1 Introduction

Obesity has recently been recognized as a significant public health issue because it is a known risk factor for chronic diseases such as type Ⅱ diabetes, hypertension, and coronary heart disease (Wu et al., 2013). Obesity is the result of an imbalance between energy intake and metabolic expenditure and causes marked increases in the weight of adipose tissue (Galani et al., 2007). Many researchers have demonstrated that the current approaches for treating obesity, including pharmacotherapy and bariatric surgery, feature a number of important limitations, such as adverse reactions and high rebound rates (Kushner, 2014).

Control of energy intake through the diet and increased metabolic expenditure through exercise are two major programs that can suppress body fat accumulation (Adegboye and Linne, 2013). Many food ingredients, such as saponins, can effectively alleviate the development of obesity (Wang et al., 2018). Sea cucumber is a traditional Asian food, and sea cucumber saponins (SCS) are important secondary metabolites. Holothurin A (HA) and echinoside A (EA) are two major saponins in sea cucumber (Fig. 1) (Wang et al., 2014). Previous studies revealed that dietary supplementation with SCS significantly suppresses adipose tissue accumulation and ameliorates obesity-induced inflammation (Zhao et al., 2018). Additionally, a number of researchers have indicated that exercise can normalize adipose tissue weight, body weight, and inflammation in diet-induced obesity mice; however, some Chinese sports and health surveys have suggested that the participation rate of Chinese adults in regular physical exercise is only 10% (Ye et al., 2016). It is important to encourage Chinese adults to attend more physical exercises in addition to paying attention to nutrition. Thus exploring the combined effects of dietary supplementation with SCS and exercise on obesity is a worthwhile endeavor.

In the present study, the body weight and fat accumulation of high-fat diet-fed mice were analyzed following treatment to evaluate the combined effects of dietary supplementation with SCS and exercise on obesity. The possible underlying mechanism was also described.

2 Materials and Methods 2.1 Materials

Acetonitrile and ammonium bicarbonate were purchased from Sigma-Aldrich Corp. (St Louis, MO, USA). Deionized water was obtained from a Milli-Q gradient system (Millipore, Bedford, MA, USA). Sea cucumber (Pearsonothuria graeffei) was purchased from Nanshan Aquatic Market (Shandong, China). All other reagents used in this work were of analytical grade.

2.2 Preparation and Analysis of Sea Cucumber Saponins

Saponins were extracted from sea cucumbers according to a reported method (Hu et al., 2010). Briefly, the body walls of air-dried sea cucumbers were pulverized and then extracted four times with 60% ethanol (v/v) at room temperature. The solvent-to-material ratio was 4:1. The ethanol extracts were subsequently extracted in water and chloroform. The aqueous layer was extracted with n-butanol. The obtained n-butanol extract was dissolved in water, placed on a HP-20 resin column, and eluted with water and 80% ethanol. The obtained 80% ethanol extract was loaded onto a normal-phase silica gel column and eluted with chloroform: methanol: water (10:1:0.1–7:3:0.3, v/v/v). Finally, the eluate (7:2.5:0.2, v/v/v) was mixed and dried to obtain the SCS sample. Column chromatography with an ODS silica gel column (YMC-Pack, Japan) was used to separate the pure EA and HA standard substances by gradual elution using methanol and H₂O.

HPLC-UV (Agilent 1260 Infinity system) analysis with an Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm; Agilent, Palo Alto, CA, USA)was employed to evaluate the contents of EA and HA at 205 nm. The column temperature was maintained at 30℃, and the mobile phase consisted of acetonitrile (A) and 0.1% ammonium bicarbonate (B). Gradient elution was performed as follows: 0 – 5 min, 30% A; 5 – 30 min, 60% A; 30 – 35 min, 30% A. The flow rate was 1 mL min⁻¹, and the injection volume was 20 μL. The purity of the obtained SCS sample was 90.2%, including 21.6% HA and 68.6% EA.

