Elsevier

European Journal of Pharmacology

Volume 727, 15 March 2014, Pages 115-124
European Journal of Pharmacology

Pulmonary, gastrointestinal and urogenital pharmacology
SIRT1 activation by methylene blue, a repurposed drug, leads to AMPK-mediated inhibition of steatosis and steatohepatitis

https://doi.org/10.1016/j.ejphar.2014.01.035Get rights and content

Abstract

Sirtuins maintain energy balance. Particularly, sirtuin 1 (SIRT1) activation mimics calorie restriction and nutrient utilization. However, no medications are available for the up-regulation of SIRT1. Methylene blue (MB) had been in clinical trials for the treatment of neurological diseases. This study investigated the effect of MB on sirtuin expression in association with the treatment of steatosis and steatohepatitis, and explored the underlying basis. The effects of MB on mitochondrial function, molecular markers, pharmacokinetics, and histopathology were assessed using hepatocyte and/or mouse models. Immunoblotting, PCR and reporter assays were done for molecular experiments. After oral administration, MB was well distributed in the liver. MB treatment increased NAD+/NADH ratio in hepatocytes. Of the major forms, MB treatment up-regulated SIRT1, and thereby decreased PGC-1α acetylation. Consistently, hepatic mitochondrial DNA contents and oxygen consumption rates were enhanced. MB treatment also notably activated AMPK, CPT-1 and PPARα: the AMPK activation relied on SIRT1. Activation of LXRα and the induction of SREBP-1c and its target genes by T0901317 were diminished by MB. In addition, MB treatment antagonized the ability of palmitate to acetylate PGC-1α, and increase SERBP-1c, FAS, and ACC levels. In mice fed on a high-fat diet for 8 weeks, MB treatment inhibited excessive hepatic fat accumulation and steatohepatitis. The ability of MB to activate SIRT1 promotes mitochondrial biogenesis and oxygen consumption and activates AMPK, contributing to anti-lipogenesis in the liver. Our results provide new information on the potential use of MB for the treatment of steatosis and steatohepatitis.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the hepatic constituent of metabolic syndrome, and may result in hepatitis and/or facilitate the progression of more severe liver diseases such as fibrosis and cirrhosis (Marra et al., 2008). Aberrant hepatic fat accumulation is provoked mainly by decreases in mitochondrial fuel oxidation and increases in liver X receptor α (LXRα)-dependent lipogenesis. Excessive fat accumulation and fatty acid oxidation within hepatocytes cause oxidative stress in the liver and promote the production of proinflammatory cytokines (Mantena et al., 2008, Mantena et al., 2009). The consequent oxidative stress triggers a robust production of lipid peroxides that form adducts to macromolecules, and chronically results in damage to organelles such as mitochondria (Marra et al., 2008).

Sirtuin (SIRT) maintains energy balance in the cell (Rodgers et al., 2008); the activation of SIRT mimics several metabolic aspects of calorie restriction that enhance selective nutrient utilization and mitochondrial oxidative function. A line of studies support the role of SIRT1 in homeostatic energy metabolism under metabolic disorders including fatty liver, insulin resistance, and atherosclerosis; SIRT1 regulates peroxisomal proliferators-activated receptor alpha (PPARα) and PPARγ coactivator (PGC-1α), the molecules that govern mitochondrial fuel oxidation and lipid homeostasis (Lagouge et al., 2006). Since SIRT1 is the key molecule that activates AMPK (Lan et al., 2008), it may be an attractive target for the treatment of metabolic disorders. However, no medications are currently available for the induction of SIRT1.

Methylene blue (MB) regulates electron transfer chain reaction within the mitochondria, and enhances mitochondrial respiration by exchanging electron at complex I and increasing the activity of cytochrome c oxidase (Wen et al., 2011). So, MB has been utilized as a redox indicator. Recently, MB has been in clinical trials for the treatment of pathologic conditions such as methemoglobinemia, ischemic-reperfusion injury, and cyanide poisoning (Wen et al., 2011). Moreover, it may also be applied for the treatment of Alzheimer disease, a disease highly associated with mitochondrial dysfunction (Atamna et al., 2008, Eckert et al., 2012); MB treatment protects from the cognitive decline inflicted by inhibitors of complex IV through its redox activity (Callaway et al., 2002). Nevertheless, the effect of MB on sirtuin expression had never been explored.

This study investigated whether MB has an effect on sirtuin expression, and if so, whether it has therapeutic efficacy for the treatment of NAFLD. To accomplish this, we used in vitro and in vivo models (i.e., hepatocyte-derived cell line; primary rat hepatocyte; mouse pharmacokinetics; and feeding of a high fat diet, HFD). Here, we report that MB has the ability to activate SIRT1 and promote mitochondrial biogenesis and oxidation of free fatty acids (FFAs). Our findings demonstrate the beneficial effect of MB on metabolic homeostasis in conjunction with the inhibition of liver fat accumulation, implying that MB may be utilized as a repurposed drug for the treatment of steatosis and steatohepatitis.

Section snippets

Materials

MB and methylene violet [internal standard (IS) for ultra-performance liquid chromatography tandem mass-spectrometric (UPLC-MS/MS)] were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Acetonitrile and heptafluorobutyric acid were obtained from Fisher Scientific Co. (Seoul, Korea), and all other chemicals and reagents used for pharmacokinetic studies were of analytical grade. T0901317 (T090) was obtained from Calbiochem (San Diego, CA, USA). The antibodies recognizing SREBP-1,

Increase in NAD+/NADH ratio in hepatocytes

MB may alter the biochemical activity of specific mitochondrial components: it facilitates the electron transfer efficiency on the complex I of electron transport chain through its redox potential by receiving electrons and being reduced to form MBH2 (Atamna et al., 2012). To find the basis for the increase in mitochondrial function, we wondered whether MB treatment facilitates NADH conversion to NAD+ (Fig. 1A). In HepG2 cells, MB treatment significantly increased the NAD+/NADH ratio and NAD+

Discussion

MB, a potent cationic dye, serves as a redox indicator (i.e., an artificial electron donor to complexes I, II, III and IV of mitochondria), or is used as a staining dye for nucleic acids. Since MB has a potential to enhance mitochondrial respiratory chain by exchanging electrons within mitochondria, it had been applied for the treatment of neuropathies (i.e. Alzheimer’s dementia or Parkinson’s disease). Accumulating evidence indicates that mitochondrial dysfunction plays a role in the

Acknowledgments

This work was supported by the National Research Foundation of Korea grant funded by the government of Korea (MEST) (No. 2007-0056817), and in part by the Ministry of Health & Welfare (No. A111641).

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