Bleiswijk, The Netherlands). Fetal bovine serum (FBS) was purchased from Bodinco (Alkmaar, The Netherlands). Additional cell culture supplements, 100 × protease inhibitor cocktail, phenylmethanesulfonyl fluoride (PMSF), DL-dithiothreitol (DTT), 3-[(3cholamidopropyl) dimethyl-amonio]-1-propanesulfonate (CHAPS), αcyano-4-hydroxyl-cinnamic acid (CHCA), trifluoroacetic acid (TFA) and acetonitrile (ACN) were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Immobilized pH gradient (IPG) buffer (pH 3–11, nonlinear), Dry-Strip cover fluid and immobiline Dry-Strips (pH 3–11, nonlinear, 24 cm) were obtained from GE Healthcare (Diegem, Belgium).

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2.2. Cell culture

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Human Simpson–Golabi–Behmel syndrome (SGBS) cells were obtained from Prof. Dr. M. Wabitsch (University of Ulm, Germany) [17] and cultured as described [13]. Ninety percent confluent preadipocytes (2.2 ± 0.36 × 106, mean ± SD, n = 3) were differentiated into mature adipocytes during 14 days as described [13]. On average 78% of the preadipocytes differentiated into mature adipocytes. To determine cell numbers, adipocytes were counted with a raster ocular. Preadipocytes were trypsinized and counted with a hemocytometer.

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2.3. CR experiments

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Dose–response experiments were performed to define the optimal glucose concentration to study the effect of CR on the adipocyte secretome. Mature adipocytes (14 days differentiated) were cultured with 66 nM insulin and with different glucose concentrations (0, 0.2, 0.55 and 1 mM glucose) for a period of 96 h. The control cells reflected the time point of the beginning of the CR intervention. As such, the control adipocytes were cultured for only 14 days after the initiation of differentiation. On day 12 of the differentiation period the non-calorie restricted adipocytes were washed twice and incubated with phenol red free DMEM/F12 medium containing 17.5 mM glucose (normal glucose condition) and 66 nM insulin for 48 h. These cells are indicated as non-CR adipocytes. The effect of CR on the adipocytes was measured by intracellular TG contents and glycerol release. Mature adipocytes (14 days differentiation) cultured with 66 nM insulin and the normal medium glucose concentration of 17.5 mM glucose was used to demonstrate the maximum TG accumulation within the 96 h incubation period. In the subsequent experiments, 14 days differentiated adipocytes were washed twice and cultured with 0.55 mM glucose and 66 nM insulin in phenol red free DMEM/F12 medium for 4 days. No cell death was observed.

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2.5. Western blotting

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CR-mediated altered expression of hormone sensitive lipase (HSL), adipose tissue triglyceride lipase (ATGL), peroxisome proliferatoractivated receptor γ coactivator 1-alpha (PGC-1α) and SIRT1 was measured by Western blotting. Total cell lysates were prepared either in ureum buffer (8 M urea/2% w/v CHAPS/65 mM DTT/protease inhibitor cocktail) for HSL and ATGL or in RIPA buffer (25 mM Tris–HCl pH 7.6/ 150 mM NaCl/1% NP-40/1% deoxycholate/0.1% SDS/protease inhibitor cocktail) for PGC-1α and SIRT1. Protein concentrations were determined by either a Bradford-based protein assay kit (Bio-Rad Laboratories, Veenendaal, The Netherlands) (ureum buffer samples) or by a Pierce BCA protein assay (Thermo Scientific, Etten-Leur, The Netherlands) (RIPA buffer samples). Proteins were separated by SDS– PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk and probed with polyclonal rabbit antibodies against human HSL (kind gift from Prof. Dr. C Holm, Lund University, Sweden), ATGL, PGC-1α (Cell Signalling Technology, Leiden, The Netherlands) or SIRT1 (Calbiochem, Merck Millipore, Amsterdam, The Netherlands). Subsequently, the membranes were subjected to polyclonal swine anti rabbit IgG/HRP (DAKO, Glostrup, Denmark) as secondary antibodies. Bound antibodies were visualized with the SuperSignal West Dura or West Femto chemiluminescent substrates (Thermo Scientific). Since general housekeeping proteins (e.g. β-actin) were found differentially expressed during CR, Ponceau S staining per protein lane on the nitrocellulose membrane (see Supplement 2) was quantified and used as reference for protein sample load by using Image Lab software (Bio-Rad Laboratories).

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2.6. 2-DE

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Preparation of secretome samples was done similarly as described before [13]. Protein samples derived from 2.75 × 106 adipocytes were used for 2-DE analysis according to Bouwman et al. [18] but with different IPG strips (pH 3–11, nonlinear, 24 cm). One hundred micrograms of total protein in a volume of 450 μl containing 0.5% (v/v) IPG was loaded onto the IPG strips. For protein profiling, 4 independent replicates were made for CR and non-CR adipocytes. The gels were stained with Flamingo fluorescent gel stain according to the manufacturer's protocol (Bio-Rad Laboratories). Proteins spots were visualized by a Molecular Imager FX scanner (Bio-Rad Laboratories).

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2.7. Image analysis and protein identification

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Stained gels were processed by PDQuest 8.0 software (Bio-Rad Laboratories). Data were normalized with respect to the total density of gel image. CR and non-CR groups were formed from samples of 4 independent experiments with the same treatment. Protein spots were regarded as significantly differentially expressed if the average spot intensity between the CR and non-CR groups differed more than 1.5-fold with p b 0.05 (Student's T-test). The criteria for indicating a trend were a spot intensity difference of more than 1.5-fold with 0.05 b p b 0.1. For subsequent protein identification, gels with differentially expressed spots were re-stained with SYPRO Ruby Protein Stain according to the manufacturer's protocol (Bio-Rad Laboratories). Identification of differentially expressed protein spots derived from the 2-DE gels was done by in-gel digestion and LC–ESI–MS/MS essentially as described before [13].

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Intracellular TG contents in and glycerol release from CR and non-CR SGBS adipocytes was determined as previously described [13]. Adiponectin concentrations in secretion media were measured by a radio-immunoassay (Human adiponectin RIA kit, Millipore, St. Charles, MO, USA).

