Notes
Oleanolic acid induces lipolysis and antioxidative activity in 3T3-L1 adipocytes
2021 年 27 巻 3 号 p. 511-519
詳細
2021 年 27 巻 3 号 p. 511-519
Oleanolic acid (OA), a natural triterpenoid found in various edible plants possessing anti-adiposity and antioxidative activities, could be exploited against obesity, a serious global public health problem. To confirm these properties of OA and understand the underlying mechanisms, this study investigated its effects on lipolysis and antioxidative activities in 3T3-L1 adipocytes. The results demonstrated that OA stimulated lipolysis and phosphorylation of hormone-sensitive lipase (HSL) in 3T3-L1 adipocytes, which was blunted by a PKA inhibitor H-89. In addition, OA induced the expression of heme oxygenase-1 (HO-1) and reduced intracellular oxidative stress. HO-1 inhibitor treatment suppressed the antioxidative effect of OA but did not affect its lipolytic activity. These results indicate that OA exerts lipolytic activity through the PKA pathway and possesses antioxidative properties. These findings suggest the potential of OA as a useful candidate for the treatment of obesity and its related oxidative stress.
Obesity, a serious metabolic disorder, has become a global health problem in today's world (Malik et al., 2013). Obesity increases oxidative stress and the production of inflammatory cytokines in adipose tissue, causing systemic metabolic disorders such as dyslipidemia, insulin resistance, and hypertension (Furukawa et al., 2004; Singla et al., 2010). Therefore, much attention has been paid to its therapeutic and preventive strategies, and the dietary strategy has been explored as a promising approach to reduce the health risk of obesity both at the pre-clinical and clinical stages. Therefore, there is an urgent need to search for new biologically active natural compounds from food materials that could reduce lipids in adipose tissues.
Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA) is a naturally occurring triterpenoid widely present in many plant species, such as olive leaves, olive pomace, grape skin, and mistletoe sprouts (Liu, 1995; Zhang et al., 2004; Pollier and Goossens, 2012). OA has been reported to exhibit various biological properties such as antioxidant, anti-inflammatory, anti-diabetic, anti-hyperlipidemic, and anti-cancer activities (Pollier and Goossens, 2012; Ayeleso et al., 2017; Sharma et al., 2018).
Triterpenes have been shown to have several potentials to regulate physiological activities such as differentiation, lipolysis, and redox regulation in adipocytes. Sung et al. (2010) reported that OA downregulated the key transcription factors for adipogenesis, peroxisome proliferator-activated receptor γ (PPARγ), and cytidine-cytidine-adenosinethymidine (CCAAT) enhancer-binding protein α (C/EBPα), and suppresses the differentiation of 3T3-L1 adipocytes. Ursolic acid and 18β-glycyrrhetinic acid have also been reported to stimulate lipolysis and induce phosphorylation of hormone-sensitive lipase (HSL) at Ser 563, the rate-limiting enzyme to hydrolyze triglycerides into monoglyceride and free fatty acids, and translocation of HSL to the surface of the lipid droplet in adipocytes (Li et al., 2010; Moon et al., 2012). However, the lipolysis effect of OA in 3T3-L1 adipocytes is mostly unclear.
Furthermore, several studies have reported the antioxidative properties of OA. For instance, OA has been shown to inhibit an environmental pollutant, polychlorinated biphenyls (PCBs)-induced oxidative stress and insulin resistance through HNF1b-mediated regulation of redox homeostasis in 3T3-L1 adipocytes (Su et al., 2018). In addition, in rat smooth muscle cells, OA and maslinic acid have been shown to protect the cells from oxidative stress through the induction of heme oxygenase-1 (HO-1) expression, an enzyme that degrades heme into biliverdin, free iron, and carbon monoxide, which can protect cells from oxidative damage (Feng et al., 2011; Qin et al., 2014). On the contrary, the studies opposing the antioxidative activities of OA have also been reported. OA and its related compounds have been reported to increase cellular oxidative stress, which might be involved in its anticancer activity (Chu et al., 2017; Castrejón-Jiménez et al., 2019). A derivative of OA has been shown to induce oxidative stress, consequently inhibiting cell proliferation and inducing apoptosis in breast cancer cells (Chu et al., 2017). In addition, it has also been reported that an increase in oxidative stress is associated with lipolysis (Ji et al., 2011; Hashimoto et al., 2013; Issa et al., 2018). Issa et al. (2018) have shown that NADPH oxidase (NOX), which produces superoxide and hydrogen peroxide, is induced in mature 3T3-L1 adipocytes stimulated with cytokines. The increased NOX activity consequently promotes lipolysis by regulating phosphorylation of HSL. Thus, changes in oxidative stress levels and redox state may affect lipolytic activity in 3T3-L1 adipocytes. However, it is unclear whether OA induces oxidative stress in 3T3-L1 adipocytes, as shown in cancer cells, and whether the modulation of cellular oxidative stress levels or redox state by OA affects OA-induced lipolysis in adipocytes.
