Anti-obesity effects of Celastrus orbiculatus extract containing celastrol on canine adipocytes

Abstract

From 50 to 60% of companion animals in the United States are overweight or obese and this obesity rate is rising. As obesity is associated with a number of health problems, an agent that can help weight loss in pets and assist in clinically managing obesity through veterinary prescription foods and medication would be beneficial. Many studies have shown that celastrol, a phytochemical compound found in Celastrus orbiculatus extract (COE), has anti-obesity and anti-inflammatory effects, although these effects have not yet been determined in canine or canine-derived cells. The objective of this study was to investigate the effects of celastrol on the adipogenic differentiation and lipolysis of canine adipocytes. Primary preadipocytes were isolated from the gluteal region of a beagle dog and the primary adipocytes were differentiated into mature adipocytes by adipocyte differentiation media containing isobutylmethylxanthine, dexamethasone, and insulin. In a water-soluble tetrazolium (WST) assay, the cell viability of mature adipocytes was decreased after treatment with COE (0, 0.93, 2.32, and 4.64 nM celastrol) in a concentration-dependent manner, although preadipocytes were not affected. Oil Red O (ORO) staining revealed that COE inhibited the differentiation into mature adipocytes and lipid accumulation in adipocytes. In addition, treatment with COE significantly reduced triglyceride content and increased lipolytic activities by 1.5-fold in canine adipocytes. Overall, it was concluded that COE may enhance anti-obesity activity in canine adipocytes by inhibiting lipid accumulation and increasing lipolytic activity.

Introduction

Obesity is the most common nutritional and preventable disorder in companion animals. In 2018, the Association for Pet Obesity Prevention estimated that 60% of cats and 56% of dogs in the United States were overweight or obese and these obesity rates are gradually rising (petobesityprevention.org). Obesity is usually the result of either excessive dietary intake or lack of exercise, which cause a state of positive energy balance and weight gain (1,2). Obesity has a harmful effect on the health and longevity of companion animals and can lead to many types of disease, such as type-2 diabetes, abnormalities in circulating lipid profiles, heart disease, kidney disease, reproductive disorders, neoplasia, and hormonal changes (3–5).

As obesity in pets is usually caused by excessive food intake or lack of exercise, treatment options include dietary management and increased physical activity (6). It is therefore necessary to discover and investigate an agent that can help weight loss in pets, as well as clinically manage obesity with veterinary prescription food and medication. As a serious concern in veterinary medicine, there is a need to raise awareness of obesity in companion animals.

Celastrol is the main chemical component of Celastrus orbiculatus and Tripterygium wilfordii (Thunder god vine) (7). It is a pentacyclic triterpenoid that is 24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid, bearing an oxo substituent at position 2, a hydroxy substituent at position 3, and 2 methyl groups at positions 9 and 13 [PubChem, National Center for Biotechnology Information of United States National Institutes of Health (NIH)]. Celastrol has been used in traditional Oriental medicine as a natural remedy to treat inflammation-related diseases (8).

Celastrol has recently been shown to have obesity-controlling effects in in-vitro and in-vivo studies. It was demonstrated that interleukin 1 receptor 1 (IL1R1)-deficient mice are resistant to celastrol-induced leptin sensitization and anti-obesity, which means that celastrol increases the expression of IL1R1, resulting in anti-obesity effects via a pro-inflammatory signaling pathway (9).

In a previous study using the hyperleptinemic diet-induced obese mice model, celastrol suppressed food intake and blocked the reduction of energy expenditure, resulting in a weight loss of 45%, which indicates that celastrol is a leptin sensitizer and may be a promising drug for the pharmacological treatment of obesity (10). Moreover, celastrol induced hypophagia-driven weight loss (11) and showed a melanocortin 4 receptor (MC4R)-independent signaling antiobesity effect (12). In a study at the cellular level, celastrol showed an inhibitory effect, mediated by proliferator-activated receptor-γ (PPARγ), on adipogenic differentiation of human adipose-derived stem cells (13). Taken together, these studies indicate that celastrol has the potential to exert potent anti-obesity effects, as well as anti-inflammatory effects (10,13).

