Sinapic acid prevents adipogenesis by regulating transcription factors and exerts an anti-ROS effect by modifying the intracellular anti-oxidant system in 3T3-L1 adipocytes

Document Type : Original Article

Authors

Department of Biomedical Sciences, Faculty of Biomedical Sciences and Technology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai – 600116, Tamil Nadu

10.22038/ijbms.2022.62590.13847

Abstract

Objective(s): In this study, we tested the hypothesis that sinapic acid (SA), a naturally occurring hydroxycinnamic acid found in vegetables, cereal grains, and oilseed crops with various biological activities suppresses adipogenesis in 3T3-L1 adipocytes by down-regulating adipogenesis transcription factor. 
Materials and Methods: 3T3-L1 adipocytes were treated with SA and evaluated by Oil Red O staining, triglyceride estimation, lipolysis, and reverse transcription-polymerase chain reaction. 3T3-L1 adipocytes were treated with various concentrations of SA (100 to 1000 μmol) during differentiation. 
Results: SA prevented an increase in adipocytes by reducing preadipocyte clonal expansion. ORO staining analyses revealed that SA reduced cytoplasmic lipid droplet accumulation in 3T3-L1 by 57% at the highest concentration of 1000 μmol without affecting cell viability. Furthermore, SA down-regulated the expression of peroxisome proliferator-activated receptor-gamma, CCAAT/enhancer-binding protein alpha, sterol regulatory element-binding protein 1c, and fatty acid synthase. ROS generated during adipogenesis was also attenuated by SA treatment by increasing antioxidant enzymes superoxide dismutase, catalase, and the cellular antioxidant glutathione. SA demonstrated no in vivo toxicity in the Drosophila melanogaster model. 
Conclusion: These results suggest that SA exerts anti-oxidant and anti-adipogenic effects and could be used as a functional nutraceutical ingredient in combatting obesity-related diseases.

