Secreted protein acidic and rich in cysteine (SPARC) regulates thermogenesis in white and brown adipocytes

Molecular and Cellular Endocrinology 506 (2020) 110757

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Secreted protein acidic and rich in cysteine (SPARC) regulates thermogenesis in white and brown adipocytes

T

Sulagna Mukherjeea, Min Ji Choia, Sang Woo Kimb, Jong Won Yuna,∗ a b

Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk, 38453, Republic of Korea Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do, 25601, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Adipocytes Anti-obesity Fat browning SPARC Thermogenesis

SPARC, also known as osteonectin, is well known for its physiological roles in bone formation and tissue remodeling, as well as in cancer pathology; however, evidence regarding its function in adipocytes is lacking. The present study explored the physiological role of SPARC in cultured 3T3-L1 white and HIB1B brown adipocytes of murine cell lines. Treatment of recombinant SPARC upregulated the fat browning marker proteins and genes in white adipocytes and activated brown adipocytes. Conversely, knockdown of Sparc markedly reduced these genes and proteins in both cell lines. In addition, recombinant SPARC inhibited expression of adipogenic and lipogenic proteins but elevated lipolytic and fatty acid oxidation proteins. Furthermore, in silico analysis revealed that SPARC directly interacted and regulated VEGF in adipocytes. In conclusion, SPARC acts as a regulatory protein in both white and brown adipocytes by controlling thermogenesis and is thus regarded as a possible therapeutic target for treatment of obesity.

1. Introduction The biological factors arising from a set of complex interactions are responsible for governing metabolism in body which intervenes with several metabolic disorders (Campbell, 2016; Narciso et al., 2019). SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or BM-40), is one of such factors and has related roles in various pathological conditions including diabetes and cancer (Kos and Wilding, 2010; Harries et al., 2013; Aseer et al., 2017). SPARC belongs to the matricellular family of secreted proteins and although it was initially found to be secreted from bone (Termine et al., 1981), it is expressed in most tissues and is one of the extracellular matrix protein found in adipose tissue (Chavey et al., 2006; Khan et al., 2009; Aseer et al., 2015). SPARC has been found to be ubiquitously expressed, and its expression increases during bone formation, wound healing, and tissue remodeling (Nagaraju and Sharma, 2011). Many loss-of-function studies have been conducted to identify pathophysiological roles of SPARC. These have shown that absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction (Schellings et al., 2009) enhanced carcinogenesis and progression in cancer (Said et al., 2013; Nozaki et al., 2006), reduced ocular neovascularization (Thomas et al., 2015), promoted macrophage activation and phagocytosis in brain tumors (Atorrasagasti et al., 2013), attenuated liver fibrinogenesis (Bradshaw et al., 2002), ∗

and accelerated cutaneous wound closure (Nie et al., 2008). Moreover, our recent study indicated that SPARC deficiency alleviates superoxidemediated oxidative stress, apoptosis, and autophagy in diabetogenic hepatocytes (Aseer et al., 2017). SPARC has recently attracted increased interest because of its proposed roles in obesity. SPARC inhibits adipogenesis in vitro (Nie et al., 2008) and in vivo (Nie and Sage, 2009) by inhibiting mitotic clonal expansion of preadipocytes at an early stage of adipogenesis (Nagaraju and Sharma, 2011; Nie et al., 2011). However, SPARC expression was upregulated in adipose tissues of obese mice and in 3T3-L1 fibroblasts during adipocyte differentiation (Tartare-Deckert et al., 2001; Shen et al., 2014). Consistent with these results, plasma SPARC levels showed a positive correlation with body mass index (BMI) in humans, while plasma SPARC concentrations were significantly elevated in age- and BMI-matched subjects with coronary artery disease (Takahashi et al., 2001). Elevated plasma SPARC levels have also been shown to be associated with insulin resistance, dyslipidemia, and inflammation in gestational diabetes mellitus (Xu et al., 2013). Earlier reports demonstrated that SPARC expression in human adipose tissue was correlated with fat mass and was higher in subcutaneous adipose tissue (Kos et al., 2009). With regard to obesity, absence of SPARC was found to increase adiposity without inducing a significant difference in overall body weight in mice fed a normal diet (Bradshaw et al., 2002). However, when fed a high fat diet, SPARC-null mice exhibited significantly

Corresponding author. Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk 712-714, Republic of Korea. E-mail address: [email protected] (J.W. Yun).

https://doi.org/10.1016/j.mce.2020.110757 Received 23 November 2019; Received in revised form 7 February 2020; Accepted 8 February 2020 Available online 10 February 2020 0303-7207/ © 2020 Elsevier B.V. All rights reserved.