Therefore, in this study, we investigated the effects of OA on lipolysis in 3T3-L1 adipocytes and examined its underlying mechanism. In addition, the study also investigated the antioxidative properties of OA. Here, we revealed that OA induces lipolysis through the PKA pathway and increases HO-1 expression and antioxidative activity. These results suggest that OA could improve the metabolic disorders associated with obesity and/or overweight.
Reagents Dexamethasone (DEX) and human recombinant insulin were purchased from FUJIFILM Wako Chemicals (Osaka, Japan). 3-Isobutyl-1-methylxanthine (IBMX) was obtained from Sigma-Aldrich (St. Louis, MO). OA and zinc protoporphyrin-9 (ZnPP) were obtained from Cayman Chemical (Ann Arbor, MI). H-89, a cell-permeable cAMP-dependent protein kinase inhibitor, and a primary antibody against HO-1 were acquired from Enzo Life Sciences (Farmingdale, NY). Primary antibodies against HSL and phospho-HSL (Ser563) were purchased from Cell Signaling Technology (Danvers, MA). A primary antibody against β actin antibody (AC-15) was obtained from Novus Biologicals (Littleton, CO). All other reagents were of analytical grade.
Cell culture and differentiation Mouse 3T3-L1 preadipocytes (Human Science Research Resources Bank, Osaka, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO) supplemented with 10 % bovine calf serum until confluent. Two days post-confluence, the medium was changed to DMEM supplemented with 10 % fetal bovine serum (FBS), 10 µg/mL insulin, 250 nM DEX, and 0.5 mM IBMX. After 3–4 days (Day 0), the medium was replaced with DMEM supplemented with 10 % FBS and 5 µg/mL insulin and changed every 2 days for the next 6 days (Day 6).
Glycerol release assay 3T3-L1 preadipocytes were differentiated in a 24-well culture plate (5 × 104 cells/well) as described above. The differentiated cells (Day 6) were then washed with PBS and incubated with 50 µM OA in DMEM for the indicated times in figure legends. The media in each well were collected and stored at −20 °C until further analysis. Glycerol concentrations in each medium were determined using a quantitative enzymatic colorimetric assay kit (Triglyceride E-Test, FUJIFILM Wako Chemicals) according to the manufacturer's instructions. The level of released glycerol in each medium was normalized to that of the protein content of the cell lysate.
LDH-release assay We tested whether OA induces damages in the differentiated 3T3-L1 adipocytes through lactate dehydrogenase (LDH)-release assay. Briefly, the differentiated cells (Day 6) were treated with various concentrations (0–50 µM) of OA. After 24 h incubation, the media were collected, and LDH activities in the media were determined using an LDH activity analysis kit (Cytotoxicity Test Wako, FUJIFILM Wako Chemicals) according to the manufacturer's instructions. The level of cell damage in each treatment was expressed as a relative value with the LDH activity in the media from cells treated with 0.1 % Tween 20.
Determination of ROS production Intracellular ROS levels were assessed by measuring the oxidation of 2,7-dichlorofluorescein diacetate (DCFH-DA) according to a previously described method (Nagami et al., 2017). Briefly, the differentiated cells (Day 4) were pretreated with 50 µM OA in DMEM supplemented with 10 % FBS and 5 µg/mL insulin in the absence or presence of 10 µM ZnPP for 48 h in a 24-well black plate (5 × 104 cells/well, lumox multiwell, Sarstedt, Nümbrecht, Germany), and then labeled with 10 µM DCFH-DA (Sigma-Aldrich) in DMEM supplemented with 0.1 % bovine serum albumin (BSA) for 45 min. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using an Infinite F200PRO plate reader (TECAN, Grödig, Austria).
