Developing tools to understand molecular regulation of fasting in marine mammals

Marine mammals such as elephant seals (Mirounga angustirostris) are unique in their ability to fast from food for months while undergoing energetically demanding processes of molting and reproduction on land. To gain insights into energy provisioning during fasting, we are studying gene and protein expression and evolution using energy-rich blubber tissue collected from elephant seals at the beginning and end of their month-long molting fast. We are using real-time quantitative PCR (qPCR) to quantify changes in expression of seven metabolic regulator genes identified as differentially expressed during stress in a previous blubber transcriptome study: DKK1, CEBPD, DDIT4, LPL, PPARG, FOXO1, and FABP4. By comparison with the reference genes NONO and YWHAZ, we expect lipid metabolism genes to be upregulated during fasting. We are also examining the sequence of leptin (LEP), which suppresses appetite in terrestrial mammals when fat levels are elevated and may play a role in controlling energy balance during periods of fasting in seals. We are comparing elephant seal LEP sequence with that of other mammals to obtain insights into leptin function and evolution. We found that the longest LEP mRNA transcript previously identified by transcriptome sequencing contained 1453 nucleotides, which was translated into a peptide sequence with 337 amino acids. This sequence aligned with the human LEP gene on Chromosome 7, which contains 3 exons, and had highest sequence similarity to Weddell seal LEP. We are also examining changes in the entire elephant seal blubber proteome during fasting using orbitrap mass spectrometry. We are developing tissue lysis and protein extraction methods for the first pipeline for marine mammal blubber shotgun proteomics. We were able to identify and annotate over 100 proteins in blubber, which include Toll-interacting proteins used in the IL-1 and Toll-like receptor signaling pathways, and alpha-2-macroglobulin, a carrier of signaling molecules in blood and tissues