Adipogenesis, lipolysis, and extracellular matrix remodeling are early modulated by a triterpene-enriched natural extract in a mouse cell line and primary human adipocytes

Chronic Inflammation has a key role in the development of insulin resistance and type 2 diabetes. Previously, we demonstrated that OBE100, a natural extract from the leaves of Eucalyptus tereticornis, has anti-inflammatory properties. Three pentacyclic triterpenoids, ursolic acid, oleanolic acid, and ursolic acid lactone are the major compounds present in OBE100. These molecules have shown multiple biological activities. In this study we analyzed how the compounds of OBE100 modify adipocyte gene expression using RNA sequencing. Triterpenoids regulate the inflammatory program in differentiated adipocytes, inhibiting the expression of many cytokines, chemokines, and inflammatory mediators. However, the OBE100 extract has a more powerful immunomodulatory effect than the triterpene mixture increasing the number of genes regulated, both in mouse and human models. Our study shows that OBE100 is a promising extract for the treatment of diabetes that can break the link between inflammation and insulin resistance. Overall design: 3T3-L1(CL-173TM) mouse pre-adipocytes were purchased from ATCC (Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 2mM glutamine, 1% penicillin/streptomycin, and 25 mM glucose (Growth Medium 2 – GM2) at 37°C and 5% CO2. Differentiation was induced two days postconfluence by adding GM2 containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 µM dexamethasone, 2 µM Rosiglitazone, and 1 µg/ml insulin. After two days of incubation, the medium was replaced with GM2 containing 1 µg/ml insulin. Two days later, the medium was replaced by GM2 and incubated for 24 hours with the different treatments. Human primary adipocyte culture was obtained from the abdominal fat of young women. Pre-adipocytes were induced into adipocytes after 100% confluence cultured in a differentiation medium (DMEM 25 mM glucose, 10% FBS, 0.5 mM IBMX, 5 µM dexamethasone, 10 µM insulin, and 2 µM Rosiglitazone) for 7 days. The cells were incubated with DMEM, 25 mM glucose, 10% SFB, and different treatments for another 24 hours.Crude extract OBE100, M1, UA, OA, and UAL were reconstituted in dimethyl sulfoxide (DMSO) at 25 mg/ml (stock solution). The final concentration of the compounds in the culture medium was OBE100 50 µg/ml, M1 39 µg/ml (UA+OA+UAL mix), UA 23.8 µg/ml, OA 7 µg/ml, and UAL 8.2 µg/ml. The concentration used for the three different triterpenes, both in the mixture called M1 and individually, were those determined in the fraction called OBE100. Three independent experiments were performed, each in triplicate. We then performed RNA seq profiling using data obtained from RNA-seq. We compared differentiated adipocytes againts each control (OBE100, M1, UA, OA, UAL) and we also included an adipocyte without fat as control, to verify that the differentation was successfull