Progress 02/01/12 to 01/31/16
Outputs Target Audience:Local, national and international audiences interested in health and nutrition and non-alcoholic fatty liver disease. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student was actively engaged in this project and trained for various molecular and cellular techniques. Also, opportunities for oral and poster presentation were given to the student. How have the results been disseminated to communities of interest?The results of this project were submitted for publication andpresented at local and international conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts What was accomplished under these goals?
Activation of hepatic stellate cells (HSCs) is critical for the development of liver fibrosis. Previously, we showed that astaxanthin (ASTX), a xanthophyll carotenoid, has anti-fibrogenic effects in LX-2 cells, a human HSC cell line. The objectives of this study were to determine the effect of ASTX on HSC activation and inactivation, and to identify molecular mediators that play critical roles in the processes.Quiescent mouse primary HSCs and human primary HSCs were incubated with ASTX. Normal human livers and those with several pathological conditions, such as primary biliary cirrhosis (PBC), were analyzed for histone deacetylase 9 (HDAC9) expression. HDAC9 was knocked down in LX-2 cells to evaluate its role in transforming growth factor b1 (TGFβ1)-mediated HSC activation. ASTX prevented the activation of mouse primary HSCs, as evidenced by attenuated induction of procollagen type I a1 (COL1A1). In human primary HSCs, ASTX decreased TGFβ1-induced fibrogenic gene expression. Among 11 classical HDACs, changes in HDAC9 expression were most evident during HSC activation while ASTX significantly decreased the expression of HDAC9 and its transcriptional regulator myocyte enhancer factor 2 (MEF2). HDAC9 and MEF2 expression in human PBC livers was significantly higher than normal livers and other liver pathologies also induced HDAC9 in human liver specimens. HDAC9 knockdown in LX-2 cells decreased TGFβ1-induced fibrogenic gene expression.ASTX inhibits HSC activation and facilitates HSC inactivation, which is attributable to its inhibitory action on HDAC9 expression. The objective of this study was to determine if astaxanthin (ASTX), a xanthophyll carotenoid, can prevent obesity-associated metabolic abnormalities, inflammation and fibrosis in diet-induced obesity (DIO) and nonalcoholic steatohepatitis (NASH) mouse models. Male C57BL/6J mice were fed a low fat (6% fat, w/w), a high fat/high sucrose control (HF/HS; 35% fat, 35% sucrose, w/w), or a HF/HS containing ASTX (AHF/HS; 0.03% ASTX, w/w) for 30 wk. To induce NASH, another set of mice was fed a HF/HS diet with 2% cholesterol (HF/HS/HC) or ASTX-supplemented HF/HS/HC (AHF/HS/HC; 0.015% ASTX, w/w) for 20 wk. Compared to LF, HF/HS significantly increased plasma total cholesterol, triglyceride and glucose, which were lowered by ASTX. ASTX decreased the mRNA levels of liver macrophage markers and fibrosis in both models. The effect of ASTX was more prominent in NASH than DIO mice. In the epididymal fat, ASTX also decreased macrophage infiltration and M1 macrophage marker expression, and inhibited hypoxia-inducible factor 1-a and its downstream fibrogenic genes in both mouse models. ASTX significantly decreased tumor necrosis factor a mRNA in lipopolysaccharide-stimulated splenocytes from DIO mice, compared with those from control mice fed a HF/HS diet. Additionally, ASTX significantly elevated the levels of genes that regulate fatty acid b-oxidation and mitochondrial biogenesis in the skeletal muscle, compared with control obese mice, whereas no differences were noted in adipose lipogenic genes. Our results indicate that ASTX inhibits inflammation and fibrosis in the liver and adipose tissue and enhances the skeletal muscle's capacity for mitochondrial fatty acid oxidation in obese mice.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
M. Bae, J. Lee. Astaxanthin attenuated the expression of fibrogenic genes induced by high glucose alone and in combination with transforming growth factor beta 1 in hepatic stellate cells. FASEB J
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Astaxanthin prevents and reverses the activation of hepatic stellate cells via the modulation of histone deacetylase 9. Liv Int
