Expression of Caveolin 1 Is Enhanced by DNA Demethylation during Adipocyte Differentiation. Status of Insulin Signaling

Abstract

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.

Effect of TNF-Alpha on Caveolin-1 Expression and Insulin Signaling During Adipocyte Differentiation and in Mature Adipocytes

“…In agreement with previous results [22], the baseline level of Cav-1 phosphorylation was significantly increased in mature adipocytes (day 21) and the response to insulin was small and not significant, while, on the other hand, the IR and AKT-2 insulin response was enhanced in the mature cells (day 21). The addition of TNF-α during the differentiation process significantly reduced Cav-1 baseline phosphorylation, but maintained its capacity to respond to insulin.…”

“…Enhanced Cav-1 expression is a hallmark of adipocyte differentiation [35], which is accompanied by IR, AKT-2 and Glut-4 gene induction (Fig. 5A and 7A), as we have reported previously [22]. Surprisingly, the presence of TNF-α did not significantly reduce the mRNA levels of any of these genes, despite slowing down and limiting the 3T3-L1 maturation process.…”

“…We have reported previously that Cav-1 is phosphorylated and activated during adipogenesis [22], which explains why, after 21 days of differentiation, although IR and AKT-2 insulin responses are markedly increased, insulin has little effect on Cav-1 activation (Fig. 5C).…”

“…In this regard, it is important to note that TNF-α has been previously reported to cause a total depletion of the Glut-4 protein in the adipocyte membrane [36,37], retaining it in an inactive form in intracellular membrane compartments. The differences observed in control cell glucose uptake during adipocyte differentiation were discussed in a previous study by our group in relation to the facilitative insulin-independent glucose transporter-1 (Glut-1) [22]. Nonetheless, insulin-stimulated AKT-2 phosphorylation was not affected by TNF-α.…”

“…On the other hand, the region of the IR gene studied encompasses from 249 bp 5' to 545 bp 3' of the ATG codon, including 40 CpG dinucleotides located in the first exon and the first intron. Both Cav-1 and IR gene sequences have been described in detail in a previous work of our group [22]. Genomic DNA was extracted and purified from 3T3-L1 cells using the QIAamp DNA kit (Qiagen, Hilden, Germany) at checkpoints established for both experimental designs, days 0, 7 and 21 for the long-term and days 7 and 9 for the short-term TNF-α treatment.…”