Although the anti-obesity effects of celastrol have been shown in a number of previous studies, these effects have not yet been determined in canine or canine-derived cells. The objective of this study was therefore to investigate the effects of celastrol on the adipogenic differentiation and lipolysis of canine adipocytes. We hypothesized that treatment with Celastrus orbiculatus extract (COE) containing celastrol could stimulate anti-obesity activity in canine adipocytes. To test this hypothesis, changes in the cell viability of primary canine adipocytes were evaluated under continuous culture with various concentrations of COE and the effects of COE on adipogenesis, lipid accumulation, and lipolytic activity were investigated.

Materials and methods

Preparation of Celastrus orbiculatus extract

Dried Celastrus orbiculatus extract (COE) was obtained from JEIL Feed (Daejeon, Republic of Korea). The amount of celastrol in COE was quantified by high performance liquid chromatography (HPLC) (Shimadzu Corporation, Kyoto, Japan) conducted by the Korea Quality Testing Institute (Suwon, Republic of Korea).

The operating conditions were as follows: UV/Vis Detector; wavelength: 425 nm; column oven temperature: 30°C; injection volume: 20 μL; mobile phase: Isocratic, 0.2% phosphoric acid:acetonitrile = 15:85. The celastrol content was 4.18 mg celastrol/100 g COE. Raw data for the analysis of celastrol using HPLC are shown in Figure 1.

Figure 1.

Analysis of content of celastrol in Celastrus orbiculatus (COE) using high performance liquid chromatography (HPLC). The amount of celastrol in COE was quantified by high performance liquid chromatography (HPLC) conducted by the Korea Quality Testing Institute. The operating conditions were as follows: UV/Vis Detector; wavelength: 425 nm; column oven temperature: 30°C; injection volume: 20 μL; mobile phase: Isocratic, 0.2% phosphoric acid:acetonitrile = 15:85. The celastrol content was calculated as 4.18 mg celastrol/100 g COE.

Approximately 5 g of dried powder of Celastrus orbiculatus root was finely ground in a sterile mortar, 100 mg of the ground Celastrus orbiculatus was put in a 1.5-mL microtube, and 1 mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, Missouri, USA) was added, then vigorously vortexed for 1 min. It was then extracted at room temperature for 24 h and this extract was filtered in turn onto 0.45-μm and 0.20-μm syringe filters. This extract was serially diluted to 0.5%, 1%, and 2.5% in DMSO and stored at room temperature before use in the experiments. The amount of COE added into culture media at different concentrations of celastrol is provided in Table I.

Table I.

Concentration of celastrol in Celastrus orbiculatus and amount of celastrol in medium.

Concentration of celastrol in Celastrus orbiculatus (%)	Amount of celastrol in medium (nM)
0.0010	0.93
0.0025	2.32
0.0050	4.64

Isolation of primary canine adipocytes

Approximately 50 g of adipose tissue from the gluteal region of a beagle dog (female, 3 y old, 9 kg) removed during a regular surgical procedure was received from the Veterinary Teaching Hospital of Chungbuk National University. This adipose tissue was transported in phosphate-buffered saline (PBS) with 1% penicillin-streptomycin (100 U/mL and 100 μg/mL respectively in media; Biowest, Nuaillé, France) (Figure 2A). The animal protocol to isolate canine adipose tissues was approved by the Institutional Animal Care and Use Committee (CBNUA-1337-20-02).

Figure 2.

Isolation and culture of primary canine adipocytes. A — Canine adipose tissues were isolated from gluteal region of a beagle dog and transported in PBS containing 1% penicillin-streptomycin. B — The tissues were collagenase-digested with collagenase type I and centrifuged to divide into 2 different phases; the supernatant contains primary adipocytes and the lower layer is the fluid portion containing stromal vascular fractions (SVFs). C — The minced and enzymatically digested adipose tissue was transferred to 75T flask fully filled with growth medium. The ceiling-side of the flask was coated with 0.1% gelatin and the adipocytes slowly moved to this side and adhered to it for 1 wk at 37°C in the humidified CO₂ incubator. The flask was turned over again and the 15 to 20 mL of medium remained and the rest was aspirated.

