Anti-Obesity Effect of Dyglomera® Is Associated with Activation of the AMPK Signaling Pathway in 3T3-L1 Adipocytes and Mice with High-Fat Diet-Induced Obesity

Dyglomera® is an aqueous ethanol extract of the fruit pods of Dichrostachys glomerata, a Cameroonian spice. Several studies have shown its anti-diabetic and anti-obesity effects. However, the underlying mechanisms for such effects remain unclear. Thus, the objective of this study was to investigate the anti-obesity effect of Dyglomera® and its underlying mechanisms in mice with high-fat diet-induced obesity and 3T3-L1 adipocytes. Our results revealed that Dyglomera® inhibited adipogenesis and lipogenesis by regulating AMPK phosphorylation in white adipose tissues (WATs) and 3T3-L1 adipocytes and promoted lipolysis by increasing the expression of lipolysis-related proteins. These results suggest that Dyglomera® can be used as an effective dietary supplement for treating obesity due to its modulating effect on adipogenesis/lipogenesis and lipolysis.


Introduction
Obesity is a disease associated with metabolic disorders such as nonalcoholic fatty liver disease (NAFLD), insulin resistance, hyperlipidemia, and type 2 diabetes [1]. Obesity is a complex and multifactorial chronic disease and is a serious health problem in many countries. The incidence of obesity is multifactorial. Dysregulation of lipid and glucose metabolism is considered to be an important factor [2,3]. Obesity is caused by excessive energy intake causing lipid synthesis (lipogenesis) and differentiation of pre-adipocytes into mature adipocytes (adipogenesis) in the adipose tissue [4,5]. White adipose tissue (WAT) can store excess energy as triglycerides (TG) and mobilize energy in the form of free fatty acids (FFA). High plasma lipid levels can also cause FFA not to be efficiently used by adipocytes for storage, resulting in the hepatic accumulation of lipids [6]. Therefore, lowering plasma FFA levels could be potential strategies for metabolic syndrome management. Moreover, various treatments for improving obesity include exercise therapy, diet therapy, drug therapy, and surgery. Although treatment with chemically synthesized anti-obesity drugs has high efficacy, it causes many side effects. For this reason, there is a growing interest in the use of natural plant materials that are safe and that have a low risk of side effects.
Dichrostachys glomerata is a plant of western Cameroon. Its fruits and seeds are edible. The fruit is a dried pod commonly used as a spice in "Nah poh", a traditional Cameroonian soup [7]. D. glomerata have been found to contain various components such as flavonoids, phenolic compounds, alkaloids, tannins, saponins, and terpenoids [8]. Recent studies have shown that D. glomerata has antioxidant activity [9]. Other studies have shown that D. glomerata has antioxidant activity in vitro and in vivo, along with low-density lipoproteins' (LDL) antioxidant properties. D. glomerata has been shown to be able to lower fasting serum glucose levels and glycated hemoglobin in laboratory diabetic rats [10,11]. In addition, D. glomerata has been reported to improve oxidative stress in obese and type 2 diabetes patients [12,13]. Dyglomera ® is a standardized powder prepared by extracting D. glomerata fruit pods with aqueous ethanol followed by concentration and drying. Dyglomera ® also has anti-inflammatory and fat regulation activity in obese patients with metabolic syndrome [14,15]. Also, Dyglomera ® has been reported to be safe in a subchronic study in rats and genotoxicity tests [16].
However, there are few reports on the effects of the Dyglomera ® on obesity, underlying mechanisms, and related metabolic disorders. Therefore, the aims of the current study were to investigate the anti-obesity effects of Dyglomera ® and possible underlying mechanisms on 3T3-L1 adipocytes and high-fat diet induced obese mice.

Composition of Dyglomera ®
The marker compounds in Dyglomera ® were confirmed by high-performance liquid chromatography (HPLC) analysis. Quantitative analysis of Dyglomera ® revealed that Myricetin and Luteolin content was determined at approximately 1.6% and 1.0%, respectively (Figure 1), and the optimized Dyglomera ® was used for the following experiments.

