*Purpose: This study aimed to define characteristics and mechanisms leading to nonalcoholic fatty liver disease and metabolic syndrome in a fatty liver mouse model that mimics the state of marginal steatotic allografts in liver transplantation(LTx), and presents a growing challenge by limiting organ availability and increasing risk of liver graft failure from severe ischemia reperfusion injury(IRI).

*Methods: A C57BL/6 mouse model for fatty liver disease was established. Control mice were fed a standard chow diet; fatty mice a lard-based high fat diet(HFD) for at least 16 weeks. Weights were monitored and intra-peritoneal glucose tolerance test(IPGTT) assessed the development of diabetes mellitus(DM). MRI quantified visceral fat and changes in fat composition. Mice were sacrificed at 29-34 weeks old and serum liver chemistries were measured. Livers were sectioned and processed for immunohistochemistry(IHC) and immunoblotting. A cohort of mice underwent partial warm IRI for 45 minutes and were sacrificed 24 hours later.

*Results: HFD mice showed significant increases in body weight over time, averaging a weight of 42g versus 25g in control. Likewise, liver weights were drastically different- 2.7g in HFD versus 1.6g in control. This was further corroborated by IHC in which the HFD group consistently scored for steatotic changes in >66% of liver parenchyma and many cells with balloon degeneration. Based on IPGTT, HFD mice had a higher propensity of developing DM as their final blood glucose levels were markedly higher and abnormal. MRI confirmed that HFD mice accumulate more visceral and hepatic fat. There was no difference in serum liver transaminases between the two groups, however with IRI, transaminases in both groups were noted to trend upwards. AST was significantly higher in HFD with IRI mice versus HFD alone(p=0.027). Next, we hypothesized that increased expression of perilipin 2(PLIN2) has a pivotal role in lipid accumulation in fatty IRI livers and dysbalance of glucose homeostasis. Immunoblotting of liver lysates showed a significant increase of PLIN2 in conjunction with a decrease of insulin receptor substrate 1(IRS1) and the inactive phosphorylated form of glycogen-synthase kinase 3β(GSK3β) in HFD mice, indicating that in HFD IRI increased sequestration of PLIN2 by lipid droplets results in severe diabetogenic changes through increased degradation of IRS1 and increased activity of GSK3β, both key players in glucose metabolism.

*Conclusions: Changes in IRS1 and GSK3β expression regulated by PLIN2 have crucial implications in HFD-induced metabolic syndrome and may further support a proinflammatory state in fatty liver disease that is enhanced by IRI. Translation of these findings to human LTx may allow for targeted intervention to better utilize marginal organs and prevent graft failure.

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