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2.4. Measurement of intracellular TG content, glycerol release and 132 adiponectin secretion 133

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Previously, we showed a positive effect of resveratrol (RSV) on the SGBS adipocyte secretome [13]. RSV can mimic CR in obese persons [14,15], which results in improved insulin resistance, decreased levels of blood glucose, TG and cytokines as well as increased intramyocellular lipid levels, decreased intrahepatic lipid content and decreased systolic blood pressure. These RSV-induced beneficial health effects are also thought to be mediated by SIRT1 activation [16]. This suggests that CR and RSV may exert their beneficial effects on adipocytes by a common mechanism. It prompted us to compare the data from this CR study with data from our previous RSV study with respect to the effect of both treatments on the human adipokine profile.

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Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

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Glycerol release and TG accumulation were analyzed by a one-way ANOVA with repeated measures. Western blot data and adiponectin secretion were analyzed by a two-tailed paired T-test. Significant differences (p b 0.05) between groups are indicated with asterisks.

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3. Results

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3.1. Low-glucose CR-induced lipolysis in SGBS adipocytes

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CR-induced lipolysis in SGBS cells was determined by measurement of intracellular TG content and glycerol release under low-glucose conditions (0, 0.2, 0.55 and 1.0 mM). Compared to non-CR SGBS adipocytes a significant reduction of intracellular TG was observed in SGBS adipocytes incubated for 96 h with 0.2 mM (reduction of 25 ± 3.2%, n = 4) and 0.55 mM (reduction of 17 ± 6.0%, n = 4) glucose (Fig. 1A and B). A significant decrease of glycerol release was observed with 0 mM (reduction of 88 ± 7.5%, n = 4), 0.2 mM (reduction of 90 ± 5.7 %, n = 4) and 0.55 mM (reduction of 81 ± 14.2%, n = 4) glucose compared to the control (Fig. 1C). To investigate the maximum CR-induced secretome changes with minimal cell death-related effects of mature SGBS adipocytes, we chose a 0.55 mM glucose concentration. A concentration of 0.55 mM glucose equals 3% of the high DMEM medium glucose concentration (17.5 mM) and is about 10% of the normal physiological glucose concentration. CR effects of 0.55 mM glucose were further investigated by measuring the expression of HSL and ATGL, two key lipolytic enzymes [23]. Western blot results from 4 independent experiments showed that after 96 h of 0.55 mM glucose treatment HSL expression was down-regulated, although not significantly, while ATGL expression was significantly down-regulated compared to non-CR adipocytes (Fig. 1D).

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3.2. Measurement of SIRT1 and PGC-1α expression

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Previously we showed an RSV-induced expression of SIRT1 in SGBS adipocytes [13]. Since RSV mimics CR we investigated the SIRT1 expression under low-glucose conditions. PCG-1α expression was also measured as this protein can be activated by SIRT1 and by reduced TG contents. [24]. Fig. 2 shows that SIRT1 is significantly up-regulated in SGBS adipocytes by 0.55 mM glucose while PCG-1α expression is also induced, although not significantly.

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3.4. Measurement of adiponectin secretion

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Adiponectin secretion inversely correlates with adipocyte size and plasma adiponectin concentrations increase during weight loss [25]. As such, to confirm a CR-induced adiponectin secretion we measured the adiponectin concentration in medium derived from CR and non-CR SGBS adipocytes. Fig. 4 shows that the amount of secreted adiponectin in medium derived from SGBS adipocytes treated with 0.55 mM glucose is significantly higher compared to non-CR SGBS adipocytes.

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3.5. Secretome validation

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Comparison of the secretome data sets with recent human and rodent adipokine studies revealed several new adipocyte-secreted proteins: 3-hydroxyisobutyryl-CoA hydrolase; cDNA FLJ54471, highly similar to complement C1r subcomponent; cDNA FLJ59142, highly similar to epididymal secretory protein E1; cDNA FLJ60094, highly similar to F actin capping protein subunit β; HP protein and ubiquitin carboxyl-terminal hydrolase isozyme L3 [3,13,26–30]. Together, this validation analysis revealed 6 novel identified adipocyte secreted proteins in this study.

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CR-induced secretome changes were analyzed by 2-DE. Representative gels of the non-CR and the 96 h low-glucose CR condition together with the master gel are shown in Fig. 3. From a total of 511 matched spots, 141 spots were either significantly (p b 0.05: 92 spots) or as trend (p b 0.1; 49 spots) differentially expressed. These spots were excised from the gels and analyzed by LC–ESI–MS/MS. One hundred twenty-seven of the 141 spots were identified. Subsequent analysis revealed 89 unique proteins (Fig. 3 and Supplement 1). SignalP 3.0 and SecretomeP 2.0 analysis of the 89 proteins revealed 34 proteins as classical secreted proteins, 22 proteins as non-classical secreted and 33 proteins as intracellular proteins (Supplement 1). Identical proteins could be identified in different spots containing either one or several different proteins. As such, proteins with similar accession numbers can be related to different ID-numbers dependent on the number of different proteins within one spot. Therefore, protein identifications derived from one protein per spot were listed in Table 1. From those proteins a clear CR-mediated regulation pattern could be obtained. The regulation pattern of proteins identified from spots containing more than one protein remains elusive. These are listed in Table 2. Next to secretory proteins intracellular proteins were also detected in non-single proteins spots, these are listed in Supplement 1. The category of classical secreted proteins was further sub-categorized into extracellular matrix (ECM) proteins (9), processing (4), regulation/signaling proteins (18) and immune regulation proteins (3) (Tables 1 and 2, Supplement 1). Analysis of the regulation patterns of proteins with similar functions revealed that a large group of structure-related proteins changed considerably by CR. As such, most of the subunits and isoforms of type I, III, IV, V and VI collagens and fibronectin were found to be down-regulated. In contrast, all isoforms of collagen 2(I) were induced by CR. In addition, the structural proteins including cofilin 1, transgelin and vimentin were also induced by CR. Spots of processing proteins as well as endoplasmin were up-regulated by CR. CR also changed the secretion of proteins related to regulation and signaling. As such, most of the identified subunits and/or isoforms of apolipoprotein E (ApoE), haptoglobin, pigment epithelium-derived factor (PEDF) and zinc-α-2-glycoprotein (ZAG) were down-regulated by CR (Table 1). In addition, many adipocyte-specific proteins including adiponectin, angiopoietin-related protein 1, angiotensinogen, calumenin, follistatin-related protein 1, plasminogen activator inhibitor 1 (PAI-1) were identified after the CR intervention (Table 2). However, these proteins were found in a mixed spot, which makes their CR-mediated regulation pattern less clear.

