Sudachitin and Nobiletin Stimulate Lipolysis via Activation of the cAMP/PKA/HSL Pathway in 3T3-L1 Adipocytes

Polymethoxyflavones are flavonoids that are abundant in citrus fruit peels and have beneficial effects on human health. Previous studies have demonstrated that the polymethoxyflavones, namely sudachitin and nobiletin, ameliorate obesity and diabetes in humans and rodents. Although nobiletin induces lipolysis in adipocytes, lipolytic pathway activation by sudachitin has not been clarified in adipocytes. In this study, the effect of sudachitin on lipolysis was elucidated in murine 3T3-L1 adipocytes. Glycerol release into the medium and activation of the cyclic AMP (cAMP)/protein kinase A (PKA)/hormone-sensitive lipase (HSL) pathway was evaluated in 3T3-L1-differentiated adipocytes. Treatment with sudachitin and nobiletin for 24 and 48 h did not induce cytotoxicity at concentrations of up to 50 μM. Sudachitin and nobiletin at concentrations of 30 and 50 μM increased intracellular cAMP and medium glycerol levels in 3T3-L1 adipocytes. Western blotting revealed that sudachitin and nobiletin dose-dependently increased protein levels of phosphorylated PKA substrates and phosphorylated HSL. Sudachitin- and nobiletin-induced glycerol release, phosphorylation of PKA substrates, and HSL phosphorylation were suppressed by pharmacological inhibition of adenylate cyclase and PKA. These findings indicated that sudachitin, similar to nobiletin, exerts anti-obesogenic effects, at least in part through the induction of lipolysis in adipocytes.


Introduction
Overweight and obesity are global public health challenges that have increased worldwide [1]. White adipose tissue (WAT) is the primary tissue responsible for lipid storage. Increased consumption of Western-style diets results in overnutrition, thereby leading to excess lipid accumulation in WAT [2]. Hypertrophic adipocytes accumulate in obese adipose tissue and cause systemic metabolic dysfunction mediated by insulin resistance and inflammatory adipokine secretion [3].

Oil Red O Staining
Oil red O staining for the quantification of intracellular lipids was performed as described previously, with some modifications [37]. At day 8, mature 3T3-L1 adipocytes were treated with 50 µM sudachitin and nobiletin. After 48 h, adipocytes were washed thrice with phosphate-buffered saline (PBS) and fixed with 10% neutral-buffered formalin at 25 • C for 20 min. After washing thrice with PBS, the cells were incubated with oil red O solution at 25 • C for 20 min. After washing thrice with PBS, representative images of the stained adipocytes were captured using a BioZero BZ-X810 microscope (Keyence, Osaka, Japan). For quantification, the absorbance of extracted oil red O was detected using a microplate reader (iMark Microplate Reader, BioRad, Hercules, CA, USA) at 490 nm.

LDH Activity Assay
LDH release to the culture medium was measured to assess cell damage [38]. 3T3-L1 adipocytes on day 8 were treated with different sudachitin and nobiletin concentrations (1-50 µM). Adipocytes treated with 0.1% DMSO served as vehicle controls. After 24 and 48 h, the media were collected from each well, and LDH activity was measured using the LDH-Cytotoxic Test Wako (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) according to the supplier's instructions. Briefly, 50 µL of coloring solution was added to 50 µL of the collected media and incubated at 25 • C for 45 min. After dispensing 100 µL of 1 M HCl, the absorbance of the mixture was immediately measured at 570 nm using an iMark. Cytotoxicity was calculated using the following formula: Cytotoxicity (%) = (Abs in medium of cells treated with sudachitin or nobiletin)/(Abs in medium of cells treated with DMSO) × 100. LDH activity was calculated relative to the absorbance of DMSOtreated adipocytes.

Glycerol Release Assay
3T3-L1 adipocytes on day 8 were treated with 0.1% DMSO, 50 µM sudachitin, 50 µM nobiletin, or 10 µM isoproterenol (positive control) for 3 h. In pharmacological inhibition experiments, the cells were incubated for 1 h with 100 µM SQ22536 or 20 µM H-89 before treatment with sudachitin, nobiletin, or isoproterenol. At the end of each experiment, media were collected from each well to determine the concentration of free glycerol using the EnzyChrom TM Glycerol Assay Kit (BioAssay Systems, Hayward, CA, USA) according to the supplier's instructions. Briefly, 100 µL of working reagent containing glycerol kinase and glycerol phosphate oxidase was added to 10 µL of the collected media and incubated at 25 • C for 20 min. The absorbance of the reaction mixture was detected at 570 nm using an iMark.

