In the present study, we examined the anti-obesity effect of CAF in 3T3-L1 cells by measuring lipid accumulation and by analyzing changes in adipocyte differentiation, which modulates adipocyte-specific gene expression and Akt phosphorylation. We demonstrated that CAF treatment inhibited lipid accumulation and the differentiation of 3T3-L1 preadipocytes into adipocytes in a dose-dependent manner. CAF treatment decreased the expression of key adipocyte differentiation regulators, including C/EBPβ, C/EBPα and PPARγ, and down-regulated Akt phosphorylation. These results suggest that CAF plays a critical role in preventing adipogenesis and the accumulation of cytoplasmic lipid droplets during the differentiation in 3T3-L1 cells.
Adipocyte differentiation and fat accumulation are associated with the occurrence and development of obesity . Hyperplastic obesity is caused by an increase in the number of fat cells relative to the increase in adipose tissue mass. A reduction of adiposity is related to the inhibition of angiogenesis along with a reduction of adipocyte numbers and the lipid content of adipocytes. The differentiation of preadipocytes into adipocytes is regulated by a complex network of transcription factors. At the center of this network are nuclear receptor PPARγ and members of C/EBP family, which initiate the entire adipogenic program and regulate the process of terminal differentiation . The expression of C/EPBα and PPARγ cross-regulate each other through a positive feedback loop and transactivate downstream target genes, such as aP2, LPL, and FAS, that are adipocyte specific and are involved in maintaining the adipocyte phenotype [6, 9]. In the present study, CAF treatment remarkably reduced the level of Oil-red O staining in a dose-dependent manner, and microscopic inspection revealed a significant decrease in the level of accumulated intracellular triglyceride. The triglyceride levels of cells treated with 50 μg/ml CAF displayed a marked reduction in adipogenesis. These results indicate that CAF inhibited the differentiation of 3T3-L1 preadipocytes into adipocytes and also inhibited the accumulation of lipid droplets in the cytoplasm, an adipocyte phenotype that follows differentiation based on lipid accumulation. Our results show that CAF treatment significantly down-regulated PPARγ and C/EBPβ at the mRNA and protein levels, compared to those in fully differentiated adipocytes. PPARγ and C/EBPβ, which are two central transcriptional regulators, are induced prior to the transcriptional activation of most adipocyte-specific genes . Moreover, PPARγ-deficient cells fail to differentiate into adipocytes, and the overexpression of PPARγ and C/EBPα accelerates adipogenesis . Therefore, these results suggest that CAF suppresses adipocyte differentiation through PPARγ and C/EBPβ in the early stages of adipocyte differentiation.
We focused on the effects of CAF on the regulation of aP2 and FAS expression during 3T3-L1 differentiation. PPARγ and C/EBPα synergistically activate the downstream promoters of adipocyte-specific genes such as aP2 and FAS. The aP2 gene is a terminal differentiation maker of adipocytes, and it facilitates the cellular uptake of long-chain fatty acids in a pathway linking fatty acid metabolism and obesity . FAS is a lipogenic enzyme that facilitates the synthesis and cytoplasmic storage of massive amounts of triglycerides . In the current study, the presence of CAF suppressed the expression of aP2 and FAS, which suggested that CAF inhibits adipogenesis through the down-regulation of C/EBPα and PPARγ. Furthermore, the down-regulation of aP2 and FAS decreased fatty acid utilization and fatty acid transport in 3T3-L1 adipocytes. However, we did not observe any change in the expression of LPL, which is also controlled by PPARγ and C/EBPα. Consistent with our results, hydroxytyrosol from olive oil inhibited lipid accumulation during adipocyte differentiation and inhibited the expression of all genes tested, except LPL . CAF stimulated lipolysis, which induced glycerol release when added to mature adipocytes, correlated to the CAF-induced down-regulation of adipogenic gene expression. Therefore, these results strongly suggest that CAF prevents adipogenesis through the inhibition of PPARγ and C/EBPα gene expression, reduces the expression of adipogenesis- and lipid metabolism-associated genes, and strongly induces lipolysis in 3T3-L1 adipocytes.