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
B. Kim, C. Farruggia, C. S. Ku, T. X. Pham, Y. Yang, Casey J. Wegner, N. J. Farrell, E. Harness, Y. Park, Sung I. Koo, J. Lee. Astaxanthin, a xanthophyll carotenoid, inhibited inflammation and fibrosis in the adipose and the liver of diet-induced obesity and liver fibrosis mice. J Nutr Biochem
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Y. Yang, M. Bae, B. Kim, Y. Park, S. I. Koo, J. Lee. Astaxanthin prevents and reverses the activation of mouse primary hepatic stellate cells. J Nutr Biochem 2016; 29:21-26.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
M. Bae, J. Lee. The effect of carotenoids on transforming growth factor ?1-induced fibrogenesis in LX-2 cells, a human hepatic stellate cell line. FASEB J
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Progress 02/01/14 to 01/31/15
Outputs Target Audience: Local, national and international audience interested in health and nutrition and non-alcoholic fatty liver disease. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Students and postdocs were trainedinconducting experiments, data anlalysis and data interpretation as wellas inpresenting data. How have the results been disseminated to communities of interest? Our findings were presented at the Keystone Symposia in March 2014 and Experimental Biology meeting in April 2013.Articles were published, submitted for publication,or arein preparation. What do you plan to do during the next reporting period to accomplish the goals? Results of the project will be presented at the Experimental Biology in March 2015 and other conferencesto disseminate our findings to scientific societies. I will continue to publish our findings in scientific journals.
Impacts What was accomplished under these goals?
Activation of hepatic stellate cells (HSC) is a major event in the development of liver fibrosis. Previously, we showed that astaxanthin (ASTX), a xanthophyll carotenoid, has a potent anti-fibrogenic effect in LX-2 cells, a human HSC cell line. We investigated if ASTX prevents the activation of quiescent HSC and also facilitates the inactivation of activated, myofibroblast-like HSC using mouse primary HSC. Incubation of ASTX with quiescent HSC inhibited their activation as evidenced by decreased loss of intracellular lipid droplets, and repressed expression of HSC activation markers, such as α smooth muscle actin (αSMA), at mRNA and protein levels. Moreover, in activated HSC, the addition of ASTX for 2 or 4 d resulted in reappearance of lipid droplets and decreased αSMA expression, suggesting that ASTX reversed HSC activation. As histone deacetylases (HDAC) have a potential role in HSC activation, we screened the effect of ASTX on the expression of all 11 classical HDAC isoforms. Notably, HDAC9 expression was significantly elevated during HSC activation, which was markedly attenuated by ASTX. Taken together, ASTX not only prevented the activation of quiescent HSC but reversed morphological changes and increased αSMA expression in activated HSC close to quiescent stage of HSC possibly via the modulation of HDAC9 expression. ASTX may be used for the prevention of liver fibrosis. Chronic inflammation and oxidative stress are associated with obesity-related metabolic diseases such as non-alcoholic fatty liver disease, type 2 diabetes, and cardiovascular disease. Astaxanthin (ASTX), a xanthophyll carotenoid, has been suggested to play anti-inflammatory and antioxidant functions. We investigated potential mechanisms of action by which ASTX exerts the anti-inflammatory and antioxidant effects in macrophages. When RAW 264.7 macrophages were stimulated by LPS (100 ng/ml) for 24 h, mRNA expression of IL-6 and IL-1β was increased. However, co-incubation with ASTX (25 mM) significantly attenuated the induction. ASTX markedly decreased LPS-induced nuclear translocation of nuclear factor kB (NFkB). In addition, LPS increased cellular reactive oxygen species (ROS), which was abolished by ASTX with a concomitant decrease in NADPH oxidase 2 (NOX2) expression. Bone marrow-derived macrophages (BMDM) isolated from wild-type and nuclear factor E2 related factor 2 (Nrf2) knockout mice were incubated with ASTX without or with LPS. In both unstimulated and LPS-stimulated BMDM, ASTX decreased cellular ROS accumulation regardless genotype, suggesting that Nrf2 is likely to play a minimal role in the reduction of cellular ROS by ASTX. Taken together, ASTX exerts both anti-inflammatory and antioxidant effects in macrophages, at least in part, by inhibiting NFkB nuclear translocation and by repressing NOX2 expression, respectively. ASTX may be a potential preventive/therapeutic agent for chronic diseases that are caused by chronic inflammation and oxidative stress.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
