Cryptotanshinone promotes commitment to the brown adipocyte lineage and mitochondrial biogenesis in C3H10T1/2 mesenchymal stem cells via AMPK and p38-MAPK signaling.

Although white adipose tissue (WAT) stores triglycerides and contributes to obesity, brown adipose tissue (BAT) dissipates energy as heat. Therefore, browning of WAT is regarded as an attractive way to counteract obesity. Our previous studies have revealed that treatment with cryptotanshinone (CT) during adipogenesis of 3T3-L1 cells inhibits their differentiation. Here, we found that pretreatment of C3H10T1/2 mesenchymal stem cells with CT before exposure to adipogenic hormonal stimuli promotes the commitment of these mesenchymal stem cells to the adipocyte lineage as confirmed by increased triglyceride accumulation. Furthermore, CT treatment induced the expression of early B-cell factor 2 (Ebf2) and bone morphogenetic protein 7 (Bmp7), which are known to drive differentiation of C3H10T1/2 mesenchymal stem cells toward preadipocytes and to the commitment to brown adipocytes. Consequently, CT treatment yielded brown-adipocyte-like features as evidenced by elevated expression of brown-fat signature genes including Ucp1, Prdm16, Pgc-1α, Cidea, Zic1, and beige-cell-specific genes such as CD137, Hspb7, Cox2, and Tmem26. Additionally, CT treatment induced mitochondrial biogenesis through upregulation of Sirt1, Tfam, Nrf1, and Cox7a and increased mitochondrial mass and DNA content. Our data also showed that cotreatment with CT and BMP4 was more effective at activating brown-adipocyte-specific genes. Mechanistic experiments revealed that treatment with CT activated AMPKα and p38-MAPK via their phosphorylation: the two major signaling pathways regulating energy metabolism. Thus, these findings suggest that CT is a candidate therapeutic agent against obesity working via activation of browning and mitochondrial biogenesis in C3H10T1/2 mesenchymal stem cells.

Wnt/ß-catenin signaling plays a distinct role in methyl gallate-mediated inhibition of adipogenesis.

The canonical Wnt/ß-catenin signaling not only features in many developmental processes but also recently emerged as an attractive negative regulator of differentiation of preadipocytes into adipocytes. Here, we show that ß-catenin signaling plays a distinct role in methyl gallate (MG)-mediated inhibition of 3T3-L1 adipocytes differentiation. We found that the expression of ß-catenin decreased after adipogenic hormonal induction, whereas incubation of the differentiating cells with a physiological concentration of MG during adipogenic hormonal induction significantly prevented ß-catenin degradation by activating Wnt signaling components such as Wnt1, Wnt10b, Fzd1, Fzd2, Lrp5, Lrp6, Dvl1, and Dvl2. Mechanistic experiments revealed that MG treatment during early adipocytic differentiation specifically inhibited degradation of ß-catenin caused by phosphorylation at serine-33. In addition, MG treatment led to phosphorylation of GSK3ß, which is one of ß-catenin-degrading enzymes. Consequently, MG treatment facilitated translocation of the stabilized ß-catenin from the cytoplasm to nucleus, and activates its target genes cyclin D1 and c-Myc. Furthermore, MG-induced stabilization of ß-catenin suppresses PPARγ expression. Moreover, pharmacological activation or inhibition of ß-catenin signaling during adipocytes differentiation decreased and increased, respectively, the level of the key adipogenic marker, PPARγ, and of its downstream targets, aP2 and adiponectin while MG treatment effectively reversed their expression level. Collectively, our data suggest that MG is a novel pharmacological stimulator of canonical Wnt/ß-catenin signaling, and therefore represents a promising therapeutic agent in obesity.

Methyl gallate, a potent antioxidant inhibits mouse and human adipocyte differentiation and oxidative stress in adipocytes through impairment of mitotic clonal expansion.

Methyl gallate (MG) is a derivative of gallic acid and a potent antioxidant. In this study, we confirmed that MG treatment effectively inhibits lipid accumulation, which occurred mostly in the early stages of adipogenesis. We also showed that shortly after adipogenic induction, MG facilitated a G0/G1 cell cycle arrest. Mechanistic studies revealed that MG treatment inhibited ERK1/2 phosphorylation, which is a key regulator of the G1- to S-phase transition. Furthermore, MG treatment prevented the adipogenic hormonal stimuli-induced inhibition of the cyclin-dependent kinase inhibitor p27Kip1 . This led to inhibition of the transcription factor E2F1 by preventing the phosphorylation of, and thereby activation of its destruction partner RB. MG treatment also downregulated factors that are upstream of RB-E2F1 signaling such as Cdk2, Cyclin E, Cdk4, and Cyclin D1 where Cyclin D3 level was unaffected. We also found that MG treatment markedly decreased the expression and phosphorylation of C/EBPß, by phosphorylating, and therefore inactivating, GSK3ß, which is a prerequisite for its DNA binding capacity, and thereby mitotic clonal expansion (MCE). Ultimately, MG treatment downregulates key terminal adipogenic transcription factors including C/EBPα, PPARγ, aP2 (Fabp4), and adiponectin. Moreover, MG also protects adipocytes from oxidative stress by alleviating intracellular reactive oxygen species and activating Nrf2, HO-1, and PRDX3. Thus, this study provides a mechanistic insight into the anti-adipogenic actions of MG. © 2016 BioFactors, 42(6):716-726, 2016.