For the isolation, approximately 5 g of adipose tissue were minced and transferred to a silicon-coated 50-mL tube (NEST Biotechnology, Wuxi, China). After mincing, 0.2% collagenase type I solution (Gibco, Waltham, Massachusetts, USA) per gram of tissue was added for tissue digestion. The mixture was incubated at 37°C, 5% carbon dioxide (CO₂), for 30 min in a shaking incubator while vigorously shaking every 10 min. To stop enzyme activity, Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone Laboratories, Logan, Utah, USA) was added with components of heat-inactivated 10% (v/v) fetal bovine serum (FBS) (Rocky Mountain Biologicals, Missoula, Montana, USA), 1% penicillin-streptomycin (Biowest), and 1% HEPES.

The mixture was pipetted up and down to further disintegration of the adipose tissue. The digested tissue was centrifuged at 200 rpm for 5 s and the isolated adipocytes are shown in Figure 2B. Primary adipocytes floating in the white upper layer were transferred to a 0.1% gelatin-coated 75T flask (SPL Life Science, Seoul, Republic of Korea) at a cell density of 6 × 10⁴ cells/cm². Preadipocytes were isolated by centrifuging the fluid portion containing stromal vascular fractions (SVFs) for 10 min at 1000 rpm. These 2 types of primary cells were incubated at 37°C in the humidified CO₂ incubator.

Culture of primary canine adipocytes

For the culture of primary adipocytes, we refer to the method described in previous studies with slight modification (14,15). As shown in Figure 2C, primary adipocytes obtained by enzymatically digesting canine adipose tissue were transferred to a 75T flask completely filled with DMEM containing 10% FBS and the flask was then turned over. The adipocytes slowly moved to the 0.1% gelatin-coated ceiling side and adhered to the ceiling side for 1 wk. The flask was turned over again and the 15 to 20 mL of medium remained and the rest was aspirated. The primary adipocytes were incubated with DMEM containing 10% FBS at 37°C in the humidified CO₂ incubator. Preadipocytes isolated from the fluid portion were cultured with DMEM containing 10% FBS in 100-mm cell culture dishes.

Maturation of primary canine adipocytes

An experimental method for differentiating into mature adipocytes has been referred to in previous studies with slight modification (16–18). The primary adipocytes cultured in 75T flasks were collected with a cell scraper and resuspended using a 1-mL micropipette and these cells were seeded into 6-well plates or 96-well plates (SPL Life Science) for subsequent experiments.

To mature the adipocytes that formed lipid droplets, the following MDI (methylisobutylxanthine, dexamethasone, and insulin) medium and insulin medium were prepared. The MDI medium was prepared by adding 500 μM isobutylmethylxanthine (IBMX) (Sigma-Aldrich), 1 μg/mL insulin (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), and 10% FBS to DMEM. Insulin medium was prepared by adding 1 μg/mL insulin and 10% FBS to DMEM. The canine primary adipocytes isolated from adipose tissue of a beagle dog were cultured in MDI medium for 2 d and then cultured in insulin medium for 2 d to differentiate into adipocytes that form lipid droplets. The matured canine adipocytes were cultured in DMEM containing 10% FBS.

Cell viability assay (WST assay)

A water-soluble tetrazolium (WST) assay was carried out to measure the cell viability of adipocytes, as referred to in previous studies (19–21). The primary adipocytes or preadipocytes were seeded at a density of 10 000 cells/well in 96-well plates (SPL Life Science). The primary adipocytes were matured with MDI medium and insulin medium at 37°C in the humidified CO₂ incubator. The following treatment groups were prepared for each run of the experiment immediately before use: vehicle control, 0.93, 2.32, and 4.64 nM of the COE.

After 1 d of incubation for cell adhesion, the new medium containing COE was added to each well of the 96-well plate. The medium was replaced with fresh medium containing COE after 3 d and the cells were then incubated for an additional 3 d, for a total of 6 d incubation. All medium was then removed and EZ-Cytox (DoGen, Seoul, Republic of Korea), which produces WST, was added and incubated for 1 h at 37°C in the humidified CO₂ incubator. After incubation, the absorbance was measured at 450 nm using a microreader (Epoch; BioTek Instruments, Winooski, Vermont, USA).