Citrus fruits are abundant sources of compounds that help prevent lifestyle-related diseases such as diabetes, high blood pressure, and cancer. CAL contains many flavonoid components, such as naringin, hesperidin, poncirin, isosinnesetin, hexamethoxyflavone, sinestin, nobletin, heptamethoxyflavone, tangeretins, and hydroxypentamethoxyflavone [22, 23]. Polymethoxylated flavones from citrus improve lipid and glucose homeostasis and increase adiponectin in fructose-induced insulin-resistant models . Citrus depressa Hayata extracts show antiobesity effects in high-fat diet-induced obese mice . Other studies have reported that dietary flavonoids, which include catechin, quercetin, kaempferol, and genistein, inhibit adipogenesis in 3T3-L1 adipocytes [29, 31]. Mercader et al. showed that a Citrus aurantium extract exhibited lipolytic activity in human adipocytes, providing the basis for an anti-obesity effect .
Many studies using natural substances and herbal compounds focus on the activity of the PI3K/Akt signaling pathway in preventing obesity; hormones and growth factors that are specific to adipogenesis act via their receptors to transduce external differentiation signals through a cascade of intracellular events in the PI3K/Akt signaling pathway . Thus, Akt activation has been identified as a major target for the control of obesity and diabetes . Akt plays a critical role in the insulin signaling pathway, and the insulin-stimulated phosphorylation of Akt via PI3K is an important indicator of proper insulin function . Constitutively active Akt causes the spontaneous differentiation of 3T3-L1 cells in the absence of insulin stimulation . The Akt signal cascade is important for adipogenesis, and it activates PPARγ and C/EBPα during 3T3-L1 adipocyte differentiation . Moreover, Akt regulates adipogenesis via the phosphorylation and inactivation of substrates such as Foxo1 and GSK3β, which directly regulate PPARγ, C/EBPβ, C/EBPα, and GS [15, 16]. Therefore, to investigate the molecular mechanism underlying the anti-adipogenesis stimulated by CAF, we studied the effects of CAF on the activation of Akt. Our results demonstrate that CAF caused a marked and dose-dependent attenuation of the Akt phosphorylation (Ser473) induced by insulin. However, DMII induction of the 3T3-L1 cells increased Akt activation, which is consistent with enhanced Akt phosphorylation. Our results strongly support the conclusions of a previous study showing that naringenin, which is derived from Citrus
aurantium, inhibits phosphotidylinositide-3-kinase activity and glucose uptake in 3T3-L1 adipocytes . Intriguing, CAF also decreased insulin-induced GSK3β (Ser9) phosphorylation in a dose-dependent manner in 3T3-L1 adipocytes. The results of another study demonstrated that the Ser9 phosphorylation of GSK3β is increased following insulin treatment, and its activity is repressed by insulin and lithium chloride (LC) . Lithium mimics insulin in its stimulation of glucose transport. LC treatment of 3T3-L1 cells inhibited PPARγ expression and adipocyte differentiation . In addition, a study of Akt-deletion mice showed that Akt is essential for adipocyte differentiation and for the induction of PPARγ expression . Therefore, our results indicate that the inhibition of Akt phosphorylation and activation by CAF blocks hormone-induced adipocyte differentiation in 3T3-L1 preadipocytes. These results imply that there is an important association between PI3K/Akt/GSK3β-mediated signaling and the transcription factors, PPARγ and C/EBPα, in 3T3-L1 adipocyte differentiation induction. These results identify one possible mechanism of CAF action, suggesting that CAF-induced inhibition of Akt suppresses adipogenesis by inhibiting other signaling cascades that include C/EBPs and PPARγ during the process of 3T3-L1 adipocyte differentiation.