Effects of Dyglomera ® on Body Weight and Food Intake in Mice with High-fat Diet-induced Obesity
We investigated the effect of Dyglomera ® on obesity in high-fat diet (HFD)-induced obesity in mice. As shown in Figure 2A, the body weight gain of the HFD group was significantly increased (11.42 ± 2.31 g) when compared with the normal-fat diet (ND) group after seven weeks of feeding. In contrast, the administration of Dyglomera ® 25, 50, and 100 mg/kg resulted in a significant decrease in body weight gain: 8.87 ± 1.43 g (p < 0.05), 8.40 ± 2.18 g (p < 0.05), and 6.81 ± 0.88 g (p < 0.01), respectively ( Figure 2A). On the other hand, food intake and water intake did not display significant group wise differences ( Figure 2B,C). Effects of Dyglomera ® on body weight and food intake in mice with high-fat diet-induced obesity. (A) body weight, (B) food intake, and (C) water intake. ## p < 0.01 between the normal diet (ND) group and the high-fat diet (HFD) group. * p < 0.05 and ** p < 0.01 between HFD group and Dyglomera ® treated group. Data are presented as mean ± standard deviation (n = 5/group).

Effects of Dyglomera ® on Plasma Biochemical Levels in Mice with High-Fat Diet-Induced Obesity
Plasma biochemical parameters such as glucose, total-cholesterol (T-CHO), triglyceride (TG), free fatty acid (FFA), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), HDL-c/LDL-c ratio, alanine transaminase (ALT), aspartate transaminase (AST), adiponectin, leptin, and blood urea nitrogen (BUN) were investigated. As shown in Table 1, serum adiponectin level and HDL-c/LDL-c ratio were decreased (p < 0.01), whereas serum glucose, T-CHO, FFA, LDL-c, ALT, AST, leptin, and BUN levels were increased (p < 0.05) in the HFD group. However, glucose, T-CHO, ALT, AST, and leptin levels in groups administered with Dyglomera ® 25 (p < 0.05), 50 (p < 0.05), and 100 (p < 0.01) were significantly decreased as compared to those in the HFD group. TG, FFA, and BUN levels were significantly decreased (p < 0.01) in groups treated with Dyglomera ® . LDL-c levels I groups administered with Dyglomera ® were also decreased (p < 0.05), whereas HDL-c showed a tendency to increase in the group administrated with Dyglomera ® . HDL-c/LDL-c ratio and adiponectin levels were increased (p < 0.01) in the group administrated with Dyglomera ® . Table 1. Effects of Dyglomera ® on plasma biochemical levels in mice with high-fat diet-induced obesity. Data are presented as mean ± standard deviation (n = 5/group). # p < 0.05, ## p < 0.01 between the normal diet (ND) group and the high-fat diet (HFD) group. * p < 0.05 and ** p < 0.01 between the HFD group and the Dyglomera ® treated group.  (Figure 3A). Moreover, subcutaneous adipose tissue, epididymal adipose tissue, peritoneal adipose tissue, and total WATs weight were also measured to determine the effect of Dyglomera ® on organ weights. The weight of WATs was markedly increased (p < 0.01) in the HFD group compared to that in the ND group. Subcutaneous adipose tissue weight decreased (p < 0.05) in the Dyglomera ® 50 and 100 groups. Epididymal adipose tissue weight decreased (p < 0.05) in the Dyglomera ® 100 group. However, peritoneal adipose tissue only showed a tendency to decrease without showing statistical significance. Total WATs weight was significantly decreased by administration of Dyglomera ® in a dose-dependent manner (in Dyglomera ® 25 and 50 groups, p < 0.05: in 100 group, p < 0.01) ( Figure 3B).

Figure 4.
Effects of Dyglomera ® on expression of adipogenesis and lipogenesis related proteins in epididymal WATs. ## p < 0.01 between normal diet (ND) group and high fat diet (HFD) group. ** p < 0.01 between HFD group and Dyglomera ® treated group. Data are presented as mean ± standard deviation of three independent experiments.

