Anti-obesity effects of agar (Gelidium amansii)-derived oligosaccharides in high-fat diet-treated C57BL/6N mice due to differential regulations of lipogenesis and lipolysis

ABSTRACT

To investigate the effects of agar oligosaccharides (AO) on lipid metabolism, changes in obesity phenotypes and related molecular factors were evaluated in C57BL/6N mice fed a high-fat diet (HFD). When HFD-induced obese mice were fed AO, they lost weight. Also, fat accumulation in abdominal and liver tissues was lower in the AO groups than in the Vehicle group. Lipid droplet sizes in tissue sections were reduced by AO, and these observations were mirrored by serum lipid contents. To evaluate the effects of AO on lipid metabolism, lipogenesis and lipolysis-related factors were analyzed. The mRNA expressions of genes involved in lipogenesis, such as adipocyte-protein 2 (aP2) and fatty acid synthase (FAS), were reduced by AO administration, and the expressions of lipolysis-associated proteins, including perilipin, hormone-sensitive lipase (HSL), and fat triglyceride lipase (ATGL), were increased. Taken together, our results suggest that AO should be considered a valuable natural agent that inhibits obesity.

Graphical Abstract

Agar oligosaccharides (AO) improve lipid metabolism in high-fat diet (HFD)-treated mice by regulating lipogenesis and lipolysis.

anti-obesity, agar oligosaccharide (AO), high-fat diet (HFD), lipid metabolism

Obesity is characterized by excessive accumulation of adipose tissues and induces metabolic disorders such as type 2 diabetes and hyperlipidemia, which can result in deadly complications, including optic neuropathy, renal failure, and heart disease (Heyn, Corrêa and Magalhães 2020). Undeniably, the incidence of obesity is increasing at a tremendous rate worldwide, and thus, interest in the development of foods that are satiating but do not increase body fat accumulation is increasing. Seaweeds contain large amounts of indigestible fiber, which is known to maintain satiety and suppress excessive nutrient absorption, and recently have been spotlighted as a health food that can prevent obesity (Lange et al.2015).

Agar is a linear polysaccharide composed of repeated agarobiose units and is industrially produced using red seaweeds included in the Gelidium genus (Din et al.2019; Fabra et al.2021). Agar, alginate, and carrageenan are the three major algal polysaccharides found in the cell walls of red seaweed (Li, Li and Zhu 2014; Lee et al.2017). These agar polysaccharides are underutilized in the industry despite their various functionalities due to limitations imposed by their poor solubilities and bio-availabilities (Louala and Lamri-Senhadji 2019; Chen et al.2021). However, agar oligosaccharide (AO) is considered a material that may overcome the limitations of agar polysaccharides in the food industry (Cheong et al.2018; Chen et al.2021). AO is prepared by randomly cleaving glycosidic bonds using acid in the industry. Recently, some seaweed-derived oligosaccharides have been reported to inhibit weight gain, improve lipid and glucose homeostasis, and lower inflammatory indicators and blood pressure levels (Cheong et al.2018; Li, He and Wang 2019; Chen et al.2021). Interestingly, AO has been reported to attenuate obesity by regulating short-chain fatty acid (SCFA) and bile acid (BA) metabolism-related pathways in the intestine of high-fat diet (HFD)-fed obese rodent models as prebiotics (Chen et al.2019; Higashimura et al.2016).

Although AOs are recognized as effective remedies for metabolic disorders, little research has been conducted on the anti-obesity effects of AO or the mechanism involved under HFD conditions. Therefore, we first established AO-producing conditions with excellent solubility and analyzed the molecular weight (MW) and degree of polymerization (DP) of AO. Next, we investigated the beneficial effects of AO on lipid metabolism in HFD-induced obese mice and the influence of AO on obesity-associated lipid metabolism by examining its effects on the expressions of molecular factors associated with lipogenesis and lipolysis pathways.

Materials and methods

Preparation of AO

Standardized agar (Gelidium amansii) specimens were provided in powdered form by the National Federation of Fisheries Cooperatives (Jeju, Korea) and its nutritional components are shown in Table 1. The voucher specimens were stored in the functional materials bank of the PNU-Wellbeing RIS center at Pusan National University (WPC-19-008).

Table 1.

