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. 2021 Feb 6;30(2):299–304. doi: 10.1007/s10068-020-00859-8

Mineral-rich Jeju lava sea water suppresses lipid accumulation in 3T3-L1 adipocytes and ameliorates high-fat diet-induced obesity in C57BL/6 J mice

Ye Ji Hyun 1, Ju Gyeong Kim 2, Min Jeong Kim 2, Sung Keun Jung 2, Ji Yeon Kim 1,✉
PMCID: PMC7914322  PMID: 33732520

Abstract

This research aimed to evaluate the potential inhibitory effect of mineral-rich Jeju lava sea water (JLSW) on lipid accumulation. This study optimized the calcium (Ca): magnesium (Mg) ratio (5:1, 2.5:1, 1:1) of JLSW and evaluated the effect on lipid accumulation in 3T3-L1 cells using Oil Red O staining. JLSW with a high Ca:Mg ratio (5:1) suppressed lipid accumulation in 3T3-L1 adipocytes. Based on these in-vitro results, the effects of JLSW on lipid accumulation were investigated in C57BL/6 J mice fed high-fat diets for 14 weeks. Epididymal adipose tissue weight was significantly decreased in mice that received JLSW with a hardness of 800 or 100 mg/L compared to HFD. Adipocyte size was significantly reduced in mice treated with JLSW with a hardness of 20 mg/L in comparison with HFD. Thus, long-term intake of JLSW may be expected to have anti-obesity effects due to the reduction of lipid accumulation.

Keywords: Jeju lava sea water, Adipogenesis, 3T3-L1, High-fat diet, Adipocyte size

Introduction

Adipose tissue is composed of adipocytes, also known as lipocytes or fat cells. Energy balance is controlled by the adipocytes in adipose tissues (Kwon et al., 2015). In adipose tissues, excess energy is stored in the form of triglycerides, which is decomposed into fatty acids when energy is needed (Yu and Ginsberg, 2005). Adipose tissue grows via two mechanisms: hyperplasia, which refers to an increase in the number of adipocytes, or hypertrophy, which is an increase in adipocyte size. The number of adipocytes is generally established early in life. Thereafter, ingestion of excess calories results in adipocyte size expansion (Ghaben and Scherer, 2019). When adipocytes increase in size and new adipocytes are formed, lipids accumulate abnormally in adipose tissue, leading to obesity. Obesity is a growing problem in modern society and can cause chronic diseases such as type 2 diabetes, hypertension, and cancer (Jang et al., 2019).

Intracellular calcium (Ca2+) regulates insulin signaling and the production of new fat cells by inhibiting insulin-regulated dephosphorylation (Hwang et al., 2009a; 2009b; Song et al., 2004; Worrall and Olefsky, 2002; Yang et al., 2000). Magnesium (Mg) acts as a cofactor in carbohydrate metabolism, which affects insulin and glucose metabolism. Therefore, Mg sufficiency has a beneficial effect on obese diabetics (Paolisso et al., 1992; Song et al., 2004). Accordingly, deep-sea water rich in minerals, such as Ca and Mg, is reportedly effective in inhibiting pre-adipocyte differentiation. In-vitro studies have shown that deep-sea water containing Ca and Mg effectively inhibits adipocyte differentiation (Hwang et al., 2009b; Noh et al., 2010), and in vivo studies have demonstrated that it has anti-obesity and anti-diabetes effects (Ha et al., 2013; Hwang et al., 2009a). Jeju lava sea water (JLSW) is naturally filtered by the Jeju volcanic rock, and it is rich in minerals, including Mg, Ca, potassium, zinc, and selenium, as well as nutrients (Jung et al., 2017). Thus, JLSW is a valuable drinking water source, and there is a need to evaluate its functionality. In the present study, we investigated the effects of JLSW in 3T3-L1 cells and high-fat diet (HFD)-induced obese C57BL/6 J mice by measuring weight of body weight and epididymal adipose tissue and adipocyte size.

