Research ArticleArginase 2 deficiency results in spontaneous steatohepatitis: A novel link between innate immune activation and hepatic de novo lipogenesis
Introduction
Non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease affecting both adults and children, and is strongly associated with obesity and insulin resistance [1], [2]. One in three adults and one in ten children or adolescents in the United States have hepatic steatosis, a stage within the spectrum of NAFLD, that is characterized by triglyceride accumulation in liver cells and follows a benign non-progressive clinical course [3], [4]. Non-alcoholic steatohepatitis (NASH) is defined as lipid accumulation with evidence of cellular damage, inflammation and varying degrees of scarring or fibrosis [5]. NASH is a serious condition, as approximately 25% of these patients progress to cirrhosis and its known complications that include portal hypertension, liver failure and hepatocellular carcinoma [6], [7], [8].
NAFLD pathogenesis, and specifically disease progression to NASH, has been the subject of intense investigation over the past decade, as both diagnostic and prognostic markers and treatment options for patients with this disease remain limited [9]. A multi-hit model of NAFLD pathogenesis has emerged in recent years in which intrahepatic lipid accumulation in the setting of insulin resistance is postulated to be the first hit, which sets the stage for multiple “second hits” resulting in inflammation, hepatocellular damage and eventually fibrosis [10]. Growing evidence supports the concept that innate immune activation and increased oxidative stress in the liver are key “second hits” involved in disease progression to NASH. Activation of liver resident macrophages, or Kupffer cells, at least in part by an increase in exposure to gut-derived bacterial wall components such as endotoxin, result in an increased production of proinflammatory cytokines as well as inducible nitric oxide synthase (iNOS) and has been proposed to play a pivotal role in this process [11], [12]. Expression of iNOS in cell types such as macrophages and hepatocytes is induced by proinflammatory cytokines [13], and iNOS have been implicated in the pathophysiology of NAFLD [14], [15], [16]. Mammals express two isoforms of arginase, an enzyme that competes with iNOS for its substrate, arginine, designated as types 1 and 2 [17], [18], [19]. Arginase 1 is mainly expressed in hepatocytes, and mice with a disruption of the arginase 1 gene (Arg1) die soon after birth. Arginase 2 is poorly expressed in hepatocytes, and most highly expressed in kidney, prostate, and immune cells, such as monocyte/macrophages [17], [19]. An imbalance in iNOS/arginase ratio in favour of the former may play a crucial role in regulating immune responses and macrophage activation towards a classically “M1” activated, proinflammatory state [20], [21], [22], [23]. The aim of our study was to test the hypothesis that arginase 2 deficiency in mice would result in progression from isolated hepatic steatosis typically seen in diet induced obesity (DIO) to steatohepatitis. Our data identify a novel link between Kupffer cell activation and hepatic steatosis and suggest that in the presence of arginase 2 deficiency, altered immune responses precede, and are responsible for lipid deposition in hepatocytes mainly by activation of de novo lipogenesis pathways in the liver. These findings have important implications for the pathogenesis of NAFLD and potential development of novel treatment strategies for patients with this condition.
Section snippets
Animal studies
The experimental protocol has been approved by the Institutional Animal Care and Use Committee at Cleveland Clinic. Male C57BL/6 wild type (WT) were purchased from Jackson Laboratory. The generation of Arg2−/− mice has been described previously [24]. Arg2−/− mice are viable and indistinguishable from WT mice. Mice were fed either a diet consisting of 5% fat (TD 2918, Harlan Laboratories, Madison, WI) or a high fat (HFAT), Western-type diet (consisting of 42% of Kcal from fat, TD88137, Harlan
Arg2−/− mice develop spontaneous liver injury and hepatic steatosis
Arg2−/− and WT mice showed similar trends in weight gain for up to 14 weeks of age (Fig. 1A) and no evidence of systemic metabolic derangements with plasma assays of fasting triglycerides, free fatty acids, glucose and insulin (Fig. 1B). However, serum levels of alanine aminotransferase (ALT) were elevated in Arg2−/− mice compared to WT animals (110 vs. 35 U/L, p <0.05; Fig. 1D) suggesting the presence of liver injury. Indeed, H&E and ORO stained liver sections revealed notable inflammation and
Discussion
The principal findings of this study relate to the role of arginase 2 in the development of hepatic steatosis and the progression to steatohepatitis. The results demonstrate that arginase 2 deficiency leads to spontaneous development of hepatic steatosis that is independent of weight gain and systemic metabolic changes and associated with increased hepatic de novo lipogenesis and liver Kupffer cell/macrophage activation towards a proinflammatory M1 phenotype. Selective depletion of Kupffer
Financial support
This work was supported by NIH grants to AEF (DK076852, DK082451), SG, and SCE (HL103453, HL109250, HL60917).
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Authors’ contributions
LAN, AW, SCE, AEF: study concept and design. LAN, AW, DP, MPB, AE, BGP, SG: acquisition of data. LN, AW, DP, SCE, AEF: analysis and interpretation of data. LAN, AW, DP, SCE, AEF: drafting of the manuscript. LAN, AW, AEF: statistical analysis. SG, SCE, AF: obtained funding. MPB, AE, BGP: administrative, technical, or material support. SCE, AEF: study supervision. All authors: critical revision of the manuscript for important intellectual content.
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These authors contributed equally to this work.