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MS/MS spectra and raw data files were processed as described [13]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository [19] with the data set identifier “PXD000301 and DOI http://dx.doi.org/10. 6019/PXD000301.” Mascot DAT files were processed by PRIDE Converter software [20] and submitted via ProteomeXchange (http:// www.proteomexchange.org) to the PRIDE database [21], project accession number: 29652-29779. MS data can be visualized using PRIDE Inspector [22] (http://tinyurl.com/csffalc) and Scaffold .sf3 free viewer (https://proteomecommons.org/tool.jsp?i=1009). For verification of secreted protein candidates an amino acid sequence analysis was performed with SignalP 3.0 and SecretomeP 2.0 (CBS, Technical University Copenhagen, Denmark, URL: www. cbs.tdu.dk) as described before [13]. Furthermore, identified proteins were screened for their association with exosomes by the Exocarta database (www.exocarta.org).

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Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

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Fig. 1. Glycerol release and TG accumulation studies of calorie restricted adipocytes. A: Images with a Nikon TE 200 eclipse phase contrast microscope of 14 days differentiated adipocytes which were not calorie restricted (non-CR) or calorie restricted for 96 h with different concentrations of medium glucose. From left to right: non-CR, 0, 0.2, 0.55, 1.0 and 17.5 mM medium glucose. B: Intracellular TG accumulation determined by spectrophotometry after similar conditions as in A. C: Glycerol release in the culture medium determined after similar conditions as in A. Reported values are means ± SEM of quadruple measurements from 2 biological replicates. D: HSL and ATGL protein levels in non-CR SGBS adipocytes and in SGBS adipocytes treated 96 h with 0.55 mM glucose (0.55 mM gluc). Western blotting analysis was performed with 10 μg of total protein lysate. Reported values are means ± SEM of at least 4 independent biological replications and * indicates differences with p b 0.05.

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To the best of our knowledge this is the first detailed proteomics study of CR-induced secretome changes of in vitro human adipocytes. As such, we compared our data with data from human trial studies in which CR-induced adipokine changes were determined. Klempel [31] and Varady [32] reviewed in vivo studies of CR-induced human adipokine changes, which revealed a relatively small set of plasma circulating adipokines including adiponectin, IL 6, IL-8, leptin, monocyte chemotactic protein-1, resistin, retinol-binding protein and tumor necrosis factor-α. From these proteins only adiponectin, interleukin (IL)

6, IL-8, leptin and retinol-binding protein 4 are known as human adipocyte-secreted proteins. All other described proteins are secreted from other cells in the WAT. Comparison with our data revealed only adiponectin as a shared protein [31,32].

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3.6. Comparison of the secretion profile of CR- and RSV-treated adipocytes 320 Since RSV mimics CR we compared the data described in this study 321 with data from our recently published study on RSV-mediated changes 322

Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

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4. Discussion

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Changes in lifestyle, especially dietary changes, are known to prevent age and obesity-associated diseases, which results in improved life quality and a longer lifespan [6,7]. CR beneficially influences circulating adipokine levels [31,32]. However, effects of CR on the secretome of in vitro human adipocytes are largely unknown. Therefore, we subjected human SGBS adipocytes to low-glucose CR, which revealed a positive impact on the SGBS adipocyte secretion profile. In addition, 6 novel adipocyte-secreted proteins were identified. Recently we showed RSVmediated changes of the human adipocyte secretome [13]. RSV induces protective effects against metabolic diseases [33] and has life extending properties [34]. This has been attributed to the CR-mimicking effects of RSV [9,15,34]. As such, we compared results from this CR study with our previous RSV study with respect to effects on the adipocyte secretome. TG reduction by low calorie diets is associated with increased intracellular lipolysis [35,36]. Here, a non-significantly reduced HSL and a significantly reduced ATGL expression were detected after lowglucose CR. This is in line with the study of Jocken et al. [36] in which a significant down-regulation of HSL and ATGL mRNA and protein expression was observed in subcutaneous fat biopsies from obese persons subjected to a hypocaloric diet. Interestingly, mRNA levels of HSL and ATGL were increased during differentiation of human hMADS adipocytes [37]. This suggests that expression of these two lipolytic enzymes per se in human adipocytes is induced during TG accumulation with no further increase, or even decrease, during conditions of TG reduction. Alternatively, HSL is activated by phosphorylation, while ATGL interacts

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in the human adipocyte secretome [13] (Table 3 and Fig. 5). From all identified secreted proteins from this CR study 17 proteins were also present in the RSV data (Fig. 5A), including adipokines like adiponectin and PAI-1 identified in spots containing more than one protein. To gain more insight in regulation patterns only proteins identified as single protein per spot were compared (Table 3). As such, 12 proteins were identified in the CR as well as RSV data sets. Furthermore, this comparison revealed an additional set of 20 unique proteins from the RSV study and 24 unique proteins from this CR study (Fig. 5B and Table 3).

