Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver diseases in Western countries( Reference Angulo 1 ). It is characterised by excessive fat accumulation over 5–10 % of the total liver weight in patients with little or no history of alcohol consumption( Reference Ludwig, Viggiano and McGill 2 ). NAFLD is closely associated with increased fat deposition in the liver due to elevated lipolysis in the adipose tissue of obese subjects( Reference Rector, Thyfault and Wei 3 ). In approximately 10–25 % of NAFLD patients, the disease progresses to non-alcoholic steatohepatitis (NASH), which is characterised by hepatocyte damage, inflammatory cell infiltration and fibrosis( Reference Ludwig, Viggiano and McGill 2 , Reference Marra, Gastaldelli and Svegliati Baroni 4 ). NASH can further progress to cirrhosis and hepatocellular carcinoma( Reference Brown, Fujioka and Wilson 5 ), increasing mortality in patients with liver diseases( Reference Vernon, Baranova and Younossi 6 ). Currently, there is no effective therapy available for NASH patients( Reference Marchesini, Bugianesi and Forlani 7 ). The challenge to the development of NASH therapy is largely the complexity of the disease, which is closely related to visceral obesity, dyslipidaemia, insulin resistance and hyperglycaemia( Reference Rector, Thyfault and Wei 3 ).
Astaxanthin (ASTX) is a xanthophyll carotenoid that is mainly present in marine animals, such as salmon and crustaceans( Reference Hussein, Sankawa and Goto 8 , Reference Pashkow, Watumull and Campbell 9 ). ASTX acts a potent antioxidant by functioning as a powerful reactive oxygen species quencher as well as a free radical scavenger( Reference Guerin, Huntley and Olaizola 10 ). It is known that ASTX can also scavenge peroxyl radicals and destroy peroxides, thereby protecting fatty acids and biological membranes from lipid peroxidation( Reference Goto, Kogure and Abe 11 , Reference Kurashige, Okimasu and Inoue 12 ). Studies have shown that ASTX has a protective effect against oxidative stress, inflammation and metabolic disorders, such as type 2 diabetes and CVD, in animals( Reference Yang, Seo and Nguyen 13 – Reference Uchiyama, Naito and Hasegawa 15 ) and humans( Reference Karppi, Rissanen and Nyyssonen 16 , Reference Yoshida, Yanai and Ito 17 ). In our previous study, apoE knockout (apoE − / − ) mice fed an atherogenic diet supplemented with ASTX exhibited decreases in the plasma concentrations of total cholesterol (TC) and TAG( Reference Yang, Seo and Nguyen 13 ). Furthermore, ASTX also increased the expression of nuclear factor erythroid 2-related factor 2 (NRF-2)-responsive antioxidant genes and decreased the concentrations of an oxidative stress marker, glutathione disulphide, in the liver( Reference Yang, Seo and Nguyen 13 ). The study demonstrated that ASTX supplementation improves lipid metabolism as well as enhances antioxidant defence mechanisms in apoE − / − mice, supporting that ASTX may prevent the development of NAFLD.
In the present study, we aimed to determine the effective dietary supplementation dosage of ASTX at which it could counteract metabolic dysfunctions as well as to evaluate whether ASTX supplementation can improve metabolic and inflammatory parameters that are disturbed by a high-fat (HF) diet in diet-induced obesity (DIO) mice.