B. Kim, C. S. Ku, T. X. Pham, Y. Yang, Casey J. Wegner, N. J. Farrell, E. Harness, C. Farruggia, Y. Park, J. Lee. Astaxanthin, a xanthophyll carotenoid, improved metabolic and inflammatory abnormalities in diet-induced obesity mice.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Y. Yang, B. Kim, Y. Park, S. I. Koo, J. Lee. Astaxanthin prevents transforming growth factor ?1-induced pro-fibrogenic gene expression by inhibiting Smad3 activation in hepatic stellate cells. Biochimica Biophysica Acta 2014; 1850:178-185
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Y. Yang, J. Lee. Astaxanthin, a xanthophyll carotenoid, prevented and reversed the activation of mouse primary hepatic stellate cells via the modulation of histone deacetylases. Keystone Symposia Conference: Fibrosis: From the bench to bedside 2014; 1052
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
B. Kim, C. S. Ku, T. X. Pham, Y. Yang, C. Wegner, Y. Park, J. Lee. Supplementation of astaxanthin, a xanthophyll carotenoid, improved metabolic and inflammatory abnormalities associated with obesity in diet-induced obese mice. FASEB J 2014; 645.8.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Y. Yang, J. Lee. Astaxanthin, a xanthophyll carotenoid, inhibited and reversed the activation of mouse primary hepatic stellate cells via the modulation of histone deacetylases. FASEB J 2014; 39.1.
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Progress 02/01/13 to 01/31/14
Outputs Target Audience: Local, national and internationalaudience interested in health and nutrition and non-alcoholic fatty liver disease. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Students and postdoc were trained forconducting experiments, data anlalysis anddata interpretation as well asfor presenting data. How have the results been disseminated to communities of interest? Our findings were presented at the Experimental Biology meeting in April 2013 and manuscripts were published or are in preparation. What do you plan to do during the next reporting period to accomplish the goals? Results of the project will be presented at the Experimental Biology in April 2014 and at the Keystone Symposia Conference in March 2014 to disseminate our findings to scientific societies.
Impacts What was accomplished under these goals?
Activation of hepatic stellate cells (HSC) is a major event in the development of liver fibrosis. Previously, we showed that astaxanthin (ASTX), a xanthophyll carotenoid, has a potent anti-fibrogenic effect in LX-2 cells, a human HSC cell line. We investigated if ASTX prevents the activation of quiescent HSC and also facilitates the inactivation of activated, myofibroblast-like HSC using mouse primary HSC. Incubation of ASTX with quiescent HSC inhibited their activation as evidenced by decreased loss of intracellular lipid droplets, and repressed expression of HSC activation markers, such as α smooth muscle actin (αSMA), at mRNA and protein levels. Moreover, in activated HSC, the addition of ASTX for 2 or 4 d resulted in reappearance of lipid droplets and decreased αSMA expression, suggesting that ASTX reversed HSC activation. As histone deacetylases (HDAC) have a potential role in HSC activation, we screened the effect of ASTX on the expression of all 11 classical HDAC isoforms. Notably, HDAC9 expression was significantly elevated during HSC activation, which was markedly attenuated by ASTX. Taken together, ASTX not only prevented the activation of quiescent HSC but reversed morphological changes and increased αSMA expression in activated HSC close to quiescent stage of HSC possibly via the modulation of HDAC9 expression. ASTX may be used for the prevention of liver fibrosis. Elevated plasma triglyceride (TG) and cholesterol concentrations are risk factors for cardiovascular diseases. We previously reported that astaxanthin (ASTX), a xanthophyll carotenoid, lowers