Keywords


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    Several phytochemicals are known to inhibit the anti-adipogenic effect through their anti-oxidant activity [46]. Herein, the in vitro antioxidant activity of SA was demonstrated through DPPH, ABTS, NO, and OH radical scavenging activity (Table 2). The effective antioxidant property of SA is due to its aromatic phenolic ring (Figure 1) that delocalizes and stabilizes unpaired electrons within its ring structure, thereby acting as free-radical scavengers [47]. Increase in the number of methoxy substitutions in positions ortho to the OH in monophenols like SA increases greatly and enhances the electron-donating properties in the 4- or 4’-position [48]. The other hydroxycinnamic acids, such as p-coumaric, caffeic, and ferulic are also known to be excellent antioxidants. The antioxidant activity of SA in 3T3-L1 adipocytes was examined to further determine its antioxidant ability. Intense adipogenesis in obesity is strongly correlated with oxidative stress which leads to production of ROS [49]. ROS in adipocytes is generated by NOX4 during adipogenesis causing insulin resistance and cell damage [50]. NOX4 is especially expressed in adipocytes and acts as a switch between proliferation and differentiation of adipocytes [51]. Adipogenesis leads to low levels of endogenous antioxidant enzymes such as SOD, CAT, and GSH [27]. Therefore, inhibiting ROS production in adipocytes can be a potential target for improving obesity. SA effectively reduced ROS levels during adipogenesis (Figures 6A & B) compared with DC and NAC. This could be due to the effective antioxidant ability of SA. As shown in Figure 7A, SA increased GSH, SOD, and CAT in a concentration-dependent manner compared with DC. Previous studies have shown that phytocompounds like resveratrol [52]but long chain polymers produce flocs by a bridging mechanism which overcomes electrostatic repulsions. Quantitative relationships are developed between the optimum concentration of flocculant and the rates of flocculation, subsidence, and particularly rate of filtration through the filter cake. INTRODUCTION”,”author”:[{“dropping-particle”:””,”family”:”Vigilanza”,”given”:”Paola”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Aquilano”,”given”:”Katia”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Baldelli”,”given”:”Sara”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Rotilio”,”given”:”Giuseppe”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Ciriolo”,”given”:”Maria Rosa”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””}],”container-title”:”Journal of Cellular Physiology”,”id”:”ITEM-1”,”issue”:”8”,”issued”:{“date-parts”:[[“2011”]]},”page”:”2016-2024”,”title”:”Modulation of intracellular glutathione affects adipogenesis in 3T3-L1 cells”,”type”:”article-journal”,”volume”:”226”},”uris”:[“http://www.mendeley.com/documents/?uuid=b9e67033-f913-454a-abe4-dc367c1f53ec”]}],”mendeley”:{“formattedCitation”:”[57]”,”plainTextFormattedCitation”:”[57]”,”previouslyFormattedCitation”:”[59]”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}, esculetin [53], and dibenzoylmethane [54] reduce ROS levels in murine adipocytes. To gain further insight into the mechanism of SA-mediated ROS inhibition, the mRNA expression levels of NOX4, the major pro-oxidant in 3T3-L1 adipocyte was studied (Figure 6C). The NOX4 mRNA level of NAC-treated adipocytes was greatly increased whereas a decrease was observed in cells treated with 1000 µmol SA. Inhibition of ROS generation in the NBT and DCF-DA assay is not completely reflected by the NOX4 gene. This suggests a much more complex ROS regulation mechanism with involvement with other genes that control ROS production in adipocytes. Therefore, SA can inhibit adipogenesis-induced intracellular ROS production through its antioxidant activity and by up-regulating the cellular antioxidant defense mechanisms. 
    The fruit fly, D. melanogaster is a reliable model for assessment of toxicity of food or chemical structures [55]to mwh single spots. Recording of the frequency and the size of the different spots allows for a quantitative determination of the mutagenic and recombinogenic effects. This and earlier studies with a small set of well‐known mutagens indicate that the test detects monofunctional and polyfunctional alkylating agents (ethyl methanesulfonate, diepoxybutane, mitomycin C, Trenimon and an alternative method to the use of the animal model [56,57]000 chemicals in commercial use today, and the approximately 2000 new chemicals introduced each year, makes development of sensitive and rapid assays to screen for neurotoxicity paramount. In this article I advocate the use of Drosophila in the modern regimen of toxicological testing, emphasizing its unique attributes for assaying neurodevelopment and behavior. Features of the Drosophila model are reviewed and a generalized overall scheme for its use in toxicology is presented. Examples of where the fly has proven fruitful in evaluating common toxicants in our environment are also highlighted. Attention is drawn to three areas where development and application of the fly model might benefit toxicology the most: 1. Since the eukaryotic genome of Drosophila has more than 80% homology with disease-related loci in humans, the results obtained are highly specific and translational [58]which we have summarized by disease category. This breakdown into disease classes creates a picture of disease genes that are amenable to study using Drosophila as the model organism. Of the 548 Drosophila genes related to human disease genes, 153 are associated with known mutant alleles and 56 more are tagged by P-element insertions in or near the gene. Examples of how to use the database to identify Drosophila genes related to human disease genes are presented. We anticipate that cross-genomic analysis of human disease genes using the power of Drosophila second-site modifier screens will promote interaction between human and Drosophila research groups, accelerating the understanding of the pathogenesis of human genetic disease. The Homophila database is available at http://homophila.sdsc.edu.”,”author”:[{“dropping-particle”:””,”family”:”Reiter”,”given”:”L T”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Potocki”,”given”:”L”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Chien”,”given”:”S”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Gribskov”,”given”:”M”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Bier”,”given”:”E”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””}],”container-title”:”Genome research”,”id”:”ITEM-1”,”issue”:”6”,”issued”:{“date-parts”:[[“2001”,”6”]]},”language”:”eng”,”page”:”1114-1125”,”title”:”A systematic analysis of human disease-associated gene sequences in Drosophila  melanogaster.”,”type”:”article-journal”,”volume”:”11”},”uris”:[“http://www.mendeley.com/documents/?uuid=cd425ca8-976c-44b0-9ce4-73a9f0abb63b”]}],”mendeley”:{“formattedCitation”:”[63]”,”plainTextFormattedCitation”:”[63]”,”previouslyFormattedCitation”:”[63]”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}. DNA shearing and fragmentation are indicative of the first-line toxicity of a compound [59]. In the present study, SA did not show any DNA shearing or fragmentation in both parental and F1 generations. Whereas, DNA smearing was observed with EMS treatment, which is indicative of activation of caspase-3 [60]. This suggests that SA is non-toxic to the eukaryotic genome during long-term application of the compound.  
Conclusion
We present in vitro the anti-adipogenic potential of SA by reducing the preadipocyte clonal population and inhibiting adipocyte differentiation by down-regulating adipogenic transcription factors and lipogenesis. SA showed improved lipolysis and cellular antioxidants thereby preventing intracellular ROS accumulation. Also, SA was a non-toxic Drosophila model on long-term exposure. These results suggested that SA could be used as a possible candidate for development of clinically effective anti-obesity agents. 
Acknowledgments
The results presented in this paper were part of a doctoral thesis. This work was carried out in the Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai - 600116. It was partly supported by the NPV Ramasamy Udayar PhD fellowship grant, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai – 600116, Tamil Nadu, India.
Authors’ contributions
SA provided study conception or design, prepared the draft manuscript, and helped with visualization. CMJ helped with data processing and collection and performed experiments. CMJ and SA prepared the draft manuscript and helped with visualization. CMJ analyzed and interpreted the results . CMJ and SA critically revised or edited the article. SA approved the final version to be published. SA supervised, and helped with funding acquisition.

