Binge drinking results in transient steatosis in over 90% of people. Once considered benign, repeated episodes of alcohol-induced steatosis are now thought to be a "first hit" that primes the liver for more severe forms of alcoholic liver disease (ALD). Aside from abstinence, there is no treatment for alcoholic steatosis. Thus, it is important to understand the molecular basis for this condition. Activation of the sterol response element binding proteins (SREBPs) and the unfolded protein response (UPR) is observed in multiple etiologies of faty liver disease (FLD). Some data indicates that these two pathways might be interrelated, however, whether SREBPs and the UPR collaborate to induce alcoholic steatosis is currently unknown. This research proposal is based on preliminary data that suggests depletion of a key mediator of the UPR, ATF6, reduces alcoholic steatosis in larval zebrafish, a novel tool for studying ALD. The long-term objective of this work is to dissect the interplay between ATF6 and SREBPs in alcoholic steatosis. The relationship between SREBPs and ATF6 will be analyzed through two independent avenues.
The first aim of this proposal assesses the individual and combined roles of Srebp1, Srebp2, and Atf6 to induce alcoholic steatosis. Each gene will be transiently blocked in zebrafish larvae by morpholino knockdown. The requirement and cooperativity of these pathways in the development of alcoholic steatosis will be determined by i) oil red O staining of whole larvae and sections and ii) quantitative, real-time PCR (qPCR) and whole mount in situ hybridization of Srebp1, Srebp2, and UPR target genes.
The second aim will determine if activation of Atf6 is sufficient for the development of steatosis in the absence of alcohol. Active, nuclear Atf6 (nAtf6) will be conditionally overexpressed in zebrafish using the CrePR/loxP-STOP-loxP system. The ability of nAtf6 to induce steatosis independent of Srebps will be assessed through use of wildtype larvae and mutants that are deficient in mbtps1, a protease required for Srebp activation. Oil red O staining, qPCR, and whole mount in situ hybridization will be performed as in the first aim. This proposal employs an in-depth means to addres a mechanism of steatosis development that is currently unclear. The epistasis analysis described will determine whether Srebps and Atf6 work together or in parallel to induce steatosis in response to alcohol. The ultimate goal of this proposal is to identify pathways that can be targeted by ALD therapies.
Binge drinking causes transient fatty liver (steatosis) in virtually all consumers. Aside from abstinence, there is no treatment for alcoholic steatosis. The goal of this project is to identify genetic pathways that can be targeted by therapies.
|Howarth, Deanna L; Lindtner, Claudia; Vacaru, Ana M et al. (2014) Activating transcription factor 6 is necessary and sufficient for alcoholic fatty liver disease in zebrafish. PLoS Genet 10:e1004335|
|Vacaru, Ana M; Di Narzo, Antonio Fabio; Howarth, Deanna L et al. (2014) Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish. Dis Model Mech 7:823-35|
|Howarth, Deanna L; Yin, Chunyue; Yeh, Karen et al. (2013) Defining hepatic dysfunction parameters in two models of fatty liver disease in zebrafish larvae. Zebrafish 10:199-210|
|Tsedensodnom, Orkhontuya; Vacaru, Ana M; Howarth, Deanna L et al. (2013) Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease. Dis Model Mech 6:1213-26|