Materials and methods

Materials

Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and penicillin–streptomycin were purchased from Biowest (Nuaillé, Cholet, France). Bovine calf serum (BS) and DMEM powder were obtained from Gibco (Rockville, MD, USA). Insulin from bovine pancreas, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), 2-propanol, and Oil Red O (ORO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of JLSW

JLSW was supplied by Orion Jeju Yongamso (Jeju, Korea) and was used at various Ca:Mg ratios in this study. In order to use the provided JLSW for cell culture, DMEM powder was dissolved in JLSW. The control group dissolved DMEM powder in diluted water instead of JLWS. For in vitro experiments, JLSW was prepared with Ca:Mg ratios of 5:1 (containing 580 μg/L Zn, 5.6 μg/L Mn, 5.9 mg/L I, 15.5 mg/L SiO2, 12 μg/L Ge, and 4.1 μg/L V), 2.5:1 (containing 652 μg/L Zn, 5.6 μg/L Mn, 7.2 mg/L I, 13.6 mg/L SiO2, 12 μg/L Ge, and 3.6 μg/L V) and 1:1 (containing 690 μg/L Zn, 8.6 μg/L Mn, 5.8 mg/L I, 10.5 mg/L SiO2, 14 μg/L Ge, and 2.4 μg/L V), while for in vivo experiments, it was prepared by diluting to different hardness levels (800, 400, 210, 100, and 20 mg/L) with a Ca:Mg ratio of 7:1 (containing 560 μg/L Zn, 5.6 μg/L Mn, 5.6 mg/L I, 16.8 mg/L SiO2, 12 μg/L Ge, and 4.4 μg/L V). The Ca:Mg ratio used in vivo was based on the in-vitro results.

Cell culture and differentiation

3T3-L1 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The composition of the medium was made according to various differentiation process: Preadipocyte growth medium is DMEM containing 10% BS and 1% penicillin–streptomycin; Differentiation medium is DMEM containing 10% FBS, 10 μg/mL insulin, 115 μg/mL IBMX, 1 μM DEX, and 1% penicillin–streptomycin; Differentiation maintain medium is DMEM containing 10% FBS, 10 μg/mL insulin, and 1% penicillin–streptomycin; The 3T3-L1 preadipocytes were cultured in preadipocyte growth medium at 37 °C and in an atmosphere of 5% CO2. 3T3-L1 preadipocytes were seeded in 6-well plates at 1 × 105 cells/mL with in preadipocyte growth medium and cultured until 90%–100% confluence. Differentiation was performed using insulin to induce differentiation of 3T3-L1 cells into adipocytes. Thereafter, the cells were further cultured in differentiation medium with JLSW or diluted water for 6 days. To maintain the adipocytes, 3T3-L1 cells were cultured in maintain medium dissolved in JLSW or diluted water for 6 days. The medium was changed every 2–3 days.

ORO staining

After washing with DPBS, the 3T3-L1 adipocytes were fixed with 4% paraformaldehyde for 1 h. The stock solution was dissolved in 0.5% isopropanol and diluted with diluted water to prepare 60% ORO as a working solution. The working solution was filtered through filter paper (Whatman No. 3, USA) and added to the adipocytes for 30 min. After washing with diluted water, isopropanol was added to extract the stain from the stained adipocytes. The absorbance at 510 nm was measured using a spectrometer (BioTek Instruments Inc., Winooski, VT, USA), in a 96-well plate.

