Roles of extrahepatic lipolysis and the disturbance of hepatic fatty acid metabolism in TNF-α -induced hepatic steatosis

Abstract

Our previous study showed that both Kupffer cell eliminator (GdCl₃) and tumor necrosis factor α (TNF-α) receptor antagonist (etanercept) could partially attenuate binge drinking-induced liver steatosis. Herein, we extended the study by directly investigating the roles of TNF-α on the hepatic fat levels in mice and in HepG2 cells, and explored the underlying mechanisms. SPF male ICR mice were exposed to TNF-α (0.166 mg/kg body weight) with or without phenylisopropyl adenosine (PIA, an anti-lipolytic drug) for 1.5, 3, 6, and 24 h. We found that TNF-α treatment resulted in hepatic triglyceride (TG) elevation at 6 h time point, which was blocked by PIA. TNF-α led to the activation of extrahepatic lipolysis demonstrated by the increase of serum free fatty acid (FFA) level, and the increased protein levels of adipose triglyceride lipase (ATGL) and phosphorylated hormone-sensitive lipase (HSL) in mice epididymal adipose tissues, but had no significant effects on the protein levels of sterol regulatory element binding protein 1c (SREBP-1c) and peroxisomal proliferator activation receptor α (PPAR-α) in mice liver. The in vitro study showed TNF-α treatment could not result in elevation of TG in HepG2 cells, although it indeed brought about a slight activation of SREBP-1c. These results support the hypothesis that TNF-α might make a small contribution to ethanol-induced fatty liver by stimulating extrahepatic lipolysis.

Introduction

Alcoholic liver disease (ALD) remains to be a worldwide health problem (Singal and Anand, 2013; Wang et al., 2014). ALD encompasses a spectrum of progressively aggravated liver injury ranging from steatosis, to steatohepatitis, fibrosis, and finally cirrhosis. Alcoholic fatty liver (AFL), manifested by the accumulation of triglyceride (TG) in liver, is the earliest phenotype of ALD. Epidemiological studies showed that AFL developed in about 90% of individuals who drank more than 60 g/day of ethanol, but might also occur in some sensitive individuals who drank less (O’Shea et al., 2010). Now, it is generally accepted that fatty liver is a pathogenic condition as fatty liver is more vulnerable to other hepatotoxicants such as acetaminophen and arsenic (Kucera et al., 2012; Tan et al., 2011). Notably, AFL could progress into more advanced ALD despite abstinence (Leevy, 1962; Sorensen et al., 1984). AFL is considered as the optimal intervention stage to block the progression of ALD, as steatosis is usually benign and reversible. Therefore, it has long been the research focus to elucidate the underlying mechanisms and to screen potential therapeutic agents for ethanol-induced steatosis (Zhao et al., 2017).

The pathogenesis of AFL is complicated and may be attributed to many factors including the changes of redox condition, transportation impairment of the synthesized lipid, inhibition of fatty acid oxidation, the enhancement of the lipogenesis, and the increased lipolysis of extrahepatic adipose tissue (Livero and Acco, 2016; Purohit et al., 2009; Zeng and Xie, 2009; Zhong et al., 2012). Several lines of evidence suggested that tumor necrosis factor α (TNF-α), a well-known pro-inflammatory cytokine secreted by Kupffer cells, might play some roles in the pathogenesis of AFL, which was denied by results of other studies. Ethanol-induced hepatic steatosis is accompanied by the elevation of serum and hepatic TNF-α levels (Lin et al., 1998; Pritchard et al., 2007; Zeng et al., 2016). Notably, ethanol-induced hepatic steatosis in mice could be significantly attenuated by depletion of Kupffer cell (Adachi et al., 1994; Koop et al., 1997) and by genetic knockout of TNF-α receptor 1 (TNF-R1 ^−/−) (Bergheim et al., 2006; Yin et al., 2001, 1999), suggesting that a causal role of TNF-α in the development of AFL. However, results of some other studies argued that TNF-α might play negligible role in AFL. For example, Ji et al. found that TNF-α made a moderate contribution to ethanol-induced elevation of alanine transaminase (ALT), necroinflammation, apoptosis, but only a small contribution to the fatty liver (Ji et al., 2004). Similarly, the study by Iimuro et al. revealed that TNF-α antibody significantly attenuated hepatic inflammation and necrosis but not steatosis (Iimuro et al., 1997). Therefore, much more work are needed to clarify the roles of TNF-α in the pathogenesis of AFL.

Our recent study showed that both Kupffer cell eliminator (GdCl₃) and TNF-α receptor antagonist (etanercept, a genetically engineered, soluble, systemic TNF-α receptor fusion protein) could partially but significantly attenuate binge drinking-induced fatty liver in mice (Zhao et al., 2017). Results from both in vivo and in isolated epididymal adipose tissues showed that TNF-α could stimulate the phosphorylation of hormone sensitive lipase (HSL) and the secretion of free fatty acid (FFA), which suggested that ethanol-induced KCs activation might promote binge drinking-induced fatty liver possibly by TNF-α-mediated extrahepatic lipolysis (Zhao et al., 2017). Herein, we extended the study by directly investigating the roles of TNF-α on the hepatic fat levels in mice and in HepG2 cells and explored the potential mechanisms.