Defining Hepatic Dysfunction Parameters in Two Models of Fatty Liver Disease in Zebrafish Larvae

Abstract

Fatty liver disease in humans can progress from steatosis to hepatocellular injury, fibrosis, cirrhosis, and liver failure. We developed a series of straightforward assays to determine whether zebrafish larvae with either tunicamycin-or ethanol-induced steatosis develop hepatic dysfunction. We found altered expression of genes involved in acute phase response and hepatic function, and impaired hepatocyte secretion and disruption of canaliculi in both models, but glycogen deficiency in hepatocytes and dilation of hepatic vasculature occurred only in ethanol-treated larvae. Hepatic stellate cells (HSCs) become activated during liver injury and HSC numbers increased in both models. Whether the excess lipids in hepatocytes are a direct cause of hepatocyte dysfunction in fatty liver disease has not been defined. We prevented ethanol-induced steatosis by blocking activation of the sterol response element binding proteins (Srebps) using gonzo mbtps1 mutants and scap morphants and found that hepatocyte dysfunction persisted even in the absence of lipid accumulation. This suggests that lipotoxicity is not the primary cause of hepatic injury in these models of fatty liver disease. This study provides a panel of parameters to assess liver disease that can be easily applied to zebrafish mutants, transgenics, and for drug screening in which liver function is an important consideration.

Zebrafish: An Important Tool for Liver Disease Research

“…UPR activation (typically used interchangeably with the term “ER stress”) can be a harbinger of secretory pathway dysfunction in ALD. 99 We found strong upregulation of UPR markers 24,48,100 and a decrease in a marker of hepatocyte secretion 24 in zebrafish after ethanol exposure, indicating that the hepatic secretory pathway is dysfunctional in ethanol-treated zebrafish. In zebrafish genetic studies, aft6 was found to be necessary and sufficient for ethanol-induced steatosis 82 (Figure 4) and UPR activation was found to cause ethanol-induced liver disease.…”

“…Moreover, cells in zebrafish livers appear similar to mammals (see Figure 3 B [ inset ]) and perform many of the same functions as their mammalian counterparts, including bile secretion, 23 glycogen and lipid storage, 24,25 insulin responsiveness, xenobiotic and ammonia metabolism, and secretion of serum proteins such as complement and clotting factors, transferrin, and albumin-like protein. 26 Importantly, all hepatic functions evaluated thus far are found in larvae as early as 5 days postfertilization (dpf).…”

“…These tools have been used to study morphological changes in stellate cells during development of ALD, 27,33 after hepatocyte ablation, 33 and during acute induction of hepatic endoplasmic reticulum (ER) stress. 24 Additionally, cell-specific promoters are useful for expressing functional proteins that alter liver biology. When combined with inducible or conditional genetic expression approaches, these approaches enable lineage tracing experiments to identify cells that contribute to liver regeneration 34,35 and can be applied to other liver and biliary disease models to advance our understanding of how populations of cells function, interact, and respond to pathological insults.…”

Getting the Inside Tract: New Frontiers in Zebrafish Digestive System Biology

“…Remarkably, the pathology of zebrafish with similar disorders often mirrors human diseases, making this an outstanding system to study hepatobiliary, gastrointestinal, and pancreatic diseases. For instance, many of the features of fatty liver disease, which in humans is typically caused by metabolic syndrome or alcohol abuse, are similar in zebrafish, as described in this issue by Howarth et al 13 The zebrafish has also been a useful model for investigating the mechanisms underlying intestinal injury and inflammation. Oehlers and colleagues describe in this issue, diverse methods for inducing and evaluating intestinal injury, providing new models of human inflammatory bowel disease.…”

Dexamethasone-induced hepatomegaly and steatosis in larval zebrafish

“…The transparency of the larvae enables in vivo visual observation of internal organs including the liver under a microscope. Moreover, hepat-ic steatosis upon exposure to some toxins in larvae can be observed by oil red staining (Howarth et al, 2013). The zebrafish has a single gene of the glucocorticoid receptor that is expressed in the liver and intestine by 5 dpf (Bertland et al, 2007).…”