Nutritional compositions of agar oligosaccharide (AO)

Content	Concentration (/100 g)
Calories	38.71 Kcal
Protein	0.21 g
Total Fat	0.07 g
Saturated Fat	0 g
Carbohydrate	88.13 g (Glu : Gal : Fruc = 2.18 :176.61 : 1:00)
Dietary Fiber	78.82 g
Sodium	509.98 mg
Moisture	11%
Ash	0.59%

We produced AO under four conditions and compared the solubility among them. The detailed experimental conditions are shown in Figure 1. The prepared AO was dissolved in distilled water (dH₂O) to 50 mg/mL, and diluted with 1X phosphate buffered saline (PBS) to the required concentration.

Figure 1.

Schematic diagram of various agar oligosaccharide (AO) producing conditions.

Molecular weight determination by gel permeation chromatography (GPC)

The molecular weight of the solution was evaluated and determined by gel permeation chromatography (GPC) with Waters Breeze Systems (Waters Corporation, USA) equipped with an ultrahydrogel linear column (500, 250, and 100). The detailed operation conditions were mobile phase: 0.02 N NaNO₃; flow rate: 0.8 mL/min; column temperature: 30^oC; injection volume: 20 µL; running time: 70 min. To construct calibration curves, the standard polyethylene glycol used 20600, 12600, 6690, 4290, 1400, 960, 430, and 106 (Mp: peak molecular weight basis).

Design of animal experiment

The animal protocol used in this study was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval Number PNU-2018-2061). All C57BL/6 mice used in the experiment were handled at the Pusan National University-Laboratory Animal Resources Center, which is accredited by the Korea Ministry of Food and Drug Safety (MFDS) (Accredited Unit Number-000231) and AAALAC International (Accredited Unit Number; 001525).

We purchased male C57BL/6N mice that were 7 weeks old from Samtako Biokorea Inc. (Osan, Korea) and provided them with ad libitum access to water and a standard irradiated chow diet (Samtako Biokorea Inc.). During the experiment, mice were maintained in a specific pathogen-free state under a strict light cycle (lights on at 08:00 h and off at 20:00 h) at 23 ± 2°C and 50 ± 10% relative humidity.

Experimental mice were acclimatized to a normal diet for 1 week and after subdivided as follows (7 mice/group): normal diet (ND), HFD plus Vehicle (200 µg of dH₂O), HFD plus 200 µg of 5% AO (low concentration of AO, LAO), and HFD plus 200 µg of 10% AO (high concentration of AO, HLO). AO was administered orally once daily for 8 weeks.

The HFD containing 60% kcal fat was purchased from Research Diets (Research Diets, Inc., New Brunswick, USA), and was fed to all mice of the subset groups treated with HFD for 8 weeks. After 24 h of the final AO treatment, all mice were euthanized using CO₂ gas, after which the tissue samples were acquired and stored in the tubes at −70 °C until assay.

Measurement of body and organ weight

Throughout the experimental period, the body weight of mice treated with Vehicle and AO were measured daily at 10:00 am using an electronic balance (Mettler Toledo, Greifensee, Switzerland) according to the National Institute of Food and Drugs Safety Evaluation (NIFDSE) guidelines. In addition, the weights of liver and abdominal fat (visceral and epididymis fat) weight collected from the sacrificed C57BL/6N mice were determined using the same method employed to measure the body weight.

Body mass index (BMI) was calculated using the weight and length (nose-to-anus [cm]) of animals at week 8 by the following formula (Novelli et al.2007).

$$\begin{equation*} {\rm{BMI}} = {\rm{body}}\,{\rm{weight}}\left( g \right)/{\rm{lengt}}{{\rm{h}}}^2\left( {{\rm{c}}{{\rm{m}}}^2} \right) \end{equation*}$$

Serum biochemical analysis

After the final treatment of AO, whole blood was collected from the abdominal veins of all C57BL/6N mice after fasting for 18 h. Serum was obtained from the blood by centrifugation at 1500 × g for 15 min. The concentration of low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG), total cholesterol (TC), and glucose were analyzed by the automatic chemical analyzer (BS-120 Chemistry Analyzer; Mindray, Shenzhen China). All assays were conducted in duplicate using fresh serum.