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FGF21 fibroblast growth factor 21 HSL hormone-sensitive lipase Lhx8 gene encoding LIM/homeobox protein Lhx8 PGC-1α/Ppargc1α peroxisome proliferator-activated receptor gamma co-activator 1-alpha/encoding gene PPAR peroxisome proliferator-activated receptor PRDM16/Prdm16 PR domain-containing 16/encoding gene SPARC secreted protein acidic and rich in cysteine SREBP-1c sterol regulatory element-binding transcription factor 1 Tbx1 gene encoding T-box protein 1 Tmem26 gene encoding transmembrane protein 26 UCP1/Ucp1 uncoupling protein 1/encoding gene VEGF vascular endothelial growth factor Zic1 gene encoding zinc finger protein ZIC1

Abbreviations ACC acyl-CoA carboxylase ACOX peroxisomal acyl-coenzyme A oxidase AMPK AMP-activated protein kinase ATGL adipose triglyceride lipase Cidea gene encoding cell death-inducing DFFA-like effector a Cited1 gene encoding Cbp/p300-interacting transactivator 1 Cd137 tumor necrosis factor receptor superfamily member 9 C/EBP/Cebp CCAAT/enhancer-binding protein/encoding gene CPT1 carnitine palmitoyltransferase 1 CYT-C cytochrome C Eva1 gene encoding myelin protein zero-like 2 FAS fatty acid synthase

2.2. Quantitative real-time PCR

greater weight gain relative to their wild type counterparts because of increased accumulation of adipose tissue (Nie et al., 2011). Suppression of white adipose tissue (WAT) expansion and activation of brown adipose tissue (BAT) have recently been considered as potential therapeutic targets for treatment of obesity (Lee et al., 2014; Bargut et al., 2019). In particular, recent advances have been made to utilize BAT function to increase oxidative metabolism at the expense of fat storage, thereby dissipating energy as heat (Calderon-Dominguez et al., 2016). Induction of the brown fat phenotype in WAT (browning or beiging) represents another promising strategy for the treatment of obesity, and numerous phytochemicals with fat browning function have been reported (Azhar et al., 2016; Jang et al., 2018; Silvester et al., 2019). Fat browning is not only induced by phytochemicals, but also by cold exposure, exercise, β-adrenergic stimulation, several hormones (e.g. irisin, FGF-21, follistatin, β-aminoisobutyric acid, myostatin, leptin or meteorin-like) as well as PPARγ agonists (Rodríguez et al., 2017). Transition from white to beige adipocytes involves transcriptional regulation of multiple brown fat-associated gene products (Harms and Seale, 2013). Apart from classical regulators such as PRDM16, PPARγ, and PGC-1α (Lo and Sun, 2013), numerous transcriptional modulators responsible for browning have recently been identified (Muller, 2016). Previous studies investigating elevated SPARC expression by introduction of recombinant SPARC technology into cellular models (Lee et al., 2013) have revealed functional relationships between SPARC and AMPK/PGC-1α-activated mitochondrial biogenesis and energy metabolism (Haber et al., 2008). These results led us to hypothesize that SPARC may be linked to regulation of thermogenesis in adipocytes. Therefore, we report here for the first time an unidentified function of SPARC demonstrating that SPARC promotes browning in white adipocytes and activates brown adipocytes, thereby stimulating thermogenesis.

