Reduced methylation of PP2Ac promotes ethanol–induced lipid accumulation through FOXO1 phosphorylation in vitro and in vivo

Highlights

• PP2Ac methylation is a target of ethanol action in the liver.

• Lowering of PP2A methylation induced by ethanol promotes phosphorylation of FOXO1 and consequent fat accumulation.

• The reduction in PP2A/Bα, as a result of decreased PP2Ac methylation by ethanol, is responsible for FOXO1 phosphorylation.

Abstract

Although disturbance of the methionine cycle and sequent decrease in hepatic methylation capacity are known to be important factors in the development of alcoholic liver injury, the underlying mechanisms are not fully understood. Here, we investigated the importance of the methylation of protein phosphatase 2A (PP2A) in alcoholic liver disease (ALD). We found that the severity of ethanol-induced liver injury and the extent of demethylation of PP2A catalytic C subunit (PP2Ac) were reduced after treatment with betaine, a methyl donor involved in the methionine-homocysteine cycle. These results suggest that PP2Ac methylation is decreased due to a broad decrease in hepatic methylation capacity after exposure to ethanol. Moreover, we found that the reduction in PP2Ac methylation led to increased degradation of the regulatory Bα subunit, thus promoting the phosphorylation and nuclear exclusion of Forkhead box O1 (FOXO1) and reducing FOXO1 transcriptional activity. Ultimately, the reduced activity of FOXO1 led to increased expression of TXNIP, which caused hepatic lipid accumulation. Our findings suggest that the reduction of PP2A methylation, a result of decrease hepatic methylation capacity, played an important role in ethanol–induced lipid accumulation via down-regulation of PP2A/Bα and FOXO1 phosphorylation.

Introduction

Excessive alcohol consumption leads to alcoholic liver disease (ALD), which is the leading cause of death among all chronic liver diseases (Liangpunsakul et al., 2016). The development of alcoholic fatty liver is the earliest response of the liver to chronic alcohol consumption and can progress to more extensive liver injuries such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (Gao and Bataller, 2011). (Gao and Bataller, 2011). Although this general disease progression is well established, the underlying molecular mechanisms by which ethanol causes liver steatosis are still not completely understood.

Accumulating evidence has indicated that alterations of the methionine cycle and the reduction of the ratio of S-adenosylmethionine (SAM):S-adenosylhomocystein (SAH) play a fundamental role in the pathogenesis of ALD (Kharbanda et al., 2013; Fujii and Kawada, 2014; Williams et al., 2014). A decrease in the SAM:SAH ratio affects hepatic methylation capacity by affecting SAM-dependent methyltransferase reactions, including methylation of protein phosphatase 2A (PP2A) (Sutter et al., 2013; Laxman et al., 2014). PP2A encompasses a large family of Ser/Thr phosphatases that all have a catalytic subunit (C), a structural scaffolding subunit (A), and one of many possible regulatory B subunits that provide substrate specificity to the holoenzyme (Shi, 2009). Fifteen B subunit genes encoding 26 different isoforms give rise to more than 100 combinations of the PP2A holoenzyme, enabling a wide spectrum of biological activities. The PP2A catalytic C subunit (PP2Ac) is methylated on Leu-309 by SAM-dependent leucine carboxyl methyltransferase-1 (LCMT-1) (Bottiglieri et al., 2012). Changes in the methylation state of the C subunit critically modulate the recruitment of specific regulatory B subunits to the (AC) core enzyme, thereby influencing the substrate specificity of the PP2A holoenzyme (Sontag and Sontag, 2014; Sontag et al., 2014). The importance of PP2Ac methylation has been underlined by the observation of its changing methylation status in SAMe and methionine-treated hepatocytes, suggesting a potential role in liver steatosis due to altered interactions with TORC1 (Zubiete-Franco et al., 2016).

Forkhead box O1 (FOXO1) belongs to a family of transcription factors related to stress response pathways. Of note, FOXO1 plays a major role in regulating hepatic lipid metabolism, and the liver is one of its most important sites of action (Deng et al., 2012; Valenti et al., 2008; Tikhanovich et al., 2013). FOXO1 is regulated through reversible phosphorylation, which is catalyzed by kinases and phosphatases. The phosphorylation of FOXO1 leads to nuclear exclusion and thus inhibition of its transcriptional activity (Xie et al., 2012). A growing body of evidence has suggested that PP2A is a physiologically important phosphatase for FOXO1 (Yan et al., 2012, 2008). Of note, a recent study showed that FOXO1 levels were reduced in the livers of patients with alcoholic hepatitis (AH) and in animal models of ALD, supporting the inhibitory effect of ethanol on FOXO1 (Heo et al., 2019). Nonetheless, the question of whether alcohol consumption promotes a change in FOXO1 phosphorylation and, if so, how FOXO1 phosphorylation is related to the progression of ALD have not been explored to date. Given that PP2A is affected by ethanol–induced hypomethylation and that it regulates FOXO1 phosphorylation, we hypothesized that PP2A may be part of the link between ethanol–induced hypomethylation and FOXO1 dysregulation in the progression of ALD.

The present study investigated the mechanisms by which reduced methylation of PP2Ac is related to the phosphorylation FOXO1 in ALD. Using betaine, a methyl donor, we first demonstrated that decreased hepatic methylation capacity is an important factor that causes reduced PP2Ac methylation. Then, we found that ethanol–induced demethylation of PP2Ac contributes to the reduction of PP2A/B55α, leading to hyperphosphorylation of FOXO1 and subsequent accumulation of triglyceride (TG) via upregulation of thioredoxin-interacting protein (TXNIP).