Bioactivity of Astaxanthin in the Prevention of Hepatic Inflammation

Non-Technical Summary:<br/>
Astaxanthin (ASTX) is a carotenoid known to have a potent antioxidant property. However, its bioactivity has been understudied compared with other carotenoids. Food and Drug Administration (FDA) awarded generally recognized as safe (GRAS) status to ASTX extracted from Haematococcus pluvialis (H. pluvialis), a green alga, in early 2010. ASTX consumption as a food component, therefore, is expected to largely increase. Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease associated with the increasing prevalence of obesity epidemic. Steatosis can progress to non-alcoholic steatohepatitis (NASH), i.e., inflamed fatty liver. Lipotoxicity and oxidative stress due to increased free fatty acid (FFA) influx to the liver are known to be among the primary causes for the disease progression. We observed that ASTX induced the expression of genes involved in the beta-oxidation of fatty acids (FA) possibly via the activation of peroxisome proliferator activated receptor alpha (PPAR alpha) in vivo and in vitro. Additionally, the expression of nuclear factor E2 related factor 2 (Nrf2)-dependent endogenous antioxidant genes was induced by ASTX, resulting in improved hepatic glutathione (GSH) redox status. In this proposal, we aim at further investigating the potential mechanisms underlying the bioactivity of ASTX as a regulator of fat metabolism and antioxidant defense system to prevent hepatic inflammation. Information generated from the proposed work will lead to the development of dietary recommendations for ASTX and foods containing ASTX to lower the risk of NASH and other inflammatory condition associated with obesity.
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Approach:<br/>
In aim 1, male C57BL/6J mice at age of 6 weeks will be fed a LF control, a HF/HS control or HF/HS with ASTX supplementation (0.003, 0.01, 0.03%, wt/wt) for 10 weeks. Each dietary group will be subdivided into two groups for regular or 0.2% sucrose-containing drinking water. After 10 week-dietary treatment, blood and tissue (liver, adipose, muscle) samples will be harvested for phenotypical characteristics of mice, ASTX bioavailability and hepatic steatosis. In aim 2, male C57BL/6J mice will be fed a LF control, a HF/HS control or HF/HS with ASTX supplementation at the level of ASTX that will be determined in Objective 1. Each dietary group will be subdivided into two groups for regular or 0.2% sucrose-containing drinking water. Food consumption and body weight of mice will be measured weekly and blood samples will be collected monthly. At 18 week, oral glucose tolerance test will be conducted. After 20 week-dietary treatment, blood and tissue (liver, adipose, muscle) samples will be harvested for phenotypical characteristics of mice, insulin sensitivity, hepatic steatosis, oxidative stress, systemic inflammation and NASH, apoptosis, and fat metabolism and insulin signaling in adipose and muscle. In aim 3, primary hepatocytes will be isolated from C57BL/6J mice or WT and Nrf2-/- mice depending on experiments. Cells will be treated with 0-10 microM of ASTX for 24 hr. The ASTX levels are chosen based on human studies demonstrating plasma concentrations of ASTX to be 0.1-2.2 microM. Primary hepatocytes from C57BL/6J mice will be used for the following experiments unless it is stated otherwise: role of PPAR alpha in the up-regulation of FA beta-oxidation gene by ASTX expression; regulatory mechanisms for the activation of PPAR alpha by ASTX; evaluation on the role of ASTX in the induction of endogenous antioxidant genes and ROS production; role of Nrf2 in the antioxidant effect of ASTX; and evaluation of PGC-1/PPAR alpha/Nrf2 activation by ASTX.
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Progress:<br/>
2012/02 TO 2013/01<br/>
OUTPUTS: The studies were disseminated by oral presnetation at the Korean Society for Food Science and Nutrition Annual meeting in South Korea in November 2012. Also, our findings will be presented by oral and poster presentations at the Experimental biology in April 2013. PARTICIPANTS: Yue Yang (graduate student) Tho Pham (gradaute student) Bohkyung Kim (Postdoctoral fellow) Youngki Park (reserach associate) Chai Siah Ku (gradaute student) Tyler Benn (gradaute student) Casey Wegner (graduate student) TARGET AUDIENCES: Target audiences for this project is general public and individual with non-alhoholic fatty liver disease to improve their health. PROJECT MODIFICATIONS: As hepatic stellate cells (HSC) play a critical role in the development of non-alcholic steatohepatitis, we developed techniques to isolate primary HSC from mice and have been investigating the effect of astaxanthin in the activation of HSC.
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IMPACT: We completed the mouse in vivo study. Male C57BL/6J mice were fed a high fat (HF), control diet (60% energy from fat) or a HF diet supplemented with 0.003, 0.01 and 0.03% ASTX by weight for 12 wk. Mice fed 0.03% ASTX showed a significant reduction in plasma triglyderide levels. Quantitative real-time PCR (qRT PCR) has revealed a higher mRNA expression of acyl-coenzyme A oxidase 1, suggesting there was potentially elevation in peroxisomal fatty acid beta-oxidation, which may counteract the effects of increased expressions of lipogenic genes and eventually lead to hypotriglyceridemic outcome. Compared with control, plasma high-density lipoprotein (HDL)-cholesterol levels were significantly lower in ASTX-fed mice, with no difference in plasma non-HDL cholesterol levels. The mRNA expression of low-density lipoprotein (LDL) receptor and HMG-CoA reductase implied an increase in LDL-cholesterol uptake by liver. Moreover, the expression of Nrf2 has been elevated at mRNA level in the ASTX-fed group, suggesting a potentially activated endogenous antioxidant mechanism. No differences have been found in the expressions of fibrogenic and macrophage markers between groups. In conclusion, 0.03% ASTX in diet decreased plasma TG and increased HDL-cholesterol in DIO mice and therefore, it is potentially beneficial for managing dyslipidemia and protecting liver from chronic conditions in obesity.