A Novel 2-Hit Zebrafish Model to Study Early Pathogenesis of Non-Alcoholic Fatty Liver Disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in adults. NAFLD progresses from benign liver fat accumulation to liver inflammation and cirrhosis, and ultimately leads to liver failure. Although several rodent models have been established for studying NAFLD, they have limitations that include cost, speed of disease development, key dissimilarities, and poor amenability to pharmacological screens. Here, we present a novel 2-hit zebrafish model to replicate aspects of NAFLD pathogenesis. We fed zebrafish larvae a high-fat diet (HFD) to drive liver fat accumulation (first hit). Next, we exacerbated liver-specific inflammation using a transgenic line (fabp10-CETI-PIC3) that induces the expression of proinflammatory cytokines following induction with doxycycline (second hit). These hits promoted fat accumulation and liver inflammation, as demonstrated by the high expression of inflammatory cytokines, macrophage infiltration, stress induction, and hepatic lipid droplet accumulation. Furthermore, zebrafish in this paradigm showed deranged glucose metabolism. To validate a small-molecule screening approach, we treated HFD-fed fish with pioglitazone, a drug shown to be beneficial for NAFLD in humans, and measured a sharp reduction in liver lipid accumulation. These results demonstrate new utility for zebrafish in modeling early NAFLD pathogenesis and demonstrate their feasibility for in vivo screening of new pharmacological interventions.

Leukotriene B4 receptor 2 governs macrophage migration during tissue inflammation

“…Consistent with prior studies, HFD feeding caused a significant increase in the number of macrophages observed within the liver boundaries ( Fig. 3 D ) ( 22 ). Importantly, upon HFD-feeding, the number of macrophages per sampled liver area was significantly reduced in the embryos injected with the ltb4r2a MO compared to the non-injected controls ( Fig.…”

“…In a second model of inflammation, we fed zebrafish embryos with a high-fat diet (HFD) consisting of a 5% homogenized egg solution at 3 and 4 dpf ( Fig. 3 C ), which leads to lipid accumulation and a mild inflammatory state that promotes macrophage infiltration into the liver parenchyma ( 22 ). Consistent with prior studies, HFD feeding caused a significant increase in the number of macrophages observed within the liver boundaries ( Fig.…”

“…The genetic and physiological similarities between the hepato-pancreatic systems of mammals and zebrafish make them suitable model organisms for studying metabolic conditions. Importantly, zebrafish models have been extensively utilized to study the dynamics of macrophage migration ( 17 , 31 ) and model islet and liver inflammation ( 8 , 22 , 32 ). Leveraging the well-characterized tailfin injury model and two distinct models of metabolic inflammation, our study demonstrated that zebrafish Ltb4r2a (but not Ltb4r2b ) promotes macrophage migration during different inflammatory settings, indicating its role as a contributor to inflammatory responses.…”

Altered local RAS in the liver increased the risk of NAFLD in male mouse offspring produced by in vitro fertilization

“…NAFLD can be classi ed as simple steatosis (NAFL) and nonalcoholic steatohepatitis (NASH) and has tended to become epidemic in children worldwide in the last decade [11] . Although the pathogenesis of NAFLD is still unclear, the "2-hit hypothesis" is widely accepted [12] . The theory suggests that suboptimal conditions and adaptations in early development ( rst-hit) may lay the foundation for long-term susceptibility to subsequent adverse events (second-hit), which may occur in the foetus or later life, leading to more extreme outcomes or phenotypes [13] .…”

NAFLD: From Mechanisms to Therapeutic Approaches

“…Consequently, new reliable models displaying identical liver phenotypes to humans are needed. In this issue, interesting novel preclinical models are evaluated, including a guinea pig disease model, chimeric mice with humanized livers, and a novel 2-hit zebrafish model to specifically model the early pathogenesis of NAFLD [ 9 , 10 , 11 ].…”