Caffeine inhibits adipogenesis through modulation of mitotic clonal expansion and the AKT/GSK3 pathway in 3T3-L1 adipocytes Adipogenesis;AKT;Caffeine;Glycogen synthase kinase;Mitotic clonal expansion; Caffeine has been proposed to have several beneficial effects on obesity and its related metabolic diseases; however, how caffeine affects adipocyte differentiation has not been elucidated. In this study, we demonstrated that caffeine suppressed 3T3-L1 adipocyte differentiation and inhibited the expression of CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ, two main adipogenic transcription factors. Anti-adipogenic markers, such as preadipocyte secreted factor (Pref)-1 and Krüppel-like factor 2, remained to be expressed in the presence of caffeine. Furthermore, 3T3-L1 cells failed to undergo typical mitotic clonal expansion in the presence of caffeine. Investigation of hormonal signaling revealed that caffeine inhibited the activation of AKT and glycogen synthase kinase (GSK) 3 in a dose-dependent manner, but not extracellular signal-regulated kinase (ERK). Our data show that caffeine is an anti-adipogenic bioactive compound involved in the modulation of mitotic clonal expansion during adipocyte differentiation through the AKT/GSK3 pathway.

Pancreatic cancer is characterized by a poor prognosis, with its five-year survival rate lower than that of any other cancer type. Gemcitabine, a standard treatment for pancreatic cancer, often has poor outcomes for patients as a result of chemoresistance. Therefore, novel therapeutic targets must be identified to overcome gemcitabine resistance. Here, we found that SLC38A5, a glutamine transporter, is more highly overexpressed in gemcitabine-resistant patients than in gemcitabine-sensitive patients. Furthermore, the deletion of SLC38A5 decreased the proliferation and migration of gemcitabine-resistant PDAC cells. We also found that the inhibition of SLC38A5 triggered the ferroptosis signaling pathway via RNA sequencing. Also, silencing SLC38A5 induced mitochondrial dysfunction and reduced glutamine uptake and glutathione (GSH) levels, and downregulated the expressions of GSH-related genes NRF2 and GPX4. The blockade of glutamine uptake negatively modulated the mTOR-SREBP1-SCD1 signaling pathway. Therefore, suppression of SLC38A5 triggers ferroptosis via two pathways that regulate lipid ROS levels. Similarly, we observed that knockdown of SLC38A5 restored gemcitabine sensitivity by hindering tumor growth and metastasis in the orthotopic mouse model. Altogether, our results demonstrate that SLC38A5 could be a novel target to overcome gemcitabine resistance in PDAC therapy.

Mesenchymal stem cells have the capacity to give rise to multiple cell types, such as adipocytes, osteoblasts, chondrocytes, and myocytes. However, the molecular events responsible for the lineage specification and differentiation of mesenchymal stem cells remain unclear. Using gene expression profile studies, we determined that Scavenger receptor class A, member 5 (SCARA5) is a novel mediator of adipocyte commitment. SCARA5 was expressed at a higher level in committed A33 preadipocyte cells compared to C3H10T1/2 pluripotent stem cells. Gain- and loss-of-function studies likewise revealed that SCARA5 acts as a mediator of adipocyte commitment and differentiation in both A33 and C3H10T1/2 cells. RNAi-mediated knockdown of SCARA5 in A33 cells markedly inhibited the adipogenic potential, whereas overexpression of SCARA5 enhanced adipocyte differentiation in C3H10T1/2 cells. We also demonstrated that the focal adhesion kinase (FAK) and ERK signaling pathways is associated with the SCARA5-mediated response, thereby modulating adipocyte lineage commitment and adipocyte differentiation. Additionally, glucocorticoids induced the expression of SCARA5 in differentiating adipocytes through glucocorticoids response elements (GRE) in the SCARA5 promoter. Taken together, our study demonstrates that SCARA5 is a positive regulator in adipocyte lineage commitment and early adipogenesis in mesenchymal stem cells.

Significance: Glucocorticoids are well known to play a major role in obesity, but underlying mechanisms have been obscure. We demonstrate that the small G protein Dexras1, first identified based on its dramatic induction by glucocorticoids, mediates adipogenic differentiation of preadipocytes, as well as diet-induced obesity in intact rodents. Thus, the adipogenesis of preadipocytes is abolished by Dexras1 deletion and selectively induced by Dexras1 expression. Relevance to intact animals is evident from our experiments wherein diet-induced obesity is prevented in mice with knockout of Dexras1. Thus, pharmacotherapy involving Dexras1 may afford a promising approach to the therapy of obesity.

KLF8 (Krüppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPARγ2 and C/EBPα promoter activity, suggesting that KLF8 is an upstream regulator of PPARγ and C/EBPα. The KLF8 binding sites were localized by site mutation analysis to −191 region in C/EBPα promoter and −303 region in PPARγ promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis.