Eurycomanone regulates lipid metabolism by activating the cAMP/PKA pathway

Eurycoma longifolia Jack (ELJ) contains mainly alkaloids, and quassinoids, which are the main active ingredients. Eurycomanone (EN), one of the most common quassinoids, is said to have beneficial effects on lipid and glucose metabolism. In this study, we investigated the effects of EN on lipolysis by establishing a high-fat animal model in vivo and evaluated its efficacy as a lipolytic and anti-fatty liver agent. Oil red O staining showed morphological changes of 3T3-L1 preadipocytes after EN treatment and confirmed the inhibitory effects of EN on adipocyte differentiation. The mechanism of EN promotes lipolysis in 3T3-L1 cells was analyzed by immunofluorescence, Western blotting, quantitative real-time PCR and siRNA transfection. In C57BL/6J mice fed a high-fat diet, intragastric administration of EN (5 mg/kg and 10 mg/kg) for two weeks, decreased fat droplet mass and size, and reduced fat accumulation in the liver. Furthermore, EN activated PKA and promoted the PKA/hormone sensitive lipase lipolysis signaling pathway, thereby increasing the release of glycerol and free fatty acids from adipocytes. Our findings indicate the potential of EN as a promising alternative pharmacologic agent for the prevention of obesity.


Introduction
Obesity is a complex disease mainly caused by excessive accumulation of fat.
When there is an imbalance between energy acquisition and energy consumption, the number of adipocytes gradually increases, the formation and storage of large lipid droplets. The incidence of obesity is increasing worldwide at an alarming pace and has become a major threat to public health [1]. Obesity is closely associated with type 2 diabetes, cardiovascular disease, non-alcoholic fatty liver disease and metabolic syndrome; therefore, the search for effective and safe weight-loss drugs has become a common aspiration.
Energy storage in the form of lipids in white adipose tissue (WAT) leads to WAT enlargement and eventually, to body weight gain [2][3][4][5][6]. Promoting WAT decomposition and inhibiting lipid formation can prevent excess lipid accumulation.
Inhibit the differentiation of preadipocytes and promote lipolysis are proposed to be an alternative strategy to treat obesity [7].
Lipolysis is a complex process that is highly regulated and involved in coordinating several lipases and lipid droplet (LD)-associated proteins [8]. Adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoacylglycerol lipase are the three major lipases involved in lipolysis [9]. The activity of ATGL, which is highly specific for triacyl substrates, is largely determined by its comparative gene co-activation of 58 (CGI-58), whereas G (0)/G (1) switching gene 2 (G0S2) acts as an inhibitor of ATGL activity and ATGL-mediated lipolysis [10]. It has recently been shown that ATGL is phosphorylated at Ser406 by modulation of AMPK, leading 4 to increased TAG hydrolase activity [11]. The activity of HSL is regulated by reversible post-transcriptional phosphorylation. In mouse adipocytes, PKA phosphorylates HSL at residues Ser563, 659 and 660, resulting in increased HSL translocation from the cytoplasm to the lipid droplet and lipase activity at the droplet surface [12]. In addition, AMP-activated protein kinase (AMPK) phosphorylates HSL at Ser565, thereby preventing PKA-induced HSL phosphorylation [13][14]. Activation of phosphodiesterase 3B (PDE3B) attenuates PKA activity by Akt-mediated phosphorylation of Ser273, thereby reducing HSL activation and lipolysis [15][16]. In addition to PKA-mediated phosphorylation, HSL can also be phosphorylated by other kinases, such as the extracellular signal-regulated kinase (ERK11\2), which activates HSL by phosphorylation at Ser600 [17]. Studies have also indicated that c-Jun-N-terminal kinase (JNK) plays a role in the regulation of lipolysis based on the silencing effect of Jnk1 and Jnk2 to promote lipolysis in mouse adipocytes [18].
Perilipin A forms a scaffold for lipid droplets, and plays an important role in protein coordination during lipolysis [19]. Under certain conditions, perilipin A restricts translocation of the lipase from the cytoplasm into the lipid droplets (LD), thereby reducing the rate of fat decomposition. However, PKA promotes perilipin A phosphorylation and conformational changes, which promotes the translocation of phosphorylated HSL to LDs, thereby increasing lipolysis [20]. Studies have indicated that a newly identified intracellular fat-specific phospholipase A2 (AdPLA) interferes with lipolysis by regulating the production of arachidonic acid [21,22]. Eurycomanone (EN) is a naturally occurring quassinoids derived from Eurycoma 5 longifolia Jack (ELJ) [Family: Simaroubaceae] (Figure 1). EN has been shown to exert a variety of physiological activities including anti-cancer effects, exhibiting strong dose-dependent anticancer efficacy against lung carcinoma (A-549 cells) and breast cancer (MCF-7 cells), while moderate efficacy was achieved against gastric (MGC-803 cells) and intestinal carcinomas (HT-29 cells) [23][24][25]. The pharmacokinetic properties of EN after oral administration of the pure compound and of E. longifolia extracts remain to be determined. The present method would be useful for future pharmacokinetic studies of the efficacy and safety of EN [26][27], metabolite research in vivo [28], toxicology [29][30], lipolysis and improvement of hypertension [31][32][33]. At present, there are a lot of theories related to lipolysis that have been supported in a large number of animal studies. The most common theory related to lipolysis involves the promotion of male sexual function. Sperm survival is associated with lower cholesterol levels in the testes. Increased lipolysis makes sperm more energetic, and shows synergy with other bioactive compounds that reduce body weight [34][35].
In this study, we investigated the inhibitory effect of EN on adipocyte differentiation and lipid accumulation in 3T3-L1 preadipocytes, and assessed the expression level of genes contributing to lipolysis. We found that EN promotes AKT phosphorylation in 3T3-L1 adipocytes and stimulates lipolysis by activation of the cAMP/PKA pathway. 6 The following monoclonal antibodies were used in this study: mouse The high-fat animal model was established by feeding mice for 8 weeks with a high-fat diet (HFD, 10% lard, 10% yolk, 1% cholesterol, 0.2% cholate and 78.8% standard diet, Nanjing Qinglongshan Experimental Animal Center). Mice received EN (5 mg kg -1 , 10 mg kg -1 ) by intragastric administration. Two weeks later, animals were fasted for 24 h before blood samples were collected from the orbital sinus and glycerol levels were assayed with commercial kits. Mice were sacrificed by cervical dislocation and epididymal adipose tissues and liver were isolated and stored at -80℃ for assay. Other parts of liver as well as epididymal and perirenal white adipose tissues were excised, weighed and portions of the tissues were fixed in 10% formalin for histopathology.
Images were acquired using an inverted microscope (Nikon).

Cell culture and differentiation
Murine 3T3-L1 preadipocyte (ATCC ® CL-173™) cells were cultured in DMEM with 10% FBS (10% FBS/DMEM) and antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin) maintained at 37°C under a humidified atmosphere of 5% CO 2 8 in an incubator. Adipocyte differentiation was induced one day after reaching confluence (day 0) by changing the medium to DMEM with 10% FBS and 0.5 mM IBMX, 0.25 μM DEX and 10 μg/mL insulin (differentiation medium). Two days after induction (day 3), the medium was replaced with DMEM with 10% FBS and 10 μg/mL insulin medium to enhance differentiation, and the cells were cultured for another 2 days. On day 5, the medium was replaced with DMEM with 10% FBS and 10 μg /mL insulin medium and the cells were cultured for a further 2 days cells. Then cells were cultured with DMEM with 10% FBS medium for two more days. On day 8, cells were used in glycerol release enhancement assays.

Glycerol release assay
On day 8, the medium was changed to sample-containing medium (phenol-red-free DMEM) and incubated for 6 h. In inhibitor studies, the cells were incubated with inhibitor for 1 h prior to addition of the EN, and then incubated for a further 24 h. On the day of the glycerol release enhancement assay, the medium was collected and mixed with a free glycerol reagent (Glycerol release assay kit, Nanjing Built Biotechnology Co., Ltd. China). The mixture was incubated at 37°C for 5 min and its absorbance at 550 nm was measured to quantify the amount of the released glycerol. The absorbance relative to that of the negative control was calculated.

Western blotting analysis
Cells seeded in 6-well plates (510 5 11 Total cellular RNAs were extracted using the TRIzol reagent (Invitrogen)

RNA isolation and quantitative real-time PCR (qRT-PCR)
according to the manufacturer's instructions. cDNA was synthesized from the isolated RNA by reverse transcription using RevertAid™ first strand cDNA synthesis kit (5× All-In-One RT MasterMix, ABM) according to the manufacturer's instructions in a RNase-free environment. The relative expression of genes was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) analysis using SYBR Green Supermix (TaKaRa); β-actin served as a housekeeping gene. qRT-PCR was performed using the CFX Connect system (Bio-Rad), and the relative expression of genes was analyzed using the 2 −ΔΔCT method. Three independent experiments were performed, and the primer sequences are listed in Table 1.

Statistical analysis
Data were expressed as means ± standard deviation (SD). Data were analyzed by using analysis SPSS software (SPSS Inc., Chicago). The significance of the difference among mean values was determined by one-way analysis of variance followed by Tukey's test. Comparisons between two groups were made using Student's t-test. A value of P < 0.05 was considered to indicate statistical significance. 12

EN cytotoxicity in 3T3-L1 adipocytes
The cytotoxicity of EN on preadipocytes and mature adipocytes were evaluated using MTT and Trypan blue assays. The MTT assay indicated that culturing cells with EN had no adverse effects on preadipocyte viability. The Trypan blue assay also indicated that treating mature adipocytes with EN had no adverse effects on cell viability ( Fig.   2A and B, respectively).

3T3-L1 cells
Lipid accumulation is the most prominent marker of adipogenesis, and its quantification is used to assess the extent of adipocyte differentiation. Intracellular fat accumulation was significantly decreased by treatment with EN for 4 days compared with control. The lipid content in EN-treated cells during the early phase of adipocyte differentiation was lower than that in intermediate and late stages ( Fig. 3A and B). significantly induced glycerol release (35%) (Fig. 3F).

EN promotes adipolysis via the cAMP/PKA/HSL signaling pathway
Western blotting analysis was conducted to determine the effect of EN on cellular protein levels of cAMP, p-PKA and p-HSL after treatment for 24 h. MDI stimulation enhanced TG accumulation in 3T3-L1 adipocytes; however, EN treatment increased protein expression levels of p-PKA and p-HSL, and promoted TG decomposition (Fig. 4A, B). These results were consistent with qRT-PCR analysis of these genes (Fig. 4C). ISO, a well-known lipolytic agent, was used as a positive control. In differentiated 3T3-L1 adipocytes, ISO stimulated higher levels of glycerol release than that stimulated by EN. Moreover, both ISO and EN strongly induced PKA and HSL phosphorylation, but had no significant effects on the level of total HSL protein 14 (Fig. 4D). The densitometry data are shown in Figure 4E, F. The Western blotting data were consistent with qRT-PCR analysis of these genes (Fig. 4G) Fig. 4H); this was consistent with the qRT-PCR analysis of these genes (Fig. 4I). The Western blotting results were confirmed by immunofluorescence analysis, which showed that EN treatment promoted p-HSL protein expression in adipocytes (Fig. 4J).

Effect of EN on AKT signaling pathways
The effect of EN on p-AKT protein level in cells was determined by Western blotting assay.
After EN treatment, p-PKA expression was significantly increased, whereas glycerol release was decreased (Fig. 5A-D). In the presence of the AKT inhibitor (MK), glycerol release from adipocytes was increased. However, in the presence of both EN and MK were combined, glycerol release from adipocytes was significantly inhibited ( Fig. 5E-F).

Effect of PKA-ca-silencing on lipolysis in 3T3-L1 cells
Compared with the untransfected control group, transfection of 3T3-L1 adipocytes with control siRNA had no significant effect on glycerol release (P > 0.05), whereas glycerol release was significantly increased in siRNA-mediated silencing of PKA-ca group after treatment with EN (P < 0.05). These results indicated that EN promotes the breakdown of intracellular TG (Fig. 6A). Western blot analysis showed that compared with the normal group, p-PKA protein expression was significantly inhibited by siRNA transfection (P<0.05), while p-HSL protein expression was significantly increased (P < 0.05). Immunofluorescence analysis showed that p-HSL protein expression was significantly increased compared with that in the control group (P < 0.05), while the expression of p-PKA protein was significantly increased (P < 0.05), indicating that PKA and HSL are involved in the regulation of lipolysis (Fig.   6B). Following siRNA-mediated silencing of PKA-ca, mRNA expression of cAMP, PKA-ca, PKA-cb and HSL in adipocytes treated with EN for 24 h was measured by qRT-PCR analysis. There was no significant change in PKA-cb expression, whereas PKA-ca expression was significantly inhibited. HSL expression was also inhibited.
However, there was no significant change in PKA-cb expression following the addition of EN (30 μM), whereas the expression of PKA-ca and HSL was up-regulated (Fig. 6C).

Effects of EN treatment on lipid profile in C57BL/6J mice
Serum TG levels were elevated in C57BL/6J mice fed HFD compared with control group. Intragastric administration of EN significantly decreased TG levels and increased serum glycerol release in mice fed HFD (Fig. 7A-B). Furthermore, H&E staining showed a reduced adipocyte size as well as a reduction in the number of LDs in the liver of C57BL/6J fed HFD following treatment with EN (Fig. 7C).

EN promoted cAMP/PKA activation in adipose tissues
We validated the effects of EN on the cAMP/PKA pathway in HFD-fed mice. In 16 accordance with the in vitro results, oral administration of EN promoted p-PKA induction in the adipose tissue of HFD-fed mice. In addition, PKA enzymatic activity assays showed that HFD feeding attenuated p-PKA activity in adipose tissue of HFD-fed mice, while EN administration promoted p-PKA activity (Fig. 8A-D).

Discussion
With the improvement in living standards, the incidence of obesity and related diseases has increased year by year, and the burden on families and society has also increased. Adipocyte metabolism and dysfunction play a pivotal role in obesity [36][37], which are currently focus of research. Abnormal expression of PKA phosphorylation has been identified in obese samples and closely related to lipid metabolism disorders [38][39][40]. The occurrence and development of obesity is inseparable from the accumulation level of TG, and the activation of protein kinases promotes TG decomposition in adipocytes. Adipocyte formation depends on pre-adipocyte differentiation. Thus, inhibiting the formation of adipocytes and promoting the decomposition of mature adipocytes is an effective method to induce weight loss. A recent study demonstrated that EN played a significant role in alleviating lipid accumulation and promoting lipolysis [41][42]. However, the role of EN in lipolysis and lipid accumulation is to be elucidated.  [49][50][51][52]. We speculated that the cAMP/PKA signaling pathway has a greater effect on lipolysis than the upregulation of AKT expression, such that EN promotes lipolysis. Our study also showed that EN had no effect on ATGL expression. PKA inhibition assays were conducted to verify that PKA is the major protein regulated by EN in promoting lipolysis. These studies confirmed that EN enhances the effects of H-89 inhibition.
The pathway culminates in regulation of HSL, so we visually observed the effect of EN on the expression of HSL by immunofluorescence analysis. Our experimental results are consistent with previous reports [53][54]. Finally, we performed siRNA-mediated silencing of PKA to confirm its role in the mechanism by which EN promotes lipolysis. PKA-ca expression was significantly inhibited, and HSL expression was also inhibited. There was no significant change in PKA-cb expression following the addition of EN, while expression of PKA-ca and HSL was promoted.
These results indicate that EN promotes lipolysis by acting on the PKA-ca site of PKA.

Conclusion
In this study, we showed that EN significantly attenuated lipid accumulation and differentiation in 3T3-L1 cells. Intervention with EN also promoted the expression of 19 the main transcriptional regulator PKA and HSL followed by upregulation of adipogenic-specific molecules including CAMP, ATGL, PKA-CA, PKA-CB, HSL and AKT at the mRNA and protein levels, probably via the cAMP/PKA signaling pathway. In addition, the administration of EN decreased body weight gain and adipose tissue hypertrophy induced by HFD in vivo. Moreover, supplementation of EN improved glucose clearance and decreased TG levels in HFD-fed mice.
Additionally, the development of hepatic steatosis was also significantly prevented in the obese mouse supplemented with EN. Collectively, our results strongly suggest the novel effect of EN in inhibiting adipogenesis, promoting lipolysis and the high therapeutic potential of EN in preventing the development of obesity.