Animals and diets

Five-week-old male C57BL/6 J mice were purchased from Joong-Ah Bio (Suwon, Korea). The mice were housed in an air-conditioned room (23 ± 2 °C) with a 12 h light/dark cycle. They were allowed free access to food and tap water. All animals received humane care, and the study protocol (KNU-2020–0045) was approved and performed in accordance with the guidelines for animal use and care at Kyungpook National University. The diets included a normal diet (A04; SAFE, Augy, France) and a HFD of 60% kcal (D12492; Research Diets Inc. New Brunswick, NJ, USA). Mice were acclimated for 1 week prior to the study and then randomly allocated to the following treatment groups (n = 10 mice per group, seven groups in total): normal diet and sterilized water (CON group), HFD and sterilized water (HFD group), HFD and JLSW with a hardness of 800 mg/L (H800 group), HFD and JLSW with a hardness of 400 mg/L (H400 group), HFD and JLSW with a hardness of 210 mg/L (H210 group), HFD and JLSW with a hardness of 100 mg/L (H100 group), HFD and JLSW with a hardness of 20 mg/L (H20 group). Mouse weight, food, and water intake were recorded weekly throughout the study. After 14 weeks, the animals were sacrificed and epididymal adipose tissues and livers were carefully removed and weighed.

Measurement of adipocyte size

Epididymal adipose tissue was collected, washed with physiological saline to remove the blood, and snap-frozen in liquid nitrogen and stored at −80 °C for analysis. After fixing the adipose tissues with a 10% formalin solution, the fixation solution was removed, and the tissues were treated with xylene to remove the alcohol content. The tissues were embedded in paraffin and sliced at 4 μm thickness along the central vein and stained with hematoxylin–eosin (H&E). The tissues were observed and photographed under an optical microscope, at a magnification of 200 × . Adipocyte size was measured using the NIH Image J software.

Statistical analysis

All statistical analyses were performed using SAS v. 9.4 (SAS Institute, Cary, NC, USA). All data are reported as the mean ± standard error (SE). Means were compared by analysis of variance, followed by Duncan’s multiple range tests. p < 0.05 was considered significant.

Results and discussion

Figure 1 shows the effects of JLSW on lipid accumulation in 3T3-L1 cells according to the Ca:Mg ratio of JLSW. 3T3-L1 cells are a preadipocytes, and widely used to confirm lipid metabolism in in vitro studies. Insulin was treated in 3T3-L1 cells to induce lipid accumulation and then stained with ORO to quantify lipid accumulation. The amount of accumulated lipids was expressed as % value for NC group of preadipocytes without JLSW treatment (Fig. 1A). In 5:1 group with the highest of Ca:Mg ratio, lipid accumulation was significantly decreased by 34.63% compared to the 0 group of mature adipocytes cultured without JLSW (p < 0.05). Lipid accumulation was also significantly reduced to 29.99% and 11% in 2.5:1 group and 1:1 group (p < 0.05), respectively. When ORO-stained 3T3-L1 cells were observed under the microscope, red-stained lipids were found to decrease with increasing Ca:Mg ratio (Fig. 1B). It was confirmed that the highest Ca:Mg ratio (5:1) was the highest suppression of lipid accumulation. A previous study reported that mineral-balanced deep-sea water (Ca:Mg = 3:1) prevented high glucose or free fatty acid as well as high glucose-induced cellular cholesterol accumulation (Lee et al., 2017). Although the Ca:Mg ratio in this previous study was different, it can be concluded that balanced mineral sea water exerts a beneficial effect on lipid metabolism and accumulation.

Fig. 1.

Fig. 1

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Effect of JLSW on lipid accumulation 3T3-L1 cells. (A) Lipid accumulation was quantified by ORO staining. (B) Images of ORO-stained 3T3-L1 cells. 3T3-L1 cells were induced to differentiate by treatment with insulin and were then treated with JLSW with different Ca:Mg ratios. Lipid accumulation is expressed relative to that in 3T3-L1 preadipocytes without JLSW. NC, normal control. 3T3-L1 preadipocytes without JLSW; 0, mature adipocytes without JLSW; 1:1, mature adipocytes with JLSW with a 1:1 Ca:Mg ratio; 2.5:1, mature adipocytes with JLSW with a 2.5:1 Ca:Mg ratio; 5:1, mature adipocytes with JLSW with a 5:1 Ca:Mg ratio. Values with different letters are significantly different at p < 0.05, as determined by Duncan’s multiple range test