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with ABHD5 upon lipolysis stimulation [38]. In fact, we observed a nonsignificant CR-induced phosphorylation of HSL (data not shown). Once activated, HSL and ATGL translocate and dock to intracellular lipid droplets [37,38]. As such, by phosphorylation or by binding to interaction partners (part of) the HSL and ATGL proteins may not be detected by the applied antibodies, which may result in reduced signals observed via Western blotting. Increased lipolysis and decreased TG content are associated with an elevated basal glycerol release. However, we measured a down-regulation of glycerol release after low glucose CR. This was also observed when SGBS adipocytes were treated with a low (5 μm) RSV concentration [13]. A possible explanation is that reduced intracellular TG stimulates the transcriptional coactivator peroxisome proliferators-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) [39]. Indeed, we observed an induction of PGC-1α, although not significant, in SGBS adipocytes subjected to low-glucose CR. PCG-1α triggers the expression of glycerol kinase (GK) [40]. Up-regulated GK turns glycerol into glycerol-3phosphate, which can be broken down via the glycolysis pathway [41]. We also observed a low-glucose CR-induced up-regulation of SIRT1. There are strong indications that the beneficial effects of CR and RSV are mediated via SIRT1 [34,42,43], despite SIRT1-independent effects of RSV are also reported [44]. Activation of SIRT1 stimulates fat mobilization in WAT by increased lipolysis activity and elevated free fatty acids (FFA) release, which results in reduced TG levels [9,23]. Indeed, RSV [13] as well as low-glucose CR (this study) mediated a significant reduction of TG contents of human adipocytes, which coincided induced SIRT1 expression. It is thus tempting to speculate that beneficial effects of RSV and CR on the adipocyte secretome are (partly) mediated via SIRT1. Interestingly, SIRT1 was also induced when serum from CR-treated obese subjects [45] or CR-treated animals [46] was applied to liver cells, resulting in increased hepatic cell survival. We speculate that CRinduced beneficial alterations of blood adipokine profiles may contribute to this. As shown here, reduced intracellular TG content leads to cell shrinking which induces a structural remodeling of the adipocytes [2]. Reduced cell size urges a decreased expression of cell structure components, which is in line with the down-regulation of most of the

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Fig. 2. Western blot analysis of PCG-1α (A) and SIRT1 (B) in non-CR SGBS adipocytes and in SGBS adipocytes treated 96 h with 0.55 mM glucose (0.55 mM gluc). Twenty-five micrograms of total protein lysates was used. Typical examples of blots are presented. Reported values are means ± SEM of 3 biological replicates run in duplicate and * indicates a difference with p b 0.05.

Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

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collagen isoforms and subunits identified here. However, a CR-induced secretion of all collagen α-2(I) isoforms, various vimentin isoforms and the actin remodeling protein cofilin-1 was also observed. A possible explanation of such a divergent regulation pattern might be that collagen type I isoforms are also part of so-called adiposomes, which are adipocyte-specific exosomal vesicles involved in the secretion of adipokines [47]. Indeed, the majority of the identified proteins in this study, including proteins annotated as intracellular, have been found before in exosomes (see Tables 1, 2 and Supplement 1). As such, enhanced adiposome secretion may increase the amount of collagen type I, and also several intracellular proteins, in the secretion medium. Compared to RSV, low-glucose CR seems a less stress inducing process in relation to intracellular TG reduction. This is reflected by the regulation pattern of structural proteins but also by the fact that most of the stress-associated proteins, including calreticulin, nucleobindin-1, putative heat shock 70 kDa protein 7 and the apoptosis inducing factor galectin-1 [48] were identified within the RSV data set [13]. An explanation for RSV-induced stress-related proteins might be the relative high RSV concentration (75 μM) applied or the in vitro oxidation of RSV which generates H2O2 and H2O2-induced stress responses [49]. However, increased H2O2 levels would result in decreased SIRT levels [50–52], whereas we found in an RSV-induced SIRT1 expression, which indicates a normal response. We found 6 novel adipocyte-secreted proteins that were regulated by low-glucose CR. According to UniProtKB 3-hydroxyisobutyryl-CoA

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Fig. 3. 2-DE gels of adipocyte secreted proteins. A: Representative gels of medium-derived secreted proteins of non-CR SGBS adipocytes (left) and 96 h calorie restricted with 0.55 mM glucose (0.55 mM gluc SGBS adipocytes) (right). B: Master gel with identified spots. Spot numbers refer to ID numbers of Table 1.

hydrolase and ubiquitin carboxyl-terminal hydrolase isozyme L3 are associated with ubiquitin cell regulation, signaling and synthesis processes. cDNA FLJ54471, highly similar to complement C1r subcomponent; cDNA FLJ59142, highly similar to epididymal secretory protein E1; cDNA FLJ60094, highly similar to F-actin capping protein subunit β and HP are proteins of which the functions have not been established. Their similarity to known proteins might be taken as a lead to understand their real biological function. Since CR is known to introduce beneficial effects towards a less inflammatory phenotype as well as an improvement of obesity-associated metabolic disorders [34,53] their function may be sought in this direction. We also found adipocyte-specific secretion proteins with metabolic functions of which apoE, haptoglobin, PEDF, prostaglandin-H2 Disomerase and ZAG were identified in single protein spots while adiponectin levels were determined by a RIA assay. As such their regulation may contribute to a better understanding of low-glucose CRmediated effects. ApoE regulates TG turnover and expression of genes involved in lipid synthesis [54]. In obese mice ApoE is decreased and again increased during CR [55]. In line with an ApoE induction during TG release we observed an RSV-mediated ApoE induction in SGBS adipocytes [13]. However, in this CR study the protein spot containing the full-length ApoE protein was significantly down-regulated. Huang et al. [54,55] showed that CR-induced ApoE expression is associated with increased TG mass, TG syntheses and decreased TG hydrolysis as an adipose tissue and energy homeostasis protection response.

Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444

J. Renes et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

t1:1 t1:2 Q2

7

Table 1 Single protein per spot identifications of secreted proteins from CR and non-CR SGBS adipocytes. Accession number

Protein name

ID

MW [kDa]

Fold change

p-Value

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49 t1:50 t1:51 t1:52 t1:53 t1:54 t1:55 t1:56 t1:57 t1:58 t1:59 t1:60 t1:61 t1:62 t1:63 t1:64 t1:65 t1:66 t1:67 t1:68 t1:69 t1:70 t1:71

Classical secreted Extracellular matrix P02452

Collagen α-1(I) chain

37 37 37 30 2 28 40 40 40 40 40 40 87 53 27

219.3 221.0 251.4 33.4 36.0 196.7 28.7 48.4 206.3 203.2 205.2 199.7 247.8 201.8 308.5

−2.4E + 00 −3.9E + 00 7.9E + 00 −2.9E + 00 −4.4E + 00 −1.3E + 00 6.1E + 04 2.1E + 00 7.6E + 00 5.7E + 00 2.5E + 01 1.3E + 01 −5.7E + 00 −4.8E + 00 −8.7E + 00

9.32E 4.27E 2.87E 1.83E 7.60E 2.25E 1.30E 9.20E 1.95E 5.05E 4.55E 4.82E 2.49E 2.23E 2.67E

Cathepsin L1 Metalloproteinase inhibitor 1

34 74 74

39.7 30.3 29.5

5.3E + 04 2.2E + 04 1.9E + 00

6.62E − 02 8.49E − 03 5.05E − 02

Apolipoprotein E

8 8 6 12 52 52 38 23 23 23 23 23 16 16 3 7 7 7 7

24.6 37.8 32.1 58.1 116.7 117.1 16.7 51.3 49.3 49.5 48.5 46.3 27.1 16.4 9.3 43.2 43.9 42.2 31.7

4.9E + 03 −4.1E + 00 −3.8E + 00 6.7E + 03 −2.0E + 00 −6.4E + 00 −8.8E + 00 −9.8E + 00 −2.5E + 00 −4.5E + 00 −4.0E + 00 1.1E + 01 −1.5E + 01 1.4E + 05 3.4E + 04 −9.3E + 00 −8.4E + 00 −9.0E + 04 9.2E + 04

7.00E 2.62E 2.10E 1.56E 7.80E 9.41E 6.96E 1.62E 6.77E 7.94E 1.04E 3.82E 2.15E 1.91E 7.21E 3.02E 3.92E 9.62E 1.83E

− − − − − − − − − − − − − − − − − − −

07 02 02 02 02 02 02 02 02 03 02 02 02 03 04 02 02 03 02

29 29 29 14 14 14 51

123.0 121.7 123.4 122.2 119.6 111.3 249.6

−4.0E + 00 −4.5E + 00 −2.5E + 00 −3.4E + 00 3.2E + 00 1.5E + 04 8.1E + 00

3.97E 4.59E 3.33E 4.91E 7.69E 7.17E 2.43E

− − − − − − −

02 02 02 02 02 06 03

75 78 72 10 41 90 71 84 57 55 55 55 5 4 4 4 4 4

40.1 56.5 14.8 48.5 26.6 43.2 20.7 21.5 21.6 22.1 17.1 22.1 26.9 17.1 21.1 18.6 22.1 27.2

−5.8E + 00 −4.5E + 00 2.6E + 00 6.4E + 03 1.3E + 00 −5.3E + 04 2.0E + 00 2.1E + 00 −2.3E + 00 3.0E + 04 4.6E + 00 2.0E + 00 2.9E + 00 8.1E + 00 6.7E + 01 1.9E + 00 2.5E + 00 4.9E + 03

4.59E 5.79E 2.50E 7.36E 7.28E 1.22E 7.61E 4.21E 3.74E 2.32E 5.44E 7.95E 5.71E 6.22E 5.32E 3.27E 2.04E 1.99E

− − − − − − − − − − − − − − − − − −

02 02 02 02 02 02 02 02 02 04 02 02 02 02 02 02 02 02

t1:72 t1:73 t1:74

P00738 P36955

Haptoglobin Pigment epithelium-derived factor

P41222

Prostaglandin-H2 D-isomerase

P10599 P25311

Thioredoxin Zinc-α-2-glycoprotein

P09871

E R R

cDNA FLJ54471, highly similar to complement C1r subcomponent*

N C O

Immune regulation B4DPQ0

E

Calumenin (isoform 4) Endoplasmin Gelsolin

C

D6QS48 P14625 P06396

T

Regulation/signaling P02649

D

Complement C1s subcomponent

Protein S100-A9

3-Hydroxyisobutyryl-CoA hydrolase. mitochondrial (isoform 1)* α-Enolase (isoform α-enolase) Cofilin-1 γ-Enolase Heat shock protein β-1 HP protein Phosphatidylethanolamine-binding protein 1 Proteasome subunit β type-5 (isoform 1) Superoxide dismutase [Mn], mitochondrial Transgelin

U

P06702 Non-classical secreted Q6NVY1 P06733 P23528 P09104 P04792 Q6NSB4 P30086 P28074 P04179 Q01995

P15374 P08670

Ubiquitin carboxyl-terminal hydrolase isozyme L3* Vimentin

O

R O

Collagen α-2(IV) chain Collagen α-2(VI) chain Fibronectin

P08572 P12110 P02751 Processing P07711 P01033

P

Collagen α-1(III) chain Collagen α-1(V) chain Collagen α-1(VI) chain Collagen α-2(I) chain

P02461 P20908 P12109 P08123

F

t1:3

− − − − − − − − − − − − − − −

Exosomal

02 02 02 02 02 02 06 02 02 02 02 02 03 02 03

Y

Y Y Y Y

Y Y Y

Y

Y Y Y Y Y Y

Y Y

Y

Y

Y Y Y Y Y Y Y Y (rat) Y

Y (rat) Y

Single protein per spot identifications of secreted proteins from CR and non-CR SGBS adipocytes by 2DE followed by LC–MS/MS. Proteins marked with * are identified as novel adipocyte secreted proteins. ID numbers refers to marked spots of Fig. 2B. MW of the protein spots is defined by PDQuest 8.0 software. Lines marked bolt are significant CR reactive proteins. Proteins previously found in human exosomes (Exocarta database) are indicated with Y or with Y (rat) when identified in rat exosomes.

Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

8

MW [kDa]

fold change

p-Value

15 15 15 15 18 18 18 46 66 80 15 15 15 15 21 31 66 67 1 13 25 44 45 67 80 81 88 89 67 80 81 11

34.7 217.5 248.3 248.8 34.2 31.9 35.0 47.2 243.2 249.8 34.7 217.5 248.3 248.8 50.0 30.8 243.2 254.7 35.3 125.0 74.0 31.0 28.6 254.7 249.8 248.5 200.0 200.6 254.7 249.8 248.5 44.5

−5.4E + 04 −3.4E + 00 7.4E + 03 3.1E + 04 −2.0E + 00 8.2E + 04 −2.8E + 00 4.6E + 00 5.2E + 00 1.1E + 01 −5.4E + 04 −3.4E + 00 7.4E + 03 3.1E + 04 −4.0E + 00 2.4E + 00 5.2E + 00 3.2E + 00 −5.0E + 00 −2.0E + 00 −4.9E + 04 −3.2E + 00 1.4E + 04 3.2E + 00 1.1E + 01 5.8E + 00 9.6E + 02 1.6E + 01 3.2E + 00 1.1E + 01 5.8E + 00 −4.4E + 00

4.45E 9.55E 2.29E 2.27E 5.17E 1.04E 7.07E 9.70E 9.22E 4.70E 4.45E 9.55E 2.29E 2.27E 6.02E 7.85E 9.22E 4.10E 5.13E 1.20E 4.98E 1.05E 2.56E 4.10E 4.70E 9.08E 3.49E 7.42E 4.10E 4.70E 9.08E 1.88E

60 63

35.8 47.6

2.3E + 00 3.9E + 04

5.88E – 02 2.87E – 02

Prostaglandin-H2 D-isomerase Thioredoxin domain-containing protein 5 Zinc-α-2-glycoprotein

70 19 19 31 66 77 25 1 18 18 18 19 19 77 32 9 21 22 46 48 49 66 63 76 42 22 11

13.1 36.3 31.0 30.8 243.2 40.6 74.0 35.3 34.2 31.9 35.0 36.3 31.0 40.6 36.3 46.1 50.0 49.3 47.2 42.2 47.7 243.2 47.6 42.8 27.0 49.3 44.5

8.9E + 00 −3.3E + 00 −5.9E + 00 2.4E + 00 5.2E + 00 2.5E + 00 −4.9E + 04 −5.0E + 00 −2.0E + 00 8.2E + 04 −2.8E + 00 −3.3E + 00 −5.9E + 00 2.5E + 00 −6.6E + 00 −3.3E + 00 −4.0E + 00 −2.7E + 00 4.6E + 00 6.2E + 03 −2.0E + 00 5.2E + 00 3.9E + 04 7.1E + 03 1.4E + 05 −2.7E + 00 −4.4E + 00

9.81E 3.89E 6.42E 7.85E 9.22E 6.01E 4.98E 5.13E 5.17E 1.04E 7.07E 3.89E 6.42E 6.01E 1.31E 2.32E 6.02E 7.38E 9.70E 1.97E 8.42E 9.22E 2.87E 3.47E 8.17E 7.38E 1.88E

Complement C1s subcomponent

13

125.0

−2.0E + 00

1.20E – 03

40S ribosomal protein SA cDNA FLJ60094, highly similar to F-actin capping protein subunit β* α-Enolase (Isoform α-enolase) Destrin Fatty acid binding protein. adipocyte Glutathione S-transferase P Glutathione reductase. mitochondrial

11 44 49 73 68 39 39

44.5 31.0 47.7 14.1 10.1 24.0 61.3

−4.4E + 00 −3.2E + 00 −2.0E + 00 3.9E + 00 6.5E + 04 8.3E + 00 4.3E + 03

1.88E 1.05E 8.42E 5.04E 6.37E 2.72E 5.11E

Collagen α-1(I) chain

(C-term)

(C-term) (C-term) (C-term) (C-term)

Collagen α-1(III) chain

P02461

(C-term)

(Isoform 1) (C-term)

P08123

Collagen α-2(I) chain

(N-term) (C-term) (C-term)

P08572

Collagen α-2(IV) chain

SPARC

T

D3DQH8 Processing Q9UBR2 Q15113 Regulation/signaling B4DV10 Q15848

C

Cathepsin Z Procollagen C-endopeptidase enhancer 1

Angiopoietin-related protein 1

P01019 P02649

Angiotensinogen Apolipoprotein E

Q15782 Q96AJ1 Q12841 P36955

Chitinase-3-like protein 2 Clusterin-associated protein 1 Follistatin-related protein 1 Pigment epithelium-derived factor

O

R

R

O95841

E

cDNA FLJ59142, highly similar to epididymal secretory protein E1* Adiponectin

P05121

P41222 Q8NBS9 P25311 Immune regulation P09871 Non-classical secreted P08865 B4DWA6 P06733 P60981 P15090 P09211 P00390

Plasminogen activator inhibitor 1

P

Collagen α-1(IV) chain Collagen α-1(V) chain

E

P02462 P20908

D

(C-term) (C-term)

F

ID

O

P Protein name

R O

Accession number Classical secreted Extracellular matrix P02452

C

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24 t3:25 t3:26 t3:27 t3:28 t3:29 t3:30 t3:31 t3:32 t3:33 t3:34 t3:35 t3:36 t3:37 t3:38 t3:39 t3:40 t3:41 t3:42 t3:43 t3:44 t3:45 t3:46 t3:47 t3:48 t3:49 t3:50 t3:51 t3:52 t3:53 t3:54 t3:55 t3:56 t3:57 t3:58 t3:59 t3:60 t3:61 t3:62 t3:63 t3:64 t3:65 t3:66 t3:67 t3:68 t3:69 t3:70 t3:71 t3:72 t3:73 t3:74 t3:75 t3:76 t3:77 t3:78

N

t3:3

Table 2 Identifications from non-single protein spots of secreted proteins of CR and non-CR SGBS adipocytes.

