Intermuscular pH was significantly decreased by exercise, and this decrease was inhibited by intake of astaxanthin. Levels of PGC-1α and its downstream proteins were significantly elevated in astaxanthin-fed mice compared with mice fed a normal diet.

Astaxanthin intake resulted in a PGC-1α elevation in skeletal muscle, which can lead to acceleration of lipid utilization through activation of mitochondrial aerobic metabolism.

Already have an account? Sign in here. Journal of Clinical Biochemistry and Nutrition. Online ISSN : Print ISSN : ISSN-L : Journal home Advance online publication All issues About the journal.

The astaxanthin-induced improvement in lipid metabolism during exercise is mediated by a PGC-1α increase in skeletal muscle. Po Hung Liu , Wataru Aoi , Maki Takami , Hitomi Terajima , Yuko Tanimura , Yuji Naito , Yoshito Itoh , Toshikazu Yoshikawa Author information.

Corresponding author. Keywords: astaxanthin , skeletal muscle , lipid metabolism , running exercise , PGC-1α. JOURNAL FREE ACCESS. Published: Received: December 16, Released on J-STAGE: March 01, Accepted: December 28, Advance online publication: February 19, Revised: -.

Download PDF K Download citation RIS compatible with EndNote, Reference Manager, ProCite, RefWorks. Article overview. References Over the past few decades, obesity has become globally recognized as one of the most common life-threatening chronic metabolic problems, with steadily increasing rates year by year, which are ascribed to the imbalance of energy metabolism and excessive fat accumulation in the body 1 , 2.

Obesity causes a series of abnormal metabolic complications, including lipid metabolism, oxidant stress, inflammatory responses, insulin resistance IR and steatohepatitis 3 , 4.

Notably, among patients with NAFLD, obese individuals present more severe histological phenotypes and may suffer from higher mortality and morbidity 5. Recently, according to an epidemiological survey on nutrition and health, the prevalence of obesity has gradually become appeared in younger individuals 6.

In modern society, a suboptimal diet and little exercise are among the leading causes of poor health, also increasing triacylglycerol TG , total cholesterol TC , free fatty acid FFA accumulation, and lipid peroxidation 7 , 8.

For example, systemic oxidative stress, a key factor in pathological obesity, can be induced by a high-calorie diet through various mechanisms 9 , Currently, the existing anti-obesity medications that have been developed are unsuitable for certain individuals with obesity due to potential side effects and drug tolerability 11 , There is, therefore, a key practical value to search for safe and effective functional components from natural foods to prevent obesity and related metabolic diseases when compared with synthetic drugs.

With health benefits increasing in the human diet, carotenoids, natural antioxidants distributed in numerous microbes, plants, and animals, have received considerable scholarly attention in recent years Furthermore, an unambiguous association was found between the mechanism of carotenoids regulating liver lipid metabolism and the incidence of obesity-related NAFLD, as demonstrated in many studies 13 — Astaxanthin ATX extracted from Haematococcus pluvialis is a xanthophyll carotenoid in marine organisms and is often used as a nutritious supplementary food in the daily diet.

ATX has a protective effect against oxidative stress, inflammation, and metabolic disorders, such as liver fibrosis and type 2 diabetes 16 , Furthermore, ATX also improved lipid metabolism by regulating lipid-related gene and metabolite contents. According to many previous animal experiments, consumption of ATX not only showed no signs of poisoning but also exerted a positive pharmacological effect 19 , Recently, the relationship between gut microbiota and metabolic diseases has attracted attention from the scholarly community because intestinal flora has been confirmed as a target for the prevention and treatment of obesity, metabolic syndrome and cardiovascular diseases Therefore, it is important to investigate how ATX prevents the development of hepatic steatosis and oxidative stress with the risk of metabolic disease.

The present study aims to contribute to this growing area of research by exploring the prevention effects on obesity and the development of NAFLD through long-term dietary ATX in mice.

The detection kits for alanine transaminase ALT , aspartate transaminase AST , TG, TC, high-density lipoprotein cholesterol HDL-C , and low-density lipoprotein cholesterol LDL-C , and the antioxidant assay kits for SOD, CAT, GSH, T-AOC, and malondialdehyde MDA were purchased from Nanjing Jiancheng Bioengineering Institute Nanjing, China.

Other chemicals, solvents and reagents used in the present study were of laboratory analytical grade. Astaxanthin oleoresin was provided by Shandong Jinjing Biotechnology Co.

After purification, ATX of The Institutional Animal Care and Use Committee of Shanxi Agricultural University approved all experimental protocols for animal care, handling and experimentation SXAU-EAW We also confirmed that all experiments were conducted in accordance with relevant guidelines and regulations.

