A high-fat diet or high-cholesterol diet (HCD) is a major cause of metabolic diseases, including obesity and diabetes; vascular diseases, including hypertension, stroke and arteriosclerosis; and liver diseases, including hepatic steatosis and cirrhosis. The present study aimed to evaluate the effects of deep sea water (DSW) on rats fed a HCD. DSW decreased HCD-induced increases in total cholesterol and low-density lipoprotein (LDL) cholesterol in the blood, and recovered high-density lipoprotein cholesterol. In addition, DSW decreased levels of liver injury markers, which were increased in response to HCD, including glutamate-oxaloacetate transaminase, glutamate-pyruvate transferase and alkaline phosphatase. Lower lipid droplet levels were observed in the livers of rats fed a HCD and treated with DSW at a hardness of 1,500, as compared with those in the HCD only group. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that mRNA expression levels of fatty acid synthase and sterol regulatory element binding protein-1c (SREBP-1c) in rats fed a HCD with DSW were lower compared with the HCD only group. Furthermore, quantitative RT-PCR revealed that DSW enhanced LDL receptor (LDLR) mRNA expression in a hardness-dependent manner. Combined, the results of the present study indicated that DSW may reduce HCD-induced increases in blood and liver lipid levels, indicating that DSW may protect against hypercholesterolemia and non-alcoholic hepatic steatosis. In addition, the present study demonstrated that DSW-induced downregulation of lipids in the blood and hepatic lipid accumulation was mediated by enhancement of LDLR expression and suppression of fatty acid synthase and SREBP-1c.
Hyperlipidemia is a lipid metabolism disorder, the prevalence of which has markedly increased in recent years. This disorder, which is caused by excessive consumption of food containing high levels of fat and cholesterol, is closely associated with hypertension, atherosclerosis (AS) and cardiovascular diseases (CVD) (
It is well established that amelioration of lipid concentration in the blood prevents hypercholesterolemia and hepatic lipid accumulation. Lipid metabolism in the liver is controlled by fatty acid-synthesizing and energy expenditure enzymes, with decreased energy expenditure enzymes and increased fatty acid-synthesizing enzymes generally being observed in the livers of obese animal models fed a high-fat diet (HFD) and/or a high-cholesterol diet (HCD) (
Deep sea water (DSW) is considered a potent material that has food and medical applications. DSW contains abundant minerals, including magnesium (Mg), calcium (Ca), potassium (K) and zinc, which have important roles in cellular homeostasis and physiological responses (
DSW was obtained from the Marine Deep Ocean Water Application Research Center in the Korea Institute of Ocean Science & Technology (Ansan, South Korea) from a depth of 500 m in the East Sea (Goseong, South Korea). Saline and minerals in DSW were removed and extracted by reverse osmosis filtration and electrodialysis (
Animal experiments were conducted following approval by the Animal Use and Care Committee at Dongguk University (approval IACUC-2013-001; Gyeongju, Korea). A total of 42 male 5-week old Sprague-Dawley rats (120–130 g) with a normal diet (ND; 5L57, containing no cholesterol) were obtained from Orient Bio Inc. (Seongnam, Korea). The rats were housed under a 12 h light/dark cycle at 25±2°C and a relative humidity of 50±5%. The rats received the ND and tap water ad libitum for 1 week. Subsequently, rats received a HCD (D12336, Research Diets, Inc., New Brunswick, NJ, USA) with tap water or DSW of various hardness ad libitum for 6 weeks. The composition of the HCD is presented in
TG, TC and high-density lipoprotein cholesterol (HDL-c) in the blood were enzymatically analyzed using commercial kits (AM157K, AM202K and AM203K respectively; Asan Pharmaceutical Co., Ltd., Seoul, Korea) according to the manufacturer's protocols. The LDL-c concentration was calculated using the Friedwald formula: LDL-c concentration=TC concentration-HDL-c concentration-TG/2.
Glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transferase (GPT) and alkaline phosphatase (ALP) activity in the blood were assessed as indicators of liver damage. GOT and GPT activities were measured using a commercial kit (AM101K; Asan Pharmaceutical Co., Ltd.) based on the Reitman-Frankel method (
Livers were pre-fixed with 0.1 M PBS containing 2.5% glutaraldehyde for 2 h at 4°C and subsequently washed with 0.1 M PBS three times for 15 min. The tissues were subsequently post-fixed by immersion in 2% osmium tetroxide solution for 2 h at 4°C followed by dehydration with ethanol. Tissues were embedded with epon-812 resin, sectioned at 100 nm thickness using a Leica Ultracut R (Leica Microsystems GmbH, Wetzlar, Germany) and double-stained with uranyl acetate and lead nitrate. Finally, tissues were visualized using a Hitachi H-7500 transmission electron microscope (Hitachi, Ltd., Tokyo, Japan) at 80 kV.
The expression of fatty acid synthase (FAS), carnitine palmitoyltransferase-1 (CPT-1), sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor γ (PPARγ) was analyzed by semi-quantitative RT-PCR, and qPCR was used to analyze the expression of LDLR. Livers were rapidly frozen in liquid nitrogen and stored at −80°C. Total RNA in individual liver samples was extracted using an easy-BLUE™ Total RNA Extraction kit (17061; Intron Biotechnology, Inc., Seongnam, Korea) according to the manufacturer's protocol. cDNA synthesis was performed using PrimeScript™ 1st strand cDNA Synthesis kit (6110a; Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocols and amplification of PCR products for semi-quantitative RT-PCR was performed with 2 µl of cDNA in Ex Taq DNA polymerase mixture containing 2 mM MgCl2, 200 µM dNTP (Takara Bio, Inc.) and 0.2 µM of each forward and reverse primer (Bioneer Corporation, Daejeon, Korea) with a final reaction volume of 25 µl. The PCR cycling conditions were as follows: 95°C for 10 min (initial denaturation), 22–32 cycles at 95°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec (amplification) and 72°C for 10 min (final extension). All reactions were finished during the exponential phases. PCR products and 100 bp ladder (WelGene Co., Daegu, Korea) were subjected to agarose gel electrophoresis containing 0.5 µg/ml ethidium bromide (Promega Corporation, Madison, WI, USA) and observed using i-MAX Gel Image Analysis System with CoreBio MFC software (CoreBio System Co., Ltd., Seoul, Korea). qPCR was performed using a QGreen™ SYBR Green Master Mix kit (Cellsafe Co. Ltd., Suwon, Korea) and the Eco Real-Time PCR system (Illumina, Inc., San Diego, CA, USA). The PCR cycling conditions were as follows: 95°C for 10 min followed by 45 cycles at 95°C for 10 sec, 60°C for 10 sec and 72°C for 30 sec. The relative intensity of the target genes was calculated using Eco™ software version 3.1.7 (Illumina, Inc., San Diego, CA, USA) by the ΔΔCq method (
Values were presented as the mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance with SPSS software (version no. 22; SPSS, Inc., Chicago, IL, USA) followed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
The present study monitored body weight, and food and water (tap water or DSW) intake, in rats fed a HCD. No significant differences in body weight (
Metabolic diseases, including obesity, diabetes and hypercholesterolemia, may be induced by a HCD and are associated with hepatic lipid accumulation (
Lipid accumulation in the liver, and increased blood TC and LDL-c concentration, are associated with liver injury. The present study detected the suppressive effects of DSW on hepatic lipid accumulation, and the elevation of blood TC and LDL-c concentration. Therefore, the effects of DSW on liver injury indices in the blood, including GOT, GPT and ALP, were assessed. HCD-induced increased GOT, GPT and ALP levels in the blood were significantly decreased by H1500 DSW compared with the Tap HCD group (P<0.05;
