EFFECTS OF GMG-43AC ON ADIPOCYTE DIFFERENTIATION IN THE CULTURE OF 3T3-L1 CELLS

In recent decades, obesity has become a prominent health problem in many countries. It is closely linked to cardiovascular disease, type 2 diabetes mellitus and metabolic syndrome, resulting in increasing morbidity and mortality. Hence, it is necessary to develop effective prevention and treatment of obesity. In this study, we examined whether GMG-43AC, the new experimental drug of Giuliani Sp. A. (Milan, Italy), could inhibit the differentiation induced by a hormone mixture (IBMX, DEX and insulin) or troglitazone of 3T3-L1 preadipocytes, and further explored the possible mechanism of action. 3T3-L1 cells constitute one of the most popular in vitro models for the investigation of adipogenesis. To achieve the goals proposed in this research project it was necessary to set up: a) the differentiation protocol for 3T3-L1 cells; b) the colorimetric technique for detecting adipocytes droplets, and c) the quantification assay for accumulated triglycerides. In our experimental conditions droplets of triglycerides accumulated in differentiated 3T3-L1 cells were highly positive to the Oil Red O staining. As evidenced by Oil Red O staining, GMG-43AC dose-dependent inhibited triglycerides synthesize and accumulate at concentrations of 500, 1000 and 2000 µM. Studies in adipogenic cell lines have shown that the differentiation of preadipocytes into adipocytes is accompanied by changes in gene expression: e.g., a dramatic increase in the expression of the CCAAT/enhancer-binding proteins C/EBPβ and C/EBPd followed by the expression C/EBPa and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARg). At the molecular level, the expression of transcription factors, C/EBPb, PPARy and C/EBPa, was reduced by GMG-43AC during adipogenesis. After treatment for 10 days, the mRNA levels of adipocyte-specific genes, such as FABP-4 and leptin, were also down-regulated by GMG-43AC in a dose-dependent manner. Furthermore, the exposure to GMG-43AC during the initial 48 hours of adipocyte differentiation markedly reduced C/EBPb levels in a dose-dependent manner with the exception of the dose 300 µM. Moreover, we investigated the capability of GMG-43AC to revert the adipocyte differentiation process induced by hormone stimulants or troglitazone. We observed that GMG-43AC was able to revert the adipocyte phenotype in fully differentiated adipocytes. The reverting effect of GMG-43AC treatment can be observed from concentrations of 500 µM or greater. Interestingly, the drug left in the culture medium for the period of 14 days became more effective in the reversion of differentiation with respect to the treatment for 7 days. The accurate molecular mechanisms that regulate loss of accumulated triglycerides in fully differentiated adipocytes are still unclear and will be investigated in the near future. Thus, GMG-43AC has the potential to be an innovative product for the prevention and treatment of obesity.