The design of animal experiments was based on our previous methods The mice in the ND group were fed standard rodent chow containing 3. The mice in the other group were fed a HFD containing 4.

The mice in the ND group were given distilled water, and the solvent group was gavaged with corn oil. In addition, the mice in the ATX treatment groups were gavaged with 0.

During the diet phase, all mice were given intragastric treatment once per day at a. The diets were purchased from Beijing Huafukang Bioscience Co. Supplementary Table 1 shows the ingredients of the experimental diets.

The body weight and food intake were recorded daily for 63 days. To avoid error values, the measurement of weight was repeated three times for each mouse. The energy intake was calculated as food intake × 4. Mice were fasted for 12 h after the last treatment and then euthanized by inhalation with isoflurane.

Blood samples were obtained from the retro-orbital veins on Days 0, 30, and All other organs, including the liver, heart, kidney, spleen, and adipose tissues, were immediately collected and weighed individually after sacrificing the animals. The serum TG, TC, HDL-C, and LDL-C levels and activities of AST GOT and ALT were determined using biochemical kits according to the standards and protocols provided by the manufacturer Nanjing, China.

The supernatant was collected to determine the protein and lipid levels TG and TC and enzymatic analyses T-AOC, SOD, CAT, GSH, MDA, and ROS. The dihydroethidium DHE probe method was used to qualitatively detect ROS.

Five-micron-thick sections of the liver were dyed with DHE, and incubation was performed at 37°C for 10 min in a dark environment. The samples were directly observed under a fluorescence microscope at a measuring emission of nm.

The ROS-positive cells had strong red fluorescence. Meanwhile, the frozen sections were stained with Oil Red O ORO , which was performed to further detect hepatic vacuolization, inflammatory cell infiltration, and lipid droplets. The above sections were used to examine hepatocellular apoptosis with the YF TUNEL assay apoptosis detection kit.

After the TUNEL reaction, the sections were mounted using antifade mounting medium with DAPI and observed under an inverted fluorescence microscope at and nm wavelength excitation. The negative cells were dyed with blue fluorescence intensity at nm, while the apoptotic cells exhibited green fluorescence at nm.

ImageJ software National Institutes of Health, United States was used to measure the cell counting of sections from each group. Then, cDNA was synthesized from total RNA using the PrimeScript Reverse Transcription reagent kit Takara, Dalian, China.

Quantitative polymerase chain reaction PCR was conducted in triplicate for each group to detect gene expression. The quantitative analysis of AMPK , SREBP1c , ACC , CPT-1 , PPARα , PPARγ , LXRα , SCD-1 , PGC-1 , FAS , CYP27A1 , and CYP7A1 mRNA expression in the liver was measured in triplicate for each group by quantitative PCR.

According to the SYBR Premix Ex Taq II Takara, Dalian, China , the thermal cycle of qPCR was reacted on the CFX 96 Real-Time PCR Detection system BIO-RAD, Hercules, CA, United States under the following conditions: 95°C for 10 min, then 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s.

Supplementary Table 2 shows the PCR primer sequences of each gene, and the target genes were normalized to the reference gene GAPDH. The 2 —ΔΔCt method was used to calculate relative gene expression. To investigate lipid metabolism, fresh samples were sent to MetWare Biotechnology Co.

After RNA was extracted from liver biopsy samples, liver transcriptome analysis was conducted by RNA sequencing, as described in detail previously 22 , The caecal contents were sent to Shanghai Personal Biotechnology Co.

to investigate microbial diversity through 16S rRNA analysis on the Illumina MiSeq platform. A previous study illustrated the analytical conditions and detailed parameters All experiments were biologically repeated three times, and the data were analyzed with Social Sciences SPSS Origin 9.

A dramatic increment in body weight was observed in the HFD group, while a moderate increase was observed in the ATX treatment groups Figure 1A. Body weight gain plays a pivotal role in evaluating the effect of HFD on obesity and assessing its prevention.

Table 1 presents the initial and final body weights of mice in each group. During feeding induction, the mice gained more weight in the HFD group There was no significant difference in the food efficiency ratio of the mice in each group except for the ND group Table 1.

Figure 1. Astaxanthin ATX prevented obesity and related indices in HFD-fed mice. A Body weight. B Body weight gain. C Energy intake. D Related organ weight. E Visceral adipose tissue. F Visual appearance pictures of metabolic mice and liver.

G Hepatic TG level. H Hepatic TC level. I Serum ALT level. J Serum AST level. Table 1. Effect of astaxanthin ATX supplementation on body weight, energy intake, and food efficiency ratio in high-fat diet-induced mice a. To estimate whether the HFD with ATX supplementation affected visceral organs and fat, the wet weights of adipose tissue and organs were measured in each group, especially the mouse liver Figure 1D.

There were no significant differences in the heart, spleen or kidney in each group, similar to our previous results 18 Table 2. ATX supplementation 0. Table 2. Effect of ATX supplementation on related organ weights and adipose tissue weights in high-fat diet-induced mice a.

Liver lipid indicators, namely TG and TC levels, are important parameters for obtaining an understanding of diet-induced fat deposition. The liver turned brown following the accumulation of TG and TC in the HFD group, indicating dyslipidaemia, possibly leading to other diseases.

This phenomenon was suppressed in the ATX treatment group compared to the HFD group Figure 1F. The TG and TC levels were examined to further quantify liver fat deposition, as shown in Figures 1G,H. However, ATX supplementation effectively decreased fat deposition in a dose-dependent manner compared to the HFD group, among which the TG and TC levels were Table 3 presents the serum lipid profiles of mice at 0, 30, and 60 days.

There were no obvious differences in the initial serum lipid profiles among the six groups. Such results indicated that lipid metabolism was disordered.

The mice in the 0. When compared with the HFD group, serum TG levels in the 0. Serum TC levels in the 0. Serum LDL-C levels in the 0. Therefore, based on the results mentioned above, 0. Table 3. Effect of ATX supplementation on the levels of serum TG, TC, HDL-C, and LDL-C in the HFD-fed mice.

In addition, we evaluated serum AST GOT and ALT to further explore the liver function induced by HFD and ATX consumption. The serum AST level was increased in the solvent and HFD groups compared with the ND group; however, the serum ALT level was not apparent in any group Figure 1I.

Malondialdehyde and reactive oxygen species ROS activities are crucial components contributing to the development of oxidative stress in high-fat diet-fed animals. The levels of antioxidant enzymes, including T-AOC, CAT, SOD, and GSH, were assessed in the liver and exhibited similar trends.

According to the ROS qualitative fluorography images, intense red fluorescence was observed in the HFD and the positive control groups incubated with H 2 O 2 , while the faint fluorescence in the ATX-treated samples corresponded with the quantitative results Figure 2G. Furthermore, 0.

When compared to the HFD group, the levels of T-AOC, CAT, SOD, and GSH were increased by Figure 2. Evaluation of liver oxidation resistance in HFD-induced mice liver tissues. The levels of ROS intensity A , MDA B , T-AOC C , CAT D , SOD E , and GSH F are illustrated in the panel.

In the ND group mice, hepatocytes were fairly uniform, with regularly shaped hepatic plates arranged in an ordered pattern and hepatic cords, except for slight congestion Figure 3A.

However, the HFD induced typical lesions in the mouse liver, such as hepatocyte necrosis, inflammatory cell infiltration, congestion of the central veins, ballooning, hepatic sinus expansion and chromatin condensation.

In the solvent group, the structure of hepatic plates was irregularly arranged along with fat accumulation, indicating that long-term excessive fat intake disturbed lipid metabolism in the liver. Figure 3. Pathological changes of ATX on liver and epididymal fat in HFD-induced mice.

A Liver sections stained with HE ×, ×. B Liver sections stained with Oil red O ×, ×. C HE-stained e-AT sections ×. D Steatohepatitis scores. E Percentage of the lipid droplet area assessed by Oil red O staining.

F Mean cell area of adipocyte in e-AT. To further investigate the production of lipid droplets in the liver, Oil Red O staining was performed Figure 3B. More oil red O-stained lipid droplets were observed in the liver tissue of the HFD and solvent groups than in the liver tissue of the ND group, resembling the percentage result of lipid droplets Figure 3E.

Conversely, ATX supplementation dose-dependently decreased the production of fatty droplets, in which the area of droplets was significantly lessened in the 0. These results confirmed that ATX prevented lipid accumulation and hepatic steatosis, conforming to the results of intrahepatic TG and TC levels.

As shown in the e-AT sections of HFD-induced mice Figures 3C,F , the mean adipocyte size increased almost Apoptotic cells were detected by green fluorescent TUNEL staining, and cell nuclei were stained blue DAPI.

Compared to that in the HFD group, the number of apoptotic cells stained green was reduced in a dose-dependent manner with ATX supplementation, and the apoptosis rates were decreased by To understand the mechanism s by which ATX modulates hepatic lipid metabolism in response to a high-fat diet, we analyzed the expression of genes related to lipogenesis and fatty acid β-oxidation in the liver by qRT—PCR.

These results indicated that consumption of a HFD contributed to fat synthesis and ultimately disturbed lipid metabolism; furthermore, high-dose ATX could improve the disorder of lipid metabolism by promoting cholesterol metabolism and inhibiting fat synthesis.

