Aaron Miller, Danny Sims, and Dr. Steven Wood, BYU Department of Chemistry and Biochemistry
Animal and human cell membranes are composed primarily of phospholipids that form a bi-layer around
the cell. Phospholipids play a crucial role in cell membrane function by regulating fluidity and structure. Alterations in the cell membrane composition can drastically affect cell function and metabolism. The purpose of our research study was to identify muscle cell membrane compositional changes due to exercise and muscular dystrophy. Also, we sought to identify the specific chemical changes occurring in the cell membrane by characterizing the structures of the cell membrane phospholipids using mass spectrometry.
In our study of membrane compositional changes due to exercise, we randomly assigned forty male Sprague-Dawley rats to each of three exercise groups: a sedentary, a low intensity, and a high intensity exercise regimen. Following the fourteen-day exercise training, all of the rats were euthanized, and the gastrocnemius muscle was removed and analyzed for phospholipid compositional changes. Using electrospray ionization mass spectrometry (ESI-MS) and principal component analysis (PCA), we identified a change in the phospholipid composition of the muscle cells as a result of exercise. Furthermore, we were able to use tandem mass spectrometry to characterize the structures of a common type of phospholipid found in the cell membrane. This common type of phospholipid that we characterized is called phosphatidylcholine. The characterization of the phosphatidylcholines revealed that the molecular weights of the phosphatidylcholine molecules increased in the high intensity exercise group relative to the control. The chemical change contributing to the increased molecular weight occurred predominantly at the sn-2 carbon of the phosphatidylcholine molecules. The results of our study were supported by previous research publications that revealed a change in phospholipid composition in both rats1 and humans2 due to exercise.
The observed change that occurs in cell membrane composition as a result of increased exercise
establishes a correlation between the metabolic rate and the muscle cell membrane phospholipid
composition. As exercise intensity increases, the metabolic rate increases to meet the increased need for
energy. This increased metabolic rate could be one explanation for why the cell membrane composition
changes as a result of exercise. Additionally, a different study revealed that dystrophic muscle has a
metabolic rate that is about half the metabolic rate of healthy muscle.3 Therefore, applying the assumption that the metabolic rate effects cell membrane composition, we hypothesized that the lower metabolic rate of muscular dystrophy would effect the phospholipid composition of dystrophic muscle cells.
Unfortunately, we were unable to test our hypothesis using muscle tissue due to the high cost of obtaining mice with muscular dystrophy. However, we were able to simulate the dystrophic diseased state by treating human embryonic kidney cell cultures (HEK-293) with lidocaine, which is a common local anesthetic. A previous research publication revealed that cells treated with lidocaine have a lower metabolic rate relative to untreated cells.4 To confirm the results of this study, we performed two experiments by treating HEK-293 cells with lidocaine. In the first experiment, we treated the cells with differing concentrations of lidocaine for twenty-four hours to determine if the lower metabolic rate caused by lidocaine has any effect on the phospholipid composition of the HEK-293 cells. Following this first experiment, we treated a different set of cells with 10 mmol of lidocaine for differing amounts of time to see how quickly the effects of lidocaine can alter the phospholipid composition. The cells from both of these experiments were analyzed using ESI-MS and PCA.
The results of the first experiment verified that the lower metabolic rate caused by lidocaine effects the phospholipid composition of the cells. The compositional change was most drastic in the cells treated with 10 mmol lidocaine. The PCA data from the second experiment revealed that the 10 mmol lidocaine treatment caused a change in the phospholipid composition of the cells in less than twenty-four hours. We had previously predicted that the lower metabolic rate caused by lidocaine would take days or weeks to cause a change in the cells; therefore, we were surprised that the lidocaine caused a change in the phospholipid composition in less than a day.
The results from these two experiments confirmed that the lower metabolic rate caused by lidocaine effects the cell membrane composition. With these data, we were confident that lidocaine treated cells simulated the dystrophic diseased state well enough to perform additional experiments to determine if we can re-establish a normal metabolic rate in the cells treated with lidocaine. Furthermore, we wanted to determine if a re-established normal metabolic rate would restore the phospholipid composition to normal. To experimentally re-establish the metabolic rate and reverse the effects of lidocaine, we treated HEK-293 cells with different concentrations lidocaine and fructose, and we treated another set of cells with different concentrations of lidocaine and fructose 1,6-bisphosphate. We analyzed these cells with ESI-MS and PCA, and the results revealed that the cells treated with 2.5 mmol fructose and 2.5 mmol lidocaine had a phospholipid composition similar to the normal, untreated cells. These results revealed that the fructose treatment re-established a normal metabolic rate and restored the cell phospholipid composition to normal. The fructose 1,6-bisphosphate treatment did not reverse the effects of lidocaine as effectively as the fructose.
From our study, we determined that lidocaine treated cells simulate the dystrophic diseased state. Also, we succeeded in showing that fructose reverses the effects of lidocaine and restores the cell phospholipid composition to normal. Hence, we conclude that fructose could possibly be used to stop the progression of muscular dystrophy by restoring the metabolic rate to normal. Furthermore, we speculate that fructose is not the best long-term solution for treating muscular dystrophy. Many more tests will need to be performed to confirm more definitively that fructose could be used to restore a normal metabolic rate in muscular dystrophy and halt the progression of the disease. In the future, we hope to identify a drug that effects the metabolic rate and phospholipid composition similar to fructose.
References
- Mitchell TW, Turner N, Hulbert AJ, Else PL, Hawley JA, Lee JS, Bruce CR, and Blanksby SJ. Exercise alters the profile of phospholipid molecular species in rat skeletal muscle. J Appl Physiol 97: 1823-1829, 2004.
- Andersson A, Sjodin A, Olsson R, and Vessby B. Effects of physical exercise on phospholipid fatty acid composition in skeletal muscle. Am J Physiol Endocrinol Metab 274: E432-E438, 1998.
- Wu G, Sher RB, Cox GA, and Vance DE. Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice. Biochimica et Biophysica Acta (BBA) – Molecular and Cell Biology of Lipids 1791(5) 347-356, 2009.
- 4 Karniel M, and Beitner R. Local anesthetics induce a decrease in the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, and ATP, and in the viability of melanoma cells. Molecular Genetics and Metabolism 69: 40-45 2000.