FGF21 deletion mildly exacerbates hepatic dysfunction in MASH diet and alcohol fed rats

Objective: Fibroblast growth factor 21 (FGF21) is a hepatokine that improves dyslipidemia, steatosis, inflammation, and fibrosis. FGF21 analogues are in clinical development as treatments for metabolic and alcohol-associated liver disease, creating a need for new models to help understand FGF21 physiology and drug mechanisms of action. The aim of this study was to create and initially characterize the first FGF21 knockout (KO) rat line to validate its utility as a translational animal model that recapitulates human MASH and ALD, and to provide a resource for examining FGF21-related phenotypes that are more appropriate for the rat. Methods: We generated an FGF21 KO rat model using CRISPR/Cas9 to insert an artificial STOP codon in exon 1 and exposed 6-month-old WT and KO rats to either chow (n=8 per genotype) or the GAN (Gubra Amylin NASH) diet (n=16 per genotype) for 12 weeks. We further evaluated alcohol drinking behavior and biochemistry in FGF21 KO and WT rats. In the diet model, we further analysed liver and blood biochemistry in addition to histopathological scoring of NAFLD activity score (NAS), fibrosis stage and the liver transcriptome for in-depth characterization of the model. Results: Lack of endogenous FGF21 increased plasma transaminases, liver weight, and total levels of liver TG in GAN-fed FGF21 KO rats. FGF21 deletion also increased ALT in alcohol-fed FGF21 KO rats. However, in the GAN diet model, FGF21 KO had no impact on body weight, fat mass, glycaemic traits, MASH histological endpoints including hepatic steatosis, NAS score, lobular inflammation, ballooning degeneration or fibrosis stage after 12 weeks. Similarly, there was no effect of the loss of endogenous FGF21 on the liver transcriptome in response to GAN diet feeding. Finally, we demonstrate that endogenous FGF21 does not regulate drinking behaviour in rats. Conclusion: FGF21 deficiency accelerates hepatic dysfunction in diet and alcohol-induced liver disease models in rats, providing support from a new species that FGF21 might be a hepatoprotective factor. ### Competing Interest Statement Financial support PA was supported by the Danish Diabetes Academy (PD005-20), MHN was supported by Innovation Fund Denmark (0153-00178B). Declaration of competing interest statement M.H. Nielsen, D. Oro, H.H. Hansen, and M. Feigh are employed by Gubra. M.P. Gillum is employed by Novo Nordisk. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.