Lehtola 2015 Master's Thesis

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Lehtola A (2015) Main protein component of high density lipoprotein, apolipoprotein A1, modulates leukocyte cell respiration. Master's Thesis 1-44.

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Lehtola A (2015) Master's Thesis

Abstract: The main protein component of high density lipoprotein (HDL), apolipoprotein A1 (ApoA1), has previously been shown to stimulate glycolysis and mitochondrial oxidative phosphorylation in mouse muscle cells. ApoA1 is also suggested to have a role in normal metabolism and metabolic diseases in skeletal muscle. In leukocytes, by regulating cellular cholesterol, ApoA1 is able to affect leukocyte cell activation and proliferation and through these actions, cell metabolism can be changed. However, direct effects of ApoA1 to leukocyte cell respiration have not been studied. Leukocytes have been suggested to function as predictive biomarkers of mitochondrial function under metabolic stress. Leukocyte mitochondria have previously been used to reflect physiological changes in heart muscle cells and the ultimate target is that leukocytes could be used to reflect ApoA1 related metabolic changes in muscle cells. The aim of this preliminary study was to examine whether ApoA1 has similar effect on leukocyte cell respiration, as it does on mouse muscle mitochondria. First hypothesis is that ApoA1 increases human leukocyte cell respiration and second hypothesis is that ApoA1 affects the amount of respiration complex proteins in human leukocytes. To examine the effect of ApoA1 to cell respiration, cultured human T lymphocytes were incubated for 4, 12 or 24 hours with 50 μg/ml of ApoA1. Cell respiration was studied with high resolution respirometry in intact (4h, 12h, 24h) cells and complex specific respiration was studied with permeabilized cells (12h). Glutamate, malate, succinate, ADP, oligomycin, CCCP, rotenone and antimycin A were used to induce different respiration states. Effect of ApoA1 on respiration complex proteins was determined with western blot using antibody cocktail for proteins of five different respiration complexes. Increase in cell respiration was statistically significant at ROUTINE respiration (p=0.040) and ET capacity (p=0.011) in intact cells treated with ApoA1 for 4 hours. ROUTINE respiration reflects respiration in intact cells by growth medium substrates. ET capacity reflects the maximal capacity of oxidative phosphorylation related electron transfer.

Keywords: ApoA1, Cell respiration, Leukocyte, Jurkat cells, Biomarker, High-resolution respirometry


Labels: MiParea: Respiration 


Organism: Human  Tissue;cell: Blood cells, Other cell lines, Lymphocyte  Preparation: Intact cells, Permeabilized cells 


Coupling state: LEAK, ROUTINE, OXPHOS, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k 


Abstract continued

ROUTINE control ratio was significantly decreased (p=0.035) in 4 hours ApoA1 treated intact cells. This indicates that ROUTINE respiration is operating closer to ET capacity in control cells than in ApoA1 cells. Respiration complex IV proteins were significantly decreased in ApoA1 treated cells (p=0.018). Complex specific respiration in permeabilized cells was not significantly affected by ApoA1 treatment. First hypothesis was confirmed by results obtained with intact cells. These results indicate ApoA1 increased ET capacity in leukocytes. Increased ET capacity leads to increased ROUTINE respiration and residual respiration. Second hypothesis was confirmed since significant decrease in CIV protein was discovered with western blot. Based on this study, it can be concluded that ApoA1 increases cell respiration in intact leukocytes and affects respiration complex proteins. However, this study was limited preliminary study and further experiments with larger sample size are needed to confirm the obtained results. Due to several artifacts significant results were not obtained regarding complex specific respiration in permeabilized cells. Respiration in permeabilized cells should be examined in future studies to gain knowledge of more detailed effects of ApoA1 to cell respiration. Nonetheless the results obtained here are promising for the future use of leukocytes to reflect metabolism in muscle cells.