Western blotting The differentiated cells (2.5 × 105 cells/35 mm dish, Day 6) were washed once with PBS and incubated with various concentrations of OA in serum-free DMEM for the indicated time in figure legends. The medium was removed and the cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer solution (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1 % SDS, 1 % Triton-X, 1 % deoxycholic acid, 0.5 mM Na3VO4, and 5 mM EDTA). Cell extracts were prepared by centrifugation for 10 min at 20,000 × g at 4 °C to remove cell debris. Protein concentrations were measured as described previously (Nagami et al., 2017). Equal amounts of proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The membrane was immersed in blocking buffer (5 % BSA in 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 1 mM EDTA, 0.1 % Tween 20 (TBS-T)) at room temperature for 1 h, and then incubated overnight at 4 °C with primary antibodies, followed by incubation with the appropriate secondary antibody, such as anti-rabbit, or anti-mouse IgG antibody conjugated with horseradish peroxidase at room temperature for 1 h. Immunoreactive signals were detected using ECL Western Blotting Substrate (GE Healthcare, Tokyo, Japan) and a Lumino Image analyzer (ImageQuant LAS 4000, GE Healthcare), and then analyzed with ImageJ software (NIH, Maryland, USA).
Statistical analysis Data are expressed as the mean ± standard error (SE). Data were analyzed by one-way ANOVA and Tukey's post hoc test (GraphPad InStat Software ver. 3.0a, GraphPad Software Inc., San Diego, CA, USA). Differences were considered statistically significant at p values below 0.05.
Oleanolic acid induces lipolysis in 3T3-L1 adipocytes We first investigated whether OA induces lipolysis in fully differentiated 3T3-L1 adipocytes. The cells (Day 6) were incubated with 0–50 µM OA for 24 h or with 50 µM OA for 0, 6, 12, and 24 h, and the amount of glycerol released into the medium was measured. As shown in Figs. 1A and 1B, treatment with OA increased the release of glycerol into the culture medium in a concentration- and time-dependent manner. To examine whether OA-induced glycerol release was due to cellular damage, we performed an LDH assay in the medium in which adipocytes were cultured with various concentrations of OA for 24 h. As shown in Fig. 1C, OA had no cytotoxic effect against adipocytes at these concentrations in the present study.
Effects of oleanolic acid (OA) on glycerol release and cell damage.
Differentiated 3T3-L1 adipocytes (Day 6) were incubated with various concentrations of OA (0–50 µM) in serum-free DMEM for 24 h (A) or 50 µM of OA for 0, 6, 12, and 24 h (B). Glycerol content in the media was measured using a glycerol quantification kit as described in Materials and Methods. For the cytotoxicity assay (C), the cells were treated with OA (0–50 µM) or 0.1 % Tween 20 (positive control) for 24 h. Cell damage was evaluated using an LDH activity analysis kit as described in Materials and Methods. Values are expressed as mean ± SE (n = 3–4). Significant differences between values are indicated with different superscript alphabets (p < 0.05).
Effect of oleanolic acid on phosphorylation of hormone-sensitive lipase (HSL) HSL hydrolyzes diacylglycerols to monoacylglycerols in adipocytes, and its activity has been reported to be regulated by phosphorylation (Frühbeck et al., 2014). Therefore, we investigated the effects of OA on phosphorylation of HSL in 3T3-L1 adipocytes. Treatment of the cells (Day 6) with 50 µM OA for different time points (0–24 h) followed by western blotting with anti-pHSL Ser563 and anti-HSL antibodies revealed phosphorylation of HSL within 6 h upon stimulation of OA, which was reduced thereafter until 24 h (Fig. 2A).
Time course of OA-induced HSL phosphorylation, and the effect of a PKA inhibitor (H-89) on OA-induced lipolysis.
