Abstract
Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment.
Highlights
In light of its competence in secreting hormones and cytokines, adipose tissue has been recognized as the largest endocrine organ, which can balance cellular anabolic and catabolic metabolism, a key requirement to preserve energy homeostasis and total body health [1].adipose tissue dysfunction can arise in response to overall hyper-anabolic conditions, as occurs during obesity and metabolic syndrome [1].Adipogenesis has been deeply investigated in vitro, especially thanks to 3T3-L1 preadipocyte cell culture by which the cascade of events eliciting the differentiation process can be followed
As well as the icant concentration dependent decrease in BrdU incorporation was found in DCQAtranscription factors cyclic-AMP response element-binding protein (CREB) and STAT3, which initiate the earliest phase of adipogenesis, treated 3T3-L1 cells 48 h after MDI induction (Figure 4B, blue bars)
Despite the fact that terminally developed adipocytes are unable to replicate, progenitors cells resident in adipose tissue can proliferate and differentiate, supplying new adipocytes to the tissue throughout its life [43,44]
Summary
In light of its competence in secreting hormones and cytokines, adipose tissue has been recognized as the largest endocrine organ, which can balance cellular anabolic and catabolic metabolism, a key requirement to preserve energy homeostasis and total body health [1]. Tang et al showed that MCE is an essential event for 3T3-L1 differentiation into adipocytes [3] In actuality, during such an event, post-confluent 3T3-L1 pre-adipocytes are driven to re-enter in the cell cycle by MDI, which elicits many proliferative signaling pathways [4]. Antioxidant administration, instead, caused cell cycle arrest during the MCE [9] This suggests that a significant oxidative stress condition is essential for adipogenesis. For many plant extracts and their secondary metabolites, a positive correlation between the antioxidant properties of these compounds and their anti-adipogenic effects is widely known Both coffee and cocoa extracts reduced the production of ROS as well as the lipid accumulation in adipocytes [15].
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