Categoría, Prueba Saber 11

Our working model for the role that eEF1A-1 plays in the stress response to saturated fatty acid excess in hepatocytes is summarized in Figure 4.1. Based on this model, and our evidence of increased eEF1A-1 protein in the livers of obese, leptin-deficient mice with severe hepatic steatosis (ob/ob) mice, several future experiments are warranted.

To further support our evidence of the interaction between eEF1A-1 and the actin cytoskeleton during palmitate overload, I would like to determine if eE1A-1 functions by regulating cytoskeleton dynamics, as is seen in other cell types. To do so, I would disrupt the interaction of eEF1A-1 with the actin cytoskeleton by transfecting cells with a construct encoding domain III of eEF1A-1 (the dominant actin-binding domain). This construct is expected to have a dominant-negative action on the eEF1A-1-actin interaction. To induce lipid overload and cell death, cells will be treated with palmitate and cell death will be assessed by flow cytometry.

Based on our results that treatment with didemnin B reduces palmitate-induced cell death in HepG2 cells, future studies should be directed towards testing the effectiveness of didemnin B in improving hepatocyte damage in mouse models of NAFLD. The results presented in this thesis indicate that eEF1A-1 expression is increased in the livers of leptin deficient ob/ob mice and these animals exhibited the most severe form of hepatic steatosis and ER stress. Furthermore, ob/ob mice are a well-characterized model of NAFLD (Anstee and Goldin, 2006). Thus, I would select this model for subsequent didemnin B experiments. Previous work has demonstrated that didemnin B is highly concentrated in the livers of mice following intraperitoneal administration (Beasley et al., 2005). To determine if ER stress and

hepatocyte damage is attenuated in the livers of these mice treated with didemnin B, liver samples will be analyzed for GR78 expression, PERK phosphorylation and JNK activity. These markers of ER stress are consistently exhibited in ob/ob mice (Ozcan et al., 2004). Serum measurements of ALT and AST activity will be used as additional indicators of liver damage.

The liver is composed of hepatocytes and non-parenchymal cells, which include kupffer cells, sinusoidal endothelial cells and hepatic stellate cells (Bian and Ma, 2012). Hepatic stellate cells play a key role in the hepatic fibrogenesis associated with NASH. Under normal conditions, hepatic stellate cells are maintained in a quiescent state but can undergo a phenotypic transition to become activated in response to liver injury. These cells are the major source of the excessive extracellular matrix production that characterizes fibrotic liver (Viera and Nato, 2006). It is possible that eEF1A-1 may play a role in the progression to fibrosis during the process of hepatic stellate cell activation. Future experiments in which expression of eEF1A-1 in hepatic stellate cells is reduced in obese mice may give us further insight into the role this protein plays in the progression of NAFLD.

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