2.3 Animals and Treatments

The experiment was conducted according to the guidelines provided by the Ethical Committee of Experimental Animal Care at Ocean University of China (Approval No. SPXY2015012, Qingdao, China). KM male mice (8 weeks old, 18–20 g) were purchased from Vital River (Beijing, China). All mice were cultured in individual cages with a constant temperature of 24℃, relative humidity of 65% ± 15%, and an alternating light/dark cycle of 12 h/12 h. The mice acclimated for 1 week and then randomly divided into four groups as follows: HF (high-fat diet group), HF-S (SCS group), HF-E (exercise group), HF-S + E (the combination of dietary SCS and exercise group). There are eight mice in every group. All mice in these four groups were fed a high-fat diet with 20% lard and 5% corn oil. The diets of mice in the HF-S and HF-S + E groups were supplemented with 0.06% SCS. The HF-E and HF-S + E groups were tasked to run 1 h per day using a motor-driven wheel-treadmill (YLS-10B, Shandong Academy of Medical Sciences, Jinan, China) rotating at a speed of 21 r min⁻¹ (moderate difficulty). The mouse diet was regulated on the basis of AIN-93G. After 3 weeks, the mice were fasted 10 h before sacrifice. Blood was collected and serum was obtained by centrifugation at 3500 r min⁻¹ for 15 min at 4℃. Tissues and viscera were quickly removed and stored at −80℃ until bioanalysis.

2.4 Biochemical Analyses of Serum

The corresponding enzymatic reagent kits (Biosino, Beijing, China) were used to determine serum total cholesterol (TC), triacylglycerol (TG), free fatty acid (FFA), and glycerin concentrations. High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were respectively measured using the phosphotungstic acid-Mg²⁺ precipitation and polyvinyl sulfate sedimentation methods.

2.5 Hepatic Lipid and Fatty Acid Composition Analysis

Hepatic lipids were extracted using a reported method (Folch et al., 1957). The concentrations of TC and TG were detected using the kits according to the manufacturer's instructions (Biosino). Fatty acid composition analysis was performed using an Agilent 6890 gas chromatography equipped with a flame-ionization detector and an HP-INNOWAX capillary column (30 m × 0.32 mm × 0.25 μm) (Liu et al., 2016). The detector temperature was maintained at 250℃, and the injector temperature was kept at 240℃. The column oven was heated from 170℃ to 240℃ at a rate of 3℃ min⁻¹ and then held at this temperature for 15 min. Nitrogen was used as the carrier gas and supplied at a flow rate of 1.2 mL min⁻¹.

2.6 Histological Analysis of Liver and Epididymal White Adiposes

The collected livers and epididymal white adipose tissues were stained with hematoxylin and eosin (H & E) and viewed under a light microscope equipped with a camera (Ni-E, Nikon, Japan).

2.7 RNA Extraction and Real Time (RT)-PCR

Trizol reagent (Invitrogen, USA) was used to extract the total RNA from liver and epididymal white adipose tissues (Liu et al., 2014). A total of 2 μg RNA was reversetranscribed into cDNA using random primers (TOYOBO, Osaka, Japan). SYBR Green I Master Mix (Riche, Mannheim, Germany) and an iCycler IQ5 system (Bio-Rad Laboratories, Hercules, CA, USA) were used to amplify the target genes with forward and reverse primers. The procedure of real time PCR was as follows: 1 cycle of 95℃ for 10 min and 45 cycles of 95℃ for 15 s, 55℃–60℃ for 20 s, and 72℃ for 30 s. The primers were synthesized by the Shanghai Sangon Gene Company (Shanghai, China). Changes in relative gene expression were calculated using the 2^–ΔΔCt method, and the GADPH gene was used as the reference gene. Results are reported as changes relative to the control group.

2.8 Statistical Analysis

All results were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed by oneway analysis of variance and multiple comparisons (Duncan's post hoc test) using SPSS.20 software with a confidence level of 95%. Different letters indicate significant differences among groups when P < 0.05.

