Obesity is a medical condition in which abnormal or excessive fat accumulates mainly due to sedentary behavior that is linked to lower health-related quality of life, lack of exercise, and intake of foods rich in fats and oils. Trends in overweight prevalence have been increasing rapidly in children and adults and are associated with serious mortalities, including a high hyperlipidemia incidence, type 2 diabetes mellitus, insulin resistance, heart disease, and fatty liver in addition to various types of cancer and osteoarthritis [1]. The currently available treatment options are not potent enough to prevent obesity permanently, and other effective strategies for weight loss may cause other side effects. Therefore, there is a great demand for long-term use of complementary, alternative and safe and effective medicines to treat this pandemic global obesity problem.
In the present study, we investigated the inhibitory effects of ATM on adipocyte differentiation in 3T3-L1 cell and the anti-obesity ATM activities on HFD-induced obesity rats was investigated by analyzing body and fat pad weights, adipocyte size, and blood biochemical profiles.
ATM is well-known as a functional tree with potential medicinal benefits. Diarylheptanoids [24], rhododendrol glycoside [25], and tannins [26] have been isolated from the genus Acer. Other studies have also demonstrated that ATM is rich in abundant functional compounds, including polyphenols, phenethyl glycosides, and flavonoids, suggesting that these compounds exhibit hepatoprotective activities [20, 21]. ATM exhibits potent antiangiogenic activity both in vivo and in vitro [27] and has been shown to be cytotoxic to cancer cells [28].
These results are the first to demonstrate that ATM causes inhibition of differentiation and adipogenesis of 3T3-L1 preadipocytes and lipid accumulation in mature differentiated adipocytes via regulation of adipogenic transcription factor expression and Akt signaling. In order to investigate whether ATM affects adipocyte differentiation, we measured the effects of ATM on lipid accumulation during 3T3-L1 preadipocyte differentiation. Adipogenesis is defined as the process by which preadipocytes differentiate into adipocytes. 3T3-L1 cells are induced to differentiate into adipocytes according to a coordinated program and are one of the most useful cell lines for studying adipogenesis [29]. In the present study, lipid accumulation was measured by Oil Red O staining and the TG assay. Our data demonstrated that ATM treatment caused a reduction in TG levels and a decrease in intracellular lipid content in a dose-dependent manner as quantified by Oil Red O staining in the cytoplasm of treated 3T3-L1 cells. These results demonstrate that ATM blocked adipogenesis during adipocyte differentiation via facilitating an effective decrease in lipid formation and lipid accumulation in 3T3-L1 adipocytes. To our knowledge, this is the first study showing the influence of ATM on lipid accumulation in 3T3-L1 adipocytes.
In line with these findings, we examined the effects of ATM on adipogenesis and lipid accumulation in 3T3-L1 adipocytes via regulation of adipogenic gene expression. Differentiation of preadipocytes is regulated by a complex network of transcription factors, mainly consisting of the C/EBP family and PPARγ. This cooperative function helps to maintain each of their own high expression levels. Moreover, adipocytes from mice in which the C/EBPα gene was disrupted showed defects in lipid accumulation [30]. Transgenic mice specifically lacking PPARγ in adipose tissue exhibited greatly reduced fat pad sizes [31]. PPARγ stimulates adipocytes and induces differentiation, while C/EBPα also supports the adipocyte differentiation process. Therefore, low levels of these adipocyte differentiation-specific genes are important markers of adipocyte inhibition.
In the present study, the treatment with ATM caused a decrease in mRNA expression of C/EBPβ, C/EBPα, and PPARγ in 3T3-L1 adipocytes. Western blot analysis showed that C/EBPβ, C/EBPα, and PPARγ protein levels during adipogenesis of 3T3-L1 preadipocytes demonstrated a dose-dependent reduction. These results show that the ATM treatment caused inhibition of adipocyte differentiation via inhibition of PPARγ and C/EBP family-associated adipogenesis signaling, and ATM effects on adipogenic transcription factors involved in adipocyte differentiation in 3T3-L1 cells play a critical role in adipogenesis mediation.
It is well known that adipocyte differentiation is associated with multifunctional cellular pathways and requires sequential regulation of adipogenic and lipogenic genes [32]. PPARγ and C/EBPα activate gene expressions, such as aP2, LPL, FAS and others, that are involved in adipogenesis and lipogenesis and participate in creating the adipocyte phenotype [10, 11, 33].
In the present study, the effects of ATM on the expression of aP2, FAS, and LPL were investigated during 3T3-L1 differentiation. The expression of aP2, FAS, and LPL genes is significantly down-regulated in a dose-dependent manner following ATM treatment. Together, our results demonstrate that ATM led to strong suppression of the expression of critical genes involved in creating and maintaining adipogenesis and lipogenesis via lipid storage and accumulation in 3T3-L1 adipocytes.