Histopathological analysis

Liver and abdominal tissues dissected from mice of all subset groups were fixed overnight in 10% neutral buffered formaldehyde (pH 6.8). The dehydrated tissue was then embedded in paraffin wax. Next, a series of liver and fat sections (4 µm) were cut from the paraffin-embedded tissues using a Leica microtome (#DM500, Leica Microsystems, Wetzlar, Germany). These sections were deparaffinized with xylene (#8587-4410, DAEJUNG, Gyeonggi-do, Korea), rehydrated with graded ethanol (decreasing concentrations of 100%-70%), and finally washed with distilled water. The slides with liver and fat sections were stained with hematoxylin (#MHS16, Sigma-Aldrich Co.) and eosin (#HT110332, Sigma-Aldrich Co.), washed with dH₂O, and pathological changes were assessed using the Leica Application Suite (Leica Microsystems).

Quantitative real time-PCR analysis

Frozen liver tissue was chopped with scissors and homogenized in RNA Bee solution (#CS-105B, Tet-Test Inc., Friendswood, TX, USA). Total RNA molecules were isolated by centrifugation at 16,000 × g for 15 min, and the total RNA concentration was measured by UV spectroscopy. The complementary DNA (cDNA) was synthesized by Invitrogen Superscript II reverse transcriptase (#4 376 600, Thermo Scientific, Wilmington, DE, USA). Quantitative PCR was performed with the cDNA template (1 µL) and 2× Power SYBR Green (6 µL; Toyobo Life Science, Osaka, Japan) containing specific primers. The primer sequences used for target gene expression identification were as follows: adipocyte fatty acid binding protein (aP2), sense primer: 5′-GAA CCT GGA AGC TTG TCT CCA GTG-3′ and anti-sense primer: 5′-GAT GCT CTT CAC CTT CCT GTC GTC TGC-3′; Fatty acid synthase (FAS), sense primer: 5′-GAT CCT GGA ACG AGA ACA CGA TCT GG-3′ and anti-sense primer: 5′-AGA CTG TGG AAC ACG GTG GTG GAA CC-3′; β-actin, sense primer: 5′- TGG AAT CCT GTG GCA TCC ATG AAA C-3′ and anti-sense primer: 5′- TAA AAC GCA GCT CAG TAA CAG TCC G-3′. qPCR was performed for 40 cycles using the following parameters: denaturation at 95 °C for 15 s, followed by annealing and extension at 70 °C for 60 s. Fluorescence intensity was measured at the end of the extension phase of each cycle. The threshold value for the fluorescence intensities of all samples was set manually. The reaction cycle at which the PCR products exceeded this fluorescence intensity threshold during the exponential phase of PCR amplification was considered as the threshold cycle (Cq). Expression of the target gene was quantified relative to that of the housekeeping gene β-actin, based on a comparison with the Cqs at a constant fluorescence intensity, as per the Livak and Schmittgen's method (Livak and Schmittgen 2001).

Western blot analysis

Collected liver tissue (50 mg) from each group was homogenized using PRO-PREPTM Solution (iNtRON Biotechnology Inc., Sungnam, Korea), after which total protein extracts were collected by centrifugation at 15,000 × g for 5 min. The prepared proteins were subsequently subjected to 10% SDS-PAGE for 1 h 30 min at 100 V, after which they were transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) for 2 h at 40 V in transfer buffer (25 m m Trizma-base, 192 m m glycine, and 20% methanol). Each membrane was then incubated separately at 4°C with the following primary antibodies overnight anti-perilipin antibody (#9349S, dilution, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-p-perilipin antibody (#9621S, dilution, 1:1000, Cell Signaling Technology), anti-hormone-sensitive lipase (HSL) antibody (#4107S, dilution, 1:1000, Cell Signaling Technology), anti-p-HSL antibody (#4139S, dilution, 1:1000, Cell Signaling Technology), anti-adipose triglyceride lipase (ATGL) antibody (#2183S, dilution, 1:1000, Cell Signaling Technology), and anti-β-actin antibodies (#4967S, dilution, 1:3000, Cell Signaling Technology). Next, the membranes were washed with washing buffer (137 m m NaCl, 2.7 m m KCl, 10 m m Na₂HPO₄, and 0.05% Tween 20) and then incubated with 1:1000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. Finally, membrane blots were developed using Amersham ECL Select Western Blotting detection reagent (GE Healthcare, Little Chalfont, UK). The chemiluminescence signals that originated from specific bands were detected using FluorChemi®FC2 (Alpha Innotech Co., San Leandro, CA, USA).

Statistical significance analysis

Statistical analyses were performed with SPSS for Windows, release 10.10, standard version (IBM SPSS, SPSS Inc., Chicago, IL, USA). One‑way analysis of variance followed by Tukey's post hoc test for multiple comparisons was applied to identify significant differences between groups. Data are presented as mean ± standard deviation (SD). P < .05 is considered to indicate a statistically significant difference.