3 Results 3.1 Influence of Dietary SCS and Exercise on Food Intake and Body Weight

The body weight of the mice was measured every day to determine whether dietary SCS and exercise exhibits synergistic effects on alleviating obesity. After 3 weeks, the liver weight in the HF-S + E group decreased by 20% in comparison with that in the HF group. Interestingly, no significant differences in the visceral index of the liver, heart, kidney, and spleen (Table 1) were observed among the four treatment groups. Moreover, no significant difference in daily food intake was observed among the four groups throughout the 3-week experiment (Fig. 2A). Compared with the HF group, the dietary SCS and exercise groups showed reductions in body weight gain, but the differences observed were not statistically significant. Dietary SCS combined with exercise significantly reduced body weight gain from the 7th day to the 20th day of treatment compared with that in the HF group (P < 0.05, Fig. 2B). This result suggests the synergistic effects of SCS and exercise on mitigating obesity without changing food intake and visceral index.

3.2 Influence of Dietary SCS and Exercise on Serum Parameters

Table 2 shows the influence of SCS supplementation and exercise on serum parameters, including TG, TC, HDL-C, LDL-C, glycerin, and FFA. Because serum TG is hydrolyzed into glycerol and fatty acids under the action of lipoprotein lipase (LPL), serum triglycerides were analyzed with glycerol phosphate oxidase-p-aminophenazone method (Sullivan et al., 1985). In fact, the measured glycerol concentrations reflect the contents of glycerol and triglycerides in serum and are listed as TG + glycerol in Table 2. Therefore, TG concentrations were calculated from the difference between the measured TG + glycerol level and the measured glycerol content. Compared with that in the HF group, serum triglyceride concentrations in the HF-S, HF-E, and HF-S + E groups significantly decreased (P < 0.05). Only dietary SCS combined with exercise significantly reduced LDL-C levels by 85% compared with that in the HF group. Dietary supplementation of SCS or exercise alone decreased serum cholesterol concentrations, but the difference was not significant. Interestingly, no significant changes in HDL-C level were observed among the four treatment groups. Additionally, the levels of TG hydrolysates, glycerin, and FFA increased to varying degrees among the four treatment groups; however, the exercise group exhibited the highest levels of glycerin and FFA.

3.3 Influence of Dietary SCS and Exercise on Liver Lipids and Morphology

Consumption of a high-fat diet has been associated with steatosis and metabolic liver disorders. Thus, the contents of TC and TG in the liver were determined in this study (Figs. 3A and B). Dietary SCS and exercise alone could reduce TG content in the liver by 8% and 34%, respectively, while the combination of them significantly decreased the content of TG by 55%. Compared with that in the HF group, the contents of TC in the HF-S, HF-E, and HF-S + E groups showed a downward trend, although no significant difference was observed among these groups. Because HF diets could induce fatty liver, we assayed the impact of dietary SCS and exercise on lipid accumulation and hepatic steatosis by H & E staining. Results showed intense lipid accumulation in the livers of mice in the HF group (Fig. 3C). Among the four groups, the HF-S + E group showed the least lipid accumulation (Figs. 3C–F). These results demonstrate that the combination of dietary SCS and exercise remarkably prevents HF-induced lipid accumulation and blocks the development of hepatic steatosis compared with either intervention alone.

The fatty acid composition of lipids in the mouse liver was detected using gas chromatography (GC), and the results are shown in Table 3. The combination of SCS and exercise could significantly reduce the proportion of monounsaturated fatty acids (C16:1, C18:1) and increase the proportion of saturated fatty acids (C18:0) compared with those in the other groups. Compared with those of the HF group, the liver samples of the HF-S + E group showed significant increases in arachidonic acid (C20:4) and docosahexaenoic acid (C22:6). The desaturation indices (16:1– 16:0 and 18:1–18:0) of the different groups were calculated (Sjögren et al., 2008), and the data demonstrated that the desaturation indices of the HF-E and HF-S + E groups were significantly reduced compared with those of the HF group (P < 0.05). Interestingly, the HF-S + E group showed reductions in desaturation indices of 16:1–16:0 and 18:1– 18:0 of 50% and 44%, respectively, (Table 3), which was superior to the HF-E group in reducing desaturation indices.