Figure 4. Astaxanthin significantly improved relative gene expression. B The heatmap of differential genes expression at the transcriptional level. C Regulatory effects of ATX supplementation on fatty acid and cholesterol metabolism in mice induced by HFD. Data are shown as mean ± SD of triplicate.

To explore how the hepatic lipidome is altered upon ATX intervention, RNA sequencing was used to accurately and quantitatively analyse liver transcriptional changes and lipid metabolism pathways in the liver in response to ATX supplementation.

A total of genes were differentially expressed in HFD-induced liver samples compared with ND-induced liver samples Supplementary Figures 2A,B.

However, a total of differentially expressed genes, of which were increased and 53 were decreased, were identified in the 0.

We performed a comprehensive hepatic lipidomic analysis to evaluate whether differences in lipid content or composition may account for differences in hepatic lipid disorders between the HFD group and ATX group.

A total of 1, lipid species were identified in liver samples, which belong to six primary classes of lipids, including glycerophospholipids GPs , glycerides GLs , fatty acyls FAs , sphingolipids SLs , sterol lipids STs , and prenol lipids PRs Supplementary Figure 3.

Based on the abovementioned results, we screened and 91 lipid biomarker candidates by applying volcano plots for such distinctions in ND vs. HFD and HFD vs. Figure 5.

Astaxanthin regulated lipid metabolites in HFD-fed mice. A OPLS-DA score plot left and permutation plot right.

B Venn diagram depicting the overlap of significantly changed metabolites between experimental groups. The volcano plot analysis of ND vs. HFD group C and HFD vs.

Analysis of lipid metabolism pathway of ND vs. HFD E and HFD vs. G Heatmap of 34 significantly altered metabolites in ATX-treated HFD-fed mice. Blue: downregulated metabolites. Red: upregulated metabolites.

H The associated heatmap of significantly changed metabolites. According to the Venn diagram, we found that the accumulated lipid species were significantly different between the ND and HFD groups, while ATX intervention patently changed the levels of 91 lipid species, including 24 ordinary species, compared to the levels in HFD-fed alone Figure 5B.

Furthermore, in our present study, we found that 8 of the other 20 most relevant metabolites 3 BAs, 2 CARs, 2 BMP, and 1 TG were remarkably downregulated after ATX supplementation; however, there was no significant difference in the ND vs.

HFD group. We observed a significantly positive correlation among these 34 metabolite levels associated with lipid metabolism Figure 5H. Thus, these results indicated that the 22 metabolites, including 4 FFAs, 8 TGs, 2 DGs, 3 BAs, 2 CARs, and 2 BMPs, might be potential biomarkers accountable for alleviating the steatohepatitis induced by lipid disturbance.

The KEGG database was used to perform pathway analysis of differentially expressed metabolites. The pathways were considerably disrupted in the HFD group, including glycerolipid metabolism, insulin resistance, cholesterol metabolism, fat digestion and absorption, and regulation of lipolysis in adipocytes, when compared with the ND group; however, 0.

Of the 8, OTUs visualized in the experimental groups, 4. In addition, the number of other OTUs in the ND group, HFD group and 0. The Goods coverage values had no obvious differences in each group Figure 6B. To assess community similarity among samples, we applied principal coordinates analysis PCoA to represent the relative abundance of OTUs in each community by two different analyses.

The PCoA plot showed that the structure and compositions of gut microbiota in the HFD group Axis 1, Figure 6. Astaxanthin regulated the gut microbiota. A The Venn diagram. Data were analyzed using a one-way ANOVA and are expressed as the mean ± SD.

C PCoA of unweighted UniFrac distance from beta diversity analysis. D Phylum abundance graph genus levels. E Genus abundance graph. F Species taxonomy branch map based on LEfSe analysis. G The heatmap of the 30 bacterial genera with the largest differences in abundance were selected, according to the unweighted UniFrac distance of the intestinal content samples.

H Predicted the abundance map of MetaCyc secondary functional pathways. X-coordinate: the abundance of functional pathways, Y-coordinate: the MetaCyc secondary functional pathway.

I Analysis of differences in metabolic pathways left and species composition in different MetaCyc pathways right. At the phylum level, the taxonomic profiles of the gut microbiomes showed significant differences according to increasing ATX supplementation and developing obesity severity, within which Firmicutes , Bacteroidetes , and Proteobacteria were the dominant phyla.

At the genus level, the abundance of genera, including Bacteroides , Allobaculum , Desulfovibrio , Akkermansia , Oscillospira , Ruminococcus , Parabacteroides , Adlercreutzia , Alistipes , and Bilophila , was significantly altered by a high-fat diet compared with the normal diet and moderately inverted by 0.