Oil Red O (ORO) staining

Oil Red O (ORO) staining was used to detect lipid accumulation using a method referred to in previous studies with slight modification (22,23). The adipocytes were seeded in a 6-well plate and the cells were matured with MDI medium and insulin medium. The mature adipocytes were exposed to COE for 6 d. After washing and conditioning the cells with 60% isopropanol, 0.3% ORO (Sigma-Aldrich) working solution was added and incubated for 15 min. Lipid droplets stained in ORO were photographed under the phase-contrast microscope (IX73; Olympus, Tokyo, Japan).

For quantitative analysis of lipid droplets, 100% isopropanol was added to the cells stained with ORO and ORO was then extracted from adipocytes for 30 min using a plate shaker at room temperature. After the ORO extracted in isopropanol was transferred to a new 96-well plate, the absorbance was measured at 515 nm using a microreader (BioTek Instruments). The 100% isopropanol was used as a blank. The absorbance was normalized to cell viability data (WST assay) from each of the same treatment groups.

Triglyceride contents

The adipocytes were seeded in a 96-well plate and the cells were matured with MDI medium and insulin medium. The mature adipocytes were exposed to COE for 6 d. The triglyceride content in the adipocytes was analyzed using the Triglyceride Colorimetric Assay Kit (Cayman, Ann Arbor, Michigan, USA) according to the manufacturer’s protocol. Adipocytes were collected with a scraper and placed in a 1.5-mL tube, followed by resuspension with 1 mL of cold standard diluent. Cell suspensions were sonicated for 10 s and centrifuged at 10 000 g, 4°C for 10 min.

After removing the supernatant, the remaining solution was diluted by half with a standard diluent. Each concentration of triglyceride standard or cell lysates was loaded on a 96-well plate and triglyceride enzyme mixture was added to each well. The lid of the 96-well plate was closed, shaken gently, and incubated at room temperature for 15 min. The absorbance was measured at 540 nm using a microreader (BioTek Instruments) and normalized to cell viability data (WST assay) from each of the same treatment groups.

Measurement of lipolytic activity of adipocytes

The adipocytes were seeded in a 96-well plate and the cells were matured with MDI medium and insulin medium. The mature adipocytes were exposed to COE for 6 d. Lipolytic activity of adipocytes was analyzed using the Lipolysis Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, California, USA) according to the manufacturer’s protocol. Adipocytes cultured in a 96-well plate were washed twice with 100 μL/well of Adipocyte Wash Buffer and then centrifuged at 500 g for 10 min to remove the wash buffer. Adipocyte Lipolysis Buffer (150 μL/well) and 10 μM of isoproterenol (1.5 μL/well) were added and lipolysis in the adipocytes was activated for 3 h in an incubator at 37°C; 50 μL of this reaction solution and 50 μL of reaction mix were added to each well in a new 96-well plate. The plate was wrapped with foil and incubated for 30 min at room temperature. The absorbance was measured at 570 nm using a microreader (Epoch) and normalized to cell viability data (WST assay) from each of the same treatment groups.

The lipolytic activity was also measured by quantifying the content of free glycerol produced from adipocytes and analyzed similarly to the method previously described for using the Free Glycerol Colorimetric/Fluorometric Assay Kit (BioVision).

Statistical analysis

All experiments were run at least 3 times and all data presented as means ± standard error of the mean (SEM). Data from the experiments were statistically analyzed by 1-way analysis of variance (ANOVA), followed by a post-hoc Dunnett’s test using the GraphPad Prism 5.01 software (GraphPad Software, San Diego, California, USA). P-values < 0.05 were regarded as statistically significant and this data was marked with an asterisk.

