Temperature has a profound effect on the physiology of animals. One modification that occurs in response to a decrease in temperature is an increase in mitochondrial density in some tissues. This process is critical for maintaining aerobic metabolic capacity in animals, yet questions remain about how it is regulated, and why it occurs in some tissues, yet not others. This project will address these questions, using the threespine stickleback Gasterosteus aculetaus as a model organism. This investigation will test the hypothesis that reactive oxygen species (ROS) are produced by mitochondria in response to cold temperature and are required to induce the expression of genes controlling mitochondrial biogenesis. Membrane fluidity decreases as temperature declines and may disrupt the activity of the electron transport chain proteins of the mitochondrion, leading to the formation of ROS. These ROS may directly regulate mitochondrial-biogenic gene expression, or alternatively, high levels of ROS may stimulate the activity of nitric oxide synthase which produces nitric oxide and triggers mitochondrial biogenesis. Both of these possibilities will be tested. Sticklebacks will be maintained at 20 degrees C in the laboratory and then shifted to 5 degrees C and held over a period of six weeks. Throughout the acclimation period, animals will be harvested for measuring levels of ROS, mitochondrial proteins, gene expression, and nitric oxide synthase activity. These parameters will be measured in several tissue types, including those that undergo mitochondrial biogenesis (oxidative skeletal muscle), and those that may not (liver). The differences in response to cold temperature among different tissue types may elucidate the mechanisms underlying the tissue specificity of mitochondrial biogenesis. If ROS and/or NO levels increase during the early stages of cold acclimation, then animals will be treated with inhibitors of ROS and/or NO formation. If either of these free radical species are required to induce mitochondrial biogenesis, then inhibiting their production should prevent increases in mitochondrial density in response to cold temperature. The results from this work will lead to a greater understanding of the molecular mechanisms governing mitochondrial biogenesis. This is a vital process, essential to the health of all organisms. This research will involve undergraduate and graduate Alaska Native students through a collaboration with the Alaska Native Science and Engineering Program at the University of Alaska. These students will also help to develop an outreach program at a local rural school which engages students in learning about the variety of strategies that animals use to survive at cold temperature, with an emphasis on physiological, cellular and molecular adaptations. Students will learn the important role of animals, even those found in local environments, for addressing fundamental questions in biology.
The aim of this research was to determine how metabolism is altered in response to cold temperature in fish. As ectotherms, a fish’s body temperature is equivalent to that of its environment. Consequently, as temperature decreases, body temperature also decreases, slowing rates of all metabolic processes. In order to maintain function at lower temperatures, and successfully avoid predators and capture prey, many fish adjust their metabolism to maintain energy production. In many fish species, including the threespine stickleback used in this study, remodeling involves building mitochondria through the process of mitochondrial biogenesis. Mitochondria are considered the powerhouse of the cell because they convert the energy stored in the foods we eat into a form (ATP) usable by the body. We sought to understand how mitochondrial biogenesis is controlled in fish in response to cold temperature in order to increase our understanding of how fish survive at different temperatures, and to increase our understanding of mitochondrial biogenesis, a process essential to the health of all organisms. We determined that neither nitric oxide nor reactive oxygen species trigger increases in mitochondrial density in muscle of stickleback in response to cold temperature, as they do in mammals. However, the formation of reactive oxygen species increases in liver in response to cold temperature and may be important for regulating changes in metabolism in this tissue. We also determined that the protein considered the master regulator of mitochondrial biogenesis in mammals, PGC-1a, does not control mitochondrial biogenesis in fish. Rather, the protein, NRF-1, seems to play a greater role. Our study is the first to examine how mitochondrial membranes are formed during mitochondrial biogenesis in fish, and our results suggest the enzyme glycerol-3-phosphate-acyltransferase may play a role. Previous studies have shown that exposing fish (and other organisms) to increases in temperature, results in an increase in molecular chaperones, which fold proteins that have been denatured by heat. Our study is the first to show that exposure of fish to cold temperature also results in an increase in molecular chaperones, suggesting decreases in temperature also denature proteins. We sought to engage Native Alaskans in research through this project. We collaborated with a high school teacher at Barrow High School, located off the road system in Alaska at 71°N latitude. A total of 10 Barrow H.S. students were involved in research over the course of the award period. The Principal Investigator and graduate students visited Barrow, bringing equipment and supplies, and taught students a variety of molecular biological techniques. Students presented their results at the Barrow high school science fair. Five students went on to compete in Alaska state science fairs. Two were awarded first place prizes in the animal science division in the Anchorage State Science Fair and one was awarded a 2nd place in the Alaska State High School Science Symposium in Fairbanks. One H.S. student from Fairbanks also competed in this science fair and placed 4th, and attended the National Junior Science and Humanities competition in San Diego. This project also provided funding for five M.S. students, including one Native Alaskan, three Native Alaskan undergraduates, and three additional Native Alaskan H.S students through the Rural Alaskan High School Honors Institute summer program.