Total RNA was isolated from mature cells (4–8 days) using a total RNA isolation kit (RNA-spin, Intron Biotechnology, Seongnam, Korea). The isolated RNA (1 μg) was then converted to cDNA using Maxime RT premix (Intron Biotechnology). Power SYBR green (Roche Diagnostics Gmbh, Mannheim, Germany) was employed to quantitatively determine transcription levels of genes with RT-PCR (Stratagene 246 mix 3000p QPCR System, Agilent Technologies, Santa Clara, CA, USA). PCR reactions were run in duplicate for each sample and samples were normalized against β-actin. Sequences of primer sets used in this study are listed in Supplementary Table 1. 2.3. Silencing of Sparc by siRNA Commercially available siRNA specific for Sparc (a pool of three target-specific 25 nucleotides of siRNA designed to knock down gene expression) from Invitrogen was used for gene silencing in 3T3-L1 and HIB1B cells (Supplementary Table 2). Briefly, post confluent 3T3-L1 cells in six-well culture dishes were washed twice with transfection medium overlaid by a previously prepared mixture of siRNA and transfection reagent (Roche). The transfection process was continued for 4–6 h, at which time the differentiation medium was added and kept for 48 h, followed by addition of maturation media, which was maintained for 72 h. Matured cells were collected for further experiments. 2.4. Treatment of recombinant SPARC Recombinant mouse SPARC protein (rSPARC) with a His tag with 96% purity of SPARC was purchased from Sino Biological Inc. (Wayne, PA, USA). 3T3-L1 and HIB1B preadipocytes were seeded at 1.5 × 105 cells/well in 6-well plates after trypsinization and grown to 70% and 100% confluency, respectively, for 1 or 2 days, then treated with 200 μM of rSPARC with or without differentiation media for 48 h followed by maturation media for 72 h in both 3T3-L1 and HIB1B cells. Transfection efficiency was maintained at > 50% reduction in expression of SPARC for both protein and gene.

2. Materials and methods 2.1. Cell culture and differentiation 3T3-L1 and HIB1B preadipocytes of murine cell lines were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM, Thermo, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories, Pashing, Austria) and 100 μg/ml of penicillinstreptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO2 incubator. Confluence was maintained in differentiation induction medium consisting of 1.7 mM insulin (Sigma, St. Louis, MO, USA), 0.25 μM dexamethasone (Sigma), and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma) in DMEM for 48 h, followed by maturation in DMEM containing 10% FBS and 1.7 mM insulin for 72 h.

2.5. Oil Red O staining Preadipocytes of both 3T3-L1 and HIB1B were seeded in a 6-well plate and allowed to reach 100% confluency. Cells were then treated with either rSPARC or Sparc siRNA in differentiation and maturation media. After 4–6 days of treatment, cells were washed with phosphatebuffered saline (PBS), fixed with 10% formalin for 1 h at room temperature, and then washed again three times with deionized water. A mixture of Oil Red O (ORO) solution (0.6% Oil Red O dye in isopropanol) and water at a ratio of 6:4 was layered onto cells for 20 min, after which they were washed four times with deionized water. 2

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Fig. 1. rSPARC treatment stimulates browning of white adipocytes and activates brown adipocytes. Exogenous SPARC induction by rSPARC treatment (A) consequently increases brown fat marker proteins in a dose-dependent manner (B), as well as beige-specific genes (C) in 3T3-L1 white adipocytes. rSPARC treatment elevates SPARC induction (D) and brown fat marker proteins in a dose-dependent manner (E), as well as brown fat-specific genes (F) in HIB1B brown adipocytes. Data are presented as the mean ± S.D., and differences between groups were determined by ANOVA followed by Tukey's post-hoc tests using the Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS Inc., Chicago, IL, USA) program. Significance between control and rSPARC is indicated as *p < 0.05 or **p < 0.01.

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Fig. 2. Sparc deficiency attenuates browning in white adipocytes and decreases thermogenic activity of brown adipocytes. Specific knockdown of Sparc in 3T3-L1 (A) and HIB1B adipocytes (B) significantly reduces expression of brown fat marker proteins in 3T3-L1 cells (C) and in HIB1B cells (D). Sparc-deficient cells attenuate expression levels of beige-specific genes in 3T3-L1 (E) and brown fat-specific genes in HIB1B cells (F). Data are presented as the mean ± S.D., and differences between groups were determined by ANOVA followed by Tukey's post-hoc tests using the Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS Inc., Chicago, IL, USA) program. Significance between control and Sparc siRNA-treated cells is indicated as *p < 0.05 or **p < 0.01.

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Fig. 3. SPARC inhibits adipogenesis in 3T3-L1 and HIB1B adipocytes. rSPARC treatment reduces adipogenic marker proteins (C/EBPα and PPARγ), whereas Sparc deficiency enhances expression of the adipogenic markers in 3T3-L1 cells (A). Representative images of Oil Red O staining of 3T3-L1 were taken at 20 × magnification (scale bars = 50 μm) and 40 × magnification (scale bars = 100 μm). The lipid content was quantified by extracting Oil Red O stain bound to cells with 100% isopropanol in 3T3-L1 adipocytes (B). Abundance of SPARC decreases adipogenic markers proteins, whereas Sparc deficiency enhances the expression of the adipogenic markers in HIB1B cells (C). Representative images of Oil Red O staining of HIB1B were taken at 20 × magnification (scale bars = 50 μm) and 40 × magnification (scale bars = 100 μm). The lipid content was quantified by extracting Oil Red O stain bound to cells with 100% isopropanol in HIB1B adipocytes (D). Data are presented as the mean ± S.D., and differences between groups were determined by ANOVA followed by student's t-tests using the Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS Inc., Chicago, IL, USA) program. Significance between control and rSPARC as well as control and Sparc siRNAtreated cells is indicated as *p < 0.05 or **p < 0.01, respectively.