3.4 Influence of Dietary SCS and Exercise on Adipose Mass and Morphology

As shown in Figs. 4A and B, supplementation with SCS and exercise alone significantly decreased high-fat dietinduced fat accumulation in white adipose tissue, including epididymal fat, perirenal fat, mesenteric fat, visceral white fat, and subcutaneous white fat. The decrease was accompanied by a parallel reduction in body weight (P < 0.05). Interestingly, the combination of dietary SCS and exercise demonstrated excellent effects on reducing fat weight and index compared with those obtained from dietary SCS or exercise alone; no significant difference in adipose weight and index was observed between the HF-S and HF-E groups. Compared with the HF group, the HF-S + E group showed decreases of 60%, 66%, 65%, 63%, 69%, and 63% in epididymis fat, perirenal fat, mesenteric fat, visceral white fat, subcutaneous white fat, and white adipose tissue weight, respectively. Adipose tissue cells are closely associated with lipid metabolism. The morphology of the adipose tissues from the different treatment groups was analyzed by H & E staining. Compared with the HF group, the HF-S + E group showed significant reductions in the size of adipocytes in the epididymal white adipose tissue. These reductions were greater than those observed in the HF-S and HF-E groups.

3.5 Influence of Dietary SCS and Exercise on mRNA Expression Associated with β-Oxidation and Lipogenesis in Liver

The transcription level of genes associated with lipogenesis and β-oxidation was determined to assess the mechanism of SCS and exercise in reducing triacylglycerol levels. The regulation of lipogenesis in the liver is controlled by sterol regulatory element-binding protein (SREBP). SREBP-1c is essential for TG synthesis because it regulates the expression of downstream target genes, such as fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and stearoyl-CoA desaturase-1 (SCD1). As shown in Fig. 5A, the expression of FAS and SCD1 decreased in the treatment groups compared with that in the HF group (P < 0.05), but no downtrend in the mRNA expressions of SREBP-1c and ACC1 was observed. The expressions of FAS and SCD1 in the HF-S + E group significantly decreased by 83% and 80%, respectively. SCD is the key enzyme responsible for converting saturated fatty acids into monounsaturated fatty acids, and changes in SCD1 expression were consistent with changes in the desaturation indices of the liver (Table 3). The mRNA expression levels of ME and G6PDH, which provide NADPH for the de novo synthesis of fatty acids, were not affected by dietary SCS or exercise. PPARα regulates mitochondrial and peroxisome oxidation to promote the oxidative decomposition of fatty acids, thereby regulating the expression of its target genes in downstream pathways, including CPT1a, CPT2, ACOX1, and ACAA1. As shown in Fig. 5B, the three treatment groups showed increased mRNA expression of PPARα and its target gene ACOX1 in comparison with the HF group, but the differences observed were not significant. The expressions of CPT1a, CPT2, and ACAA1 did not change in the treatment groups relative to that in the HF group.

3.6 Influence of Dietary SCS and Exercise on Expression of mRNA Associated with Lipogenesis and Lipolysis in Epididymal White Adipose Tissue