Materials and methods
Animal feeding and care
Male C57BL/6J mice aged 6 weeks were obtained from Jackson Laboratory. After 1 week of acclimation period, mice were randomly assigned to one of the following four groups: group fed a HF control diet (35 %, w/w); group fed a HF diet supplemented with 0·003 % of ASTX; group fed a HF diet supplemented with 0·01 % of ASTX; group fed a HF diet supplemented with 0·03 % of ASTX (by weight) (n 8 per group). AstaREAL® L10, an extract from the microalgae Haematococcus pluvialis containing 10 % of ASTX and approximately 90 % of TAG, was kindly provided by Fuji Chemical Industry Corporation Limited. Based on body surface normalisation to a 70 kg individual( Reference Reagan-Shaw, Nihal and Ahmad 18 ), ASTX supplementation dosages of 0·003, 0·01 and 0·03 % in mice would be equivalent to approximately 20, 66 and 200 mg/d in humans. Diet composition is given in Table 1. Mice were housed in polycarbonate cages under a 12 h light–12 h dark cycle with free access to food and water for 12 weeks. Body weight and food intake were measured weekly. At the end of 12 weeks of feeding, mice were anaesthetised with ketamine/xylazine (120/6 mg/kg; Henry Schein Animal Health) after 8 h of fasting and then killed by cardiac puncture and cervical dislocation. Blood samples were collected in EDTA-containing vacutainers (BD Vacutainer®) and centrifuged at 1500 g at 4°C for 15 min to remove erythrocytes. Liver and epididymal and retroperitoneal adipose tissue samples were snap-frozen in liquid N2 and stored at − 80°C until use. A small portion of the tissue samples was fixed in 10 % formalin for histological analysis. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Connecticut.
* ASTX supplementation dosages by weight.
† The HF control diet contained 35 % fat by weight.
Plasma chemistry and hepatic lipid quantification
Lipid from liver samples was extracted using the Folch method as described previously( Reference Rasmussen, Blobaum and Jesch 19 ). Plasma and hepatic lipids were quantified using enzymatic assays for TC, TAG, and HDL-cholesterol, and plasma glucose was quantified using the Liquid Glucose (Oxidase) Reagent Set (Pointe Scientific, Inc.)( Reference Yang, Park and Cassada 20 ). Plasma insulin was quantified using ELISA (Alpco) according to the manufacturer's instructions. Plasma alanine transaminase (ALT) and aspartate transaminase (AST) were quantified using the Cholestech LDX System (Cholestech Corporation).
Gene expression analysis
Total RNA was isolated from liver and adipose tissue samples as described previously using TRIzol Reagent (Invitrogen)( Reference Rasmussen, Blobaum and Park 21 ). Quantitative real-time PCR analysis was carried out as described previously using the SYBR Green procedure and CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad)( Reference Park, Rasmussen and Ehlers 22 ). Primer sequences were designed according to the GenBank database using the Beacon Designer software (PREMIER Biosoft), and primer sequences are available upon request. Ribosomal protein, large, P0 was used as an internal control for data normalisation.
Primary splenocyte isolation and treatment
Primary splenocytes were isolated from freshly isolated spleens as described previously( Reference Ku, Pham and Park 23 ). The splenocytes were resuspended in RPMI-1640 medium containing 10 % fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin and were then stimulated with 100 ng/ml lipopolysaccharide (LPS) for 18 h for gene expression analysis.
Histological evaluation of liver samples
Formalin-fixed liver samples were sliced into 5 μm-thick sections and stained with haematoxylin and eosin by the Connecticut Veterinary Medical Diagnostic Laboratory. The stained tissue sections were examined using a Zeiss Axio Observer.A1 microscope (Carl Zeiss Microscopy), and images were obtained at 20 × magnification with an AxioCam MRc camera (Carl Zeiss Microscopy).
One-way ANOVA or Student's unpaired t test was carried out using GraphPad Prism 6 (GraphPad Software) to identify statistically significant differences between the groups with a two-tailed level of significance at P< 0·05. Normality and equity of variance were tested using D'Agostino–Pearson omnibus normality test and Bartlett's test, respectively. Data are expressed as means with their standard errors.
Body and tissue weights and plasma chemistry
There were no significant differences in food intake (data not shown), body weight, body weight gain and retroperitoneal adipose tissue weight between the control and 0·03 % ASTX-supplemented groups (Table 2). However, the 0·03 % ASTX-supplemented group exhibited a significantly higher epididymal adipose tissue weight when compared with the control group. There were no significant differences in plasma glucose and insulin concentrations between the control and 0·03 % ASTX-supplemented groups.