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org).

Intracellular lipid accumulation was quantified by ORO staining. Briefly, the stained lipid droplets were visualized using an inverted microscope and the intracellular lipid content was quantified from standard expression of lipid droplets on an absorbance at 520 nm.

2.9. Statistical analysis All data are presented as the means ± SD of at least three independent experiments. Statistical significance among multiple groups was determined by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test or two-tailed Student's t-test using the Statistical Package of Social Science (SPSS) software version 17.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was indicated as either p < 0.05 or p < 0.01.

2.6. Immunoblot analysis Cell lysates were prepared by homogenization in RIPA buffer (Sigma) followed by centrifugation at 13,000×g for 30 min. Cell extracts were then diluted in 5 × sample buffer (50 mM Tris at pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.1% bromophenol blue) and heated at 95 °C for 5 min before 8, 10, or 12% SDS-polyacrylamide gel electrophoresis (PAGE). Following electrophoresis, samples were transferred onto a polyvinylidene difluoride membrane (PVDF, ATTO Technology, Amherst, NY, USA) and then blocked for 1 h with TBS-T (10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) containing 5% skim milk (Sigma) or BSA (Rocky Mountain Biologicals, Missoula, MT, USA). The membrane was subsequently rinsed three times consecutively with TBS-T buffer followed by incubation at room temperature for 1 h with 1:1000 diluted primary polyclonal antibodies, including anti-ATGL, anti-ACC, anti-pACC, anti-β-actin, anti-PPARγ, anti-AMPK, anti-pAMPK, anti-UCP1, anti-PGC-1α, anti-CPT1, antiACOX1, anti-C/EBPα, anti-FAS, anti-FGF21, anti-SPARC, anti-COX4, anti-CytC, anti-p53 (Santa Cruz Biotechnology), anti-PRDM16, antipHSL, and anti-VEGFA (Abcam, Cambridge, UK) in TBS-T buffer containing 1% skim milk or BSA. After three washes, the membrane was incubated with horseradish peroxidase-conjugated anti-goat IgG, antirabbit IgG or anti-mouse IgG secondary antibody (1:1000, Santa Cruz Biotechnology) in TBS-T buffer containing 1% skim milk or BSA at room temperature for 1 h. Next, immunoblots were developed with enhanced chemiluminescence and captured using an ImageQuant LAS500 system (GE Healthcare Life Sciences, Marlborough, MA, USA). Band intensities were quantified with the ImageJ software (NIH, Bethesda, MD, USA).

3. Results 3.1. rSPARC treatment stimulates browning of white adipocytes and activates brown adipocytes After confirmation of feasible penetration of rSPARC into the 3T3L1 white adipocytes during differentiation (Suppl. Fig. 1), we investigated corresponding expression levels of cellular SPARC and core brown fat-specific markers (PGC-1α, PRDM16, and UCP1) as well as beige-specific genes by exogenous rSPARC induction in 3T3-L1 cells. rSPARC treatment led to dose-dependent increases in protein levels of SPARC (Fig. 1A) and core brown fat marker proteins (Fig. 1B). Additionally, beige-specific genes were upregulated upon rSPARC induction in 3T3-L1 cells (Fig. 1C). Similarly, differentiating HIB1B brown adipocytes were treated with rSPARC and the expression levels of brown fat-specific proteins and genes were determined. Exogenous rSPARC treatment elevated cellular levels of SPARC (Fig. 1D) as well as brown fat marker proteins (Fig. 1E) and genes in HIB1B cells (Fig. 1F). These results demonstrate that SPARC participates in the induction of white fat browning and activation of brown adipocytes, at least at the cellular levels. 3.2. Sparc deficiency attenuates browning in white adipocytes and decreases thermogenic activity of brown adipocytes