Compared to the mice induced by HFD alone, the mice supplemented with ATX had significantly upregulated abundances of Akkermansia and Parabacteroides to Additionally, to explore high-dimensional biomarkers and identify significant differences at the species level, LEfSe with default parameters was used between the microbial communities compared.

The 65 most abundant OTUs were observed at the taxonomic level in the samples, among which beneficial bacteria were significantly reduced in the HFD group compared with the ND group, revealing a serious gut microbial disorder in HFD-fed mice Figure 6F.

Furthermore, 9 of the 30 most prevalent bacterial genera were upregulated and 21 bacterial genera were downregulated in the HFD-fed mice compared with the mice fed a normal diet, while these genera were partially promoted to their original relative abundance levels after ATX supplementation Figure 6G.

To characterize the functional role of the related abundant bacterial genera, we found 47 secondary functional pathways from the MetaCyc database of metabolic pathways that are relevant to lipometabolism, including the fatty acid and lipid biosynthesis pathway abundance value: 16, Obesity and obesity-related complications are classic health problems worldwide.

A long-term high-fat diet and an imbalance in energy expenditure are important causes for concern In both obese individuals and animal models of NASH, it could be characterized by excessive intracellular lipid accumulation combined with inflammation, which can ultimately progress into hepatic insulin resistance, mitochondrial dysfunction and cellular injury 27 , Emerging evidence shows that ATX, a natural functional food, has been used as a dietary supplement for treating obesity and liver injury and maintaining health 18 , Importantly, when compared to vitamin E, ATX was more effective at lipid peroxidation and preventing NASH.

In the present study, our results showed that ATX supplementation could prevent obesity and the development of NAFLD by meditating lipid metabolism and gut microbiota. Alternatively, ATX consumption also prevents oxidative stress in the liver and lipid peroxidation by improving antioxidant enzyme activity.

According to experimental results, dietary ATX not only significantly decreased body weight gain, adipose tissue weight, and serum TG, TC, and LDL-C levels but also ameliorated abnormal hepatic metabolism following the reduction of liver weight and hepatic TG and TC levels in HFD-induced mice.

No significant difference in the food efficiency ratio or serum HDL-C levels was observed in the HFD group with long-term ATX intake. From the physiological and biochemical profiles, ATX exhibited a better preventive effect on dyslipidaemia and abnormal liver function than our previous results Over the past decade, numerous pieces of evidence have shown that oxidative stress caused by a high-fat diet and specific products of ROS are involved in the development of obesity and fatty liver 31 , Thus, balancing the liver oxidative reaction is an important aspect of preventing the development of NAFLD.

Studies have shown that oxidative stress is closely related to endoplasmic reticulum ER stress in the development and progression of NAFLD and other diseases, while ATX can directly or indirectly moderate ER through antioxidant activity 33 , Interestingly, previous study has confirmed that ATX significantly reduced the levels of oxidative stress marker thiobarbituric acid-responsive substances TBARS in the liver of NASH mice In our results, both the ROS levels evaluated by the DHE probe and the levels of MDA measured, a lipid peroxidation product, were significantly increased in liver tissues in each experimental group.

HFD might have contributed to the increase in these oxidative stress indices and the decrease in antioxidant enzymes, including T-AOC, SOD, CAT, and GSH levels.

Our results are consistent with previous studies showing that HFD seriously damaged the antioxidant defense system 32 , Regardless of the dose, the MDA levels of all ATX-supplemented groups were reduced, suggesting that ATX suppresses overproduction of ROS induced by obesity.

In addition, with dose-dependent increases of the ATX in the diet, the activities of antioxidant enzymes remarkedly improved and were close to normal levels in mice fed HFD.

Multiple studies have confirmed that cell apoptosis induced by excessive endogenous cholesterol is associated with increased ROS in tissues 36 , As previously discussed, long-term HFD intake advanced total cholesterol and disturbed the oxidative balance in the liver, which was attributed to hepatocellular apoptosis.

Based on the TUNEL assay results, we found a large number of apoptotic liver cells in the HFD group, whereas ATX alleviated the degree of necrosis. Nevertheless, the precise intracellular mechanism responsible for this phenomenon was unclear in this study. Moreover, the pathological results showed that ATX could effectively prevent fat accumulation and hepatic steatosis in a dose-dependent manner.

Whether for obesity or the development of NAFLD, one of the root causes is the perturbation in lipid metabolism As reported in previous studies, excessive fat intake induced abnormal bile secretion and disturbed cholesterol levels In addition, FFAs usually trigger the accumulation of DGs and TGs by mediating insulin signal and sensitivity in liver tissue To demonstrate the function of ATX in lipid metabolism, lipidomic analysis revealed that the total levels of hepatic FFAs, TGs, and DGs were noticeably increased in HFD group mice, indicating that a high-fat diet partly supported our previous results.