Lipid homeostasis in the liver is governed by the balance of expression between fatty acid-synthesizing enzymes and energy expenditure enzymes. Numerous studies have detected fat accumulation in the livers of HFD- and/or HCD-fed rodents (
The present study demonstrated that serum TC and LDL-c levels were decreased in response to DSW in rats fed a HCD. Circulating serum cholesterol is primarily absorbed in the liver through hepatic LDLR-mediated endocytosis and is subsequently metabolized (
Several studies have demonstrated the importance of minerals, including Mg and Ca, in lipid metabolism. For example, increased Mg intake was demonstrated to prevent hypercholesterolemia, lipid oxidation and oxidative damage (
Increased levels of blood lipid components, including TG, TC and LDL-c, induced by a HFD and/or HCD may lead to liver fat accumulation. The hepatic accumulation of fat may be prevented by lowering blood levels of these lipid components. Previous studies (
Increased levels of liver injury indicators are associated with liver fat accumulation and increased serum TC and LDL-c. Chen
PPARγ and SREBP-1c are transcriptional regulators of lipid metabolism enzymes. Previous studies (
Previous studies (
In conclusion, the present study assessed the effects of DSW on HCD-induced hepatic lipid accumulation and hypercholesterolemia in rats. The results demonstrated that DSW decreased TG, TC, LDL-c, GOT, GPT and ALP levels in the blood, and reduced lipid accumulation in the liver. Furthermore, the mRNA expression levels of FAS and SREBP-1c were downregulated, whereas the expression of LDLR was upregulated by DSW. Combined, these results indicated that DSW may have the potential to prevent hepatic lipid accumulation and may exert blood cholesterol-lowering activity via the inhibition of fatty acid synthesis in the liver and enhancement of LDL-c clearance in the blood, caused by increased hepatic LDLR expression. The present study indicated that DSW is a candidate for the prevention of hypercholesterolemia and hepatic lipid accumulation.
This work was financially supported by the National R&D Project ‘Development of New Application Technology For Deep Sea Water Industry’ supported by the Ministry of Oceans and Fisheries of the Republic of Korea.
Effects of DSW on body weight, and total food and water intake, in rats fed a HCD. (A) Body weight of each rat was measured every 2–3 days. Values are presented as the mean ± standard deviation, n=6. (B) Total food intake and (C) total tap water or DSW intake, was measured every 2–3 days. Values are presented as the sum of the amount of food, and volume of water, consumed in each group for 6 weeks. DSW, deep sea water; HCD, high-cholesterol diet; Tap, tap water; H, hardness.
Effects of DSW on levels of serum lipid components. Serum (A) TC, (B) LDL-c, (C) HDL-c and (D) TG concentrations were measured in rats fed a HCD with tap water or DSW of various hardness for 6 weeks. Values are presented as the mean ± standard deviation, n=6. *P<0.05 and **P<0.01 vs. the Tap HCD group. DSW, deep sea water; TC, total cholesterol; LDL-c, low-density lipoprotein cholesterol; HDL c, high-density lipoprotein cholesterol; TG, triglyceride; HCD, high-cholesterol diet; Tap, tap water; H, hardness.
Effects of DSW on lipid accumulation in the liver. Lipid droplets in the liver were observed by electron microscopy. Arrows indicate lipid droplets. Representative images (magnification, ×2,000) from five independent experiments are presented. DSW, deep sea water; Tap, tap water; ND, normal diet; H, hardness; HCD, high-cholesterol diet.
Effects of DSW on liver injury indicators. Serum (A) GOT (B) GPT and (C) ALP activities were measured in rats fed a HCD with tap water or DSW of various hardness for 6 weeks. Values are presented as the mean ± standard deviation, n=6. *P<0.05 vs. the Tap HCD group. DSW, deep sea water; GOT, glutamate-oxaloacetate transaminase; GPT, glutamate-pyruvate transaminase; ALP, alkaline phosphatase; HCD, high-cholesterol diet; Tap, tap water; H, hardness.
