Cloning and expression of hypoxia-inducible factor 1alpha from the hibernating ground squirrel, Spermophilus tridecemlineatus.

Mammalian hibernation is associated with apnoic breathing patterns and a hypoxia-hypothermia connection has been suggested as part of the mechanism by which body temperature is reduced as animals enter torpor. Hence, we hypothesized that changes in the expression of the hypoxia inducible factor (HIF-1) may potentially be involved in regulating hibernation-responsive gene targets. The expression of the alpha subunit of HIF-1 was quantified at both gene and protein levels in four organs of the thirteen-lined ground squirrel, Spermophilus tridecemlineatus. Reverse transcription-PCR showed no change in hif-1alpha transcript levels in the liver, lung, skeletal muscle or brown adipose tissue of euthermic versus hibernating animals but HIF-1alpha protein levels were elevated by 60-70% in the two organs responsive for thermogenesis (brown adipose and skeletal muscle). Furthermore, assessment of DNA binding by HIF-1 in nuclear extracts from brown adipose revealed 6-fold higher levels in hibernator tissue than in euthermic controls suggesting increased expression of HIF-1 responsive genes during hibernation. The complete nucleotide sequence of hif-1alpha from ground squirrels, the first hif-1alpha sequence amplified from a hibernating mammal, was obtained using PCR amplification and 3' and 5' RACE. Amino acid sequence analysis revealed 90-95% identity with the HIF-1alpha protein from other mammals. Several unique amino acid sequence substitutions were identified that may affect protein conformation and could possibly function to counteract low temperature effects on HIF-1alpha conformation at near 0 degrees C body temperatures during torpor.