Gene and Protein Profiling of Effects of Tart Cherry Anthocyanins in Preadipocytes

Abstract

Several dietary bioactive compounds possess anti‐inflammatory and anti‐obesity properties and could potentially reduce obesity‐associated cardiovascular diseases, diabetes and other metabolic inflammatory diseases. We are specifically interested in tart cherry anthocyanins (ACY) and understanding mechanisms mediating their beneficial effects in metabolic diseases. Hence, we hypothesized that tart cherry ACY reduce obesity‐associated inflammation through direct anti‐inflammatory and lipid metabolizing effects in adipocytes. We first analyzed major ACY in tart cherry by Liquid Chromatography Mass Spectrometry (LC‐MS) and identified cyanidin‐3‐O‐glucoside (C3G), cyanidin‐3‐O‐rutinoside (C3R), cyanidin as well as the polyphenol p‐Coumaric acid with C3G being the most abundant ACY in tart cherry. To further gain insight into global effects of tart cherry ACY on adipocyte metabolism, we conducted RNA sequencing (using Illumina Hi‐Seq platform) and proteomics (using LC‐MS). We used 3T3‐L1 preadipocytes and treated them for 4hrs with culture media supplemented with or without tart cherry extracts (~18ug ACY/ml). Cells are then treated for 18hrs with media containing 200ng/ml Lipopolysaccharide (LPS) to stimulate inflammation in the absence or continuous presence of the tart cherry extracts. Media were used to determine changes in secreted adipokines by Enzyme‐Linked Immunosorbent Assay (ELISA). Tart cherry significantly reduced LPS‐induced secretion of the inflammatory cytokine (Interleukin‐6, IL‐6). Whole genome mRNA sequencing identified several genes differentially regulated by tart cherry ACY. Among 36,919 expressed Mus musculus genes, 2359 genes were identified as differentially expressed at the fold change of >2 at p≤0.05 and 95% confidence level. Of these, 14 genes were up regulated and 2345 genes were down regulated by tart cherry ACY. Using Ingenuity Pathway Analysis (IPA®), we observed predicted inhibition of inflammatory, immune and oxidative stress‐related pathways and genes by tart cherry. Signaling pathways regulated by tart cherry included Protein Kinase, TNFR2 and B cell activating factor signaling. Differentially expressed genes by tart cherry included Casp14, 14‐3‐3, c‐Fos, and c‐Jun. Using proteomics, we identified 62 differentially regulated proteins. Among these, inflammatory, oxidative stress and apoptosis‐related proteins were significantly down‐regulated by tart cherry at p≤0.05 (e.g. Nrf2, Gstp1, Asns, and Hsp90α). In conclusion, our results demonstrate that tart cherry ACY reduce expression of preadipocyte genes, proteins and pathways that are primarily associated with inflammation and oxidative stress. Global gene and protein profiling approaches in preadipocytes and other adipose cells may help identify novel pathways that may potentially serve as new targets for obesity and inflammation.