Effects of DSW on hepatic lipid metabolism-regulating gene expression. Levels of hepatic lipid metabolism-regulating genes (A) FAS, (B) SREBP-1c, (C) CPT-1 and (D) PPARγ were assessed by semi-quantitative RT-PCR and the densities were normalized to GAPDH, which was used as an internal control. To perform semi-quantitative RT-PCR, an equal amount of six individual total RNA samples in each group were pooled. Values are presented as the mean ± standard deviation, n=3. *P<0.05 and **P<0.01 vs. the Tap HCD group. DSW, deep sea water; FAS, fatty acid synthase; SREBP-1c, sterol regulatory element binding protein-1c; CPT-1, carnitine palmitoyltransferase-1; PPARγ, peroxisome proliferator-activated receptor γ; RT-PCR, reverse transcription-polymerase chain reaction; Tap, tap water; HCD, high-cholesterol diet; H, hardness.
Effects of DSW on hepatic LDLR expression. The relative expression levels of hepatic LDLR were determined by RT-qPCR. To perform RT-qPCR, an equal amount of six individual total RNA samples in each group were pooled. GAPDH was used as an internal control. Values are presented as the mean ± standard deviation, n=3. *P<0.05 vs. the Tap HCD group. DSW, deep sea water; LDLR, low-density lipoprotein receptor; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Tap, tap water; HCD, high-cholesterol diet; H, hardness.
Composition of high-cholesterol diet.
Ingredient | Amount (g/kg) |
---|---|
Casein | 75 |
Soy protein | 130 |
DL-methionine | 2 |
Corn starch | 275 |
Maltodextrin 10 | 150 |
Sucrose | 30 |
Cellulose | 90 |
Soy bean | 50 |
Cocoa butter | 75 |
Coconut oil | 35 |
Mineral mix | 35 |
Calcium carbonate | 5.5 |
Sodium chloride | 8 |
Potassium citrate | 10 |
Vitamin mix V10001 | 10 |
Choline bitartrate | 2 |
Cholesterol | 12.5 |
Sodium cholic acid | 5 |
Total calories (cal/kg)=4,128. (D12336; Research Diets, Inc., New Brunswick, NJ, USA).
Sequences of primers used for semi-quantitative RT-PCR and RT-qPCR.
A, Semi-quantitative PCR | ||||
---|---|---|---|---|
Gene | F/R primer | Primer sequence | Annealing temperature (°C) | Cycle number |
FAS | F | 5′-CTGGACTCGCTCATGGGTG-3′ | 60 | 25 |
R | 5′-CATTTCCTGAAGCTTCCGCAG-3′ | |||
CPT-1 | F | 5′-AACCTTGGCTGCGGTAAGACTA-3′ | 60 | 22 |
R | 5′-AGTGGGACATTCCTCTCTCAGG-3′ | |||
SREBP-1c | F | 5′-GATGCCAACCAGATTCCCTAAG-3′ | 60 | 29 |
R | 5′-TCAGTTGTTTCTTTGCCTTCCA-3′ | |||
PPARγ | F | 5′-TTCAGTTTGGAGACTTCGGACC-3′ | 60 | 32 |
R | 5′-TAGGCTCCTGCCAGATTACTCC-3′ | |||
GAPDH | F | 5′-AACTTTGGCATCGTGGAAGG-3′ | 59 | 22 |
R | 5′-TACATTGGGGGTAGGAACAC-3′ | |||
B, RT-qPCR | ||||
Gene | F/R primer | Primer sequence | Annealing temperature (°C) | Cycle number |
LDLR | F | 5′-CAGCTCTGTGTGAACCTGGA-3′ | 58 | 45 |
R | 5′-TTCTTCAGGTTGGGGATCAG-3′ | |||
GAPDH | F | 5′-AACTTTGGCATCGTGGAAGG-3′ | 58 | 45 |
R | 5′-TACATTGGGGGTAGGAACAC-3′ |
RT-PCR, reverse transcription-polymerase chain reaction; RT-qPCR, quantitative RT-PCR; F, forward; R, reverse; FAS, fatty acid synthase; CPT-1, carnitine palmitoyltransferase-1; SREBP-1c, sterol regulatory element binding protein-1c; PPARγ, peroxisome proliferator-activated receptor γ; LDLR, LDL receptor.