Mechanisms of adipose tissue extracellular matrix alterations in an in vitro model of adipocytes hypoxia and aging.

Fibrosis has been considered as a hallmark of dysfunctional adipose tissue (AT), however the role and mechanisms of fibrosis in the age related AT dysfunction are not yet well characterized. The aim of the study was to investigate the mechanisms of extracellular matrix (ECM) alterations and the role of caveolins, using an in vitro model of adipocyte aging and hypoxia. Hypoxic adipocytes, but also aged adipocytes, were characterized by a significant increase in gene expression of pro-inflammatory cytokines and ECM components. Immunofluorescence analysis confirmed an increase in collagen VI-A3 in hypoxic and also in aged adipocytes. However aged adipocytes were characterized by only a slight increase in HIF1α immunofluorescence and by a more relevant increase in senescence compared to hypoxic and mature adipocytes, with an increase in p-53 protein and a decrease in SIRT 1 protein. Immunofluorescence and western blot analysis revealed a significant decrease in caveolin-1 expression in hypoxic adipocytes and even more in aged adipocytes. In conclusions, aging adipocytes are associated to alteration of ECM and fibrosis, by modulation of the caveolins through complex mechanisms where inflammation, hypoxia and cellular senescence are coexisting.

In vivo transfection rat small intestine K-cell with pGIP/Ins plasmid by DOTAP liposome.

Gene therapies to produce insulin in diabetic patients have been considered for several years. Genetic engineering of ectopic insulin production and secretion in autologous non-beta-cells has been tested in different tissues. Recently, gut K-cells have been shown to express glucokinase, the glucose sensor of pancreatic beta-cells. K-cells are responsible for the secretion of the glucose-dependent insulinotropic peptide (GIP). Transfection of K-cells by a specific plasmid to produce insulin correlated to glucose level is being considered. Cationic liposomes are non-viral gene delivery to lung, spleen, liver and intestinal cells. DOTAP-GIP/Ins plasmid complex was used for transfection of K-cells in vivo. RT-PCR assay of human insulin mRNA revealed that the transfection of insulin gene by DOTAP liposome is an efficient tool. The genetic engineering of ectopic insulin production and secretion in autologous non-beta-cells is an appropriate method. The potential of the transmission of a constructed plasmid, which contains human insulin gene under the control of GIP promoter, to gut K-cells could be considered for treatment of diabetes.