Interestingly, our results suggested that ATX not only decreased the levels of FFAs and TGs but also specifically reduced the levels of BAs and acyl-carnitines, indicating that both cholesterol metabolism and fatty acid oxidation were improved in mouse livers.

Moreover, SREBP1c , along with its downstream genes ACC , SCD1 and FAS , is an important component in the energy metabolic system and plays a key role in regulating the FFA and TG synthesis mentioned above 38 , According to transcriptome analysis, gene expression signatures were profoundly distinguished among the experimental groups.

Considering the degree and diversity of gene expression changes, only genes associated with the target pathway were screened in this study. AMPK , a key molecule in the regulation of biological energy metabolism, is involved in diabetes and metabolism-related diseases Peroxisome proliferator activated receptor PPARα and peroxisome proliferator-activated receptor gamma coactivator-1α PGC-1 play an important role in regulating the homeostasis of adipose tissue by jointly regulating the balance between fatty acid synthesis and oxidation The expression of PPARα , which is negatively correlated with the severity of NASH, is significantly reduced in NAFLD ATX alleviated the gene expression associated with EIF-2 signaling in NASH rather than improved the expression of gene related to mitochondrial dysfunction In present study, the results revealed that dietary 0.

As our previous manuscript shown, the interaction between PPARα and PGC-1α promoted the oxidation of fatty acids and inhibited the expression of SREBP1c to a certain extent During lipid metabolism, CPT-1 is a key rate-limiting enzyme that accelerates the entry and β-oxidation of long-chain fatty acids into mitochondria A high-fat diet suppresses the expression of PGC-1α , and the mitochondrial respiration rate decreases in the absence of PGC-1α , ultimately leading to a decrease in fatty acid oxidation capacity.

In present study, our results found 0. A previous study confirmed that the suppression of SCD-1 could effectively attenuate HFD-induced insulin resistance and hepatic steatosis

ROS generated by palmitate are also known to cause insulin resistance in various cells by activating a number of stress kinases. Palmiate generates ceramide, which triggers mitochondrial oxidative stress and insulin resistance, but also regulates nicotinamide adenine dinucleotide phosphate oxidase activity involving in generation of ROS such as superoxide in L6 cells Several studies have revealed that ceramide produced by metabolizing saturated fatty acid promotes muscle insulin resistance through Akt and protein phosphatase 2A PP2A or ROS derived from mitochondria, leading to JNK phosphorylation 45 , Accordingly, antioxidants abolish the effects of ceramide-induced ROS production from mitochondria and improve insulin signaling It would be very interesting to investigate whether astaxanthin can inhibit the activation of these pathways.

Our results show that the different extents of recovery on GLUT4 translocation or glucose uptake, which are impaired by TNFα or palmitate, even though both generate similar level of ROS evaluated by the DCFH-DA staining method, and astaxanthin decreases them to the same extent Figure 6 A.

It is plausible that astaxanthin could not sufficiently respond to the palmitate-induced specific ROS at specific areas, which were not detectable by the DCFH-DA staining. Thus, it is very intriguing to elucidate the astaxanthin-insensitive insulin signaling molecules regulated by palmitate as well as localization and source of ROS generated by distinct oxidants.

Collectively our data demonstrate that astaxanthin effectively decreases levels of cellular ROS generated by TNFα or palmitate in L6 muscle cells, thus restoring insulin sensitivity, albeit partially Figures 6 and 7.

Another significant effect of astaxanthin that emerged in this report is that it enhanced insulin-stimulated Akt phosphorylation and glucose uptake in the absence of TNFαor palmitate Figures 1 and 2.

Of note, change in the level of ROS was not detectable by the DCFH-DA staining in the basal state. There are 2 explanations for this phenomenon. In fact, astaxanthin has been reported to decrease nitric oxides NO with little information about its mechanism Because NO induces insulin resistance via enhancement of insulin-stimulated IRS-1 serine phosphorylation but decreased IRS-1 tyrosine phosphorylation in skeletal muscle cells 50 , it would be plausible that astaxanthin enhances insulin signaling via the suppression of NO directly or indirectly.

Because nitric oxide synthase appears to localize to lipid rafts in endothelial cells 51 , astaxanthin may also inhibit the activity of this enzyme directly. An extended study is required for the elucidation of this intriguing possibility.

