Currently, 1 in 3, or 34% of adult Americans are diagnosed with Metabolic Syndrome6. In addition, more people are developing type II diabetes earlier in life2. It has been suggested that there is a parallel increase in metabolic syndrome with type II diabetes4. Therefore it is
becoming even more important to diagnose the risks factors and find the underlying cause of type II diabetes. Moreover, the need to address the causes for metabolic syndrome has become increasingly important due to the changing demographic. Americans are becoming even more obese, and thus increasing their risk for metabolic syndrome. However, obesity alone cannot explain the presence of metabolic syndrome as there are individuals who are obese but do not have the syndrome. It has been proposed that in addition to obesity, insulin resistance may play a role in the development of metabolic syndrome, and type II diabetes. Although the underlying cause for insulin resistance is not known, it has been suggested that the lipid intermediate content within the cell plays a role. It is now known that increased lipid intermediate content within the cell is due to an imbalance between fatty acid delivery and intracellular fatty acid oxidation and lipid storage9. Thus, in an attempt to gain a clearer understanding of how fatty acids are shuttled to various pathways after entering the cell, this study sought to determine the amount of multiplicity of infection (MOI) necessary to attain equal ACSL1 activity between wild- type, mitochondria, and ER targeted adeno-viruses. Equal infection of cells was necessary in order to later compare fatty acid oxidation and incorporation rates.
The effect of MOI on ACSL1 activity in virus infected hepatocytes, brown adipocytes and cardiomyocytes was studied. Cells were infected with adenoviruses containing green
fluorescent protein (Ad-GFP) (control), wild-type ACSL1 (Ad-Acsl), ACSL1 targeted to the ER- only (Ad-F4-Acsl1) or ACSL1 targeted to the mitochondria-only (Ad-T70-Acsl1). In virus-infected hepatocytes, brown adipocytes and cardiomyocytes, a higher MOI resulted in higher activity. In the brown adipocytes, a MOI of 50 for Ad-F4-Acsl1, and a MOI of 30 for Ad-T70-Acsl1 showed equal enzyme activity. Moreover, the activities of Ad-F4-Acsl1 and Ad-T70-Acsl1 were similar to
the activity for Ad-Acsl. However, the ACSL1 activity in the Ad-GFP cells infected with a MOI of 50 was lower than the ACSL1 activity in wild type cells not infected with virus. Therefore, we concluded that GFP alters metabolism in brown adipocytes, and that brown adipocytes cannot be used in later experiments. In primary hepatocytes, a MOI of 40 for Ad-F4-Acsl1, and a MOI of 10 for Ad-T70-Acsl1 showed equal enzyme activity. In addition, MOIs of 30 and 50 for Ad- Acsl showed enzyme activities that were similar to the enzyme activities for Ad-F4-Acsl1 cells infected with a MOI of 40 and Ad-T70-Acsl1 cells infected with a MOI of 10. In order to
determine whether a MOI of 30 or a MOI of 50 is the optimal MOI for Ad-Acsl, additional experiments must be done. However, due to GFP changing metabolism in brown adipocytes, we concluded that brown adipocytes should not be used to assess fatty acid oxidation and incorporation rates, and that further experiments using brown adipocytes would not be conducted. Furthermore, optimal MOIs were confirmed using western blot by showing equal ACSL1 expression between Ad-GFP, Ad-F4-Acsl1, and Ad-Acsl1 samples. The western blot for hepatocytes showed that there was equal protein expression between Ad-GFP infected with a MOI of 30, Ad-Acsl1 infected with a MOI of 5, and Ad-F4-Acsl1 infected with a MOI of 50. The optimal MOIs chosen based on the enzyme activities for hepatocytes infected with GFP and Ad- F4-Acsl1 were confirmed by protein expression (a MOI of 30 and a MOI of 50 respectively). However, in hepatocytes a MOI of 5 for Ad-Acsl showed lower enzyme activity when compared to the activity for Ad-F4-Acsl1 infected with a MOI of 50, but showed equal protein expression when compared to the expression for Ad-GFP, Ad-Acsl, and Ad-F4-Acsl1. Therefore, additional experiments must be conducted in order to determine an optimal MOI for Ad-Acsl. Moreover, in hepatocytes a MOI of 30 for Ad-T70-Acsl1 showed equal enzyme activity when compared to the activity for Ad-F4-Acsl1 infected with a MOI of 50, but showed lower protein expression when compared to the expression for Ad-F4-Acsl1. Therefore, additional experiments must be conducted in order to determine an optimal MOI for Ad-T70-Acsl1. However, due to the conclusion that brown adipocytes would not be used for further experiments, we also
determined that hepatocytes would not be used. Thus, determination of optimal MOIs for the various adeno-constructs in brown adipocytes and hepatocytes was not achieved.