U

t3:1 t3:2

J. Renes et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

Exosomal

– 02 – 02 – 03 – 05 – 02 – 02 – 02 – 02 – 02 – 03 – 02 – 02 – 03 – 05 – 02 – 08 – 02 – 02 – 02 – 03 – 02 – 02 – 02 – 02 – 03 – 02 – 02 – 02 − 02 – 03 – 02 – 02

– – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – –

02 02 02 08 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 03 02 02 02 02 02 02 02

02 02 02 02 02 02 02

Y

Y

Y Y

Y

Y

Y Y (rat) Y

Y Y Y

Y

Y

Y Y Y Y Y Y Y Y

Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

J. Renes et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

9

Table 2 (continued) t3:79

Accession number

P Protein name

ID

MW [kDa]

fold change

p-Value

t3:80 t3:81 t3:82 t3:83 t3:84 t3:85 t3:86 t3:87 t3:88

Q6NSB4 P40926 P30044 Q08257 P60174 P08670

HP protein* Malate dehydrogenase, mitochondrial Peroxiredoxin-5, mitochondrial Quinone oxidoreductase Triosephosphate isomerase (isoform 1) Vimentin

35 85 70 85 62 9 11 26 76

44.9 36.6 13.1 36.6 26.2 46.1 44.5 72.7 42.8

−8.7E + 04 3.5E + 01 8.9E + 00 3.5E + 01 1.4E + 04 −3.3E + 00 −4.4E + 00 −6.6E + 01 7.1E + 03

1.75E 1.35E 9.81E 1.35E 1.50E 2.32E 1.88E 6.63E 3.47E

– – – – – – – – –

Exosomal 02 02 02 02 02 02 02 02 02

Y Y Y Y Y Y

Multi-protein per spot identifications of secreted proteins from CR and non-CR SGBS adipocytes by 2DE followed by LC–MS/MS. Proteins with the identical ID number are related to the spots with the same protein identifications and proteins marked with * are identified as novel adipocyte secreted proteins. ID numbers refer to marked spots of Fig. 2B. MW of the protein spots is defined by PDQuest 8.0 software. Lines marked bolt are significant CR reactive spots. Proteins previously found in human exosomes (Exocarta database) are indicated with Y, or with Y (rat) when identified in rat exosomes.

445 446

Similarly, we previously showed an induction of ApoE in mature SGBS adipocytes compared to SGBS preadipocytes [3]. As such, a downregulation of ApoE in parallel to reduced TG levels in SGBS cells after low-glucose CR would be in line with an ApoE-induced energy homeostasis mechanism. Another interesting protein is the adipokine ZAG which is a lipidmobilizing factor in adipose tissue [56] and several types of tumors [57]. ZAG is down-regulated in obesity and is negatively correlated with plasma insulin, C-reactive protein and monocyte chemotactic protein-1 levels, which are all related to obesity-related metabolic disorders [58,59]. Therefore, CR-induced ZAG expression might promote beneficial effects with respect to the metabolic syndrome. Our CR-induced adipocyte secretome data demonstrated that 3 of the 4 identified single protein ZAG spots were down-regulated while the up-regulated ZAG single protein spot was related to a smaller MW compared to the other 3 ZA identifications. Reduced MW is mostly associated with protein modification/fragmentation and/or different isoforms. Different studies of secreted ZAG in relation to obesity revealed inconsistent results. Yeung et al. [60] showed increased circulating ZAG levels while Gong et al. [61] and Selva et al. [62] demonstrated decreased circulating ZAG levels in obesity. These contradictive findings may be due to differentially expressed and secreted ZAG isoforms. Our data indeed showed different secreted ZAG isoforms that differ in pI and molecular weight. In addition, RSV had no effect on ZAG secretion [13]. As such, ZAG needs further investigation to understand its part in obesity and obesity-associated disorders. CR has anti-oxidative [63] and anti-inflammatory [10,64] effects. Several adipokines with metabolic and inflammatory properties were influenced by low-glucose CR: haptoglobin, prostaglandin-H2 D-isomerase, PEDF and adiponectin. Haptoglobin is up-regulated

463 464 465 466 467 468 469 470 471 472 473 474

Fig. 4. Adiponectin levels in secretion medium from non-CR SGBS adipocytes and SGBS adipocytes treated 96 h with 0.55 mM glucose (0.55 mM gluc). Reported values are mean ± SEM of quadruple measurements from 3 biological replicates. * indicates a difference with p b 0.05.

D

P

R O

O

during inflammation and obesity [65]. Here we observed a CRmediated down-regulation of this adipokine. Haptoglobin knockout mice show improved insulin resistance and hepatosteatosis via increased glucose tolerance, insulin sensitivity and increased adiponectin expression [66]. As such, a CR-mediated down-regulation of haptoglobin appears beneficial for obesity-associated metabolic complications. The association of prostaglandin-H2 Disomerase with obesity and metabolic syndrome is still under debate. Elevated levels of prostaglandin-H2 D-isomerase reduce adipogenesis and decrease the risk for glucose intolerance and insulin resistance [67,68]. Here, two isoforms were identified which are significantly up- and down-regulated. This might be linked to lowglucose CR-specific reactions towards an improvement of obesityassociated metabolic status as previously described [67,68]. The adipokines adiponectin and PEDF are related to insulin resistance and a pro-inflammatory status in obese patients [69,70]. As such, a low-glucose-induced adiponectin release and a decreased PEDF release are another argument for the positive impact of low-glucose CR on obesity-associated inflammatory status, insulin resistance, glucose intolerance and WAT dysfunction. A comparison with our previous RSV secretome study [13] revealed that both RSV and low-glucose CR lead to a less inflammatory phenotype and a phenotype reflecting improvement of metabolic complications; even the secretion patterns differ from each other as shown in Fig. 5. RSV treatment resulted in reduced PEDF and PAI-1 and an induced adiponectin and ApoE secretion, which reverse the human adipocyte secretion profile towards an improved inflammatory phenotype and a more insulin-sensitizing pattern [71,72]. Except for the elevated adiponectin and reduced PEDF secretion, CR treatment did not clearly demonstrate such protein regulation pattern. PAI-1 was only identified in multi-protein spots and ApoE was even down-regulated by lowglucose CR. In addition, RSV induced secretion of complement factor D, which was not observed in this study. Complement factor D induces lipolysis and TG release [73] and as such, it may enhance the positive effects of RSV treatment. Taken together, RSV and low-glucose CR induce SIRT1 and have a positive impact on the human adipocyte secretome. Comparison of the RSV and CR data sets indicate that compared to CR RSV treatment induces stronger beneficial effects towards obesity-associated metabolic complications and a relieved inflammatory phenotype. However, the CR-mediated regulation pattern of adiponectin, haptoglobin, PEDF and prostaglandin-H2 D-isomerase as well as the reduced secretion of stress-related proteins also indicates a clear CR-induced positive impact on obesity-associated disorders as was also shown by Varady et al. [10]. Despite the mutual SIRT1 induction differences between CR- and RSV-regulated adipocyte secretomes indicate additional signaling responses and regulation mechanisms. This is also reflected by different isoforms of similar proteins, which have been found regulated in both studies. This finally results in the idea that CR is less forceful than RSV but appears to reach its positive effects with minor cellular stress.