Figure 5.
Effects of Dyglomera ® on lipolysis-related proteins in epididymal WATs. # p < 0.05, ## p < 0.01 between normal diet (ND) group and high fat diet (HFD) group. ** p < 0.01 between HFD group and Dyglomera ® treated group. Data are presented as mean ± standard deviation of three independent experiments.

Effects of Dyglomera ® on Expression of Adipogenesis and Lipogenesis Related Proteins in 3T3-L1 Adipocytes
To investigate the effects of Dyglomera ® on adipogenesis and lipogenesis, proteins were extracted from 3T3-L1 adipocytes and epididymal WATs, respectively. AMPK is a major regulator of cellular energy homeostasis. It regulates carbohydrate and fat metabolism in order to maintain cellular energy balance. SREBP-1c is a target protein directly regulated by AMPK. It has been identified as one of the transcription factors involved in adipogenesis/lipogenesis. SREBP-1c is also known to induce the expression of PPARγ. To determine the association between Dyglomera ® and the activation of AMPK, we determined whether Dyglomera ® could induce AMPK phosphorylation during 3T3-L1 differentiation. It was found that the pAMPK/AMPK level was markedly increased in Dyglomera ® 50, 100, and 200 µg/mL treated 3T3-L1 cells by 17.09%, 124.42%, and 177.79%, respectively (p < 0.01).