plasma total cholesterol and TG concentrations in apolipoprotein E knockout mice. To investigate if ASTX exerts hypolipidemic effects in the diet-induced obesity (DIO) mice and if so, to determine an effective supplementation level, male C57BL/6J mice were fed a high fat (HF) control diet (60% energy from fat) or a HF diet supplemented with 0.003%, 0.1% or 0.03% ASTX by weight for 12 wk. Only mice fed 0.03% ASTX showed a significant reduction in plasma TG levels, suggesting this level of supplementation is necessary for ASTX to have a TG-lowering effect. Compared with control, plasma high density lipoprotein cholesterol (HDLc) levels were significantly lower in 0.03% ASTX-fed mice without a difference in plasma non-HDLc levels. The ASTX supplementation decreased plasma aspartate aminotransferase levels, suggesting it protects against high fat-induced tissue damages. Sterol regulatory element binding protein 1c mRNA was decreased in ASTX-fed mice livers, which may contribute to the hypotriglyceridemic effect of ASTX. In conclusion, 0.03% ASTX in diet decreased plasma TG and increased HDLc in DIO mice and therefore, it is potentially beneficial for lowering CVD risk. We previously reported that astaxanthin (ASTX), a xanthophyll carotenoid, exerted hypolipidemic and antioxidant properties in vivo. The objective of this study was to determine protective effects of ASTX against obesity-associated metabolic and inflammatory abnormalities. Male C57BL/6J mice were fed a high fat/high sucrose control (35% fat, 35% sucrose by wt), or ASTX diet (0.03% ASTX by wt) for 30 wk. ASTX-fed mice showed lower plasma total cholesterol, triglycerides, and alanine aminotransferase than control. ASTX tended to decrease mRNA of lipogenic and fibrogenic genes in the liver, and significantly increased genes for fatty acid b-oxidation and mitochondria biogenesis in muscle. Epididymal fat pad weights were not significantly different between groups. However, the expression of F4/80, a macrophage marker, was significantly lower in the epididymal fat pad of ASTX mice, and markedly less macrophage infiltration in ASTX group was confirmed by histological analysis. Ex vivo analysis of splenocytes isolated from mice on a study diet showed that tumor necrosis factor α mRNA was significantly reduced upon lipopolysaccharide stimulation in ASTX mouse splenocytes. In conclusion, our results suggest that ASTX supplementation prevents obesity-associated metabolic dysfunctions and inflammation possibly by improving capacity of muscle in handling excess energy.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Y. Yang, T. X. Pham, C. J. Wegner, B. Kim, C. S. Ku, Y. Park, J. Lee. The hypotriglyceridemic and potential hepatic protective effects of astaxanthin supplementation in diet-induced obese mice. Eur J Nutr 2014
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2014
Citation:
2. B. Kim, C. S. Ku, T. X. Pham, Y. Yang, C. Wegner, Y. Park, J. Lee. Supplementation of astaxanthin, a xanthophyll carotenoid, improved metabolic and inflammatory abnormalities associated with obesity in diet-induced obese mice. FASEB J 2014
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2014
Citation:
Y. Yang, J. Lee. Astaxanthin, a xanthophyll carotenoid, inhibited and reversed the activation of mouse primary hepatic stellate cells via the modulation of histone deacetylases. FASEB J
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Y. Yang, T. X. Pham, C. Wegner, B. Kim, C. S. Ku, Y. Park, J. Lee. Astaxanthin supplementation lowers plasma triglyceride concentrations in diet-induced obese C57BL/6J mice. FASEB J 2013; 27: 638.14.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Y. Yang, J. Lee. Astaxanthin inhibits TGF-?1-induced fibrogenic gene expression by inhibiting the activation of Smad3 and KLF-6 pathways in LX-2 and primary mouse hepatic stellate cells. FASEB J 2013; 27:32.3
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Progress 02/01/12 to 01/31/13
Outputs 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.
Impacts 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.
Publications
- Y. Yang, T. X. Pham, C. Wegner, B. Kim, C. S. Ku, Y. Park, J. Lee. Astaxanthin supplementation lowers plasma triglyceride concentrations in diet-induced obese C57BL/6J mice. FASEB J 2013; (Abstract).
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