The cells (Day 6) were treated with 50 µM OA for 0, 6, 12, 18, and 24 h. Cell lysates were prepared as described in Materials and Methods. The changes in phosphorylated HSL levels in response to OA treatment were analyzed by western blotting with anti-pHSL Ser563 and anti-HSL antibodies (A). The cells (Day 6) were treated with 50 µM OA alone or in combination with 20 µM H-89. After 6 h of treatment, cells were lysed, and the phosphorylated HSL level was analyzed by western blotting (B). After 24 h of treatment, glycerol content in the media was quantified (C). Values are expressed as mean ± SE (n = 4). Significant differences between values are indicated with different superscript alphabets (p < 0.05).
Activation of the protein kinase A pathway is involved in oleanolic acid-induced lipolysis in 3T3-L1 adipocytes cAMP-dependent protein kinase (PKA) can regulate HSL activity by phosphorylation at serine residues, thereby promoting the translocation of HSL from the cytosol to the lipid droplet surface to induce hydrolysis of lipids (Holm, 2003; Martin et al., 2009). Therefore, as a next step, to investigate whether activation of the PKA pathway is involved in OA-induced lipolysis, differentiated 3T3-L1 adipocytes were treated with 50 µM OA in the absence or presence of 20 µM H-89, a PKA inhibitor, and phosphorylation of HSL and glycerol contents in the media were analyzed. As shown in Figs. 2B and 2C, treatment with H-89 suppressed OA-induced phosphorylation of HSL and reduced the released glycerol level in the media. These results indicate that OA induces lipolysis through activation of the PKA-dependent pathway in 3T3-L1 adipocytes.
OA induces antioxidative activity through the expression of HO-1, which seems to be not associated with its lipolytic activity Next, we investigated whether OA regulates cellular oxidative stress levels in 3T3-L1 adipocytes. Treatment with OA (0–50 µM) for 48 h induced the expression of HO-1, an anti-oxidative enzyme, in a concentration-dependent manner (Fig. 3A). Furthermore, intracellular oxidative stress was reduced by OA treatment, which was blunted in the presence of ZnPP, an HO-1 inhibitor, as shown in Fig. 3B, indicating the implications of OA in the antioxidative activity. Further, to decipher the effects of the expression of HO-1 on OA-induced lipolysis activity, the amount of glycerol released into the media was measured. As shown in Fig. 3C, there was no significant difference between the levels of released glycerol from cells treated with OA in the presence or absence of ZnPP. These results indicated that OA exerts antioxidative activity by expression of HO-1 and OA-induced lipolysis could be independent of cellular oxidative stress levels in mature 3T3-L1 adipocytes.
Effect of OA on HO-1 expression and intracellular oxidative stress, and the effect of an HO-1 inhibitor (ZnPP) on OA-induced lipolysis.
The cells (Day 4) were treated with OA (0–50 µM) for 48 h in DMEM supplemented with 10 % FBS and 5 µg/mL insulin. Cells were lysed, and HO-1 protein expression was analyzed by western blotting with anti-HO-1 antibody (A). After 48 h of treatment with OA alone or in combination with 10 µM ZnPP, intracellular oxidative stress was measured as described in Materials and Methods (B). The cells (Day 4) were treated with 50 µM OA in DMEM supplemented with 10 % FBS and 5 µg/mL insulin in the absence or presence of 10 µM ZnPP. After 48 h, the cells were treated with 50 µM OA alone or in combination with 10 µM ZnPP in serum-free DMEM. After 24 h, the media were collected, and the media's glycerol content was determined as described in Materials and Methods (C). Values are expressed as mean ± SE (n = 4–5). Significant differences between values are indicated with different superscript alphabets (p < 0.05). In (B), p values between the two groups are indicated.
The present study demonstrates that OA stimulates HSL activity through the PKA pathway, thereby inducing lipolysis in differentiated 3T3-L1 adipocytes. In addition, OA also increases the expression of HO-1, consequently upregulating the anti-oxidative activity. Although the biological effects of OA in 3T3-L1 adipocytes have not been fully investigated, treatment with OA during the differentiation process has been shown to suppress the differentiation of 3T3-L1 preadipocytes to mature adipocytes by reducing the expression of key transcription factors for adipogenesis (Sung et al., 2010). Furthermore, Loza-Rodríguez et al. (2020) have shown that OA treatment increases the expression of PPARα mRNA, a regulator of β-oxidation in mature 3T3-L1 adipocytes, while they did not observe any changes in lipolytic activity. In contrast, here we demonstrate that OA induces PKA-mediated phosphorylation of HSL, consequently increasing the lipolysis in 3T3-L1 adipocytes. The findings of the present study are consistent with the findings of Moon et al. (2012), which have shown that 18β-glycyrrhetinic acid, a related compound of OA, stimulates lipolysis in 3T3-L1 adipocytes.