* Mean value was significantly different from that of the control group (P< 0·05).
Hypotriacylglycerolaemic effect of 0·03 % astaxanthin supplementation in diet-induced obesity mice
We have previously reported that 0·03 % ASTX supplementation lowers plasma TC and TAG concentrations in apoE − / − mice fed an atherogenic diet( Reference Yang, Seo and Nguyen 13 ). To determine the effective supplementation dosage of ASTX at which it could lower blood lipid concentrations in obesity, mice were fed a HF diet supplemented with 0·003, 0·01 or 0·03 % of ASTX (w/w). The 0·03 % ASTX-supplemented group, but not the 0·003 and 0·01 % ASTX-supplemented groups, exhibited significantly lower plasma TAG concentrations when compared with the control group (Fig. 1(a)). There were no significant differences in plasma TC and HDL-cholesterol concentrations between the control and 0·03 % ASTX-supplemented groups (Fig. 1(b)). These results suggest that ASTX supplementation at a dosage of 0·03 % is required to elicit a TAG-lowering effect in DIO mice. Therefore, only the 0·03 % ASTX-supplemented group was used for subsequent analyses to compare with the control group.
Effect of 0·03 % astaxanthin supplementation on hepatic lipid concentrations and damage
There were no significant differences in liver weight and hepatic TAG and TC concentrations between the control and 0·03 % ASTX-supplemented groups (Fig. 2(a)). Consistently, histological analysis of haematoxylin and eosin-stained liver sections showed no noticeable difference in lipid accumulation between the two groups (Fig. 2(b)). However, a trend towards lower plasma ALT concentrations (P= 0·071) and significantly lower plasma AST concentrations (P= 0·015) was observed in the 0·03 % ASTX-supplemented group when compared with the control group (Fig. 2(c)).
Effect of astaxanthin supplementation on the hepatic expression of lipogenic, cholesterologenic, inflammatory and fibrogenic genes
To gain mechanistic insights into the TAG-lowering effect of ASTX, the expression of genes involved in lipogenesis and fatty acid β-oxidation was measured in the liver. There were no differences in the mRNA abundance of sterol regulatory element-binding protein (SREBP)-1c, a key transcription factor in lipogenesis, and stearoyl-CoA desaturase 1 between the control and 0·03 % ASTX-supplemented groups, while there was a significant increase in the mRNA abundance of fatty acid synthase and diglyceride acyltransferase 2 in ASTX-supplemented mice than in control mice (Table 3). In contrast to lipogenic genes, there was a significant increase in the mRNA expression of acyl-CoA oxidase 1 (ACOX-1), the rate-limiting enzyme in the peroxisomal fatty acid β-oxidation, in the 0·03 % ASTX-supplemented group, while there was no change in the mRNA expression of carnitine palmitoyltransferase-1α, the rate-limiting enzyme in mitochondrial fatty acid β-oxidation. Although there were no significant differences in plasma TC and HDL-cholesterol concentrations between the groups, marginal but significant increases in the hepatic expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and LDL receptor were observed in the 0·03 % ASTX-supplemented group with no difference being observed in that of SREBP-2. There were no significant differences in the expression of genes involved in HDL metabolism, including scavenger receptor class B, type 1 and apoA-I, between the control and 0·03 % ASTX-supplemented groups.
SREBP, sterol regulatory element-binding protein; FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase 1; DGAT-2, diglyceride acyltransferase 2; ACOX-1, acyl-CoA oxidase 1; CPT-1α, carnitine palmitoyltransferase-1α; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR, LDL receptor; SR-B1, scavenger receptor class B, type 1; α-SMA, α-smooth muscle actin; Col1A1, procollagen type I, α1; TGFβ1, transforming growth factor-β1.
