Abstract
Depletion of protein phosphatase-1 catalytic subunit beta (PPP1CB), a serine/threonine protein phosphatase and potent adipogenic activator, suppresses the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Therefore, PPP1CB is considered as a potential therapeutic target for obesity. We screened 1033 natural products for PPP1CB inhibitors and identified chebulinic acid, which is abundantly present in the seeds of Euphoria longana and fruits of Terminalia chebula. Chebulinic acid strongly inhibited the hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate by PPP1CB (IC50 = 300 nM) and demonstrated potent antiadipogenic effects in 3T3-L1 preadipocytes in a concentration-dependent manner. Additional studies have demonstrated that chebulinic acid suppresses early differentiation by downregulating key transcription factors that control adipogenesis in 3T3-L1 cells. These results suggested that chebulinic acid may be a potential therapeutic agent for treating obesity by inhibiting PPP1CB activity.
Highlights
Obesity is a global health problem [1]
To find the optimal affinity tag for the purification and enzymatic assays of protein phosphatase 1 catalytic subunit (PPP1CB), we utilized maltose-binding protein (MBP), glutathione-S-transferase (GST), and hexahistidine (6xHis) tags. Both GST- and MBP-tagged PPP1CBs were purified in a single step (Figure S2A,B)
For determining kinetic constants, purified PPP1CB was added to the reaction buffer containing various concentrations of difluoro-4-methylumbelliferyl phosphate (DiFMUP)
Summary
Obesity is a global health problem [1]. A World Health Organization report (2020) showed that 13% and 39% of adults aged 18 years and above are obese and overweight, respectively [2]. A previous study by our team found that PPP1CB upregulated adipogenesis by modulating the early adipogenic process [19] Considering all these facts, the identification of PPP1CB inhibitors is a good strategy to find potent therapeutic medicines for obesity. Our team has been conducting research to discover natural products with inhibitory effects on protein phosphatases to ameliorate metabolic diseases, and to reveal the efficacy of natural products using cell-based assays. Chebulinic acid was selected as a PPP1CB inhibitor (IC50 = 300 nM) in screening experiments and showed anti-adipogenic effects in 3T3-L1 adipocytes in a concentrationdependent manner. Chebulinic acid mostly affected the early stages of differentiation and downregulated key transcriptional factors that regulate adipogenesis in 3T3-L1 adipocytes. These results indicate that chebulinic acid is a potential therapeutic agent for obesity
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