Results

Solubility of AO produced under various conditions

Acid hydrolysis, a type of chemical hydrolysis, is a traditional method in an industry that produces AO (Zhu et al.2021). In this study, AO samples produced under four conditions (A, B, C, and D) were dissolved in 25°C and 80°C water at 50 mg/mL (Figures 1 and 2). In this study, the pretreatment factors affecting the solubility of AO during AO production were alkaline reagents and autoclave treatment at both temperatures (25°C and 80°C). First, for alkalinity, treating a strong alkali (5% NaOH) helped to provide greater solubility of AO compared to treating a weak alkali (Ca(OH)₂). Next, at high temperature and pressure (autoclave treatment) conditions, AO was easily dissolved into a solution. Only AO prepared under condition D (treatment of both NaOH and autoclave) had shown completely soluble in dH₂O. AO of condition D was used in subsequent physiological activity studies.

Figure 2.

Solubility of agar oligosaccharide (AO) produced under various conditions. The AO solution was prepared at a concentration of 50 mg/mL.

Molecular weight of AO

The results of GPC analysis showed that number average molecular weight (Mn) and weight average molecular weight (Mw) of AO were 2,220 Da and 2,602 Da, respectively, and polydispersity (Mw/Mn) was 1.17 (Figure 3 and Table 2). Considering that the molecular weight of Glu, Gal, and Fruc, the constituent monosaccharides of the prepared AO, is 180 g/mol, the AO average molecular weight of 2,220-2,602 obtained by GPC analysis means that the DP of AO is 12-14.

Figure 3.

The average molecular weight of agar oligosaccharide (AO). Mp (Peak molecular weight) basis.

Table 2.

The molecular weight of agar oligosaccharide (AO)

Name	Retention Time (min)	Mn	Mw	Mp	Polydispersity (Mw/Mn)
AO	38.671	2220	2602	3611	1.17

Mn: number average molecular weight, Mw: weight average moleculat weight, and Mp: peak molecular weight

Effect of AO on change of body weight and BMI

Weight changes in obese mice fed HFD+Vehicle or HFD+AO for 8 weeks are shown in Figure 4a. Mean weight gain in the HFD group (17.62 ± 1.53 g) was around 5-fold higher than in the ND group (3.48 ± 1.25 g). On the other hand, the LAO (13.63 ± 1.28 g) and HAO (12.31 ± 1.46 g) groups had lower weight gains than the HFD group without a reduction in dietary intake (data not shown).

Figure 4.

Effect of agar oligosaccharide (AO) on weight gain and body mass index (BMI) in high-fat diet (HFD)-induced obese mice. (a) Body weight changes. (b) BMI values. ND, normal diet; HFD, high-fat diet; LAO, low concentration (5%) of oligo saccharide (AO); HAO, high concentration (10%) of oligo saccharide (AO). Values are shown as mean ± standard deviation (n = 7 mice).

BMI was measured using the weight and length values of animals at week 8 of dietary treatment. It was confirmed that BMI was significantly reduced by AO treatment (Figure 4b).

Effect of AO on abdominal fat accumulation

Weights and sizes of abdominal tissues were found to be significantly greater in the HFD group than in the ND group, and this increase was reduced by the AO diet (Figure 5a). The weight of abdominal tissue was 11.7 times greater in the HFD group than in the ND group but was 1.4 and 1.75-fold less in the LAO and HAO groups, respectively, than in the HFD group. The sizes of lipid droplets in the abdominal tissues of obese mice fed the HFD diet are displayed in Figure 5b. Similarly, the sizes of adipocytes in abdominal tissue were significantly increased by the HFD and these increases were significantly decreased 3-fold by AO.

Figure 5.

Effect of agar oligosaccharide (AO) on the abdominal fat deposition in high-fat diet (HFD)-induced obese mice. (a) Weight changes of abdominal fat. The epididymal and retroperitoneal fats were collected in the abdominal region in mice of all subset groups. (b) Adipocyte size of epididymal tissue. ND, normal diet; HFD, high-fat diet; LAO, low concentration (5%) of oligo saccharide (AO); HAO, high concentration (10%) of oligo saccharide (AO). Values are shown as mean ± standard deviation (n = 7 mice). *P < .05 means a significant difference compared to ND, and ^#P < .05 means a significant difference compared to Vehicle.

