Winter presents organisms inhabiting temperate regions with various stresses, including low temperature. To contend with subzero temperatures, a small group of ectotherms tolerate freezing, in part through the use of cryoprotective solutes. In the wood frog, Rana sylvatica, glucose serves an important role in freezing survival; however, the mechanisms underlying the transport and distribution of cryoprotectants such as glucose are not fully understood. This dissertation describes several studies that examined a glucose transport protein (GLUT2) and its importance in the freeze tolerance of R. sylvatica.
First, I identified and functionally characterized a GLUT2 homolog from R. sylvatica to understand its role in glucose homeostasis. The newly identified GLUT2 was structurally and phylogenetically similar to other known GLUT2s. Measurements of transport kinetics in Xenopus oocytes determined that GLUT2 from R. sylvatica is functionally similar to previously characterized GLUT2s. This protein was found in various tissues but was most abundant in liver. Finally, transport kinetics were similar among two populations of R. sylvatica, and R. pipiens; however, GLUT2 abundance was greatest in the northern R. sylvatica.
R. sylvatica relies on glucose to survive multiple winter-related stresses, and the second study examined regulation of GLUT2 in response to these stresses. GLUT2 expression increased in response to organismal freezing, hypoxia exposure, and glucose loading; whereas, experimental dehydration and urea loading had no effect. These changes were the result of transcriptional regulation, resulting in synthesis of new protein. Results of this study suggest that hepatic GLUT2 is regulated to meet the physiological need to accumulate glucose.
Subarctic populations of R. sylvatica survive freezing to lower temperatures than conspecifics from temperate regions. In the last study, I sought to determine if GLUT2 contributes to the profound freeze tolerance of northern frogs. We examined GLUT2 expression in R. sylvatica from Interior Alaska and Ohio and found that GLUT2 abundance increased in preparation for winter in both populations, but that GLUT2 abundance was greater in Alaskan frogs. In Alaskan, but not Ohioan, frogs, GLUT2 protein increased in response to experimental freezing. These results suggest that GLUT2 is important in the extreme freeze tolerance of northern wood frogs.