Inflammation with increased macrophage infiltration and elevated fibrogenesis in the liver is a major marker of NASH( Reference Tosello-Trampont, Landes and Nguyen 24 ). The mRNA expression of α-smooth muscle actin and procollagen type I, α1, which are markers of fibrosis, was not altered in the 0·03 % ASTX-supplemented group, while that of transforming growth factor-β1 (TGFβ1), a potent profibrogenic cytokine, was significantly increased (Table 3). There was no change in the mRNA expression of F4/80, a macrophage marker, in ASTX-supplemented mice.
Effect of astaxanthin supplementation on the expression of nuclear factor erythroid 2-related factor 2 and its target genes in the liver
As ASTX is a well-known antioxidant, we examined whether ASTX supplementation can alter a NRF2-mediated endogenous antioxidant defence system. There was a significant increase in the mRNA expression of NRF-2 as well as its target genes, including superoxide dismutase 1, glutamate–cysteine ligase regulatory subunit and glutathione peroxidase 1, in the liver of the 0·03 % ASTX-supplemented group than in the control group (Fig. 3).
Effect of 0·03 % astaxanthin supplementation on the inflammatory responses of splenocytes
It has been suggested that the spleen functions as a reservoir of monocytes( Reference Wang, Yu and Cao 25 ). To determine whether ASTX supplementation alters the inflammatory response of splenocytes, the primary cells isolated from control and 0·03 % ASTX-supplemented mice were stimulated with LPS. There was a significant decrease in the mRNA abundance of IL-6 in splenocytes isolated from 0·03 % ASTX-supplemented mice (P= 0·038) when compared with that in splenocytes isolated from control mice, while there were no differences in the mRNA abundance of TNFα between the groups (Fig. 4).
Lipotoxicity, oxidative stress and inflammation are commonly identified as key players in the pathogenesis of NASH( Reference Dowman, Tomlinson and Newsome 26 – Reference Tilg and Moschen 28 ). Obesity is also a major risk factor for NAFLD and NASH development( Reference Bellentani, Scaglioni and Marino 29 ). In obesity, fatty acid flux to the liver is increased due to elevated lipolysis in hypertrophied adipocytes, leading to the accumulation of excessive TAG, oxidative stress, inflammation and insulin resistance in the liver. Insulin resistance promotes de novo lipogenesis and gluconeogenesis, further contributing to the development of dyslipidaemia, hyperglycaemia and other metabolic disorders, such as type 2 diabetes and CVD( Reference Fabbrini, Sullivan and Klein 30 ). The goal of the present study was to determine the effective supplementation dosage of ASTX at which it could counteract metabolic dysfunctions, oxidative stress and inflammation induced by a HF diet in DIO mice. We found that ASTX at a dosage of 0·03 % exerted a TAG-lowering effect, increased hepatic expression of endogenous antioxidant genes with a concomitant decrease in steatosis, and attenuated the inflammatory response of splenocytes to LPS in DIO mice. In addition, it should be noted that ASTX lowered plasma ALT and AST concentrations in DIO mice. ALT is predominately found in the liver, whereas AST is present in the liver, cardiac and skeletal muscle, kidneys, brain and erythrocytes( Reference Scheig 31 ). Therefore, the reduction in plasma ALT and AST concentrations by ASTX suggests that it may prevent not only liver damage, but also potential HF-induced tissue damage in other organs.