Second, it may be possible that there are some problems in the DCFH-DA staining method for the detection of the individual ROS at a specific site. Although this method has been widely used for the detection of amount of ROS in the cells, its sensitivity largely relies on the type of ROS Similarly, lipid peroxidation has been quantified by detecting thiobarbituric acid-reactive substances such as malondialdehyde; however, the specificity of this experimental method is low Moreover, the DCFH-DA staining method could not detect small changes in redox balance properly at the specific microdomain in which astaxanthin predominantly accumulates, so we could not obtain the significant effect of astaxanthin on the amount of ROS in basal state.

Notably, it has been reported by others that uncoupling protein-3 promotes relatively small amounts of mitochondria-derived ROS in the basal state in skeletal muscle 54 , which would also result in difficulty of the detection of change in level of ROS at the specific site. Our results show some tendency of decreased ROS in the cells at the basal state with astaxanthin Figure 6 A , which might be caused by the small changes in the amount of specific ROS at specific microdomain, such as lipid rafts.

In this regard, it would be important that appropriate assessment of the effect of antioxidants on the amount of ROS in the cells should be performed by another method that is more sensitive to individual ROS. Nevertheless, we presume that ROS constantly generated in the lipid rafts, in which astaxanthin and signaling molecules including IR, IRS-1, and Shc abundantly exist, in the basal state seems to be modulated by astaxanthin.

In fact, nicotinamide adenine dinucleotide phosphate oxidase subunits are reported to localize in this domain, which indicates this local area is also tightly regulated by redox balance.

Our study indicated that astaxanthin restores insulin sensitivity at the level of IRS-1 by increasing the association of the IR with IRS-1 and decreasing that with Shc Figure 4 A.

These findings contrast with those of a previous study that showed that the most deleterious defects and the origin of insulin resistance were independent of IRS-1 in L6 cells or 3T3-L1 adipocytes Interestingly, astaxanthin enhances Akt phosphorylation but decreases ERK phosphorylation in the insulin signaling pathway Figure 2 , despite the fact that these effects are originally derived from the activation of the IR.

The association of IRS-1with IR relies on at least in part by phosphorylation of serine resides on IRS In particular, Ser , which is just next to the phosphotyrosine binding PTB domain, is phosphorylated by JNK, which decreases insulin-induced tyrosine phosphorylation of IRS-1 Protein kinase C PKC -θ, which is activated by phospholipids at membrane, is known to phosphorylate JNK, leading to phosphorylation Ser of IRS-1 in part.

Phospholipids, the main component of polyunsaturated fatty acid in the membrane, play an important role for regulating localization or activity of proteins at membrane and are predominantly susceptible to peroxidation by ROS. In fact, by-products such as phosphatydilserine hydroperoxides generated by ROS are known to activate PKC.

Furthermore, appropriate localization of PKC seems to be important for its activation because this kinase exists in lipid rafts in membrane, and its activity was diminished by disruption of rafts Similarly, it may be possible that the locally generated ROS somehow regulate the association of the NPXpY motif domain of IR with the PTB domain of IRS-1, resulting in decreased IRS-1 phosphorylation and increased Shc phosphorylation.

Because Shc also binds to the NPXpY domain of the IR through its PTB domain as does IRS-1, occupation of binding sites by increased numbers of IRS-1 molecules may physiologically inhibit Shc association with the IR under the presence of astaxanthin.

The PTB domain is known to also bind to acid phospholipids, which facilitates appropriate localization and phosphorylation of signaling molecules including IRS-1 or Shc at the membrane, although the phospholipids binding to each protein seems to be distinct This different property of each PTB domain may cause their distinct association level with IR under the modulation of phospholipids status by astaxanthin.

Although previous reports have demonstrated that there is a discrepancy between phosphorylation of Akt and Shc by a peptide binding to the IR because of the coordinated internalization of IR in myocytes 58 , exploration of whether astaxanthin influences the internalization of IR at a specific site is beyond the elucidation in the present study.

Another explanation for the distinct differences in the effects on Akt and ERK is the possible involvement of a specific phosphatase. Oxidative stress also influences phosphatases such as PP2A, which is known as a negative regulator of insulin signaling through dephosphorylation of Akt in part in 3T3-L1 adipocyte Accordingly, we observed that astaxanthin increases PP2A phosphorylation and decreases its activity at the basal state, which is concomitant with enhancement of insulin-induced Akt phosphorylation in L6 cells data not shown.

Because PP2A itself is of low sensitivity to the ROS, it appears that ROS-related activation of PP2A requires a cofactor such as the Src kinase family, which is also known to be resident at lipid raft and activated under the change in redox balance.

