CONICET | Buscador de Institutos y Recursos Humanos

The epidemic increase in obesity causing insulin resistance and type 2 diabetes has become a worldwide problem. To better understand the mechanisms that lead to metabolic disorders, it is crucial to develop a better underlying knowledge of the molecular events that regulate adipocyte differentiation. Many positive modulators of this process have been identified, such as the CCAAT/enhancer binding protein (C/EBPβ) and peptide hormones like insulin. The insulin receptor (IR) is transmembrane tyrosine kinase receptor, encoded by a single gene composed of 22 exons. Due to alternative splicing of exon 11, the gene gives rise to two protein isoforms that differ by a 12-amino-acid insertion: the IR lacking exon 11 (IR-A) and the IR containing exon 11 (IR-B). We hypothesize that IR plays an important role in regulating adipocyte differentiation process. To address this issue, we have generated two IR KO clones from the 3T3-L1 cell line, using the CRIPSR/Cas9 system, and tested it as an adipocyte differentiation model. We verified accurate genome editing through sequencing and IR protein lacking through WB. These clones were unable to differentiate under standard differentiation protocols.In order to recover the differentiation capacity in these KO clones, we re-expressed IR-A or IR-B through retroviral infection. Correct isoform expression was determined at mRNA level through RT-PCR. Preliminary results show that they recapitulated some of the molecular events typical of adipocyte differentiation. This was evaluated at the level of early adipogenic markers such as C/EBPβ and in late adipocyte-specific genes, such as the gene encoding aP2, a lipid-binding protein.Taken together, these results suggest that IR could be modulating important steps of adipocyte differentiation. Further studies are necessary to elucidate the targets of IR action.