To confirm the optimal MOI in cardiomyocytes, ACSL1 activity and health of cells were analyzed using ACSL assay, light microscopy and ALT assay. We concluded that a MOI of 50 was too low because it resulted in a low number of cells being infected but that a MOI of 500 was too high because it led to cell death. This conclusion was confirmed by the higher levels of alanine aminotransferase (ALT) in media of cells infected with a MOI of 500 (8-10 U/L) when compared to the amounts of ALT in media of cells not infected with virus (4 U/L). Furthermore, we found that cells incubated for 48 hours had higher levels of ALT in the media due to a higher observation of dead cells in the pictures taken by light microscopy. Cells were classified as dead if they were floating in the media, and looked circular and clumped under the microscope. Thus, we concluded that cells should be incubated for not more than 24 hours. We were able to further narrow down the range of optimal MOIs for each adeno-construct. We determined that a MOI range of 50-200 in Ad-GFP cells, a MOI range of 250-500 in Ad-Acsl cells, a MOI range of 100-250 in Ad-F4 cells and a MOI range of 50-100 in Ad-T70 cells showed similar ACSL1 activity. Finally, we concluded that a MOI of 250 for Ad-F4-Acsl1, and a MOI of 75 for Ad-T70- Acsl1 showed equal enzyme activity. Therefore, this project was successful in determining the optimal MOI in Ad-F4-Acsl1 and Ad-T70-Acsl1 cardiomyocytes, but not in Ad-GFP and Ad-Acsl cells.
A separate aim of this project was to establish an optimized protocol for isolation and purification of endoplasmic reticulum (ER) and mitochondria in heart and liver samples. The original purpose for developing a modified protocol was to be able to use purified ER and mitochondria to look at the effect of ACSL1 location on fatty acid oxidation and incorporation rates. However, the modified protocol was not successful in obtaining completely purified ER samples from the heart and liver. We tested variation in number of strokes, speed, and volume of PBS buffer used to homogenize samples. Initially, we tested a variation in the number of
strokes used to homogenize the heart: 10 strokes versus 40 strokes at a speed of 6. We found that 40 strokes resulted in less mitochondria contamination in the ER fraction. We then tested variation in the speed of the homogenizer: speed of 5 versus speed of 10 while using 15 up and down strokes for all samples. We concluded that a speed of 10 had less mitochondria
contamination in ER fractions. Finally, we tested variation in the amount of buffer used for homogenization: 2 mL versus 8 mL. We concluded that using 2 mL to homogenize samples resulted in less mitochondria contamination. From the series of experiments, we concluded that homogenizing samples with 40 strokes at a speed of 10 with 2 mL buffer had the least
mitochondria contamination in ER fractions. Although there was improvement (less
mitochondria contamination of ER) between each protocol, overall optimization of the protocol was not successful. Mitochondria were still present in the ER fractions. Thus, we were not able to use the modified protocol for isolation and purification of mitochondria and ER in further experiments.
Future steps will be taken to look at fatty acid oxidation and incorporation into various downstream metabolites in heart tissue using the optimal MOIs determined from this project.