Intracellular cAMP Levels Assay
3T3-L1 adipocytes on day 8 were treated with 0.1% DMSO, 30 µM sudachitin, 30 µM nobiletin, or 10 µM isoproterenol (positive control) for 15 min. After aspirating the culture medium, the adipocytes were treated with 0.1 M HCl and incubated at 25 • C for 20 min to completely lyse the adipocytes. Cell extracts were separated by centrifugation (MX-200, TOMY Seiko Co., Ltd., Tokyo, Japan) for 10 min at 1000× g, and the diluted supernatant was used in the assay. Intracellular cAMP levels were assessed using a Cyclic AMP ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the supplier's instructions.

Statistical Analysis
All data are shown as means ± standard error of the mean (SEM) and were statistically analyzed using Excel-Toukei 2010 (Social Survey Research Information Co., Ltd., Osaka, Japan). One-way or two-way analysis of variance was followed by the Tukey-Kramer multiple-comparison test. p < 0.05 indicated a statistically significant difference.

Cytotoxicity of Sudachitin and Nobiletin on 3T3-L1 Adipocytes
To determine the cytotoxicity of sudachitin and nobiletin on 3T3-L1 adipocytes, an LDH assay was performed, wherein the LDH released into the medium was quantified 24 and 48 h after treatment. There were no significant differences in LDH content in the medium among DMSO-, sudachitin-, and nobiletin-treated cells 24 and 48 h after treatment ( Figure 1B,C). LDH content in the media containing 50 µM nobiletin-treated cells was 11% higher, but it was not significantly different from that of DMSO-treated cells after 24 h of treatment ( Figure 1B). LDH content in the media containing cells treated with 10, 30, and 50 µM nobiletin was 15%, 19%, and 18% higher, respectively, than that of DMSO-treated cells after 48 h of treatment ( Figure 1C). Sudachitin and nobiletin showed no cytotoxicity against 3T3-L1 adipocytes at 1-50 µM.

Sudachitin and Nobiletin Promoted Glycerol Release from 3T3-L1 Adipocytes
The levels of lipid storage were assessed in 3T3-L1 adipocytes treated with 50 µM sudachitin or nobiletin for 48 h. The lipid droplets in 3T3-L1 adipocytes decreased after treatment with sudachitin or nobiletin ( Figure 2A). Lipid accumulation was 14% lower in adipocytes treated with nobiletin than in those treated with DMSO ( Figure 2B). Sudachitin tended to reduce lipid accumulation when compared to cells treated with DMSO (p = 0.07). To elucidate the effects of sudachitin on lipolysis, the amount of glycerol in the 3T3-L1 adipocyte medium was assessed. Treatment with isoproterenol, as a positive control, resulted in a 6.2-fold increase in the medium concentrations of glycerol when compared to those treated with DMSO ( Figure 2C). Treatment with nobiletin induced the release of glycerol from 3T3-L1 adipocytes, which is consistent with a previous report [36]. Sudachitin increased the concentration of glycerol in the medium by 2.0-fold when compared to that of cells treated with DMSO ( Figure 2C). The levels of intracellular cAMP were also significantly upregulated by treatment with isoproterenol, sudachitin, and nobiletin when compared to treatment with DMSO ( Figure 2D). glycerol from 3T3-L1 adipocytes, which is consistent with a previous report [36]. S dachitin increased the concentration of glycerol in the medium by 2.0-fold when co pared to that of cells treated with DMSO ( Figure 2C). The levels of intracellular cAM were also significantly upregulated by treatment with isoproterenol, sudachitin, and n biletin when compared to treatment with DMSO ( Figure 2D).

Time Course of Sudachitin-and Nobiletin-Induced Phosphorylation of PKA Substrates an HSL in 3T3-L1 Adipocytes
Next, the protein levels of phosphorylated PKA substrates and HSL at Ser563 a Ser660 were measured in 3T3-L1 adipocytes treated with 30 µΜ sudachitin or nobile for 0-120 min ( Figure 3A,C). A significant increase in the protein levels of phosphorylat PKA substrates and phosphorylated HSL at Ser563 and Ser660 was detected within 5 m of sudachitin and nobiletin treatment ( Figure 3B,D). Protein levels of PKA substra phosphorylated and HSL phosphorylated at Ser660 were significantly higher in 3T3adipocytes treated with sudachitin and nobiletin for 5-120 min than those treated w DMSO. Treatment with sudachitin for 60 min resulted in a 1.6-fold increase in the prote levels of HSL phosphorylated at Ser563 when compared to those treated with DMSO (F ure 3B). Treatment with nobiletin for 5-120 min showed a 2.4-to-3.3-fold increase in p tein levels of HSL phosphorylated at Ser563 when compared to those treated with DMS for the corresponding time period ( Figure 3D).

