A major challenge of long-term fasting is the provision of glucose for glucose-dependent tissues (e.g. central nervous system) while sparing protein in vital body organs. Seals provide an excellent system to investigate metabolic strategies during fasting because they undertake natural fasts during periods that elevate nutrient demands. Weaned northern elephant seals fast for 2-3 months, during which time physiological development supporting their diving ability occurs. Previous investigations in this system revealed a number of unique features of metabolism that differ from the traditional view of fasting adaptation. Despite efficient protein sparing, plasma glucose levels in elephant seals are high throughout the fast. Levels of glucose production fail to exhibit the suppression with fasting that is seen in other species. Breakdown of protein and fats provides only a small portion of this glucose production and, therefore, the source of this endogenous glucose production has not been identified. This research project will investigate glucose production and disposal during long-term natural fasts in northern elephant seals. Isotopic tracer techniques and respirometry will be combined to 1) examine the contribution of various substrates to glucose production 2) examine the metabolic strategies for glucose clearance 3) examine variation in fuel use over fasting period and 4) examine the activity of several key metabolic cycles that may regulate glucose production. This study will provide important information about the metabolic strategies that enable long-term fasting during development. Increased understanding of the novel features of carbohydrate regulation in fasting-adapted animals, including maintenance of high blood glucose while fasting, avoidance of ketoacidosis, protein sparing despite high rates of glucose production, and high rates of glucose disposal despite low insulin levels and insulin resistance can potentially inform our understanding of human pathologies of carbohydrate metabolism. This research will also promote research training and education to undergraduate and graduate students, especially underrepresented minority students, through participation in exciting research on a unique wildlife system.
Most organisms fast from food or water during some portion of their life history. Some animals have lives that require extreme feats of fasting for extended periods during critical periods. These periods, like reproduction, lactation and development, can require inputs of large amounts of nutrients for energy or synthesis of things like milk or blood. The metabolic adaptations that allow extended, high energy fasts are not well understood. In general, animals that are good at fasting are fat and maintain long periods of food deprivation by running off that fat. However, animals have a very limited ability to store carbohydrates in their bodies and the real challenge for extended fasting is to provide a continuous supply of carbohydrates to tissues that depend on them, like the central nervous system and red blood cells. During extended fasts animals are required to make sugars from other stored molecules through a process called gluconeogenesis (GNG). One source of substrates for GNG is stored body protein. It is this loss of protein from vital organs to make sugars that ultimately limits fasting ability. Our previous investigations on elephant seals have shown that unlike many animals they continue to make sugars at high rates while fasting. However less than 15% of these sugars were made from proteins or glycerol molecules mobilized from stored body fat. Our current studies were an attempt to use cutting edge metabolic tracer technologies from the biomedical field to better understand the sources of glucose production in fasting elephant seals and the ultimate fate of the glucose being produced. We used a combination of several different tracer technologies, respirometry (measuring gas exchange) and metabolomics (measuring all of the compounds possible in the blood) to achieve this goal. We confirmed that high rates of sugar production in fasting seals was not from triglycerides, proteins, or stored carbohydrates (glycogen). We found that ~95% of glucose was produced from a molecule called phosphoenolpyruvate (PEP) that was maintained by high rates of pyruvate cycling, together with high rates of the tricarboxylic acid (TCA) cycle, a cycle involved in burning fatty acids. Virtually every 6-carbon molecule of sugar produced was converted to a smaller 3-carbon molecule of pyruvate and only a small amount of this molecule was burned for energy. Much of the remaining pyruvate was converted to lactic acid, which was then converted back into sugar by the liver. This 'recycling' of glucose enables elephant seals to manufacture glucose at high rates while minimally depleting vital organs. This system also allows seals to burn primarily fat for energy while avoiding making ketones, a byproduct of fat metabolism that can become toxic. We found that despite having very low insulin levels, like diabetics, insulin was the primary regulator of the process of glucose production and recycling. In addition to these major studies, this NSF grant supported numerous graduate and undergraduate projects. One project showed that there were critical sex differences in fasting metabolism in developing seals, with males being better at preserving muscle and females having lower metabolic rates and use of fat. We found that seals were able to maintain levels of water soluble vitamins across extended months-long fasts, despite no dietary input. We found that seals defended high levels of 'good' HDL cholesterol while depleting other kinds of cholesterol across the fast. We found that pups develop the ability to suppress their metabolism during breath-holds while on-shore fasting. This information lets us better understand how pup body size at weaning influences the ability to develop diving ability and survive at sea. We completed one of the first field studies of animal skin temperature using thermography in wildlife systems and showed that adult male elephant seals overheat after fighting and modify their behavior based on their ability to cool down using the wind. We found that two key hormones, insulin and cortisol, regulate mobilization of fats in lactating female elephant seals and control the energy content of the milk produced by mothers. Using the samples acquired from the procedures for these studies, we completed several investigations of how trace elements and contaminants were mobilized and transferred from mother to pup. Hundreds of undergraduates participated in the field work on our project and dozens of students completed senior thesis research under support of the project.