2.7. Immunofluorescence

To investigate the effects of Sparc deficiency on thermogenic activity in both adipocytes, specific knockdown of Sparc was conducted with an efficiency of greater than 60% in 3T3-L1 (Fig. 2A) and HIB1B adipocytes (Fig. 2B) using commercially available siRNA. Sparc deficiency gave rise to a significant reduction in brown-fat marker proteins (PGC-1α, PRDM16, and UCP1) in 3T3-L1 cells (Fig. 2C) and HIB1B cells (Fig. 2D). In addition, Sparc deficiency led to decreased expression of brown and beige-specific genes (Cidea, Pppargc1a, Prdm16, Tbx1, Tmem26, and Ucp1) in 3T3-L1 adipocytes (Fig. 2E), as well as decreased expression of brown-fat signature genes, including Eva, Lhx8, Ucp1, and Zic in HIB1B brown adipocytes (Fig. 2F). These data suggest that Sparc is an important gene that stimulates the thermogenic program in both adipocytes and modulates the browning process in white adipocytes.

Immunocytochemistry was performed on formaldehyde (4%)-fixed cells. These cells were incubated with anti-SPARC and anti-UCP1 (dilution 1:1000, Santa Cruz Biotechnology) primary antibody at 4 °C overnight followed by incubation with appropriate FITC goat antimouse secondary antibody at room temperature for 4 h. For staining of mitochondria, MitoTracker®Red (1 mM, Cell Signaling Technology) was directly added to PBB-T (PBS + 1% BSA and 0.1% Tween 20) at a concentration of 200 nM. Cells were then incubated at 37 °C for 2 h. After incubation, tissues were washed with PBS and subjected to immunostaining. Morphological findings were observed using a light microscope at X20 magnification. 2.8. In silico analysis Computational analysis was performed using STRING (version 11.0), GeneMania (3.5.1 version), and PrePPI (1.2.0 version, Honig Lab) softwares. STRING is a functional enrichment analysis tool used for identification of protein-protein interaction networks, and has the data sources from the ELIXIR core data resources and the results are integrated based on the confidence score calculated from all the interactions of all the proteins (https://string-db.org/). GeneMANIA is generally used for finding genes related to a set of input genes and uses a very large set of functional association data collected from BioGRID and PathwayCommons database (https://genemania.org/). PrePPI is the database of predicted and experimentally determined protein-protein interactions (PPI) and the predictions are determined by the Bayesian framework combining the evolutionary, structural, functional, and expression information (http://honig.c2b2.columbia.edu/preppi). The data for PrePPI is retrieved from Uniprot database (www.uniprot.

3.3. SPARC inhibits adipogenesis and lipogenesis in 3T3-L1 and HIB1B adipocytes Next, we investigated the effects of SPARC on other lipid metabolisms related to the thermogenic program. rSPARC treatment markedly decreased the expression levels of two transcription factors that are essential for adipogenesis (C/EBPα and PPARγ), whereas Sparc deficiency significantly increased their expression levels (Fig. 3A). These findings were confirmed by the lipid accumulation measured by Oil Red O staining (Fig. 3B). Moreover, the patterns observed in the HIB1B cells were identical to those in the 3T3-L1 adipocytes (Fig. 3C and D). Taken together, these data indicate that lipid droplet formation in adipocytes after the maturation stage was blocked in the abundant presence of SPARC, but effectively elevated in the absence of SPARC. To elucidate the role of SPARC in lipid synthesis in both adipocytes, 6

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SPARC.

we measured the expression levels of the major lipogenic markers in the abundant presence and deficiency of Sparc in both cells. Induction of exogenous rSPARC significantly suppressed major lipogenic markers such as ACC, FAS, and SREBP-1c in 3T3-L1 adipocytes. Conversely, a lack of Sparc led to increased fatty acid synthesis mediated by attenuated AMPK activation (Fig. 4A). Similar results were observed in HIB1B adipocytes (Fig. 4B), suggesting a possible anti-obesity role of

3.4. SPARC regulates lipid catabolism We further clarified the role of SPARC in lipid catabolism by determining the expression levels of key markers responsible for lipid breakdown and oxidation. The major lipolytic markers, ATGL and the

Fig. 4. Negative regulation of SPARC for lipid synthesis in white and brown adipocytes. Cells with high levels of rSPARC regulate expression of lipogenic marker proteins by reducing ACC, AMPK, FAS, and SREBP-1c while increasing the expression of p-ACC and p-AMPK in 3T3-L1 white adipocytes (A), as well as in HIB1B brown adipocytes (B). Data are presented as the mean ± S.D., and differences between groups were determined by ANOVA followed by student's t-tests using the Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS Inc., Chicago, IL, USA) program. Significance between control and rSPARC as well as control and Sparc siRNA-treated cells is indicated as *p < 0.05 or **p < 0.01, respectively. 7

