Life in the presence of oxygen involves the regular production of reactive oxygen species (including free radicals) and other highly reactive molecules. Some of these compounds can initiate lipid peroxidation (LPO), a series of reactions that can significantly alter the chemical, physical, and functional properties of biological membranes. Although LPO may damage cells, a certain level appears necessary for a variety of cellular processes. Poikilothermic animals (i.e., animals that live at a variety of body temperatures) represent the vast majority of animals that live on earth. In order to keep membranes at or near an optimal fluidity, poikilotherms alter the chemical compositions of their biological membranes with changes in body temperature. At low temperatures, poikilotherms have membranes particularly rich in the phospholipid phosphatidylethanolamine and omega-3 polyunsaturated fatty acids. In addition, their cells often have higher contents of energy-producing mitochondria, the primary source of reaction oxygen species. These changes could make animals at cold temperatures more vulnerable to LPO than their warm-bodied counterparts. At the same time, low temperature slows rates of LPO and production of reactive oxygen species. Using a fish model (the striped bass, Morone saxatilis), this work will test the central hypothesis that membrane susceptibility to LPO is maintained over a range of body temperatures. Other objectives include quantification of LPO products and antioxidant defenses. Recently developed fluorescent probes and chromatographic techniques will be used to address hypotheses. LPO is intensively studied in biomedicine and aging biology, yet the significance of membrane peroxidation, within the contexts of temperature physiology, has yet to be elucidated. Studies aimed at determining susceptibilities to LPO are necessary to clarify how poikilotherms avoid potentially damaging peroxidation of biological membranes. This research will contribute to a mechanistic understanding of biochemical factors that make life possible over a range of temperatures. The project will incorporate mentorship and training of graduate, undergraduate, and high school students (including research at a marine laboratory), collaboration with a faculty member from a predominantly undergraduate institution, consultation with an established researcher in the field of biomedical research, and outreach designed to educate children about how organisms adapt to life at different temperatures.
Background. There are two common, and nearly universal, physiological responses during acclimation to cold temperatures. The first response includes changes in the composition of phospholipids, which make up the matrix of biological membranes. Organisms (cells) at cold temperatures experience a significant increase in the amount of phospholipids with polyunsaturated fatty acids (PUFA). In contrast, at warm temperatures, organisms have more saturated fatty acids present in their membranes. This increase in PUFA in membrane phospholipids ensures the appropriate fluidity for membrane function at cold temperature. The second response to cold temperature is a proliferation in the amount of mitochondria within the cell. The mitochondrion represents the organelle that is the primary source of reactive oxygen species (ROS), including free radicals. Both of these responses are likely to correspond to an increase in the susceptibility of biological membranes to potentially damaging lipid peroxidation (LPO). Objectives. This project explored how changes in the composition of membrane phospholipids with temperature variation in a cold-blooded model organism (the striped bass, an animal that is tolerant of a wide range in body temperatures) affect the likelihood of incurring oxidative damage to biological membranes. Since LPO has both negative as well as beneficial consequences for organisms (cells), we tested the hypothesis that animals maintain a particular level of LPO over a range of body temperatures in order to preserve cellular processes, such as cell signaling, that require products of LPO. We also quantified products of LPO, low molecular weight antioxidants, and antioxidant enzymes. Findings and Outcomes. Our major finding was that although the susceptibility of biological membranes was not affected by temperature acclimation, the amount of LPO product (specifically phospholipid hydroperoxides) is constant in animals at both cold and warm body temperatures. These results indicate that lipid quantity rather than quality is most important in setting level of LPO. We also found that membrane antioxidants (e.g., vitamins E and C) are significantly elevated at warm body temperatures compared with animals acclimated to cold temperatures. These latter results suggest that LPO is a greater threat at warm than at cold temperatures, and that organisms meet the challenges associated with elevated temperatures by incorporating more antioxidants for protection. We published three papers in major scientific outlets including two papers in Journal of Experimental Biology, one in Comparative Biochemistry and Physiology. We currently have an additional paper that is in revision for American Journal of Physiology. Undergraduate and graduate students were lead authors or co-authors on all of these publications. Intellectual Merits and Broader Impacts. The work adds to our knowledge about the physiological responses of organisms to temperature variation. Since most organisms live at body temperatures that fluctuate with environmental temperatures, and experience changes in temperature either seasonally or diurnally, it is important to understand the physiological, biochemical, and molecular processes that make possible life over a wide range in temperatures. Undergraduate and graduate student training in physiology and biochemical techniques was provided under the award. In addition, we developed an in-class exercise (called "The Fats of Life") aimed at 5th and 6th graders, which illustrates the physical properties of membrane phospholipids at different body temperatures. This exercise was taken to classrooms in southeastern Ohio and introduced at a teacher workshop. The response was highly favorable from both students and teachers.