Astaxanthin may activate this kinase through modification of redox balance at the membrane. Likewise, because the phosphorylation of Shc is regulated by the tyrosine phosphatase PTPϵ in mammary tumor cells, astaxanthin could also regulate this phosphatase, resulting in decreased Shc phosphorylation in addition to decreased association to the IR A possible effect of astaxanthin near the membrane was also shown in previous reports by others that this antioxidant regulates carnitine palmitoyltransferase-1, promoting incorporation of fatty acid to mitochondria, which is resident in the mitochondrial membrane in mice Importantly, as supportive findings to the present report, this antioxidant improved insulin resistance, possibly through the JNK-IRSAkt axis also in liver in mouse fed a high-fat diet This suggests that astaxanthin regulates insulin signaling partly through common mechanism in distinct insulin target tissue.

Because insulin-induced stress kinases suppressed by astaxanthin also involves in cell proliferation, glycogen synthesis, or protein generation, the effect of this antioxidant in vivo must be elucidated minutely to clarify its benefit or disadvantage in a whole-body level.

α-Lipoic acid has been the subject of many studies regarding its antioxidant effect, although it is also well known to induce insulin autoimmune syndrome, promoting abnormal glucose metabolism It is produced in animals and humans and is also highly reactive to ROS 62 ; it augments glucose uptake by positive regulation of insulin signaling molecules, which might be caused by an alteration of the thiol reactivity of redox components of the insulin singling pathway 63 by ROS generation.

Because astaxanthin itself did not generate ROS in this study Figure 6 A , distinct antioxidants might have distinct mechanisms for regulating insulin signaling.

Notably, high concentrations of α-lipoic acid have been shown to enhance ERK phosphorylation by generating ROS in cardiomyoblasts In the case of astaxanthin in L6 cells, we observed hardly any ROS generation or enhancement of ERK phosphorylation data not shown or JNK phosphorylation Figure 8 C at a higher concentration 60 μM.

Furthermore, using various model systems, α-lipoic acid has been reported to exert prooxidant effects in vitro and to promote the mitochondrial permeability transition that results in mitochondrial dysfunction In contrast, astaxanthin does not act as a prooxidant, presumably because of its possession of a keto group with favorable electron acceptor properties.

Accordingly, a randomized, double-blind, placebo-controlled study demonstrated that astaxanthin can be safely consumed by healthy adults The decreased impact of α-lipoic acid on JNK phosphorylation at lower concentrations also indicates that it is inferior to astaxanthin Figure 8 C. We suggest that the enhancement of ERK phosphorylation and the impairment of Akt phosphorylation associated with α-tocopherol are because of its prooxidant effects, although the mechanism should be clarified in the future.

Collectively, these data indicate that astaxanthin has an advantage over other antioxidants. Excess antioxidant perturbs normal homeostasis within cells through the modulation of natural redox balance.

Antioxidants such as vitamin C, which largely exists in cytosol, have been shown to have inhibitory effects on the physiological induction of antioxidants such as superoxide dismutase-1 and glutathione peroxidase-1 in terms of their mRNA level.

As a result, exercise tolerance is decreased because of increased ROS production in response to a decrease in the levels of these antioxidants On the contrary, when cells were exposed to astaxanthin for a longer period, we did not observe its inhibitory effect on these enzymes in their mRNA level in muscle cells data not shown , presumably due to its specific localization or action.

This reveals that astaxanthin appears to have less impact on positive effects on physiological cellular function, although we should be very careful to evaluate plausible disadvantages of this antioxidant in detail because it presumably modulates various signaling molecules through regulation of redox balance described in this report.

This is the first report to reveal divergent effects of an antioxidant on insulin signaling in vitro. Our results indicate that we should be very careful when using antioxidants with the expectation of improving metabolism because of their multifaceted effects on intracellular events.

Astaxanthin can be expected to play a positive role in insulin action as an enhancer of glucose metabolism under insulin resistant conditions.

Further studies should explore other possible effects of this fascinating antioxidant in vitro and in vivo. This work was supported by an unconditional grant from AstaReal Co, Ltd Toyama, Japan and by a grant from the Toyama First Bank to I.

Disclosure Summary: The authors declare that this study was partially supported by an unconditional grant from AstaReal Co, Ltd Toyama, Japan , which produces astaxanthin. and H. are employed by AstaReal Co, Ltd. All other authors declare that there is no duality of interest associated with this manuscript.

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Endocrine Society Journals. Advanced Search. Po Hung Liu , Wataru Aoi , Maki Takami , Hitomi Terajima , Yuko Tanimura , Yuji Naito , Yoshito Itoh , Toshikazu Yoshikawa Author information. Corresponding author. Keywords: astaxanthin , skeletal muscle , lipid metabolism , running exercise , PGC-1α.

JOURNAL FREE ACCESS. Published: Received: December 16, Released on J-STAGE: March 01, Accepted: December 28, Advance online publication: February 19, Revised: -.

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