Regulación de la expresión y activación de la caveolina-1 durante la adipogénesis, y su relación con la señalización insulínica. Efecto de TNF-a y glucosa

Abstract

Knowledge of the regulatory mechanisms involved in adipocyte differentiation and hypertrophy is essential to understand the nexus between obesity and insulin resistance and will help to design new therapeutic approaches for obesity and diabetes. Previous studies published by our group pointed to caveolin-1 (Cav-1) as one of the genes upregulated in rat visceral white adipose tissue in response to a high fat diet. Cav-1 is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. The major objectives of the this work were: to determine changes in expression and activation of Cav-1 during adipocyte differentiation and the role of DNA methylation in the expression changes; to analyze the effect of such changes in the transmission of insulin signaling; to study the effects of external conditions typically associated with obesity, such as inflammation (TNF-α) or high glucose (hyperglycemia), on the expression and activation of Cav-1 and their influence on insulin signaling, both during adipocyte differentiation and in mature adipocytes. In this work, we have found that, during adipocyte differentiation of 3T3-L1 cells, the promoter and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, an event that would help to facilitate the maintenance of insulin sensitivity by these fully mature adipocytes. TNF-α-mediated chronic low-grade inflammation of adipose tissue is associated with obesity and insulin resistance. In our study, TNF-α slowed down the differentiation program, hindering the expression of some insulin signaling intermediates without fully eliminating insulin-mediated glucose uptake. In mature adipocytes, TNF-α did not compromise lipid-storage capacity but downregulated the expression of the insulin signaling intermediates, totally blocking insulin-mediated glucose uptake. Insulin sensitivity correlated with the level of activated phospho-Cav-1 in both situations, strongly suggesting the direct contribution of Cav-1 to the maintenance of this physiological response. Finally, adipocytes exposed to high glucose concentrations exhibited impaired metabolic functions, including an increase of oxidative and proinflammatory factors that might favour the development of insulin resistance. Our results demonstrated that both, long-term high glucose exposure during adipogenesis, and short-term incubation of mature adipocytes with high glucose, promoted triglyceride accumulation in 3T3-L1 cells. The short-term exposure of mature adipocytes to high glucose significantly reduced the sensitivity to insulin of Cav-1, IR and protein kinase B (AKT-2) phosphorylation, as well as insulin-induced deoxyglucose uptake. Adipocytes differentiated in the presence of high glucose lost Cav-1 and IR response to insulin-stimulated phosphorylation, but maintained the insulin sensitivity of AKT-2 phosphorylation and deoxyglucose uptake. Moreover, these cells showed an increase of Cav-1, IR and AKT-2 protein content, pointing to an adaptive response induced by the long-term high glucose exposure.