Time Course of Sudachitin-and Nobiletin-Induced Phosphorylation of PKA Substrates and HSL in 3T3-L1 Adipocytes
Next, the protein levels of phosphorylated PKA substrates and HSL at Ser563 and Ser660 were measured in 3T3-L1 adipocytes treated with 30 µM sudachitin or nobiletin for 0-120 min ( Figure 3A,C). A significant increase in the protein levels of phosphorylated PKA substrates and phosphorylated HSL at Ser563 and Ser660 was detected within 5 min of sudachitin and nobiletin treatment ( Figure 3B,D). Protein levels of PKA substrates phosphorylated and HSL phosphorylated at Ser660 were significantly higher in 3T3-L1 adipocytes treated with sudachitin and nobiletin for 5-120 min than those treated with DMSO. Treatment with sudachitin for 60 min resulted in a 1.6-fold increase in the protein levels of HSL phosphorylated at Ser563 when compared to those treated with DMSO ( Figure 3B). Treatment with nobiletin for 5-120 min showed a 2.4-to-3.3-fold increase in protein levels of HSL phosphorylated at Ser563 when compared to those treated with DMSO for the corresponding time period ( Figure 3D).

Induction of Phosphorylation of PKA Substrates and HSL by Treatment with Sudachitin and Nobiletin in a Dose-Dependent Manner
We investigated whether sudachitin or nobiletin dose-dependently induced phosphorylation of PKA substrates and HSL at Ser563 and Ser660. Cells treated wit dachitin and nobiletin showed dose-dependent increases in the protein levels of PKA strates phosphorylated and HSL phosphorylated at Ser563 and Ser660 ( Figure 4A). In L1 cells treated with 20, 30, and 50 µΜ sudachitin, protein levels of phosphorylated  substrates were 3.2-, 3.5-, and 4.8-fold higher, respectively, than those from cells treated with DMSO ( Figure 4B). Treatment with 5, 10, 20, 30, and 50 µM nobiletin resulted in 2.4-, 3.2-, 4.4-, 4.8-, and 5.0-fold increases in the amount of phosphorylated PKA substrates, respectively. At 20 and 30 µM, the protein levels of phosphorylated PKA substrates were 39 and 38% higher, respectively, in adipocytes treated with nobiletin than in those treated with sudachitin. Protein levels of HSL phosphorylated at Ser563 increased 3.1-and 3.7-fold in cells treated with 30 and 50 µM sudachitin, respectively, when compared to those treated with DMSO ( Figure 4B). Protein levels of HSL phosphorylated at Ser563 also increased 5.3-, 7.8-, and 8.5-fold in adipocytes treated with 20, 30, and 50 µM nobiletin, respectively, when compared to those treated with DMSO. At 20, 30, and 50 µM, protein levels of HSL phosphorylated at Ser563 were 123%, 154%, and 130% higher, respectively, in adipocytes treated with nobiletin than in those treated with sudachitin. Protein levels of HSL phosphorylated at Ser660 increased 2.6-and 3.4-fold in adipocytes treated with 30 and 50 µM sudachitin, respectively, when compared to those treated with DMSO ( Figure 4B). Protein levels of HSL phosphorylated at Ser660 also increased by 2.6-, 4.6-, 5.9-, and 6.6-fold in adipocytes treated with 10, 20, 30, and 50 µM nobiletin, respectively, compared to those in adipocytes treated with DMSO. At 20, 30, and 50 µM, protein levels of HSL phosphorylated at Ser660 were 109%, 124%, and 97% higher, respectively, in adipocytes treated with nobiletin than in those treated with sudachitin.