E

461 462

T

459 460

C

457 458

E

455 456

R

453 454

R

451 452

N C O

449 450

U

447 448

F

t3:89 t3:90 t3:91 t3:92

Please cite this article as: J. Renes, et al., Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrolinduced secretome effects, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.023

475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525

10

Table 3 Comparison of single spot identifications from secretome of CR and RSV-treated SGBS adipocytes.

t5:3

Accession number

t5:4 t5:5 t5:6 t5:7 t5:8 t5:9 t5:10 t5:11 t5:12 t5:13 t5:14 t5:15 t5:16 t5:17 t5:18 t5:19 t5:20 t5:21 t5:22 t5:23 t5:24 t5:25 t5:26 t5:27 t5:28 t5:29 t5:30 t5:31 t5:32 t5:33 t5:34 t5:35 t5:36 t5:37 t5:38 t5:39 t5:40 t5:41 t5:42 t5:43 t5:44 t5:45 t5:46 t5:47 t5:48 t5:49 t5:50 t5:51 t5:52 t5:53 t5:54 t5:55 t5:56 t5:57 t5:58 t5:59 t5:60 t5:61

Classical secreted Extracellular matrix P27797 P02452 P02461 P20908 P12109 P08123 P08572 P12110 Q12805 P02751 P11047 Q14112 Regulation/signaling P08253 Q15848 P02649 D6QS48 P14625 P09382 P06396 P00738 Q02818 P36955 P05121 P41222 P30101 P02787 Q8NBS9 P25311 Immune regulation B4DPQ0 P09871 A6XNE2 P06702 Non-classical secreted Q6NVY1 P06733 P09496 P23528 Q13011 P09622 P15090 P09104 P04792 Q6NSB4 P07195 Q06830 P32119 P30086 P28074 P50454 P04179 P00441 Q01995 P15374 P08670

Protein name

This study ID

Calreticulin Collagen α-1(I) chain Collagen α-1(III) chain Collagen α-1(V) chain Collagen α-1(VI) chain Collagen α-2(I) chain Collagen α-2(IV) chain Collagen α-2(VI) chain EGF-containing fibulin-like extracellular matrix protein 1 Fibronectin Laminin subunit γ-1 Nidogen-2

26 57

F

27

R O

8 6 12

E

D

P

52 38

T

cDNA FLJ54471, highly similar to complement C1r subcomponent complement C1s subcomponent Complement factor D Protein S100-A9

C

3-Hydroxyisobutyryl-CoA hydrolase α-Enolase Clathrin light chain A Cofilin-1 Δ(3,5)-Δ(2,4)-Dienoyl-CoA isomerase Dihydrolipoyl dehydrogenase Fatty acid-binding protein γ-Enolase Heat shock protein β-1 HP protein L-lactate dehydrogenase B chain Peroxiredoxin-1 Peroxiredoxin-2 Phosphatidylethanolamine-binding protein 1 Proteasome subunit β type-5 Serpin H1 Superoxide dismutase [Mn] Superoxide dismutase [Cu–Zn] Transgelin Ubiquitin carboxyl-terminal hydrolase isozyme L3 Vimentin

23

RSV study13 ID

2 17 6

37 30 2 28 40 87 53

O

78 kDa glucose-regulated protein Adiponectin Apolipoprotein E Calumenin Endoplasmin Galectin-1 Gelsolin Haptoglobin Nucleobindin-1 Pigment epithelium-derived factor Plasminogen activator inhibitor 1 Prostaglandin-H2 D-isomerase Protein disulfide-isomerase A3 Serotransferrin Thioredoxin Zinc-α-2-glycoprotein

31 12 27 4 3 15 9 8

5 30 14 33 38

16 25 41 3 7 29 14 45 51 75 78 1

E

72

R

46 49 32

O

R

10 41 90

71 84

37 51 19 43 56

C N

t5:1 t5:2

J. Renes et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

57 28 55 5 4

11

Comparison of secretion profiles from CR and RSV treated SGBS adipocytes (see Ref. [13]). All proteins shown are related to single protein spot identifications. Proteins presented in this table also contain different isoforms, which are shown in Tables 1 and 2 for this study and for RSV in Rosenow et al. [13]. ID numbers of this study refer to marked spots in Fig. 3B. For RSV the ID numbers refer to marked spots in Fig. 2 of our previous study [13].

526

5. Conclusion

Acknowledgements

535

527 528

We showed a low-glucose CR-induced adipocyte TG reduction associated with a change of the adipocyte secretion profile, in which 6 novel adipocyte-secreted proteins were discovered. Changes in the adipocytesecretion pattern indicate a positive effect of low-glucose CR with respect to obesity-associated inflammatory phenotype, WAT dysfunction and metabolic status. Compared to RSV the low-glucose CR-induced effect on the adipocyte secretome was associated with a milder cellular stress response.

Prof. Dr. Martin Wabitsch, University Ulm, Germany is kindly acknowledged for providing the SGBS cells. We thank Freek Bouwman and Sonja Kallendrusch, and Jos Stegen, Department Human Biology, Maastricht University, The Netherlands and Eric Royackers, Biomedical Research Institute, Hasselt University, Belgium for their practical support. Dr. Gert Schaart, Department of Movement Sciences, Maastricht University is acknowledged for providing the PCG-1α and SIRT1 antibodies and Dr. Johan Jocken, Department Human Biology, Maastricht

536 537

529 530 531 532 533 534 Q3

U

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Fig. 5. A: Venn diagram showing the comparison of all identified secreted proteins from this CR study and our previous RSV study [13]. B: Venn diagram of the distribution of identified secreted proteins as single spots from this CR study and the previous RSV study.

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