Results

Isolation and adipogenesis of canine primary adipocytes

Primary adipocytes isolated from canine adipose tissues are able to produce lipid droplets relatively uniformly and consistently through the process of further maturing these cells. Primary adipocytes obtained from a beagle dog were transferred to a gelatin-coated 75T flask, filled with DMEM, and inverted. As shown in Figure 3, immediately after the cells were transferred to the flask, lipids and primary adipocytes floated (Figure 3A), but after 2 to 3 d, adipocytes stably adhered to the gelatin-coated ceiling side. At this time, adipocytes of various sizes could be observed under a microscope and morphology of typical adipocytes was confirmed with a small amount of lipid found inside the cells. The adipocytes were seeded into 6-well plates or 96-well plates for subsequent experiments and differentiated into mature adipocytes forming lipid droplets by MDI medium and insulin medium (Figure 3B, C). Under the microscope, spherical lipid droplets of various sizes could be found inside the mature adipocytes and these gradually accumulated in the cells. Meanwhile, the primary preadipocytes obtained from SVFs showed a small spindle-like morphology and did not spontaneously form lipid droplets. The preadipocytes were cultured in 100-mm dishes.

Figure 3.

Morphological identification of the mature adipocytes forming lipid droplets. The primary adipocytes obtained by enzymatically digesting of canine adipose tissue were transferred to a 75T flask completely filled with DMEM containing 10% FBS and the flask was then turned over. A — Floating lipids and adipocytes in 75T flask were observed using phase-contrast microscope (Olympus). The cells were adhered to the flask and cultured. The adipocytes cultured in the flask were collected with a cell scraper and seeded into a 6-well plate or 96-well plate. B — The primary adipocytes were cultured in MDI medium for 2 d and then cultured in insulin medium for 2 d to differentiate into mature adipocytes that form lipid droplets in a 6-well plate. C — The primary adipocytes were also able to stably mature in a 96-well plate and form lipid droplets. Scale bars = 200 μm (A) or 100 μm (B,C).

Effects of COE on cell viability of mature canine adipocytes

To confirm the effects of COE on the cell viability of canine adipocytes, a WST assay was conducted after exposing COE (0, 0.93, 2.32, and 4.64 nM celastrol) to either mature adipocytes or preadipocytes for 6 d. The cell viability of mature adipocytes was decreased by the treatment of COE in a concentration-dependent manner (Figure 4A). At celastrol concentrations of 2.32 nM and 4.64 nM, COE decreased the cell viability of mature adipocytes by about 35% compared to vehicle control (Figure 4A). In contrast, preadipocytes did not show statistical change in cell viability after COE treatment (Figure 4B). These results implied that COE may be more effective at inhibiting cell growth of the mature adipocytes that can produce lipids.

Figure 4.

Comparison of cell viability of mature adipocytes and preadipocytes after treatment with COE. A — Canine mature adipocytes, or B — Preadipocytes were seeded into 96-well plates at a density of 10 000 cells/well. These 2 types of cells were then exposed to 4 different concentrations of Celastrus orbiculatus extract (COE) (0, 0.93, 2.32, and 4.64 nM) for a total of 6 d. After the incubation period, cell viability was measured using the WST assay after treating the cells with 10% EZ-Cytox after the cells were treated with COE. The value of the control containing 0.1% DMSO as a vehicle was set as 100%. The data in the graphs were obtained from at least 3 repeated experiments and presented as the means ± SEM. *P < 0.05 versus control.

Effects of COE on adipogenesis and lipid accumulation in canine adipocytes

To confirm the effects of COE on adipogenesis and lipid accumulation, primary adipocytes were differentiated into mature adipocytes for 6 d under the condition of continuous exposure of COE (0, 0.93, 2.32, and 4.64 nM celastrol). As shown in Figure 5A, COE inhibited the formation and accumulation of lipids in canine adipocytes in a concentration-dependent manner. In particular, even at the lowest concentration of 0.93 nM, COE inhibited adipogenesis (Figure 5A, upper panel) and reduced lipid accumulation by about 20% compared to the control, which was a statistically significant decrease (Figure 5B). These results suggest that COE may affect adipogenesis and lipid accumulation.

Figure 5.