Effects of AC and PKA Inhibition on Glycerol Release Induced by Sudachitin and Nobiletin
To better define the contribution of cAMP/PKA pathway activation to lipolysis i duced by sudachitin and nobiletin, the effects of pharmacological inhibition of AC an

Effects of AC and PKA Inhibition on Glycerol Release Induced by Sudachitin and Nobiletin
To better define the contribution of cAMP/PKA pathway activation to lipolysis induced by sudachitin and nobiletin, the effects of pharmacological inhibition of AC and PKA on glycerol release from adipocytes treated with sudachitin and nobiletin were investigated. Inhibition of AC and PKA did not affect the amount of glycerol released into the medium by DMSO-treated adipocytes ( Figure 5). Glycerol release induced by sudachitin, nobiletin, and isoproterenol was significantly suppressed by pretreatment with the PKA inhibitor H-89 and the AC inhibitor SQ22536. s 2023, 12, x FOR PEER REVIEW investigated. Inhibition of AC and PKA did not affect the amount of gly the medium by DMSO-treated adipocytes ( Figure 5). Glycerol relea dachitin, nobiletin, and isoproterenol was significantly suppressed by the PKA inhibitor H-89 and the AC inhibitor SQ22,536.

Effects of AC and PKA Inhibition on Phosphorylation of PKA Substrates by Sudachitin and Nobiletin
The effects of pharmacological inhibition of AC and PKA on PK phorylation and HSL phosphorylation at Ser563 and Ser660 induced nobiletin in 3T3-L1 adipocytes were also assessed ( Figure 6A,C). Phosp substrates and HSL at Ser563 and Ser660, stimulated by sudachitin and duced by pretreatment with SQ22,536 ( Figure 6B) and H-89 ( Figure 6D effects of pharmacological inhibition of β3-AR, an upstream receptor o PKA substrate phosphorylation and HSL phosphorylation at Ser563 an termined. Pretreatment with L-748,337, a β3-AR-selective antagonist, h dachitin-or nobiletin-induced phosphorylation of PKA substrates and Ser660, respectively (Supplementary Figure S1).