In the in-vitro study, JLSW was prepared with various Ca:Mg ratios (5:1, 2.5:1, and 1:1). Based on the in-vitro results, the Ca:Mg ratio was maximized to 7:1 for in vivo assays to maintain a high mineral content because, considering taste and smell, hardness should not be > 800 mg/L. The effect of JLSW on adipocyte size in-vivo was assessed using HFD-induced obese C57BL/6 J mice. JLSW was used at hardness of 800 mg/L (H800), 400 mg/L (H400), 210 mg/L (H210), 100 mg/L (H100), or 20 mg/L (H20). As shown in Table 1, there were no significant differences in body and liver weights after 14 weeks of HFD. The levels of food intake were slightly increased, while water intake in the H800, H210, and H100 groups significantly increased by 12.54%, 8.40%, and 20.87% (p < 0.05). As water intake increased, the weight of epididymal adipose tissue also showed a tendency to decrease significantly compared to HFD (p < 0.05). In particular, the H800 and H100 groups decreased significantly to 24.10% and 23.31% (p < 0.05). In the H400 and H210 groups, the weight of epididymal adipose tissue showed a decreasing trend by 12.75% and 12.35% compared to the HFD group.

Table 1.

Effects of JLSW on body weight, food intake, water intake, and organ weight

Obese mice Group1
CON HFD H800 H400 H210 H100 H20
Initial weight (g) 22.03 ± 3.60a 22.27 ± 1.91a 22.42 ± 1.16a 22.11 ± 1.17a 22.45 ± 0.95a 22.64 ± 1.53a 21.89 ± 1.10a
Final weight (g) 30.79 ± 2.34b 45.69 ± 5.04a 48.22 ± 3.41a 45.70 ± 5.79a 48.19 ± 4.71a 48.93 ± 6.15a 45.75 ± 3.60a
Food intake (g/day) 12.08 ± 0.66bc 11.64 ± 0.62 cd 12.45 ± 0.54b 11.77 ± 0.64 cd 12.42 ± 0.60b 12.96 ± 0.69a 11.37 ± 0.58d
Water intake (mL/day) 14.98 ± 1.20 cd 14.28 ± 0.73de 16.07 ± 0.91b 14.43 ± 0.87de 15.48 ± 0.99bc 17.26 ± 1.11a 13.67 ± 1.27e
Liver (g/100 g body weight) 3.22 ± 0.96a 3.54 ± 0.50a 3.96 ± 1.12a 3.59 ± 0.95a 3.86 ± 1.19a 4.03 ± 1.14a 3.08 ± 0.61a
Epididymal adipose tissue (g/100 g body weight) 1.92 ± 0.48c 5.02 ± 0.82a 3.81 ± 1.03b 4.38 ± 1.39ab 4.40 ± 1.10ab 3.85 ± 0.94b 5.32 ± 0.74a
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1CON, normal diet and sterilized water; HFD, high-fat diet and sterilized water; H800, HFD and JLSW with a hardness of 800 mg/L; H400, HFD and JLSW with a hardness of 400 mg/L; H210, HFD and JLSW with a hardness of 210 mg/L; H100, HFD and JLSW with a hardness of 100 mg/L; H20, HFD and JLSW with a hardness of 20 mg/L

Data are the mean ± standard error (SE) (n = 10). Values with different letters are significantly different at p < 0.05, as determined by Duncan’s multiple range test

Adipocyte size in epididymal adipose tissues of the mice was measured using NIH Image J software (Fig. 2A). HFD group significantly increased adipocyte size (81.46 μm) compared to the control (50.92 μm) (p < 0.05). In the H100 and H20 groups, adipocyte size was significantly decreased by approximately 9.02% and 9.84% (to 74.30 μm and 73.81 μm), respectively, compared to that in the HFD groups (p < 0.05). However, the H800, H400, and H210 group had no significant effect. Figure 2B shows images of adipocytes in epididymal adipose tissues. There was no visual difference in adipocyte size between HFD and H800, H400 and H210 groups, whereas adipocyte size was visibly reduced in the H100 group and, in particular, the H20 group. Thus, H20, with the lowest hardness, was most effective in reducing adipocyte size in HFD-induced obese mice.