Oil Red O staining to quantify lipid accumulation in canine adipocytes after treatment with COE. The adipocytes were seeded in 6-well plate and the cells were matured with MDI medium and insulin medium. The cells were then exposed to 4 different concentrations of Celastrus orbiculatus extract (COE) (0, 0.93, 2.32, and 4.64 nM) for a total of 6 d. After fixing the cells with 3.7% paraformaldehyde, 0.3% Oil-Red-O (ORO) was added and incubated for 15 min. A — Lipid droplets stained with ORO were photographed under the phase-contrast microscope. B — The absorbance of ORO extracted in 100% isopropanol was measured at 515 nm using a microreader. The 100% isopropanol was used as a blank. The absorbance was normalized to cell viability data (WST assay) from each of the same treatment groups. The value of the control containing 0.1% DMSO as a vehicle was set as 100%. The data in the graphs were obtained from at least 3 repeated experiments and presented as the means ± SEM. *P < 0.05 versus control. Scale bars = 100 μm.

Inhibition of triglyceride production and lipolytic activity by COE treatment in canine adipocytes

To confirm the effect of COE, triglyceride content and lipolytic activity were analyzed in mature adipocytes after continuous exposure of COE for 6 d (Figure 6). As a result, COE containing 4.64 nM celastrol inhibited triglyceride production in canine adipocytes by about 15% compared to the control (Figure 6A). In addition, COE increased lipolytic activity significantly and concentration-dependently at all experimental concentrations (Figure 6B). COE increased the level of free glycerol, which means that the glycerol was hydrolyzed from triglyceride and lipolysis was stimulated (Figure 6C). Therefore, COE may exert anti-obesity effects by promoting lipolysis of canine adipocytes.

Figure 6.

Effect of COE on triglyceride production and lipolytic activity in canine adipocytes. The adipocytes were seeded in 96-well plate and the cells were matured with MDI medium and insulin medium. The cells were then exposed to 4 different concentrations of Celastrus orbiculatus extract (COE) (0, 0.93, 2.32, and 4.64 nM) for a total of 6 d. A — The triglyceride content in adipocytes was analyzed using the Triglyceride Colorimetric Assay Kit, and the lipolytic activity of adipocytes was analyzed using B — Lipolysis Colorimetric/Fluorometric Assay Kit, or C — Free Glycerol Colorimetric/Fluorometric Assay Kit. The absorbance was measured at 540 nm (A) or 570 nm (B–C) using a microreader. The absorbance obtained from each of the 3 measurements was normalized to cell viability data (WST assay) from each of the same treatment groups. The value of the control containing 0.1% DMSO as a vehicle was set as 100%. The data in the graphs were obtained from at least 3 repeated experiments and presented as the means ± SEM. *P < 0.05 versus control.

Discussion

This study investigated whether COE can exert anti-obesity effects in canine-derived adipocytes. Treatment with COE containing celastrol decreased the cell viability of mature adipocytes, but not that of preadipocytes. When exposed to COE over a period of 6 d, adipogenic maturation, lipid accumulation, and triglyceride production in the canine adipocytes were inhibited and lipolytic activity of the adipocytes was significantly increased compared to the control.

In a study on the effects of celastrol on adipogenesis and lipolysis at the cellular level, celastrol did not significantly affect the cell viability of 3T3-L1 preadipocytes at various concentrations for 8 d (24). In contrast, it was reported that the cell viability of 3T3-L1 cells differentiated into mature adipocytes declined significantly at a concentration range of 10 to 30 μM celastrol (25). In another study, inhibition of adipogenesis decreased cell viability by inducing tumor necrosis factor-alpha (TNFα)-induced lipolysis and apoptosis in 3T3-L1 adipocytes (26), which supports the finding that the cell viability decreased by COE may have been due to inhibition of adipogenic differentiation.

These previous studies also provide evidence that COE showed a growth-inhibiting effect on canine adipocytes and did not significantly affect the cell viability of canine preadipocytes. In addition, we found that COE did not significantly change the cell viability of canine kidney cells (MDCK cells) as well as canine preadipocytes at the same concentrations tested in this study (data not shown). These results suggest that COE could act more specifically on mature adipocytes.

