Freeze tolerant frogs: cryoprotectants and tissue metabolism during freeze–thaw cycles

Abstract

Tissue-specific metabolism in the freeze tolerant frog, Rana sylvatica, was monitored over a course of 3 days of freezing exposure at −2.5 °C followed by 11 days of thawing at 3 °C. Freezing induced the rapid accumulation of glucose as a cryoprotectant with maximal levels of 257 μmol/mL in blood and 188, 140, 74, and 28 μmol/g wet weight in liver, heart, kidney, and leg muscle, respectively. Liver showed the fastest rate (20 μmol g−1 h−1 over the initial 7 h) and the shortest half time (5 h) for glucose accumulation. This plus the activation of liver glycogen phosphorylase and a 593 μmol/g decrease in liver glycogen indicated this organ as the major site of cryoprotectant production during freezing. Glucose accumulation was reversed when frogs were thawed (half time for clearance varying from 1.9 days in liver to 8.1 days in leg muscle) with glucose restored as liver glycogen. Extracellular freezing was also accompanied by a progressive accumulation of lactate and alanine as glycolytic end products indicating a reliance on fermentative metabolism during freezing and by a decline in energy status (ATP content and energy charge decrease) in tissues. Relative amounts of lactate and alanine accumulated varied dramatically between tissues: from heart, which produced only lactate, to leg muscle, which had an alanine:lactate ratio of 4.5:1. No other amino acids accumulated in tissues during freezing exposure, but alanine accumulation in leg muscle was inversely proportional to a decline in tissue aspartate, glutamate, and glutamine contents. When thawed, lactate and alanine were readily cleared from tissues and energy status was slowly restored.