Declaration of Interest Statement
The authors declare no conflicts of interest.
References
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Rosen ED, MacDougald O. Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol. 2006. 7:885–96. 
Moseti D, Regassa A, Kim WK. Molecular regulation of adipogenesis and potential anti-adipogenic bioactive molecules. Int J Mol Sci. 2016; 17:1–24. 
He Y, Li Y, Zhao T, Wang Y, Sun C. Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK Pathway. PLoS One. 2013; 8:e70135. 
Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis  in the liver. J Clin Invest. 2002; 109:1125–31. 
Bolsoni-Lopes A, Isabel Alonso-Vale MC, Isabel Alonso Vale MC. Lipolysis and lipases in white adipose tissue – An update. Arch Endocrinol Metab. 2015; 59:344–51. 
Villiers D De, Potgieter M, Ambele MA, Adam L, Durandt C, Pepper MS. The Role of Reactive Oxygen Species in Adipogenic Differentiation. Advs Exp Med Biol. 2017; 1083:125-144.
Nićiforović N, Abramovič H. Sinapic acid and its derivatives: Natural sources and bioactivity. Compr Rev Food Sci Food Saf. 2014; 13:34–51. 
Chen C. Sinapic acid and its derivatives as medicine in oxidative stress-induced diseases and aging. Oxid Med Cell Longev. 2016; 3571614.
Melo TS De, Lima PR, Carvalho KMMB, Fontenele TM, Solon FRN, Tomé AR. Ferulic acid lowers body weight and visceral fat accumulation via modulation of enzymatic , hormonal and in fl ammatory changes in a mouse model of high-fat diet-induced obesity. Braz J Med Biol Res. 2017; 50:1–8. 
Lutfi E, Babin PJ, Gutie J. Caffeic acid and hydroxytyrosol have anti- obesogenic properties in zebrafish and rainbow trout models. PLoS One 2017; 12:e0178833.
Ilavenil S, Kim da H, Srigopalram S, Arasu MV, Lee KD, Lee JC, et al. Potential Application of p -Coumaric Acid on Differentiation of C2C12 Skeletal Muscle and 3T3-L1. Molecules. 2016; 21:997.
Ansari MA, Raish M, Ahmad A, Ahmad SF, Mudassar S, Mohsin K, et al. Sinapic acid mitigates gentamicin-induced nephrotoxicity and associated oxidative/nitrosative stress, apoptosis, and inflammation in rats. Life Sci. 2016; 165:1–8. 
Kanchana G, Shyni WJ, Rajadurai M, Periasamy R. Evaluation of Antihyperglycemic Effect of Sinapic Acid in Normal and Streptozotocin-Induced Diabetes in Albino Rats. Glob J Pharacology. 2011; 5:33–9.
Karri S, Sharma S, Hatware K, Patil K. Natural anti-obesity agents and their therapeutic role in management of obesity : A future trend perspective. Biomed Pharmacother. 2019; 110:224–38. 
Wang S, Moustaid-moussa N, Chen L, Mo H, Shastri A, Su R, et al. Novel insights of dietary polyphenols and obesity. J Nutr Biochem. 2014; 25:1–18. 
Blois MS. Antioxidant Determinations by the Use of a Stable Free Radical. Nature. 1958; 181:1199–200. 
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999; 26:1231–7. 
Halliwell B, Gutteridge JM, Cross CE. Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med. 1992; 119:598–620.
Govindarajan R, Rastogi S, Vijayakumar M, Shirwaikar A, Rawat AKS, Mehrotra S, et al. Studies on the antioxidant activities of Desmodium gangeticum. Biol Pharm Bull. 2003; 26:1424–7.
Green H, Kehinde O. An established preadipose cell line and its differentiation in culture II. Factors affecting the adipose conversion. Cell. 1975; 5:19–27. 
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65:55–63.
Jiang L, Zhang N, Mo W, Wan R, Ma C, Gu Y, et al. Rehmannia inhibits adipocyte differentiation and adipogenesis. Biochem Biophys Res Commun. 2008; 371:185–90. 
Ramirez-Zacarias JL, Castro-Munozledo F, Kuri-Harcuch W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry. 1992; 97:493–7.
Furukawa S, Fujita T, Shumabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004; 114(12):1752–61. 
Koh EJ, Kim KJ, Choi J, Jeon HJ, Seo MJ, Lee BY. Ginsenoside Rg1 suppresses early stage of adipocyte development via activation of C/EBP homologous protein-10 in 3T3-L1 and attenuates fat accumulation in high fat diet-induced obese zebrafish. J Ginseng Res. 2017; 41:23–30. 
Moron MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase  activities in rat lung and liver. Biochim Biophys Acta. 1979; 582(1):67–78. 
Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys. 1984; 21(2):130–2.
Sinha AK. Calorimetric Assay of Catalase. Anal Biochem. 1972; 47(2):389–94.
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