Delphinidin, a major anthocyanin, inhibits 3T3-L1 pre-adipocyte differentiation through activation of Wnt/ß-catenin signaling.

Delphinidin (Del) is a major anthocyanin that is widely found in pigmented fruits and vegetables. Herein, we investigated the antiadipogenic effect and the molecular mechanism by which Del affects 3T3-L1 preadipocyte differentiation. We found that Del effectively reduced intracellular lipid accumulation and promoted lipolysis while regulating the expression of adipogenic transcription factors and their target genes. This lipid lowering effect of Del was largely limited to the early phase of adipogenesis, which is governed by the delayed cell cycle progression due to G1 cell cycle arrest. Subsequently, Del suppressed the expression of early adipogenic transcription factors, such as C/EBPß and C/EBPδ, as well as that of intermediate markers, C/EBPα, PPARγ, and the PPARγ-target adipocyte markers adiponectin and aP2 (FABP4). Furthermore, Del treatment activated Wnt1, Wnt10b, the Wnt receptor Fzd2, and the coreceptors Lrp5/6, while it inactivated Gsk3ß, a member of the ß-catenin destruction complex. Moreover, Del treatment stabilized cytoplasmic ß-catenin levels and promoted its nuclear translocation, with subsequent activation of the expression of its downstream target genes, c-Myc and cyclin D1. Our findings therefore suggest that Del can effectively inhibit adipogenesis followed by the stabilization of ß-catenin during early 3T3-L1 differentiation via inhibition of its destructive complexes and activation of Wnt signaling, and may thus be a promising candidate for the prevention of metabolic diseases, including obesity.

Cytoprotective Effect of Makgeolli Lees on Paraquat Induced Oxidative Stress in A549 Cells via Activation of NRF2 and Antioxidant Genes.

Makgeolli lees (ML) has several physiological effects such as antioxidant, antidiabetic, and anticancer properties, but its biological functions have not been determined definitively. Here, we tested whether ML has a cytoprotective effect on paraquat (PQ)-induced oxidative stress in the human lung carcinoma cell line A549. At 0.1 mg/ml ML, viability of PQ-exposed A549 cells was restored by 12.4%, 18.5%, and 48.6% after 24, 48, and 72 h, respectively. ML also reduced production of the intracellular reactive oxygen species (ROS) that were generated by PQ treatment. Further experiments revealed that ML treatment enhanced the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2) as well as ARE-GFP reporter activity. ML treatment also effectively increased the expression of NRF2's target genes NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase 1 (HO-1). Moreover, we found that expression of cytoprotective genes, including glutathione peroxidases (GPXs), superoxide dismutase (SOD1), catalase (CAT), peroxiredoxin 3 (PRDX3), and peroxiredoxin 4 (PRDX4), was greatly enhanced by treatment with ML during PQ exposure. Taken together, the data suggest that treatment of PQ-exposed A549 cells with ML ameliorates cytotoxicity through induction of NRF2 expression and its target genes HO-1, NQO1, and other antioxidant genes. Thus, ML may serve as a functional food applicable to ROS-mediated human diseases.

Cryptotanshinone, a compound of Salvia miltiorrhiza inhibits pre-adipocytes differentiation by regulation of adipogenesis-related genes expression via STAT3 signaling.

BACKGROUND: Cryptotanshinone (CT), a major tanshinone found in Salvia miltiorrhiza Bunge (Lamiaceae), has various pharmacological effects such as antitumor, anti-inflammatory, and antioxidant properties. Despite its well-documented benefits in a wide range of diseases, the effect of CT on adipocyte differentiation has not been well characterized. PURPOSE: The present study was designed to determine the in vitro anti-adipogenic effect and underlying molecular mechanisms of CT using 3T3-L1 murine pre-adipocytes. METHODS: We measured the levels of intracellular triglyceride accumulation and mRNA and protein expression of key adipogenic transcription factors and their target genes. RESULTS: Treatment with CT drastically reduced lipid accumulation in a dose- and time-dependent manner. Molecular assays showed that CT effectively suppressed the expression of C/EBPß, C/EBPα, and PPARγ and of their target adipocyte-specific genes aP2, adiponectin, and GLUT4 but activated the expression of anti-adipogenic genes such as GATA2, CHOP10, and TNF-α. CT treatment also inhibited the phosphorylation of STAT3 in the early phase of adipogenesis. A small-interfering-RNA-mediated knock-down of STAT3 potentiated the anti-adipogenic effect of CT. CONCLUSION: Taken together, the results suggest that CT may be a good anti-adipogenic candidate because it regulates STAT3 during early adipogenesis.