Nutrition and Health Sciences, Department of
First Advisor
Sathish Kumar Natarajan
Second Advisor
Seung-Hyun Ro
Third Advisor
Jiujiu Yu
Date of this Version
Summer 8-2019
Abstract
Mitochondrial fatty acid oxidation (FAO) plays an important role in maintaining metabolic homeostasis, especially during catabolic stress. Genetic mutation in gene that encodes for mitochondrial FAO enzymes could result in metabolic stress especially during fasting or hypoglycemic condition. Mutation in the active site residue (1528G>C) of long chain 3-hydroxy acyl CoA dehydrogenase (LCHAD) results in the loss of LCHAD enzyme activity. Children’s with LCHAD mutation were known to accumulate 3-hydroxy fatty acids such as 3-hydroxy palmitic acid and 3-hydroxy myristic acids, long chain free fatty acids, acyl carnitine and 3-hydroxy dicarboxylic acid due to their defective enzyme activity. These accumulated 3-hydroxy fatty acids and long chain free fatty acids are toxic compound to the cellular tissues and results in the development of retinopathy. Here we hypothesize that increased levels of 3-hydroxy fatty acid due to a mutation in LCHAD would induce retinal pigment epithelial cell lipoapoptosis and palmitoleate (PO) protects against 3-hydroxy fatty acid-induced retinal lipoapoptosis. Normal human immortalized retinal pigment epithelial cells (ARPE-19) were treated with 3-hydroxy fatty acid (3-HFA) for 24 hours and biochemical markers of lipoapoptosis such as characteristic nuclear morphological changes of apoptosis and caspase 3/7 activity were measured. Mitochondrial bioenergetics were assessed using Seahorse XF24 extracellular flux analyzer and lipid droplets were stained with oil red ‘O’ dye. After 24-hours of 3-HFAs treatment to ARPE-19 cells, we observed a significant increase in percent apoptotic nuclei and caspase 3/7 activity compared to the vehicle treated cells suggesting retinal lipoapoptosis. Further, percent cell survival and metabolic activity were decreased in cells treated with 3-HFA compared to vehicle treatment. 3-HFA-induced retinal cell lipoapoptosis is cJun N-terminal kinase- and caspase-dependent lipoapoptosis. Treatment of 3-HFAs to retinal cells showed decreased mitochondrial bioenergetics as evidenced by a decrease in basal respiration, ATP production, maximal respiration and mitochondrial coupling efficiency compared to vehicle (p
Advisor: Sathish Kumar Natarajan
Comments
A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Nutrition, Under the Supervision of Professor Sathish Kumar Natarajan. Lincoln, Nebraska : August, 2019
Copyright 2019 Mona Kassem Hadidi