The mRNA level of genes associated with lipogenesis and lipolysis in epididymal white adipose tissue was studied to assess the mechanism of SCS and exercise in suppressing adipose accumulation. As shown in Fig. 6A, the expression of SREBP-1c and its target genes FAS, ACC, and SCD1, all of which are related to lipogenesis, did not change in the treatment groups. By contrast, the expression of ME in the HF-S and HF-E groups declined by 38% and 41%, respectively compared with that in the HF group. Interestingly, the expression of ME decreased by 74% in the HF-S + E group; this decline is greater than those in the HF-S and HF-E groups. The expression of G6PDH did not change in the HF-E group but decreased in the HF-S + E group (P < 0.05). Moreover, the mRNA expression of FABP4 (Fig. 6B), which could enhance FFA solubility and transport to specific enzymes and cellular compartments, did not change among the three treatment groups. ATGL and HSL are the major enzymes involved in the activity of TG hydrolase in adipose tissue. In this study, the expression of ATGL in the HF-E and HF-S + E groups and that of HSL in the HF-E group significantly increased compared with the HF group. The expression of LPL, which could cause an inflow of fatty acids into adipose tissue, was not modified in any of the groups. The expression of PPARγ did not change in the HF-S and HF-E groups but increased significantly in the HF-S + E group (P < 0.05).

4 Discussion

Obesity is closely related to an individual's lifestyle and generally is considered to be resulted from a lack of physical activity, excessive intake of dietary calories, and metabolic disorders. Proper aerobic exercise can reduce obesity by increasing the body's total metabolic expenditure. Adequate exercise has clearly been proven to reduce cardiovascular risk and alleviate many cardiometabolic risk factors (Oktay et al., 2017; Swift et al., 2018). However, exercise is often ignored for different reasons. Thus, the use of dietary supplements, which requires considerably less effort than traditional behavioral changes, such as restriction of diet, has become an increasingly popular means to lose weight (Pillitteri et al., 2008). Studies on the individual beneficial effects of exercise or various food ingredients, including dietary fiber, saponins, and mulberry anthocyanins, are widely available; however, research focused on the combined effects of exercise and food ingredients on the development of obesity is limited (Wu et al., 2013; Liu et al., 2017). The results of the present study revealed that the combination of dietary SCS and exercise significantly reduces adiposity. Furthermore, SCS supplementation with exercise improves peripheral markers, such as serum parameters and hepatic TG levels (Fig. 7). Although several studies have demonstrated the independent effect of SCS on obesity, the present study further explores the anti-obesity effects of SCS under the condition of exercise (Wang et al., 2014; Guo et al., 2016). Previous studies demonstrated that the combination of dietary conjugated linoleic acids and exercise could decrease fat mass and increase lean mass in mice fed a high-fat diet (Bhattacharya et al., 2005), which supports our findings in this research. Another study demonstrated that polyunsaturated fatty acids suppressed lipogenesis by inhibiting LXRα transactivation to improve lipid metabolism in the liver via a mechanism similar to that of food-derived saponins (Takeuchi et al., 2010; Uemura et al., 2011).

Previous studies suggested that exercise may inhibit fat formation to suppress lipid accumulation in the liver (Carlson and Winder, 1999; Lavoie and Gauthier, 2006). SCS can further suppressthe lipogenesis. Indeed, the combination of exercise and SCS significantly reduced the mRNA expression of FAS and SCD1 in the liver (Fig. 5A), which is related to lipogenesis. Thus, the combination of exercise and SCS may suppress lipid accumulation by significantly decreasing the expression of lipogenesis genes in the liver. The aglycones of saponins have a steroid structure similar to that of cholesterol. Cholesterol plays a vital role in the regulation of several transcription factor involved in hepatic lipid metabolism (Uemura et al., 2011). SREBP-1c activity is regulated by the amount of intracellular cholesterol, and LXR is activated by the oxidized cholesterol (oxysterol) as an endogenous ligand. LXRα has been reported to regulate SREBP-1c transcription, which can influence de novo fatty acid synthesis-related genes, such as FAS and SCD1 (Chen et al., 2004). Exercise can increase the basal energy expenditure by increasing fat oxidation. In this study, the fatty acid β-oxidation gene expression was mediated by SCS or exercise independently, differing from previous studies. The differences might be attributed to variations in the experimental conditions, such as mice strain (KM or C57/BL6) or intensity of exercise (Meng et al., 2018).

