Involvement of glucose in freeze tolerance in the Japanese tree frog Hyla japonica

Glycerol and aquaporin 9 (aquaglyceroporin) are known to be involved in freeze tolerance in the Japanese tree frog Hyla japonica. However, the regulatory mechanisms of freeze tolerance in this species have not been fully elucidated. In the present study, we focused on the inter- and intracellular dynamics of glucose to analyze the role of glucose and glucose-related proteins such as transporter and metabolic enzymes in freeze tolerance. Serum glucose concentrations were compared among the frogs that were nonhibernating, hibernating, and thawed after freezing at -4 degrees C for 6 hr. Serum concentrations of glucose in thawed frogs were significantly higher than those in hibernating and nonhibernating, active frogs. Periodic acid-Schiff staining showed that the accumulation of glycogen in the hepatocytes increased before hibernation and decreased after freezing and thawing. Quantitative RT-PCR analysis using the liver showed that, compared with active frogs, the type 2 glucose transporter gene (glut2) was upregulated in frozen frogs, the liver glycogen phosphorylase gene (pygl) was upregulated in frozen or thawed frogs, and the type 2 glycogen synthase gene (gys2) was upregulated in hibernating frogs. Immunohistochemistry of liver sections showed that, compared with nonhibernating frogs, Glut2 proteins were clearly increased most likely on the plasma membrane of hepatocytes in hibernating frogs and further increased by freezing, then decreased after thawing. These results suggest the possibility that glucose acts as a cryoprotectant in H. japonica.