Escuela Primaria “José Ma Morelos y Pavón”

A working model for the roles eEF1A-1 and didemnin B play in the hepatocellular response to saturated fatty acid induced stress is summarized in Figure 4.1. Based on this model, and our observations in obese mice treated with didemnin B, several lines of further experimentation are warranted. However, perhaps the most critical experiment required to support the hypothesis outlined in Section 1.6, is to determine whether or not didemnin B inhibits total protein synthesis in the liver in vivo. This is a challenging experiment to perform in obese mice undergoing chronic ER stress because protein synthesis is already suppressed. However, it may be possible to measure plasma albumin levels in lean control mice upon treatment with didemnin B to address this question. It is important to note that any observed changes in this setting could also be attributed to impaired kidney function (Seimiya et al., 2014).

We have hypothesized that the reduction in food consumption observed in ob/ob animals treated with didemnin B may be due to the inhibition of ghrelin production or action. To test this hypothesis, we could assess circulating plasma ghrelin levels and ghrelin expression in tissues known to produce this hormone, such as the pancreas, stomach and intestine (Yada et al., 2008).

To further confirm the effects of didemnin B on improving clinical characteristics of NAFLD, histological sections of hepatic tissue stained with H&E could be graded using the NAFLD Activity Score (NAS) scoring system described by Takahashi and Fukusato (2014). Using this scoring system, the severity of NAFLD is evaluated based on the presence of steatosis, Mallory-Denk bodies, lobular inflammation, and hepatocyte ballooning, to accurately identify the disease from mild (benign steatosis) to severe

(NASH). Mice on a C57BL/6J background, as is the case for ob/ob mice, do not develop fibrosis with NAFLD (Anstee and Goldin, 2006), thus disease grading for this method would be limited to pre-fibrotic NAFLD.

To further support our conclusion that treatment with didemnin B improves hepatic lipotoxicity in obese mice with NAFLD, the expression of several genes could be examined by real-time quantitative polymerase chain reaction (rtQPCR). Data about metabolic gene expression would help to determine if the effects of didemnin B were associated with changes in de novo lipogenesis, lipid export, or clearance from the circulation and catabolism. Hepatic diglyceride acyltransferase (DGAT), fatty acid synthase (FAS), and SREBP-1c expression would provide further insight into the effects of didemnin B on liver triglyceride, fatty acid, and cholesterol synthesis, respectively (Nakamura et al., 2014). LDLR expression, along with lipoprotein kinetic studies, would help to elucidate whether the increased hepatic cholesteryl ester content, decreased plasma cholesterol levels and levels of cholesterol associated with LDL we observed in didemnin B treated animals were due to upregulation of LDLR and a subsequent increase in LDL clearance. Furthermore, hepatic XBP1 expression would provide us with further information about the mitigating effects of didemnin B treatment on ER stress (Henkel and Green, 2013).

Didemnin B is known to distribute to the pancreas in rodent models (Beasley et al., 2005). Due to the reduction in plasma insulin levels we observed in didemnin B treated mice, it is important to confirm whether this is solely a result of improved glucose homeostasis or due to impaired insulin production resulting from the inhibition of protein synthesis in pancreatic β-cells. To determine if treatment with didemnin B alters islet size

and insulin content relative to control and calorie restricted animals, pancreatic serial sections will be stained for insulin, and islet areas and insulin-positive islet cells will be quantified.

The chemotherapeutic activity of didemnin B was clinically tested in humans, however, these phase II clinical trials failed due to ineffective antitumor activity combined with intestinal toxicity (Benvenuto et al., 1992; Marco et al., 2004; Mittelman et al., 1999; Shin et al., 1994; Shin et al., 1991). Doses of didemnin B in these trials ranged from 3.47 mg/m2 to 9.1 mg/m2 (Shin et al., 1991), which is approximately 0.09 mg/kg to 0.25 mg/kg. Although a much lower dose (50 µg/kg) was used for the studies in this thesis, and no overt evidence of illness or functional systemic toxicity was observed in didemnin B treated animals, intestinal sections should be further assessed. Intestinal toxicity could be determined by staining serial sections of ileum and jejunum with H&E and examining sections for apparent pathology using light microscopy. In addition, sections could be stained for markers of enterocyte proliferation such as Ki67, to determine if didemnin B treatment results in abnormal cell proliferation (Bacchi and Gown, 1993; Yu and Filipe, 1993; Yu et al., 1992)

As discussed earlier, the ob/ob mouse model is resistant to NAFLD associated fibrosis (Anstee and Goldin, 2006). Therefore, to investigate the role of eEF1A-1 and the effect of didemnin B treatment on NASH and fibrosis in vivo, a different mouse model would be required. Studies using 129/SVJ mice fed a high fat diet for 6 months indicate that these animals develop obesity, hepatic steatosis, insulin resistance, and liver fibrosis (Syn et al., 2009). This diet-induced model of NAFLD and fibrosis would be more representative of the disease found in the human population and would be useful to

conduct further experiments to investigate the role of eEF1A-1 in the end stages of NAFLD.

Finally, the liver is composed of a variety of cells other than hepatocytes, including hepatic stellate cells, sinusoidal endothelial cells, and Kupffer cells (Peverill et al., 2014). Activated hepatic stellate cells are myofibroblast-like cells that produce extracellular matrix and are the principle cell type responsible for hepatic fibrosis associated with NASH (Fujii and Kawada, 2012). Our knowledge of lipotoxicity in hepatic stellate cells is limited, but it is possible that eEF1A-1 may play a role in the progression of fibrosis, especially given its function in protein synthesis under conditions of cell stress. Further experiments investigating the effects of reduced eEF1A-1 expression and activity in primary hepatic stellate cells and in a mouse model of NAFLD fibrosis may provide insight into the role(s) that eEF1A-1 plays throughout the entire liver during all stages of NAFLD progression.

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