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CPT1) was highly enhanced in the abundance of rSPARC, suggesting that SPARC has the ability to augment fat oxidation (Fig. 5C and D). However, the deficiency of Sparc failed to elevate the fat oxidation markers in both cells (Fig. 5C and D), indicating that it plays an

phosphorylated form of HSL, showed a drastic increase in expression when treated with rSPARC, whereas the knockdown of Sparc resulted in reduced levels of those marker proteins in both cells (Fig. 5A and B). Moreover, the expression of the fat oxidation markers (ACOX and

Fig. 5. SPARC regulates lipid catabolism in white and brown adipocytes. Expression of the lipolytic markers ATGL and p-HSL increases upon treatment with rSPARC, whereas Sparc deficiency leads to reduced expression in those proteins in both white and brown adipocytes (A). Abundant presence of SPARC increases expression of the fatty acid oxidation markers (ACOX and CPT1), whereas lack of SPARC results in their reduced expression (B). Data are presented as the mean ± S.D., and differences between groups were determined by ANOVA followed by student's t-tests using the Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS Inc., Chicago, IL, USA) program. Significance between control and rSPARC as well as control and Sparc siRNA-treated cells is indicated as *p < 0.05 or **p < 0.01, respectively. 8

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cells showed higher fluorescence intensity than Sparc-deficient cells (Fig. 6C) as well as greater UCP1 activity (Fig. 6D). This indicated increased mitochondrial biogenesis and UCP1-mediated thermogenesis.

important regulatory role in lipid catabolism in both white and brown adipocytes, at least at the cellular level. 3.5. SPARC promotes mitochondrial biogenesis

3.6. SPARC interacts and regulates VEGF-A expression in white and brown adipocytes

Induction of rSPARC promoted mitochondrial biogenesis of 3T3-L1 white adipocytes as a result of beiging of the white fat cells. Fig. 6A shows increased expression of mitochondrial marker proteins (COX4, CYT-C, and p53) in the presence of rSPARC, while deficiency of Sparc caused reverted results. Simultaneously, rSPARC elevated mitochondrial biogenic genes (Cox4, Nrf1, and Tfam) in 3T3-L1 cells, whereas Sparc deficiency reduced the gene expressions (Fig. 6B). Immunofluorescence images highlighted comparison between control cells, Sparc siRNA-treated cells and rSPARC-induced cells. rSPARC induced

In order to identify a novel partner protein of SPARC, we considered using the bioinformatics tools to predict linking proteins. First, we determined the protein-protein interaction with the PrePPI software to identify all the interacting pair of proteins associated with SPARC and selected VEGF protein with an SM score (or LR score based on both local structural similarity and a conserved interface) of 1.8 (Supplementary Fig. 2A) and then clarified the results with another

Fig. 6. rSPARC promotes mitochondrial biogenesis in 3T3-L1 white adipocytes. Induction of rSPARC elevated mitochondrial biogenic marker proteins while the Sparc deficiency downregulated the marker proteins (A). rSPARC also increased expression mitochondrial biogenic genes, whereas the absence of Sparc reduced the gene expressions (B). Representative images of immunocytochemistry with X20 magnification for comparison between control cells, Sparc deficient cells (KD) and rSPARC-induced cells displaying intensities for SPARC (C) and UCP1 (D) in 3T3-L1 white adipocytes. Data are presented as the means ± S.D., and differences between groups were determined by ANOVA followed by Tukey's post-hoc tests using the Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS Inc., Chicago, IL, USA) program. Significance between control and rSPARC is indicated as *p < 0.05 or **p < 0.01. 9

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(Nie and Sage, 2009). Although there is accumulating evidence of enhanced SPARC expression in obese human and animal model (Takahashi et al., 2001; Lee et al., 2014), there is an apparent contrast in the role of SPARC in relation to energy state. Previous reports showed that SPARC expression amplified in presence of surplus energy such as overfeeding in mice but reduced upon bariatric surgery or with low calorie diet (Kos et al., 2009), while we observed elevated thermogenic activity upon induction of SPARC. The maintenance of body weight in the presence of higher fat accumulation was attributed to a loss of connective tissue and osteopenia in SPARC-null mice fed a normal diet (Bradshaw and Sage, 2001). However, according to other reports, depletion of Sparc results in increased adiposity, but does not lead to an overall difference in body weight in mice fed a normal diet; however, mice fed a high fat diet showed increased body weight (Bradshaw and Sage, 2001). Additionally, SPARC-null bone marrow cells showed an increased tendency to differentiate into adipocytes rather than osteoblasts (Delany and Hankenson, 2009; Nie and Sage, 2009). Similarly, these findings supported our results of increased adipogenic effect upon depletion of Sparc in white and brown