Lipid droplets are composed of a neutral lipid core consisting mainly of triacylglycerols and cholesteryl esters surrounded by a phospholipid monolayer (27). The surface of lipid droplets consists of proteins that are involved in regulating lipid metabolism (27). Several studies using cellular and animal models have shown that celastrol inhibits the formation of lipid droplets in adipocytes (13,24,28,29). Similar to these results, in this study COE reduced the size and number of lipid droplets in canine adipocytes and inhibited intracellular accumulation of lipids. Meanwhile, while the formation of lipid droplets was inhibited by a concentration of 50 to 200 nM of celastrol in previous studies (13,24), in our study, the inhibition was shown at a relatively lower concentration (0.93 nM celastrol). It is believed that various unknown active ingredients in COE and celastrol together exerted a synergistic effect on the inhibition of lipid accumulation and thus inhibited more than celastrol treatment alone.

When cells deplete their major nutrient, usually nitrogen, the excess carbon substrate is continually assimilated by the cells and converted into stored fat (30). Recent studies reported that the size and shape of lipid droplets are closely related to the development of obesity (31–33). In adipose tissue, intracellular triglycerides are stored in cytoplasmic lipid droplets (34). The decrease in the lipid droplet directly leads to a decrease in triglyceride, which means that the accumulation of lipids in the cell is reduced (35). One study reported that celastrol reduced triglyceride accumulation by inhibiting the toll-like receptor 4 (TLR4)-mediated immune and inflammatory response in steatotic HepG2 cells (36). Another study reported that celastrol decreased hepatic intracellular triglyceride and serum triglyceride, free fatty acid, and alanine aminotransferase (ALT) in mice fed a high-fat diet (28). These results support our finding that COE reduced triglyceride content in canine adipocytes.

When lipases are phosphorylated, they break down triglycerides into fatty acids and glycerol through several stages of hydrolysis, with 1 fatty acid removed at each stage of hydrolysis. Lipase therefore catalyzes the hydrolysis of monoacylglycerol to glycerol so that lipolysis of adipocytes can be quantified by measuring the concentration of glycerol remaining in the cell (34). Increased lipolytic activity means improved anti-obesity effect. Similar to other studies demonstrating the lipolysis-stimulating effect of celastrol (24,28), in this study, it was found that COE stimulated lipolysis in canine adipocytes in a concentration-dependent manner. We have considered that COE may have anti-obesity effects by promoting lipid metabolism even at low concentrations of celastrol (0.93 nM).

Adipocytes not only play a major role in lipid metabolism and energy production, but also release several cytokines and chemokines and play a role in inflammation (4,37). Obesity is associated with the number and size of adipocytes (38), which induces the accumulation of triacylglycerol (lipid), which triggers changes in adipocyte hormone and cytokine secretion (39). These triggers cause an inflammatory response from adipocytes, which leads to several obesity-related diseases, including chronic inflammation, insulin resistance, type-2 diabetes, and cardiovascular disease (40,41).

In this study, we demonstrated that COE could inhibit the proliferation of mature adipocytes and lipid accumulation, possibly preventing these obesity-related diseases. The experimental data obtained in the present study can therefore be effectively used to prevent or treat obesity in companion animals by inhibiting the growth and adipogenic differentiation and maturation of adipocytes. Data from this study can also be used as basic research for developing canine-specific prescription foods for weight management.

In conclusion, we found that COE inhibits the formation and accumulation of lipid droplets in canine adipocytes, promotes lipolytic activity, and induces triglyceride breakdown (Figure 7). This finding is valuable in that it is the first to prove the anti-obesity efficacy of COE against canine adipocytes through in-vitro experiments. It is hoped that these findings can provide important insights into preventing and treating canine obesity.

Figure 7.

Anti-obesity effect of COE in canine adipocytes. Celastrus orbiculatus extract (COE) stimulated lipolysis of canine adipocytes to break down triglycerides into fatty acids and glycerol. COE also inhibited the formation and accumulation of lipid droplets in the adipocytes by inhibiting the production of adipose triglycerides, which means that COE exerts anti-obesity effects in canine adipocytes.

Since we have only verified the effects of COE on the functional aspects of cells by identifying the relationship between celastrol and lipases, the lipolytic activity of COE remains to be determined. Further investigation is also required into changes in the expression of obesity/anti-obesity-related genes at the tissue or gene level in order to more specifically verify the anti-obesity effect of COE. The effects of COE on obesity parameters, such as weight loss, food intake, fat content, and lipid metabolism, must also be studied in a diet-induced obese (DIO) model.