Several lines of evidence support the concept that the lipogenic pathway is localized to peroxisomes and is important for endogenous activation of PPARγ. PPARγ mainly regulates the gene network expression involved in adipogenesis and lipid metabolism [34]. PPARγ has a major role in the differentiation of preadipocytes into adipocytes involved in obesity development [35, 36]. The fact that PPARγ null mice complete lack adipose tissue clearly demonstrates that PPARγ is essential for adipocyte differentiation [37]. Focusing on lipid metabolism genes, FAS regulates fatty acid synthesis from acetyl- and malonyl-CoA and via the tricarboxylic acid cycle [38]. Therefore, reduction of FAS via ATM treatment could explain the way in which ATM extracts suppress lipid accumulation. aP2 is highly expressed in adipose tissue and plays a lipid binding protein, which reacts as a key factor in intracellular fatty acid transport and lipid metabolism [10, 11]. The expression of lipoprotein lipase (LPL) is stimulated by PPARγ, thus increasing fatty acid uptake into adipocytes [11]. These genes are activated in response to PPARγ regulation [10, 11]. In this study, ATM caused a reduction in the expressed level of FAS, LPL, and aP2, suggesting that intracellular fatty acid transport and lipid metabolism are reduced due to low lipid accumulation. The expression levels of those adipogenic and lipogenic-related genes decreased in ATM-treated 3T3-L1 adipocytes. These studies explain ATM-induced inhibition of adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes. Therefore, ATM reduced adipogenesis and lipid synthesis within differentiated adipocytes at the gene level through inhibition of genes involved in adipocyte lipogenesis and fatty acid transport and synthesis, thus leading to lipogenesis.
Akt kinases play an important role in adipogenesis and glucose transport [39]. Inhibition of Akt activation in fibroblasts displays lack of capability to differentiate preadipocytes into adipocytes, and Akt overexpression results in an increase in glucose uptake and adipocyte differentiation [40]. In the present study, our results demonstrate that ATM caused a significant and concentration-dependent decrease in the phosphorylation of Akt in 3T3-L1 adipocytes. In addition, treatment with ATM caused a marked decreased in phosphorylation of GSK3β during adipocyte differentiation of the 3T3-L1 preadipocytes. It is known that Akt phosphorylation can promote adipocyte differentiation via PPARγ up-regulation [41]. The Akt signaling cascade is considered important for adipogenesis as it appears to activate PPARγ and C/EBPα during induction of 3T3-L1 adipocyte differentiation [41]. Xu et al. showed that Akt activation enhances an important association between the Akt signaling cascade and transcription factors, PPARγ and C/EBPα, in induction of 3T3-L1 adipocyte differentiation [42]. Taken together, these results demonstrate that ATM suppressed adipocyte differentiation and lipid accumulation in 3T3-L1 cells via down-regulation of adipogenic transcription factors and the Akt signaling pathway associated with intracellular lipid accumulation.
Rodent models of HFD-induced obesity have been widely used to investigate human obesity and its related metabolic diseases. Five weeks of an HFD leads to overt obesity in rats and is characterized by body weight gain along with an increase of fat tissue weight. In this animal study, we established that HFD-induced obesity in rats caused an increase in body weight of rats and serum TG and TC levels and a decrease in HDL-C levels. However, our results showed that BW gain was lower in rats administrated the ATM extract. The weights of epididymal and perirenal adipose tissue were also markedly reduced in rats supplemented with ATM, indicating that BW loss is mainly due to decreased fat accumulation in epididymal and perirenal adipose tissues. We also observed that serum TG and TC were lower in rats fed HFD plus ATM than those of rats fed HFD, indicating that ATM is beneficial for improvements in numerous serum metabolic parameters. Thus, these data suggest that ATM suppressed lipid disorders caused by the excess lipid found in HFD-induced obese rats, thereby improving hypertriglyceridemia and hypercholesterolemia caused by HFD-induced obesity. In addition, increased adipose tissue mass, particularly the amount of visceral fat, is associated with an increased risk of metabolic diseases. In this study, ATM-fed rats showed lower masses of epididymal and perirenal adipose tissues. Thus, supplementation with ATM caused reduction in BW and fat tissue weights, thus preventing fat accumulation and improving to obesity.
Next we investigated the effect of ATM extract for the prevention on fatty liver. The liver is mostly regarded as an essential organ in lipid metabolism. Imbalance between lipid deposition and removal results in hepatic lipid accumulation, which is related to an increase in hepatic lipogenesis, augmented lipid uptake, and/or reduced TG export of β-oxidation products (43). In the present study, the liver tissue in the obese HFD-fed rats showed lipid droplet accumulation in hepatocytes, while the treatment with ATM caused a decrease in the deposits of hepatic lipids in liver tissue. These results indicate that the livers of ATM-treated rats had less adipocytes than the HFD-fed rats, thus inducing lipid droplet reduction in liver tissue. These findings highlight the critical role of ATM in reducing abnormal lipid accumulation in liver tissue in addition to causing a reduction in BW gain.