Effect of AO on liver fat accumulation

Liver weights of mice fed the HFD diet are shown in Figure 6. In the HFD group, mean liver weight was ∼0.9 g higher than in the ND group. However, liver tissue weight was significantly lower in the LAO and HAO groups than in the HFD group (Figure 6a). Furthermore, lipid droplet sizes in livers were also significantly smaller in animals fed AO. In particular, in the HAO treatment group, liver weights and lipid droplet sizes were remarkably lower than in the HFD group (Figure 6b).

Figure 6.

Effect of agar oligosaccharide (AO) on the fat deposition in liver in high-fat diet (HFD)-induced obese mice. (a) Weight changes of liver and (b) histopathological levels of lipid droplet of liver tissue sections. ND, normal diet; HFD, high-fat diet; LAO, low concentration (5%) of oligo saccharide (AO); HAO, high concentration (10%) of oligo saccharide (AO). Values are shown as mean±standard deviation (n = 7 mice). *P < .05 means a significant difference compared to ND, and #P < .05 means a significant difference compared with Vehicle.

Effect of AO on level of serum biochemical indicators

Changes in serum profiles after 8 weeks on the HFD+Vehicle or HFD+AO diets are presented in Table 3. Glucose levels were similar in the HFD+AO and HFD+Vehicle control, but all lipid serum indicators were lowered compared to the Vehicle by AO treatment. In particular, total cholesterol and LDL levels were significantly reduced by AO treatment (P < .05).

Table 3.

Serum biochemical indicators in HFD-induced obese mice

		HFD
Content	ND	Vehicle	LAO	HAO
Total cholesterol (g/mL)	71.33 ± 8.02	133.33 ± 7.00*	127.43 ± 22.05*,#	115.14 ± 25.27*,#
Total glycerol (mg/mL)	37.66 ± 7.02	44.50 ± 8.20	41.00 ± 8.41	41.14 ± 7.35
Glucose (mg/dL)	153.66 ± 8.15	297.83 ± 13.53*	353.71 ± 16.27*	318.43 ± 11.82*
HDL (mg/dL)	60.40 ± 8.12	105.25 ± 2.80*	103.77 ± 10.02*	94.53 ± 12.91
LDL (mg/dL)	5.60 ± 1.25	18.52 ± 1.17*	17.04 ± 3.76*,#	15.10 ± 2.08*,#

ND, normal diet; HFD, high-fat diet; LAO, low concentration (5%) of oligo saccharide (AO); HAO, high concentration (10%) of oligo saccharide (AO). HDL, high density lipid; LDL, low density lipid. Values are shown as mean±standard deviation (n = 7 mice). *P < .05 means a significant difference compared with ND, and ^#P < .05 means a significant difference compared Vehicle.

Effect of AO on lipogenesis factors in the liver

aP2 is involved in adipocyte differentiation, which affects the production of lipid droplets, whereas FAS participates in fatty acid synthesis, and these two proteins ultimately contribute to fat deposition in vivo. Therefore, we assessed the effects of AO on the mRNA expressions of aP2 and FAS (Figure 7). aP2 and FA2 levels were 2.3 and 1.9-fold higher, respectively, in liver tissues of the HFD group than in those of the ND group, and HAO treatment significantly reduced these increases.

Figure 7.

Effect of agar oligosaccharide (AO) on (a) FAS and (b) aP2 mRNA expression in liver tissue of high-fat diet (HFD)-induced obese mice. ND, normal diet; HFD, high-fat diet; LAO, low concentration (5%) of oligo saccharide (AO); HAO, high concentration (10%) of oligo saccharide (AO); FAS, fatty acid synthase; aP2, adipocyte protein 2. Values are shown as mean±standard deviation (n = 7 mice). *P < .05 means a significant difference compared to ND, and ^#P < .05 means a significant difference compared with Vehicle.

Effect of AO on lipolysis factors in the liver

Lipolysis provides a means of reducing total body fat. For this reason, we analyzed the protein expressions of the three major factors, viz. perilipin, HSL, and ATGL, involved in lipolysis (Figure 8). AO treatment increased the expressions of perilipin and HSL compared with HFD+Vehicle control. However, the level of ATGL was lower in AO groups than in HFD+Vehicle control. These results imply that fat hydrolysis induced by AO involves the decomposition of a diglyceride (DG) into monoglyceride (MG) by perilipin and HSL, rather than the breakdown of TG by ATGL.