HSL activity is driven by phosphorylation of the serine residue by PKA. It has been reported that several compounds from traditional medicines induce lipolysis in association with phosphorylation of HSL at Ser563 in adipocytes, and H-89, a PKA inhibitor reduces the activity of HSL (Ge et al., 2018; Lee et al., 2019). In the present study, we observed similar results in adipocytes treated with OA. Furthermore, ursolic acid, a related compound of OA, has been shown to induce lipolysis by activating the cAMP-dependent PKA pathway (Li et al., 2010). Therefore, it was speculated that OA and its related triterpenes might be similarly activating PKA. It has also been reported that betulinic acid, a related triterpene of OA, restored hippocampal cAMP levels by its inhibitory effect of phosphodiesterase (PDE) 4, a cAMP degrading enzyme (Kaundal et al., 2018). It is reported that PDE4 is expressed in 3T3-L1 cells (Oknianska et al., 2007). Thus, PDE inhibition might be involved in OA-induced lipolysis in the present study although further studies are needed to decipher the precise mechanism of PKA activation by OA.
Furthermore, it has been reported that oxidative stress induced by an OA derivative is involved in anticancer activity and oxidative stress is responsible for stimulation of lipolytic activity by cytokines in adipocytes (Chu et al., 2017; Issa et al., 2018). Therefore, in this study, we attempted to understand the association between OA-induced lipolysis and oxidative stress. The results demonstrated that OA induced HO-1 expression, and the level of intercellular ROS is not associated with OA-induced lipolytic activity. In an in vivo study, OA and glycyrrhizin have been shown to stimulate antioxidative enzymes downstream of the Nrf2 pathway, thereby ameliorating obesity-induced oxidative stress in the liver of obese model animals and decreasing adipose tissue weight (Wang et al., 2013; Abo El-Magd et al., 2018). However, whether the expressions of HO-1 and/or other antioxidative enzymes are directly involved in the decrease of adipose tissues are yet to be explored. To our knowledge, this is the first report to show HO-1 expression induced by OA, thereby exerting antioxidative activity in 3T3-L1 adipocytes. Taken together, it can be inferred that OA exerts the antiobesity effect by lipolysis through activation of the PKA pathway and antioxidative activity by HO-1 expression; therefore, it could be a beneficial compound against obesity and its associated metabolic dysregulation.
The bioavailability of OA and its related compounds is low because of poor aqueous solubility and permeability through biological membranes. A maximal plasma concentration of OA in rats has been reported to reach 0.29 ± 0.27 µM at 21 ± 17 min after oral administration of 50 mg/kg with an absolute oral bioavailability of 0.7 % (Jeong et al., 2007; Tong et al., 2008), indicating its poor absorption and possibility of extensive metabolic clearance. Meanwhile, several studies have reported that oral intake of OA regulates lipid and glucose metabolism in vivo. In obese mice, feeding pomace olive oil containing triterpenic acids such as OA and maslinic acid for 10 weeks reduced body weight, insulin resistance, and inflammation of adipose tissue (Claro-Cala et al., 2020). OA, as an isolated triterpene, has also been shown to improve glucose tolerance and reduce visceral adiposity in mice fed a high-fat diet for 15 weeks (de Melo et al., 2010). These results suggested that continuous OA intake might exert an antiobesity effect in vivo by increasing the lipolytic activity in adipose tissues, further supporting the findings of the present study.
In conclusion, OA-induced PKA-mediated phosphorylation of HSL increases the lipolysis in mature 3T3-L1 adipocytes. Furthermore, we revealed that OA enhances the expression of HO-1, thereby exerting antioxidative activity. In addition, we found that there is no significant association between modulation of intracellular ROS level by HO-1 expression and OA-induced lipolytic activity in 3T3-L1 adipocytes. Overall, these results indicate the potential of the OA to be effectively used to complement the conventional diet based therapies against obesity and oxidative stress associated with overweight. However, further animal studies are required to provide a more in-depth understanding of the effects of OA on these metabolic disorders.
