El papel del colesterol en la funcionalidad de los lipid rafts y su repercusión en la adipogénesis

Abstract

Cholesterol is an essential component of plasma membranes and is involved in important processes in mammal cells, such as cell proliferation. Actually, cholesterol deficiency has been shown to arrest the cell cycle. In promyelocytic HL-60 cells, cholesterol starvation has been shown to induce differentiation into neutrophyles. In the present work we studied the effects of cholesterol starvation on hormonally-induced differentiation of 3T3-L1 preadipocytes into mature adypocytes. We have found that treatment with different cholesterol biosynthesis inhibitors results in the reduction of lipid droplet formation and the inhibition of the expression of several transcription factors involved in adipogenesis (PPARγ, C/EBPα and LXRα) as well as adipsin and aP2, proteins that are characteristic of mature adipocytes. In order to elucidate the mechanisms involved, we firstly explored the clonal expansion, a process that is essential for the correct development of adipogenesis and precedes the expression of genes that regulate differentiation. We found that, once stimulated, 3T3-L1 cells underwent one round of cell division, independently they were treated with cholesterol biosynthesis inhibitors or not. Treatment with these inhibitors, however, resulted in the inhibition of both ERK1/2, p38 MAPK and Akt. The Inhibition of ERK1/2 apparently was the most relevant for the cessation of the differentiation process, as it resulted in the inhibition of the phosphorylation of C/EBPβ, a factor that governs the expression of several transcription factors involved in adipogenesis, which were found to be depressed by cholesterol biosynthesis inhibition. Therefore, the inhibition of C/EBPβ phosphorylation appears to be the mechanism by which cholesterol starvation inhibits experimental adipogenesis. Lipid rafts are cholesterol-rich membrane microdomains in which many receptors and acceptor molecules reside. Previous studies by others showed that membrane cholesterol extraction by means of cyclodextrins results in the destabilization of lipid rafts, and ultimately affecting both insulin-mediated signaling and 3T3-L1 differentiation. In our approach, inhibition of cholesterol biosynthesis resulted in a decrease of the cholesterol content in membranes, and the accumulation of different precholesterol sterols, depending on which enzyme was blocked. These changes in sterol composition resulted in the disruption of lipid rafts, with the lost of caveolin-1 and GM1 ganglioside, which ultimately affected the insulin-receptor signaling. This mechanism may be involved the inhibition of adipogenesis produced by cholesterol starvation. Haloperidol, a widely used antipsychotic, was reported to inhibit ergosterol synthesis in yeasts. We herein demonstrate that in mammal cells this drug inhibit different enzymes involved in cholesterol biosynthesis (∆7 -reductase > ∆8,7-isomerase > ∆14-reductase, in that order), as well as the egress of LDL-derived free cholesterol from the late endosome/lysosome compartment, thereby greatly affecting the intracellular cholesterol homeostasis. Based on these findings and considering that lipid rafts from neuronal cells have been shown to harbour both insulin and somatostatin receptors, we decided to study the effects of haloperidol in SH-SY5Y neuroblastoma cells in vitro. Treatment with this drug profoundly modified the sterol composition and disrupted lipid-raft structure, which was accompanied by the alteration of both insulin and somatostatin signalling. These effects may help in the understanding of the side effects that prolonged haloperidol treatment occasionally produces, which affect both motor activity and insulin sensitivity. Previous studies in our laboratory showed that cholesterol deprivation results in cell cycle arrest at G2/M phase and that cholesterol provision rapidly induces the expression of cyclin B1 at both mRNA and protein levels. In the present work we sought to study the mechanism involved in this action of cholesterol. By using different constructions of the human cyclin-B1 promoter we found that the region conferring the ability to respond to cholesterol was near the start of transcription point. We then identified a new possible response element for the USF factor located 77 pb upstream the transcription start site in the cyclin B1 promoter, whose mutation abolished this response. This is the first time an element on a cyclin promoter is described with the potentiality to respond to changes in the intracellular cholesterol content.