Figure 8.

Effect of agar oligosaccharide (AO) on lipolysis in liver tissue of high-fat diet (HFD)-induced obese mice. (a) Western blot analysis measured the phosphorylation or expression of several lipolysis-involved proteins, including (b) perilipin and p-perilipin, (c) HSL and p-HSL, and (d) ATGL. The intensity of each band was determined using an imaging densitometer. The relative levels of proteins were calculated based on the intensity of actin. ND, normal diet; HFD, high-fat diet; LAO, low concentration (5%) of oligo saccharide (AO); HAO, high concentration (10%) of oligo saccharide (AO); p-, phosphorylated; perilipin, HSL, hormone-sensitive lipase; ATGL, adipose triglyceride lipase. Values are shown as mean±standard deviation (n = 7 mice). *P < .05 means a significant difference compared to ND, and ^#P < .05 means a significant difference compared to Vehicle.

Discussion

HFD-induced obese mice have been widely used in the study of obesity-related mechanisms because increased lipid absorption by HFD accumulates fat in obese phenotypes, ie large amounts of lipids in the bloodstream and liver and abdominal tissues (Kim et al.2021). This HFD-induced phenotype was also observed in the present study. Mice administered the HFD for 8 weeks exhibited increased levels of fat in livers and abdomens and weight gains. However, AO treatment reduced the growth of lipid droplets in livers and abdominal tissues and lowered serum lipid levels in the HFD group. In addition, AO reduced HFD-induced increases in liver and abdominal tissue weights, body weights, and BMI values demonstrating that AO has an anti-obesity effect.

Although seaweed crude extracts have been well reported to inhibit the development of obesity, few have studied the anti-obesity effect of AO (Bermano et al.2020; Murakami et al.2021). Some studies have demonstrated the prebiotic and probiotic effects of functional AO and reported its potential use as an anti-obesity material (Li, Li and Zhu 2014; Higashimura et al.2016; Qixing Nie et al.2020).

AO is included in the non-digested oligosaccharide (NDO), which is considered prebiotics. AO is known to control SCFA and BA metabolism related to anti-obesity effects. Those two metabolic pathways and energy homeostasis are closely related in rodent models (Liu et al.2020).

The intestinal microbiota increases the intestinal SCFAs by fermenting NDOs. SCFAs stimulate the production of gut satiety hormones, peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) to affect energy intake and appetite (Dréan et al.2019).

Also, BAs form micelles, amphiphilic steroid molecules for emulsification and intestinal absorption of fat-soluble vitamins and lipids. BAs are triggers that mediate lipid metabolic signaling, generating heat in adipose tissue, increasing energy consumption, and improving the secretion of GLP-1. NDO can induce anti-obesity activity by changing BA composition (Mistry et al.2020).

The bioactivity of the oligosaccharide may vary depending on the purity, DP, and linkage characteristics (Jiang, Cheng and Liu 2021; Zhu et al.2021). Various separate interpretations of the structure-dependent physiological activity of AO appear in works of literature. This demonstrates the complexity of the composition and sequence of AOs, which are still challenges.

The tested AO of DP12-14 is larger than the DP used in other oligosaccharide studies. The possibility of the AOs of DP12-14 being decomposed into small molecular AOs by reacting with various enzymes and chemicals in the body before being applied to lipid metabolism cannot be completely excluded. In particular, agar is known as an appropriate and efficient substrate for producing galacto-oligosaccharide (GO). In the previous studies, it has been reported that the major oligosaccharides of AOs, GOs, improved the intestinal microbial environment in mice or rat, leading to improved dyslipidemia and reduced body fat by regulating bile salt hydrolase (BSH) activity and BA metabolism-related obesity (Chen et al.2019; Dai et al.2019). Also, Tran et al. reported that GOs change the gut microbiota and SCFAs of Mud Crab (Tran et al.2020). It is necessary to conduct more studies with more systematized conditions to prove the structure-activity relationship of AO in the future.

To investigate the effect of AO on HFD-induced obesity, we examined changes in molecular factors involved in lipogenesis (aP2 and FAS) and lipolysis (perilipin, HSL and ATGL) pathways to gain insight into the mode of action of AO at the molecular level.