Hypertriacylglycerolaemia, a common lipid abnormality in obese patients, is a major contributor of the development of insulin resistance( Reference Malmstrom, Packard and Caslake 32 ), type 2 diabetes( Reference Subramanian and Chait 33 ), CVD( Reference Hassing, Surendran and Mooij 34 ) and NAFLD( Reference Scaglioni, Ciccia and Marino 35 ). Hypertriacylglycerolaemia in obesity primarily occurs due to increased hepatic de novo lipogenesis consequent to elevated NEFA flux into the liver from dysfunctional adipocytes( Reference Fabbrini, Sullivan and Klein 30 ). Consistent with our previous observation that 0·03 % ASTX supplementation significantly lowers plasma TAG concentrations in apoE − / − mice( Reference Yang, Seo and Nguyen 13 ), ASTX at the same supplementation dosage also decreased plasma TAG concentrations in DIO mice in the present study. A hypotriacylglycerolaemic effect of ASTX has also been reported in humans( Reference Yoshida, Yanai and Ito 17 ). In both apoE − / − and DIO mice, we observed a significant increase in the hepatic mRNA expression of ACOX-1, while the expression of lipogenic genes was not altered or increased by ASTX supplementation. Hepatic TAG concentrations in 0·03 % ASTX-supplemented mice were lower by approximately 30 % when compared with those in control mice, although they did not reach statistical significance. Therefore, it can be presumed that elevated peroxisomal fatty acid oxidation due to increased ACOX-1 expression induced by ASTX may be responsible for the lipid-lowering effect of ASTX. The potential effect of ASTX on fatty acid β-oxidation is currently being investigated in our laboratory.
Hypertriacylglycerolaemia in obesity is frequently accompanied by other dyslipidaemic conditions, such as high TC and LDL-cholesterol concentrations( Reference Subramanian and Chait 33 ), increasing CVD risk in obese population( Reference Rashid, Watanabe and Sakaue 36 ). We have previously reported that 0·03 % ASTX supplementation exerts a cholesterol-lowering effect in apoE − / − mice fed a HF/high cholesterol diet( Reference Yang, Seo and Nguyen 13 ). In the present study, although ASTX supplementation significantly increased the hepatic mRNA expression of LDL receptor and HMGR, two of the most important cholesterol regulatory genes under the transcription regulation of SREBP-2, we did not observe any changes in plasma and hepatic TC concentrations induced by ASTX supplementation. This discrepancy in findings with regard to the cholesterol-lowering effect of ASTX between our previous study and the present study may lie in the different animal models, i.e. apoE − / − v. DIO mice, as well as dietary challenges, i.e. HF/high cholesterol v. HF, used. As in the present study, DIO mice were not challenged by dietary cholesterol, a potential cholesterol-lowering effect of ASTX may not have been initiated.
Fibrosis and necroinflammation are key events in the development of NASH, and hepatic stellate cells and macrophages play a major role in the pathogenesis( Reference Marra, Gastaldelli and Svegliati Baroni 4 ). Upon activation by oxidative stress or fibrogenic cytokines, hepatic stellate cells transform into myofibroblasts and produce extracellular matrix proteins( Reference Lee and Jeong 37 ). TGFβ1 is a cytokine that facilitates liver fibrosis( Reference Dooley, Delvoux and Lahme 38 ) and hepatocyte apoptosis( Reference Jang, Chen and Chen 39 ) and stimulates hepatic stellate cells to produce α-smooth muscle actin and procollagen type I, α1, major markers of fibrosis( Reference Bonniaud, Margetts and Ask 40 ). Therefore, the effect of ASTX supplementation on the expression of fibrogenic genes was determined. Although the mRNA expression of TGFβ1 was drastically elevated, that of α-smooth muscle actin and procollagen type I, α1 was not significantly altered in the liver of ASTX-supplemented mice. It is known that TGFβ1 is secreted as a latent form, which requires to be cleaved by proteases such as matrix metalloproteinase-2 and matrix metalloproteinase-9 and to be activated by reactive oxygen species( Reference Biernacka, Dobaczewski and Frangogiannis 41 , Reference Barcellos-Hoff and Dix 42 ). Therefore, the absence of α-smooth muscle actin and procollagen type I, α1 induction despite an increase in TGFβ1 expression induced by ASTX supplementation indicates that the activation of TGFβ1 may be perturbed in ASTX-supplemented mice. The expression of CD68 and F4/80, macrophage markers, was not significantly altered by ASTX supplementation, suggesting that it may not have an impact on hepatic inflammation in DIO mice. However, we observed that primary splenocytes isolated from ASTX-supplemented mice exhibited a decrease in the mRNA expression of IL-6 upon LPS challenge compared with those isolated from control mice. Splenocytes consist of a variety of immune cells, including T and B lymphocytes, dendritic cells, and monocytes, and IL-6 is mainly secreted by T cells and monocytes( Reference Hwang, Dasgupta and Actor 43 ). Given that LPS stimulates primarily monocyte/macrophages, the present results indicate that ASTX supplementation may decrease the sensitivity of splenic monocytes to inflammatory stimuli.
