Freeze Resistance in Rainbow Smelt (Osmerus mordax): Seasonal Pattern of Glycerol and Antifreeze Protein Levels and Liver Enzyme Activity Associated with Glycerol Production

Abstract

Rainbow smelt (Osmerus mordax) inhabit inshore waters along the North American Atlantic coast. During the winter, these waters are frequently ice covered and can reach temperatures as low as -1.9 degrees C. To prevent freezing, smelt accumulate high levels of glycerol, which lower the freezing point via colligative means, and antifreeze proteins (AFP). The up-regulation of the antifreeze response (both glycerol and AFP) occurs in early fall, when water temperatures are 5 degrees -6 degrees C. The accumulation of glycerol appears to be the main mechanism of freeze resistance in smelt because it contributes more to the lowering of the body's freezing point than the activity of the AFP (0.5 degrees C vs. 0.25 degrees C for glycerol and AFP, respectively) at a water temperature of -1.5 degrees C. Moreover, AFP in smelt appears to be a safeguard mechanism to prevent freezing when glycerol levels are low. Significant increases in activities of the liver enzymes glycerol 3-phosphate dehydrogenase (GPDH), alanine aminotransferase (AlaAT), and phosphoenolpyruvate carboxykinase (PEPCK) during the initiation of glycerol production and significant correlations between enzyme activities and plasma glycerol levels suggest that these enzymes are closely associated with the synthesis and maintenance of elevated glycerol levels for use as an antifreeze. These findings add further support to the concept that carbon for glycerol is derived from amino acids.

Seasonal Changes in Hepatic Gene Expression Reveal Modulation of Multiple Processes in Rainbow Smelt (Osmerus mordax)

“…These two processes would allow the catabolism of cell proteins to produce glycerol and antifreeze proteins, respectively, which both accumulate to high levels during the winter (Lewis et al 2004). The up-regulation of an enzyme involved in ubiquination suggests that dietary protein may not always be sufficient to accommodate the accelerated synthesis of compounds essential for winter freeze resistance in smelt.…”

“…These samples were also used for qPCR analysis of selected genes for evaluation of the microarray hybridization results. Thereafter, a more detailed examination of seasonal gene expression by qPCR was conducted with livers obtained from male smelt sampled on the following dates: 20 October 2000, 1 Nov. 2000, 20 Dec 2000, 23 Jan. 2001, and 23 March 2001(Lewis et al 2004. A detailed temperature profile for these samples is provided by Lewis et al (2004).…”

“…The rainbow smelt (hereafter referred to as smelt) produces an antifreeze protein seasonally. Glycerol also accumulates seasonally, although the cycle appears independent of that of the antifreeze protein and the two processes appear to be regulated separately (Lewis et al 2004;Liebscher et al 2006). …”

Glycerol production in rainbow smelt (Osmerus mordax) may be triggered by low temperature alone and is associated with the activation of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase

“…As the water temperature decreases further, over the next 4-6 weeks, to about 2°C, there is a sharp increase in plasma glycerol level to about 100·mmol·l -1 . As winter progresses, glycerol increases to values in excess of 250·mmol·l -1 (Treberg et al, 2002b; Lewis et al, 2004). …”

“…Temperature has a regulatory role in glycerol accumulation, as maintenance of fish at warm (8°C) temperatures over the winter suppress glycerol accumulation (Lewis et al, 2004) and premature exposure of fish to cold temperatures for 3·weeks in the fall results in glycerol accumulation (Raymond et al, 1996). In this study, we assess if the onset of glycerol accumulation is due to a temperature trigger alone or whether a shortening photoperiod is required.…”