Discussion
Obesity is a multifactorial and chronic disease caused by the excessive accumulation of lipids. Obesity represents a serious health problem worldwide as it can increase the risk of metabolic diseases. These days, many herbal plants have been studied for anti-obese properties. Among them, the fruit of Dichrostachys glomerata has received special attention as a safe and lipid-lowering plant in Cameroon [15,16].
To investigate the anti-obesity effects and underlying mechanisms of Dyglomera ® , an HFD-induced obese mouse model and 3T3-L1 cells were used in this study. They are the most widely used models for obesity studies [17,18]. 3T3-L1 adipocytes are an effective cell model for studying mechanisms of adipocyte differentiation and lipid accumulation [19,20].
It is well-known that feeding HFD can readily induce obesity such as body weight gain and weight gain of WATs [21]. Administration of Dyglomera ® was shown to lower body weight and total weight WATs markedly in HFD-administrated mice, whereas Dyglomera ® treatment did not affect the food intake and water intake of mice with HFD-induced obesity. These results indicate that Dyglomera ® has an anti-obesity effect.
Dyglomera ® supplementation significantly reduced plasma glucose, T-CHO, TG, FFA, and LDL-c levels while increasing the plasma HDL-c/LDL-c ratio in HFD-fed mice. These results suggest that Dyglomera ® can reduce hyperglycemia and hyperlipidemia caused by HFD. It is well-known that serum levels of AST and ALT, the major enzymes present in hepatocytes, are increased after hepatocellular injury [22]. Excessive intake of high fat can induce oxidative stress, which can lead to liver dysfunction and fatty degeneration. ALT is a sensitive biomarker of NAFLD [23]. Dyglomera ® administration significantly reduced serum AST and ALT levels increased by HFD. The serum levels of BUN were determined to evaluate kidney function [24]. The increase in serum BUN level was observed in HFDtreated mice. However, Dyglomera ® treatment significantly reduced serum BUN levels in HFD-induced obese mice. The findings showed administration of Dyglomera ® to HFDinduced mice has remarkably reduced plasma BUN levels, suggesting that Dyglomera ® may prevent renal injury resulting from long-term high-fat diet intake. In obesity, the accumulation of fat affects adipokines including leptin and adiponectin in adipose tissues [25]. Adiponectin is known to increases insulin sensitivity, glucose uptake, and fatty acid oxidation effects of hormone-stimulated lipolysis [26]. Leptin levels were significantly increased in obese mice. These elevated leptin levels suggest that most obese individuals develop leptin resistance, which prevents them from suppressing eating [27]. However, Dyglomera ® treatment decreased leptin levels and conversely increased adiponectin levels.
We also performed an MTT assay to confirm the cytotoxicity of Dyglomera ® . As a result, no significant toxicity was observed for Dyglomera ® at concentration of 50, 100, or 200 µg/mL. As a result of Oil-red O staining, Dyglomera ® treatment in 3T3-L1 decreased the accumulation of lipid droplets in a dose-dependent manner. To examine the underlying mechanism, we examined expression levels of adipogenesis/lipogenesis and lipolysis related proteins in WATs of mice with HFD-induced obesity and 3T3-L1 cells. AMPK plays a major role in regulating the lipid synthesis pathway and energy metabolism in adipocytes [28]. When AMPK is phosphorylated, the catabolic pathway is turned on and the ATP-consuming anabolic pathway is simultaneously turned off [29]. Activation of AMPK can suppress the expression of SREBP-1c and PPARγ in adipogenesis/lipogenesis [30]. AMPK can also stimulate mitochondrial fatty acid oxidation and lipolysis [31]. Lipolysis, the catabolic pathway of the fatty acid cycle, is also crucial to balanced fat metabolism. Enhanced lipolysis contributes to the release of fatty acids and a decrease in fat deposition [32]. Expression of pHSL, ATGL, and perilipin are key factors regulating the lipolysis pathway in adipose tissues by cleaving ester bonds to degrade triglycerides [33][34][35]. In our study, phosphorylation of AMPK was significantly increased by Dyglomera ® treatment in WATs and 3T3-L1 cells. Expression levels of adipogenesis/lipogenesis related proteins such as SREBP-1c and PPARγ were suppressed by Dyglomera ® treatment in WATs and 3T3-L1 cells. Expression levels of lipolysis-related proteins such as pHSL, ATGL, and perilipin were increased by Dyglomera ® treatment in WATs and 3T3-L1 cells.
Taken together, Dyglomera ® could inhibit adipogenesis/lipogenesis by regulating AMPK phosphorylation in WATs and 3T3-L1 cells. In addition, Dyglomera ® could promote lipolysis by increasing the expression of lipolysis-related proteins ( Figure 10). Moreover, Dyglomera ® lowered serum lipid and glucose levels. These results suggested that Dyglomera ® can be used as an effective functional food for treating obesity. In addition, further studies such as a comprehensive laboratory animal monitoring system (CLAMS) [36] are needed to evaluate the effects of Dyglomera ® in activities, even circadian rhythms. Also, it is suggested that further mechanism studies through treatment with AMPK inhibitors such as compound C are needed to elucidate the AMPK-related anti-obesity biochemical mechanism of Dyglomera ® .

Sample Preparation
Dyglomera ® , an aqueous ethanol extract of Dichrostachys glomerata fruit pods (standardized to Myricetin 1.6% and Luteolin 1.0%), was supplied by Gateway Health Alliances, Inc. (Fairfield, CA, USA). The manufacturing process was as follows: Dichrostachys glomerata fruit pods were extracted using aqueous ethanol. The resulting solution was concentrated and dried to yield Dyglomera ® . For in vitro studies, Dyglomera ® was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50, 100, or 200 mg/mL and then diluted with culture medium at concentration of 50, 100, or 200 µg/mL., respectively. Control 3T3-L1 cells were treated with culture medium containing only DMSO (final DMSO concentration 0.1%). For in vivo studies, Dyglomera ® was homogenized in 0.5 mL distilled water at a concentration of 25, 50, or 100 mg/kg, respectively and orally administered to mice. Moreover, the dose used in this study in mice represents a feasible dose in humans [37]. Control mice were given the same volume of distilled water instead of the test solution.

Oil-Red O Staining
On day six, differentiated 3T3-L1 adipocytes were washed with phosphate-buffered saline (PBS) and fixed with a 4% formalin solution (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. After fixation, cells were treated with Oil-red O solution and stained for 4 h. Each well was washed several times with distilled water and photographed with an ECLIPSE Ts2 microscope (Nikon Corporation, Tokyo, Japan). Stained lipids were then eluted with isopropanol and the absorbance was measured at 520 nm using a microplate reader (Tecan, Mannedorf, Switzerland).

