Dustin S. Hittel, Ph.D. Biology, 2001

The physiological role of differentially expressed genes and their protein products in the hibernating thirteen-lined ground squirrel Spermophilus tridecemlineatus



The role of differential gene expression in supporting the survival of the hibernation phenotype was investigated using a variety of “gene discovery” techniques. A cDNA library constructed from kidney of the thirteen-lined squirrel,Spermophilus tridecemlineatus, was differentially screened for genes that were up-regulated during hibernation. A clone encoding cytochrome c oxidase subunit 1 (Cox1) was found and confirmed as up-regulated by Northern and Western blotting. This revealed the differential expression of Cox1 mRNA in multiple organs during hibernation. It is hypothesized that hibernating mammals increase the expression of the mitochondrial genome in general and Cox1specifically during torpor, to prevent or minimize the damage caused by the cold and ischemia experienced during a hibernation bout. The up-regulation of heart and adipose type fatty acid binding proteins (FABPs) was detected during hibernation in brown adipose tissue (BAT) using a commercial rat cDNA array. Full length cDNAs encoding heart and adipose-type FABPs were subsequently retrieved from a BAT cDNA library. H-FABP mRNA transcripts increased in BAT, skeletal muscle and heart of hibernating animals whereas A-FABP transcripts, which are normally expressed exclusively in adipose tissue, increased in both BAT and heart during torpor. The translation status of differentially expressed mRNAs during hibernation was also investigated in kidney and brown adipose tissue. Polysome profile analysis revealed a significant de-aggregation of polyribosomes during hibernation and a shift of housekeeping gene mRNAs and the up-regulated organic cation transporter 2 (OCT2) mRNA to the translationally silent monosome and mRNP fractions of kidney cytoplasmic extracts. In vitrotranslation rate and immunoreactive OCT2 protein were also significantly decreased in hibernating kidney. By contrast, the increased translation status of H-FABP mRNA, increase in immunoreactive H-FABP protein and unchanging in vitrotranslation rate reflect the important role of active brown adipose tissue during hibernation. The decrease in protein synthesis and de-aggregation of polysomes in kidney but not in brown adipose tissue, is linked to the phosphorylation of eIF2 . Additionally, redistribution of Cox4 but not H-FABP mRNA to the monosome fraction in hibernating BAT may indicate a mechanism for the preferential translation of a subset of genes physiologically relevant to the survival of hibernation.