Obesity is caused by fat deposition when the energy balance between acquisition and consumption favors acquisition. This balance is determined by complex genetic variations and interactions and intracellular communications between lipogenic and lipolytic genetic factors (Kim et al.2016).

Lipid metabolic enzymes such as aP2 and FAS trigger lipogenesis (Ree et al.2021). aP2 is a key mediator of fatty acid intracellular transport and metabolism and an adipocyte differentiation marker that triggers the generation of lipid droplets in the intermediate stages of adipocyte differentiation, and thus, increases adipocyte sizes (Jang, Choi and Kim 2019). In contrast, FAS induces the production and maintenance of adipocyte phenotypes during the latter stages of lipid accumulation (Bae et al.2020; Kurek et al.2022). Therefore, these factors have been widely studied as key indicators of the potencies of anti-obesity materials. In the current study, we found that aP2 and FAS levels increased by HFD were reduced by AO (Figure 7), which suggests the gene expressions of aP2 and FAS are associated with a decrease in the size of lipid droplets in liver and adipose tissue (Figures 5 and 6). In previous studies, AOs activated AMPK and thus decreased lipids accumulation and improved lipid metabolism via regulating SREBP-1 proteins in HepG2 cells (Wang et al.2020). Also, when unsaturated alginate oligosaccharides (UAO) were administered to HFD-induced mice, the inhibition of lipogenesis was attributed to AMP-activated protein kinase (AMPK) signaling (Li, He and Wang 2019). Also, Pan et al. reported that chitosan-oligosaccharides suppressed the expressions of molecular indicators related to adipogenesis and lipogenesis and reduced lipid accumulation (Pan et al.2018). These previous reports support our findings.

In the present study, the expressions of aP2 and FAS two factors were analyzed to investigate the inhibitory effect of AO on lipogenesis. However, to further understand the effects of AO on adipogenesis at the molecular level, additional AMPK studies are needed. Because the expression of early stage adipogenic factors such as peroxisome proliferator-activated receptor and CCAAT-enhancer binding protein (PPAR and C/EBP) families regulating the expression of aP2 and FAS depends on whether AMPK has been phosphorylated.

Meanwhile, in addition to the synthesis of TG through lipogenesis, lipolysis controls total fat contents in adipose tissue (Yang and Mottillo 2020). ATGL is the initiatory enzyme of lipolysis and decomposes TG into diacylglycerides (DG) and controls the maximum rate of fatty acid mobilization (Lee et al.2019). With the assistance of phosphorylated perilipin by protein kinase A (PKA), phosphorylated or activated HSL is translocated from cytosol to lipid droplet surfaces to increase lipolytic activity (Yang and Mottillo 2020). As a result of subsequent hydrolysis reactions, DG and monocylglycerides (MG) in TG are decomposed, and fatty acids are produced (Kwon et al.2022). Our results show that AO acted more strongly on the phosphorylation of HSL and perilipin, which decomposes DG into MG than ATGL activation in lipolysis mechanisms (Figure 8). Because ATGL, unlike HSL and perilipin, is not subject to PKA phosphorylation, there may be other mechanisms whereby ATGL mediates lipolytic activity in response to AO treatment. Alternatively, ATGL may not be the major lipase stimulated by AO (Yang et al.2010; Jiang et al.2022).

In summary, AO attenuated HFD-induced increases in blood lipid indicators and reduced the sizes of adipocytes in liver and abdominal tissues, and thus, caused weight loss of tissues and body. This obesity inhibition effect is believed to be due to AO-induced reductions in the expressions of molecules related to lipogenesis and/or increases in the expressions of molecules related to lipolysis. Therefore, we suggest that AO is considered a natural anti-obesity material with the potential to prevent or treat HFD-induced obesity.

Acknowledgments

We thank Jin Hyang Hwang, the animal technician, for directing the animal care at the Laboratory Animal Resources Center in Pusan National University.

Data availability

The data used to support the findings of this study are available from the corresponding author upon request.

Author contribution

M.R.L. and J.E.K. conducted experiments, analyzed data, and wrote the manuscript. Y.J.J., Y.J.R., A.S., H.J.S., M.W.J., and J.T.H. conducted experiments and analyzed the data. M.J. and D.Y.H. designed, analyzed, and supervised the research and wrote the manuscript.

Funding

This study was supported by Company Promotion Program (R0005304) of Ministry of Trade, Industry and Energy, and the BK21 FOUR Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (F22YY8109033), Korea.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author notes

These authors equally contributed to the study.