Astaxanthin Attenuated Increased Mitochondrial Respiration And Decreased Glycolysis During the Activation of Hepatic Stellate Cells
Authors: Bae Minkyung , Pham Tho X. , Park Young‐Ki , Lee Ji‐Young 
Published: November 2023

Non‐alcoholic fatty liver disease is the most common chronic liver disease in the United States. Chronic liver injury causes inflammation, matrix deposition, parenchymal cell death, and angiogenesis, leading to liver fibrosis, a major feature of non‐alcoholic steatohepatitis. Hepatic stellate cells (HSCs) play important roles in the development of liver fibrosis as once activated, they produce excessive extracellular matrix (ECM) substances in the injured liver. We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, prevents the activation of HSCs by attenuating the expression of fibrogenic genes, such as α‐smooth muscle actin and procollagen type I α1 in LX‐2 (a human HSC cell line) as well as primary mouse and human HSCs. The objective of this study was to determine whether activated HSCs (aHSCs) have a different energy phenotype from quiescent HSCs (qHSCs), and if so, whether ASTX can ameliorate any changes in energy metabolism during HSC activation. Mouse primary HSCs were isolated from C57BL/6J mice and cultured on a non‐treated plastic dish for 7 days for spontaneous activation in the presence or absence of 25 μM of ASTX. Cells at 1 day after isolation served as qHSCs. qHSCs and aHSCs treated with or without ASTX for 7 days were plated in a Seahorse XF24 cell culture microplate for Mito Stress and Glycolysis Stress tests to determine mitochondrial respiration and glycolysis of the cells, respectively, using a Seahorse XFe24 Extracellular Flux analyzer. aHSCs showed significantly higher basal oxygen consumption rate (OCR), an indicator of mitochondrial respiration, ATP production, spare respiratory capacity, and proton leak than those of qHSCs. Also, aHSCs had significantly lower glycolysis while glycolytic capacity, maximum capacity of glycolysis, and non‐glycolytic acidification were significantly higher than qHSCs. Importantly, ASTX prevented the changes in basal OCR, ATP production, spare respiratory capacity, proton leak, and glycolysis that occurred during HSC activation. To gain insight into energy metabolism of HSCs, we measured the expression of genes involved in energy substrate utilization in mouse primary qHSCs and aHSCs treated with or without 25 μM of ASTX. The expression of mitochondrial pyruvate carrier 1, glutaminase 1, and carnitine palmitoyltransferase 1α, which are important for utilizing pyruvate, glutamine, and fatty acid as an energy source, respectively, was significantly higher in aHSCs than qHSCs. ASTX, however, decreased the expression of most of the genes in aHSCs. We also measured energy metabolism of LX‐2 cells treated with transforming growth factor β1 (TGFβ1), a potent fibrogenic cytokine. Although TGFβ1 markedly increased fibrogenic gene expression, it did not alter energy metabolism in LX‐2 cells. This indicates that energy phenotypic changes in HSCs may occur only during the transdifferentiation of qHSCs to aHSCs. In conclusion, there were drastic changes in mitochondrial respiration and glycolysis during HSC activation, which were markedly attenuated by ASTX. The anti‐fibrotic action of ASTX may be attributable, at least in part, to its inhibitory effect on the shift of energy dependency to mitochondrial respiration during HSC activation.

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NIH 1R01DK108254‐01