The antioxidant effect of ASTX may play a critical role in the aforementioned beneficial effects of ASTX in DIO mice. In our previous study in apoE − / − mice, ASTX was found to attenuate oxidative stress in the liver by increasing the expression of NRF-2 and its target antioxidant genes( Reference Yang, Seo and Nguyen 13 ). In the present study also, we found a 2-fold increase in the mRNA expression of NRF-2 and an increased expression of its target endogenous antioxidant genes in the liver of ASTX-supplemented mice when compared with that in control mice. NRF-2 plays a critical role in cellular defence against oxidative stress by inducing the transcription of antioxidant genes( Reference Aleksunes and Manautou 44 ). Under basal conditions, NRF-2 is sequestered in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of NRF-2. When cells are under oxidative stress, oxidative modifications or phosphorylations in Keap1 release NRF-2 and therefore NRF-2 enters the nucleus to activate the transcription of genes containing an antioxidant response element in their promoters( Reference Kaspar, Niture and Jaiswal 45 ). Therefore, given that ASTX is known to be a potent antioxidant( Reference Guerin, Huntley and Olaizola 10 – Reference Kurashige, Okimasu and Inoue 12 ), the increase in the expression of NRF-2 and its target genes is counterintuitive. Our observations that ASTX supplementation increased the expression of carnitine palmitoyltransferase-1α and ACOX-1 in apoE − / − mice and ACOX-1 in DIO mice led us to speculate that ASTX may activate PPARα because the genes involved in fatty acid oxidation are under the transcriptional regulation of PPARα in the liver. In support of this speculation, Jia et al. ( Reference Jia, Kim and Jun 46 ) demonstrated that ASTX is a PPARα agonist. Therefore, the activation of PPARα by ASTX could burden the mitochondrial machinery of oxidative phosphorylation due to elevated fatty acid β-oxidation, resulting in the production of reactive oxygen species. In this case, antioxidant mechanisms, possibly via the NRF-2 pathway, need to be turned on to offset the production of reactive oxygen species for the protection against oxidative damage. Further studies are required to investigate this possibility.
In conclusion, we demonstrated that 0·03 % ASTX supplementation lowered plasma TAG concentrations, induced hepatic endogenous antioxidant gene expression, and attenuated inflammatory response of splenocytes in DIO mice. The effects of ASTX indicate that it may be able to prevent the development of NASH. However, as DIO mice in the present study did not develop NASH features, we were not able to determine whether ASTX supplementation could prevent obesity-associated NASH. Another mouse study is currently underway in our laboratory, in which mice are being exposed to a severe dietary challenge for a longer period of time than in the present study to induce NASH features. Nonetheless, the present study opens the possibility that ASTX could be used for preventing obesity-associated metabolic dysfunctions and inflammation.
The present study was supported by the US Department of Agriculture AFRI (2012-67018-19290; to J.-Y. L).
The authors’ contributions are as follows: Y. Y. played a major role in conducting the experiments and contributed to manuscript preparation; T. X. P., C. J. W., B. K., C. S. K., and Y.-K. P. conducted the experiments; J.-Y. L. designed the experiments and contributed to the data analysis and manuscript preparation.
None of the authors has any conflicts of interest to declare.