computational tool GeneMania which also demonstrated subsequent network associating SPARC with VEGF as strong interacting partners (Supplementary Fig. 2B). Secondly, we verified these predicted results with STRING and build a network for SPARC and other associated proteins involved in adipocyte functions (Fig. 7A), which displayed a direct interaction with VEGF and its other isoforms (Vegfa, b and c) and the GO assimilation also depicted the connection of SPARC with VEGF signaling pathway (Fig. 7B) when compared with the other proteins. Finally, we checked the expression levels of VEGFA in vitro, after exogenous induction of rSPARC in 3T3-L1 and HIB1B cells the protein content of VEGFA increased significantly, while the absence of Sparc upon silencing drastically reduced the expression of VEGFA (Fig. 7C and D). These data support the regulatory effect of SPARC on VEGF in 3T3-L1 white as well as HIB1B brown adipocytes. 4. Discussion In the present study, we explored the unidentified functions of SPARC based on its physiological roles in adipocyte biology and obesity

Fig. 7. SPARC interacts with VEGF and regulates its expression in white and brown adipocytes. In silico analysis of protein-protein interaction using STRING shows direct interaction of VEGF with SPARC (A), as well as GO annotation displayed association of SPARC with all the isoforms of VEGF (a, b, c) signaling pathway (B). In vitro rSPARC induction increases protein content of angiogenic marker VEGFA, whereas lack of SPARC results in decreased level of VEGFA in 3T3-L1 white adipocytes (C) and in HIB1B brown adipocytes (D). Data are presented as the mean ± S.D., and differences between groups were determined by ANOVA followed by student's t-tests using the Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS Inc., Chicago, IL, USA) program. Significance between control and rSPARC as well as control and Sparc siRNA-treated cells is indicated as *p < 0.05 or **p < 0.01, respectively. 10

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exerts both angiogenic and antiangiogenic effects by switching on angiogenic properties (Jendraschak and Sage, 1996) that are tissue specific (Aseer et al., 2015). Angiogenesis is essential for hyperplasia of brown adipocytes (Tseng and Kolonmin, 2016) and SPARC inhibits angiogenesis in gastric cancer (Zhang et al., 2012) and in neuroblastoma tumors (Chlenski et al., 2002) via suppression of vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) expression, resulting in anti-cancer activity. In contrast, SPARC has potential angiogenic properties in adipocytes and has ability to modulate the expression of VEGF and matrix metalloproteinases (MMPs) (Nagaraju and Sharma, 2011; Zhang et al., 2012). An earlier study cross-linked the proteins SPARC and VEGF interactions by molecular docking aimed to identify inhibitors of VEGF-induced angiogenesis (Chandrasekaran et al., 2007) corroborates with the results of the present study. Our data supported that SPARC has interaction with VEGF and abundance of SPARC increases the expression of VEFG in both white and brown adipocytes, whereas lack of SPARC decreases its expression. However, this study is limited to in vitro analysis only and further studies are required to determine the co-expression of SPARC with VEGF and its functional role in animal models. Many conflicting studies have been considered in relation to functional role of SPARC, among which one study reported SPARC as a main inducer of adipose tissue fibrosis by influencing TGF beta which limits abdominal adipose tissue expansion (Kos et al., 2009). However, another previous study reported SPARC as an important target for reduction of fibrosis (Trombetta-eSilva and Bradshaw, 2012). Moreover, the absence of SPARC leads to disruption of the extracellular matrix structure and decreased collagen content, which makes a more permissive environment for adipocyte expansion (Bradshaw et al., 2003). In regard to cancer, SPARC has controversial reports with few studies suggesting its role in progression of tumor and metastasis (Arnold and Brekken, 2009), and also aggravating factors for breast cancer (Zhu et al., 2016). While, opposing results indicated SPARC as an inhibitor of pancreatic cancer cells (Viloria et al., 2016), as well as suppression of colon tumorigenesis (Aoi et al., 2013). Our study focused on the positive role of SPARC in regulating both white and brown adipocytes. In conclusion, we report that SPARC downregulates adipogenesis and lipogenesis while promotes browning. Thus, SPARC can be regarded as a possible therapeutic target for treatment of obesity via controlling thermogenesis in adipocytes; however, further studies are required to directly test this hypothesis in animal models.

