Caveolin 1 (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. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 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 must 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, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.

Background/Aims: Tumor necrosis factor-α (TNF-α)-mediated chronic low-grade inflammation of adipose tissue is associated with obesity and insulin resistance. Caveolin-1 (Cav-1) is the central component of adipocyte caveolae and has an essential role in the regulation of insulin signaling. The effects of TNF-α on Cav-1 expression and insulin signaling during adipocyte differentiation and in mature adipocytes were studied. Methods: 3T3-L1 cells were differentiated (21 days) in the presence TNF-α (10 ng/mL) and mature adipocytes were also treated with TNF-α for 48 hours. Cav-1 and insulin receptor (IR) gene methylation were determined as well as Cav-1, IR, PKB/AKT-2 and Glut-4 expression and activation by real time RT-PCR and western blot. Baseline and insulin-induced glucose uptake was measured by the 2-[C¹⁴]-deoxyglucose uptake assay. Results: 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. Conclusion: Cav-1 activation by phosphorylation seems to be essential for the maintenance of an active and insulin-sensitive glucose uptake.

Adipocytes exposed to high glucose concentrations exhibit impaired metabolic function, including an increase of oxidative and proinflammatory factors that might favor the development of insulin resistance. Caveolin-1 (Cav-1) is a key mediator of the insulin transduction pathway whose expression is significantly enhanced during adipocyte differentiation. In this work, we studied the effects of high glucose concentration on the regulation of Cav-1 expression and activation and its relation to the insulin signaling pathway during the adipogenic process and in long-term differentiated adipocytes. Both, long-term high glucose exposure during adipogenesis and short-term glucose incubation of mature adipocytes, 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, insulin receptor (IR) and potein 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. Although long-term high glucose exposure increased DNA methylation in Cav-1 promoter, Cav-1 expression was not affected. 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.

Type-2 diabetes is associated with a chronic low-grade systemic inflammation accompanied by an increased production of adipokines/cytokines by obese adipose tissue. The search for new antidiabetic drugs with different mechanisms of action, such as insulin sensitizers, insulin secretagogues and aglucosidase inhibitors, has directed the focus on the potential use of flavonoids in the management of type-2 diabetes. Thirty six diabetic male C57BL/6J db/db mice were fed a standard diet and randomly assigned into four experimental groups: non-treated control, (n ¼ 8); acarbose (5 mg per kg bw, n ¼ 8);helichrysum (1 g per kg bw, n ¼ 10) and grapefruit (0.5 g per kg bw, n ¼ 10) for 6 weeks. The mRNA expression in pancreas, liver and epididymal adipose tissue was determined by RT-PCR. DNA methylation was quantified in epididymal fat using pyrosequencing. Mice supplemented with helichrysum and grapefruit extracts showed a significant decrease in fasting glucose levels (p < 0.05). A possible mechanism of action could be the up-regulation of liver glucokinase (p < 0.05). The antihyperglycemic effect of both extracts was accompanied by decreased mRNA expression of some proinflammatory genes (monocyte chemotactic protein-1, tumor necrosis factor-a, cyclooxygenase-2, nuclear factorkappaB) in the liver and epididymal adipose tissue. The CpG3 site of TNFa, located 5 bp downstream of the transcription start site, showed increased DNA methylation in the grapefruit group compared with the non-treated group (p < 0.01). In conclusion, helichrysum and grapefruit extracts improved hyperglycemia through the regulation of glucose metabolism in the liver and reduction of the expression of proinflammatory genes in the liver and visceral fat. The hypermethylation of TNFa in adipose tissue may contribute to reduce the inflammation associated with diabetes and obesity.