Fig. 2.

Fig. 2

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Effect of JLSW on adipocyte size in epididymal adipose tissue. (A) Measurement of adipocyte size in epididymal adipose tissue. (B) Histological analysis of H&E-stained epididymal adipose tissues. After 14 weeks of JLSW administration, C57BL/6 J mice were sacrificed and epididymal adipose tissues were excised. Adipocyte size was determined based on H&E staining. CON, normal diet and sterilized water; HFD, high-fat diet and sterilized water; H800, HFD and JLSW with a hardness of 800 mg/L; H400, HFD and JLSW with a hardness of 400 mg/L; H210, HFD and JLSW with a hardness of 210 mg/L; H100, HFD and JLSW with a hardness of 100 mg/L; H20, HFD and JLSW with a hardness of 20 mg/L. Values with different letters are significantly different at p < 0.05, as determined by Duncan’s multiple range test

Drinking water can be a source of Ca and Mg. JLSW can be a good source of Ca and Mg in individuals whose Ca and Mg intake is limited (Sengupta, 2013). Several in-vivo studies have clearly shown that consumption of mineral-rich sea water prevents lipid accumulation in the mice liver (Chen et al., 2013; Woo et al., 2019). This study focused on Mg and Ca to investigate the health benefits of JLSW according to the Ca:Mg ratio and hardness. According to previous studies, minerals such as Mg and Ca are associated with lipid metabolism. Dietary magnesium decreased lipid accumulation in the aortic wall in in-vivo models of high cholesterol intake. High calcium diet suppressed lipid accumulation in rat adipose tissues (He et al., 2011; Ouchi et al., 1990). Intracellular Ca reportedly regulates obesity by controlling adipogenesis in 3T3-L1 adipocytes and adipose tissues of rodents (Hwang et al., 2009b; Parra et al., 2008; Worrall and Olefsky, 2002). Ca and Mg affect the taste of water (Koseki et al., 2006). Therefore, we conducted in-vivo experiments using various hardness levels. Body weights of the mice were not significantly reduced by the JLSW.

The limitation of this study is that the effect of body weight loss could not be confirmed. Considering that epididymal adipose tissue weight and adipocyte size in epididymal adipose tissues were decreased, our experimental period was too short to observe body weight loss. Long-term intake of JLSW may reduce body weight through a reduction in adipose tissue weight and adipocyte size and can be expected to have an anti-obesity effect. Therefore, this study should be further validated in-vitro and in-vivo for its anti-obesity effect and should be verified through clinical trials.

In conclusion, the effect of mineral-rich JLSW on lipid accumulation was confirmed through 3T3-L1 cells and mice induced obesity with HFD. The reduction of lipid accumulation in 3T3-L1 adipocytes was verified by ORO staining, and reduced the weight and adipocytes size of epididymal adipose tissue in obese mice.

Acknowledgements

This study was supported by Orion Jeju Yongamsoo (Jeju, Korea) and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (Grant No. 2017R1E1A1A01074320).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Ye Ji Hyun, Email: yj0706hyun@naver.com.

Ju Gyeong Kim, Email: jgrla23@naver.com.

Min Jeong Kim, Email: minjung8128@gmail.com.

Sung Keun Jung, Email: skjung04@knu.ac.kr.

Ji Yeon Kim, Email: jiyeonk@seoultech.ac.kr.

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