adipocytes. Earlier studies also reported that absence of Sparc was found to enhance diet-induced weight gain with increased size of adipocytes and bone, as well as to decrease the levels of collagen that serve as a partial barrier to enlargement of adipocytes (Nie et al., 2011). In contrast, depletion of Sparc leads to significant increases in subcutaneous fat accumulation, and thus could prevent accumulation of fat in visceral and ectopic fat depots, thereby decreasing the risk of insulin resistance (Yang et al., 2007; Nie and Sage, 2009). Results in the current study demonstrated that induction of exogenous rSPARC actively inhibits adipogenesis, which is similar to other studies with reports of SPARC blocking adipogenesis by inhibition of the adipogenic transcription factors C/EBPα and PPARγ in the later stages of adipocyte differentiation through the Wnt/β catenin pathway (Nie and Sage, 2009; Nagaraju and Sharma, 2011). Although, the difference for the mechanism of action of SPARC may vary due to the various proteolytic isoforms of SPARC (C-SPARC and N-SPARC) which induces the adipose stromal mobilization of cells in obesity (Tseng and Kolonmin, 2016). Taken together, the available data indicate that SPARC has different functions in adipogenesis and development of obesity. A current trend in study of biological mechanisms not only involves studying the downregulation of molecules but also focuses on overexpression by induction of exogenous molecules in vitro. Haber et al. (2008) previously reported elevated expression of SPARC by treatment of rSPARC into cellular models, which revealed functional relationships between SPARC and AMPK-PGC-1α that modulate mitochondrial biogenesis and support energy metabolism (Melouane et al., 2018). We discovered an important contribution of SPARC by induction of rSPARC in the adipocytes which led to browning of white to begie fat cells, in response to elevated thermogenic activity by the fat cells. Adaptive thermogenesis, which is a result of browning, has proven to be a critical process for expenditure of energy which could ultimately lead to reduction of body weight (Wu et al., 2013; Jang et al., 2018). Previous studies demonstrated that browning of white fat cells led to weight loss of obese animal models and improved energy expenditure (Bartlet and Heeren, 2013; Jeremic et al., 2017). Our observation of browning induced by rSPARC could be a possible therapy for body weight reduction, although the findings must be clarified using animal models. However, few studies have also reported certain adverse effects of browning as well including cachexia, hepatic steatosis, and immune suppression (Abdullahi and Jeschke, 2017; Tamucci et al., 2018), which is a limitation in this study as we have not yet examined these after treatment of rSPARC in adipocyets. Transition from white to beige adipocytes involves tight transcriptional regulation of multiple brown fat-associated gene products (Weiner et al., 2017). Apart from classical regulators such as PRDM16, PPARγ and PGC-1α (Lo and Sun, 2013), numerous transcriptional modulators responsible for fat browning have recently been identified (Muller, 2016). Another key factor regulating the thermogenic program determined in adipose tissues includes UCP1 protein (Porter, 2017; Fedorenko et al., 2012), and we have observed upregulation of UCP1 by rSPARC in both white and brown adipocytes. In line with our results, the study by Yan et al. (2006) showed less expression of SPARC in dormant BAT for ground squirrels and suggested role of SPARC in enhanced thermogenesis at low temperatures in BAT. Oppositely, Jespersen et al. (2019) found that SPARC is dominantly expressed in human BAT than in subcutaneous adipose tissue, which is different to rodent BAT, and reported higher adrenergic reactions in the absence of SPARC. However, SPARC has many controversial studies with a vast set of data and varying results making it a very important and interesting protein to further study. Moreover, transition of white to brown-like adipocytes (beige adipocyte) might be accompanied by switching on of an angiogenic phenotype (Seki et al., 2018). The high thermogenic activity of brown adipocytes requires a particularly high rate of blood perfusion to supply oxygen and substrates and to export heat (Seki et al., 2016). SPARC

Author contributions SM and MC carried out experiments, KSW performed in silico analysis and JWY has designed the study and wrote manuscript.

Ethics statement The cellular in vitro models used in this study were commercially available. We did not use any human or animal samples and therefore did not require approval from the Ethics Committee.

Declaration of competing interest The authors declare that they have no conflicts of interest associated with this study.

Acknowledgments This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIT) (No. 2019R1A2C2002163). 11

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Appendix A. Supplementary data

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