More than 20,000 people each year die of alcoholic liver disease, the twelfth largest cause of death in Americans. Because the liver is the major site of ethanol metabolism, it is the most susceptible organ to alcohol-induced injury. Although the progression of alcoholic liver disease is well-described clinically, the molecular basis for alcoho-induced liver injury is not understood. Our long-term goal is to understand the mechanisms that lead to alcohol-induced hepatotoxicity. This proposal is based on our findings that microtubules are more highly acetylated and more stable in ethanol-treated WIF-B cells, liver slices and livers from ethanol-fed rats, and that increased microtubule acetylation and stability can explain alcohol-induced defects in protein trafficking by impairing microtubule-based motor function. This proposal is aimed at identifying how alcohol-induced microtubule acetylation and stability directly contribute to steatosis and other liver injury. In this proposal, we ask three major, yet related, questions. Our findings that dynein/dynactin colocalizes with stalled transcytosing proteins along acetylated microtubules and that dynein binds microtubules more tightly in ethanol-treated WIF-B cells suggests to us that impaired vesicle delivery can be explained by decreased motor processivity in ethanol-treated cells. We will test that hypothesis in the experiments proposed in Aim 1. Although our previous studies have strongly correlated microtubule hyperacetylation with impaired protein trafficking and with impaired protein trafficking and motor function in ethanol-treated cells, we will test this directly using 2 approaches as described in Aim 2.
Aim 3 takes us in a new and exciting direction. Emerging evidence implicates microtubules and microtubule-based motors as important regulators of lipid droplet formation/degradation and bidirectional motility. Furthermore, acetylated microtubules are required for adipogenesis in 3T3-l1 cells. Thus, we propose that acetylated microtubules enhance ethanol-induced steatosis. We will test that possibility as described in Aim 3. In general, experiments will be initiated in polarized, hepatic WIF-B cells and confirmed when possible in livers from ethanol-fed rats. We will continue our collaboration with Dr. Dean Tuma and have garnered the support of several others to help us perform the proposed studies. I continue my appointment as Visiting Faculty in the Department of Cell Biology at Johns Hopkins University School of Medicine securing membership to the Hopkins Microscope Facility. The expansive expertise of our collaborators, the access to high-end resources coupled with our considerable expertise in hepatic cell biology situate us perfectly to perform these important mechanistic experiments. This research also suggests that modulation of the hepatocyte acetylation state may be a novel therapeutic strategy for the treatment of steatosis and other symptoms of liver disease.
Approximately 75% of all Americans consume alcohol and 100,000 deaths per year are attributed to alcohol consumption. Of those deaths, greater than 20,000 are caused by cirrhosis of the liver, the twelfth largest cause of death in Americans. Clearly, alcoholic liver disease is a major biomedical health concern in the United States.
|Doody, Erin E; Groebner, Jennifer L; Walker, Jetta R et al. (2017) Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling. Am J Physiol Gastrointest Liver Physiol 313:G558-G569|
|McAndrew, Christopher P; Tyson, Christopher; Zischkau, Joseph et al. (2016) Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA-protein complexes. Biotechniques 60:21-7|
|Tuma, Pamela L (2016) A Researcher at an AREA Grant-Eligible Institution. Cell Mol Gastroenterol Hepatol 2:260-262|
|Tyson, C; McAndrew, C; Tuma, P L et al. (2015) Automated nonparametric method for detection of step-like features in biological data sets. Cytometry A 87:393-404|
|Groebner, Jennifer L; Tuma, Pamela L (2015) The Altered Hepatic Tubulin Code in Alcoholic Liver Disease. Biomolecules 5:2140-59|
|Groebner, Jennifer L; Fernandez, David J; Tuma, Dean J et al. (2014) Alcohol-induced defects in hepatic transcytosis may be explained by impaired dynein function. Mol Cell Biochem 397:223-33|
|In, Julie G; Ihrke, Gudrun; Tuma, Pamela L (2012) Analysis of polarized membrane traffic in hepatocytes and hepatic cell lines. Curr Protoc Cell Biol Chapter 15:Unit 15.17|
|Shepard, Blythe D; Tuma, Dean J; Tuma, Pamela L (2012) Lysine acetylation induced by chronic ethanol consumption impairs dynamin-mediated clathrin-coated vesicle release. Hepatology 55:1260-70|
|Fernandez, David J; Tuma, Dean J; Tuma, Pamela L (2012) Hepatic microtubule acetylation and stability induced by chronic alcohol exposure impair nuclear translocation of STAT3 and STAT5B, but not Smad2/3. Am J Physiol Gastrointest Liver Physiol 303:G1402-15|
|Shepard, Blythe D; Tuma, Dean J; Tuma, Pamela L (2010) Chronic ethanol consumption induces global hepatic protein hyperacetylation. Alcohol Clin Exp Res 34:280-91|
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