While an abundance of small adipocytes was observed in the dietary SCS + exercise treatment group, the HF control group showed large adipocytes. Previous studies demonstrated that the consistency of adipose tissue cells is the main determinant of metabolic activity and the enhanced lipolysis or blunted lipogenesis may contribute to differences in adipocyte size (Schweiger et al., 2006; Marques et al., 2010). In this study, we found that moderate-intensity exercise and diet could inhibit body fat accumulation due to increased lipolysis. Schweiger et al. (2006) proved that adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) were the major enzymes contributing to TG breakdown in in vitro assays and organ cultures of murine white adipose tissue. The combination of SCS and exercise can increase lipolysis mainly by increasing the expressions of HSL and ATGL (Karbowska and Kochan, 2012). The fat in adipose tissue is mobilized to release FFA and glycerol into the circulation system. Serum FFA and glycerol are mainly derived from the lipolysis in adipose tissue through the catalysis of HSL (Cho et al., 2010; Zhang et al., 2014). Endurance or resistance exercise can increase the serum free fatty acid levels in obese women (Davitt et al., 2013). Our results suggest that exercise promotes lipolysis. Ohyama et al. (2014) demonstrated that exercise could activate the phosphorylation of PKA to induce the activity of HSL, thereby enhancing lipolysis. Thus, the combination of SCS and exercise may promote lipolysis by activating the phosphorylation of PKA. The combination of exercise and dietary SCS also increased the expression of PPAR-γ to regulate lipid metabolism, which was consistent with a previous study (Brunani et al., 2008).

The skeletal muscle plays an important role in the body's energy expenditure, and participatesglycolysis and lipid metabolic processes (Sylow et al., 2017). The fuel supply involves different enzymatic pathways responsible for obtaining energy from glucose and fatty acids through glycolysis and β-oxidation, respectively (Simon et al., 2002). Various stimuli, such as β-adrenergic agonists and exercise, trigger fatty acid generation (Dyck et al., 1998). In the present study, skeletal muscles may also be responsible for energy expenditure by increasing fat oxidation in the exercise group, which requires further study.

5 Conclusions

The present study is the first to confirm that dietary SCS can significantly enhance the benefits of exercise in suppressing adipose accumulation. The combination of dietary SCS and exercise can significantly reduce body weight, adipose tissue accumulation, and serum and hepatic lipids, which are consistent with the liver and adipose tissue morphology. Inhibition of adipose accumulation may be achieved by suppressing lipid synthesis in the liver and promoting lipolysis in epididymal white adipose tissue. The synergistic effects of anti-obesity food ingredients and exercise can effectively control body weight.

Acknowledgements

This work was supported by the National Key R & D Program of China (No. 2018YFD0901103) and the National Natural Science Foundation of China (Nos. 31901688 and 31571771).

Abbreviation List

ACC1, 1-aminocyclopropane-1-carboxylate 1; ACOX1, acyl-coenzyme A oxidase 1; ATGL, adipose triglyceride lipase; ACAA1, acetyl-coenzyme A acyltransferase 1; CPT1a, carnitine palmitoyl transferase 1a; CPT2, carnitine palmitoyl transferase 2; EA, echinoside A; FAS, fatty acid synthase; FABP4, fatty acid binding protein 4; FFA, free fatty acid; G6PDH, glucose-6-phosphate dehydrogenase; GC, gas chromatography; HA, holothurin A; HDL-C, density lipoprotein cholesterol; HF, high-fat diet group; HF-S, sea cucumber saponins group; HF-E, exercise group; HF-S + E, sea cucumber saponins and exercise group; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; LDL-C, low-density lipoprotein-cholesterol; ME, malic enzyme; PPARα, peroxisome proliferator activated receptor alpha; PPARγ, peroxisome proliferator-activated receptor γ; SCS, sea cucumber saponins; SCD1, stearoyl-coenzyme A desaturase 1; SREBP-1c, sterol regulatory element binding transcription factor 1; TC, cholesterol; TG, triacylglycerol.