Effects of AC and PKA Inhibition on Phosphorylation of PKA Substrates and HSL Induced by Sudachitin and Nobiletin
The effects of pharmacological inhibition of AC and PKA on PKA substrate phosphorylation and HSL phosphorylation at Ser563 and Ser660 induced by sudachitin and nobiletin in 3T3-L1 adipocytes were also assessed ( Figure 6A,C). Phosphorylation of PKA substrates and HSL at Ser563 and Ser660, stimulated by sudachitin and nobiletin, was reduced by pretreatment with SQ22536 ( Figure 6B) and H-89 ( Figure 6D). In addition, the effects of pharmacological inhibition of β3-AR, an upstream receptor of AC and PKA, on PKA substrate phosphorylation and HSL phosphorylation at Ser563 and Ser660 were determined. Pretreatment with L-748,337, a β3-AR-selective antagonist, had no effect on sudachitin-or nobiletin-induced phosphorylation of PKA substrates and HSL at Ser563 and Ser660, respectively (Supplementary Figure S1).
Herein, the effects of sudachitin on lipolysis and cAMP/PKA/HSL pathway activation in 3T3-L1 adipocytes are examined. Sudachitin and nobiletin induced glycerol release into the medium and increased the levels of intracellular cAMP, phosphorylated PKA substrates, and phosphorylated HSL. Pharmacological inhibition of AC and PKA prevented lipolysis and phosphorylation of PKA substrates and HSL stimulated by sudachitin and nobiletin. These findings support the notion that both nobiletin-and sudachitin-induced lipolysis are mediated by the activation of the cAMP/PKA/HSL pathway.
Sudachitin exerts anti-obesogenic effects in rodents and humans [25][26][27]. A previous study demonstrated that sudachitin suppresses HFD-induced obesity by stimulating mitochondrial biogenesis in myocytes. In a human study, supplementation with extracts of sudachi peel, including sudachitin, decreased the ratio of abdominal WAT to subcutaneous WAT when compared to supplementation with a placebo, thus resulting in a reduction in the risk of diabetes [25]. In humans, lipolysis and HSL activity are higher in visceral omental WAT than in subcutaneous WAT [41]. The current findings suggest that sudachitin decreased visceral fat by activating the cAMP/PKA/HSL pathway and adipocyte lipolysis.
Lipolysis is partly regulated by intracellular cAMP concentrations in response to lipolytic agents and hormones [42]. In the present study, sudachitin and nobiletin increased intracellular cAMP concentrations, which were inhibited by the AC inhibitor SQ22536 in 3T3-L1 adipocytes. These results indicate that the cAMP-dependent PKA pathway plays an important role in the lipolytic effect of sudachitin and nobiletin. However, the molecular mechanism of sudachitin-and nobiletin-induced increase in intracellular cAMP levels remains unclear. Pharmacological inhibition of β3-AR failed to reduce protein levels of phosphorylated PKA substrates and HSL in 3T3-L1 cells treated with sudachitin or nobiletin. It remains unknown whether HSL phosphorylation occurs through the activation of specific receptors or AC alone, thereby increasing cAMP levels and increasing PKA activity. Isoproterenol increases intracellular cAMP concentrations to promote lipolysis, whereas phosphodiesterase (PDE) activation hydrolyzes cAMP to inhibit PKA activity and lipolysis [42]. Nobiletin significantly reduces PDE activity at more than 100 µM in PC12D neuronal cells [43]. Insulin induces the activation of PDE3B in adipocytes in an Aktdependent manner [44]. Herein, sudachitin reduced protein levels of the phosphorylated Akt in 3T3-L1 adipocytes [23,24]. Further experiments are required to elucidate the effects of sudachitin on PDE activity in adipocytes.
We demonstrated that sudachitin and nobiletin induced lipolysis through the cAMP/ PKA/HSL pathway. ATGL is also a key lipase and regulates both basal and beta-adrenergicstimulated lipolysis and catalyzes the first step in triglyceride hydrolysis, thereby resulting in diglyceride and fatty acid formation in adipocytes [45]. Perilipins are lipid droplet-related proteins that protect triglycerides from lipolysis [46]. PKA-induced phosphorylation of perilipin A at Ser517 is necessary for ATGL-dependent hydrolysis of triacylglycerol [47]. Perilipin binds to CGI-58, a co-regulator of ATGL activity [48], resulting in the suppression of ATGL activity. PKA-mediated phosphorylation of CGI-58 releases CGI-58 from perilipin [49]. These findings suggest that ATGL and HSL are involved in sudachitin-and nobiletin-induced lipolysis in 3T3-L1 cells.
Polymethoxyflavones exert various health effects. Nobiletin reduces inflammatory responses mediated by the induction of heme oxygenase-1, which is the primary antioxidant enzyme, expressed in 3T3-L1 and RAW264.7 cells [13]. Brite adipocytes are derived from white adipocytes and have an increased capacity for fatty acid oxidation [50]. 3T3-L1 adipocytes treated with 100 µM nobiletin show a brown fat-like phenotype, with an increased expression of Ucp1 and fatty-acid-oxidation-associated genes [15]. Sinensetin (5,6,7,3 ,4 -pentamethoxyflavone), a rare polymethoxyflavone found in certain citrus fruits, induces phosphorylation of PKA and HSL by increasing intracellular cAMP concentrations in 3T3-L1 adipocytes [51,52]. These results are consistent with sudachitin-induced lipolysis mediated by the activation of the cAMP/PKA/HSL pathway. The current findings demonstrate that nobiletin is a more potent inducer of the phosphorylation of PKA substrates and HSL than sudachitin. Sudachitin is structurally similar to nobiletin, but the three methoxy groups in nobiletin are replaced by hydroxy groups [18]. The previous study demonstrated that methylation of flavonoids increases in metabolic stability and cell membrane permeability [53]. The methoxy groups at the C-5, C-7, and C-4 positions remarkably suppressed metabolic depletion in human liver homogenate and increased permeability in Caco-2 cells, the model of human intestinal absorption, when compared to the hydroxy groups at these positions [53]. These findings indicate that the methoxy groups at the C-5, C-7, and C-4 positions are involved in the enhancement of the lipolytic action of nobiletin. 5-Hydroxy-3,6,7,8,3 ,4 -hexamethoxyflavone is found exclusively in Citrus sinensis [6]. The methoxy group at the C-5 position in nobiletin was replaced with a hydroxy group, and the C-3 hydrogen was replaced with a methoxy group. 5-Hydroxy-3,6,7,8,3 ,4 -hexamethoxyflavone increases intracellular cAMP levels and PKA activity in PC12 neuronal cells [54]. Thus, the number of methoxy groups and the methoxy groups in the C-7 and C-4 positions may be associated with enhanced PKA activity induced by polymethoxyflavones in adipocytes.
In conclusion, these data demonstrate that sudachitin stimulates lipolysis in 3T3-L1 adipocytes. The induction of lipolysis by sudachitin and nobiletin was mainly mediated by the activation of the cAMP/PKA/HSL pathway. Notably, the lipolytic activity exerted by sudachitin may represent an effective mechanism for ameliorating obesity and related metabolic disorders.
Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/foods12101947/s1, Figure S1: Effects of β3-adrenergic receptor-selective antagonist on sudachitin-and nobiletin-induced phosphorylation of the PKA substrate and HSL.  Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries should be addressed to the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.