Animals and Diet
Animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Gyeonggido Business & Science Accelerator (GBSA) BIO CENTER (Suwon, Korea) (Permission No.: 2020-02-0002). Male C57BL/6J mice at five weeks old were obtained from Orient Bio Inc (Seongnam, Korea). Mice were maintained with controlled conditions (temperature of 22.0 ± 3.0 • C, humidity of 50.0 ± 20.0%, and a 12 h light/dark cycle). During acclimatization for one week, mice were given free access to water and food (Teklad Certified Irradiated Global 18% Protein Rodent Diet, 2918C, Envigo RMS, Inc., Indianapolis, IN, USA).
After acclimatization, mice were fed either a normal-fat diet (ND, n = 7) as a normal group or high-fat diet (HFD, n = 28, rodent diet with 60% kcal fat (#D12492, Research diets INC)) as an HFD-induced obesity group for seven weeks. After obesity induction, mice with obesity were further divided into the following four experimental groups (n = 7/group) and matched by body weight: HFD group; Dyglomera ® 25 group (high-fat diet + Dyglomera ® 25 mg/kg); Dyglomera ® 50 group (high-fat diet + Dyglomera ® 50 mg/kg); and Dyglomera ® 100 group (high-fat diet + Dyglomera ® 100 mg/kg). These groups were monitored for seven weeks. Body weight and food intake were measured twice a week after group separation. At the end of the experiment period, mice were fasted for 12 h and euthanized. Blood was collected and adipose tissues were rapidly removed, rinsed with physiological saline solution, weighed, and stored at −80 • C.

Biochemical Analysis
The blood collected before autopsy was stabilized in a blood collection tube containing an anticoagulant (EDTA-2k). Serum was prepared after centrifugation at 13,000 rpm for 15 min at 4 • C and then stored at −80 • C until use. Glucose, total-cholesterol (T-CHO), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) levels were measured using a blood biochemical analyzer (Hitachi 7020, Tokyo, Japan). The contents of the free fatty acids (FFA) leptin and adiponectin in the blood were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, UK).

Histological Analysis
Adipose tissues were fixed with 10% neutral formalin. Tissues were embedded in paraffin and stained with hematoxylin and eosin (H&E) stain. Stained slides were photographed under a microscope (TE2000-U; Nikon, Tokyo, Japan).

Protein Extraction and Western Blot Analysis
3T3-L1 adipocytes were lysed with CelLytic buffer (Sigma-Aldrich, MO, USA). The cell lysate was centrifuged at 13,000 rpm for 15 min at 4 • C. Epididymal adipose tissues were washed with cold PBS and homogenized with RIPA buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSE, and 1% protease inhibitor. The homogenate was centrifuged at 13,000 rpm for 15 min at 4 • C. Cells and adipose tissue supernatant were collected and protein concentration was measured with Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and were then quantified to have the same protein concentration in the same volume (20 µg/20 µL). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA). The transferred membrane was blocked with 5% skim milk for 1 h and then incubated with specific primary antibodies at 4 • C overnight. Blots were washed with tris buffered saline with 0.5% Tween 20 (TBS-T) three times and then incubated with corresponding horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G at room temperature for 1 h. The intensity of each band detected by using ECL solution (GenDEPOT, Barker, TX, USA) was measured with a LuminoGraph (Atto, Tokyo, Japan). Band images were digitized using the ImageJ program developed at the National Institutes of Health (NIH; Bethesda, MD, USA) and corrected by β-actin level. There was no significant difference between all β-actin-to-β-actin levels.

Statistical Analysis
Data are presented as mean ± standard deviation. Values were compared using Student's t-test. One-way analysis of variance (ANOVA) was performed using the software Origin 7 (Microcal Software, Northampton, MA, USA). Values of p < 0.05 and p < 0.01 indicated statistical significance.