Adaptation of peroxisome proliferator-activated receptor alpha to hibernation in bats

Abstract

Background: Hibernation is a survival mechanism in the winter for some animals. Fat preserved instead of glucose produced is the primary fuel during winter hibernation of mammals. Many genes involved in lipid metabolism are regulated by the peroxisome proliferator-activated receptor alpha (PPARα). The role of PPARα in hibernation of mammals remains largely unknown. Using a multidisciplinary approach, we investigated whether PPARα is adapted to hibernation in bats.

Results: Evolutionary analyses revealed that the ω value of Pparα of the ancestral lineage of hibernating bats in both Yinpterochiroptera and Yangochiroptera was lower than that of non-hibernating bats in Yinpterochiroptera, suggesting that a higher selective pressure acts on Pparα in hibernating bats. PPARα expression was found to be increased at both mRNA and protein levels in distantly related bats (Rhinolophus ferrumequinum and Hipposideros armiger in Yinpterochiroptera and Myotis ricketti in Yangochiroptera) during their torpid episodes. Transcription factors such as FOXL1, NFYA, NFYB, SP1, TBP, and ERG were bioinformatically determined to have a higher binding affinity to the potential regulatory regions of Pparα in hibernating than in non-hibernating mammals. Genome-wide bioinformatic analyses of 64 mammalian species showed that PPARα has more potential target genes and higher binding affinity to these genes in hibernating than in non-hibernating mammals.

Conclusions: We conclude that PPARα is adapted to hibernation in bats based on the observations that Pparα has a more stringent functional constraint in the ancestral lineage of hibernating bats and a higher level of expression in hibernating than in non-hibernating bats. We also conclude that PPARα plays a very important role in hibernation as hibernators have more PPARα target genes than non-hibernators, and PPARα in hibernators has a higher binding affinity for its target genes than in non-hibernators.

Fig. 1

Species tree of Pparα of bats. Non-hibernating bats in Yinpterochiroptera, hibernating bats in Yinpterochiroptera, and hibernating bats in Yangochiroptera are represented by orange, green, and blue colors, respectively. The N × d _N/S × d _S/ω value of each tree branch of bats is shown

Fig. 2

Expression levels of Pparα mRNA determined by real-time RT PCR. RF, HA, and MR represent hibernating R. ferrumequinum, H. armiger, and M. ricketti bats, respectively. Relative mRNA levels in torpid and active states are indicated with light blue and dark blue colors, respectively. The expression level of Pparα in active bats was set as 1.0. Data are presented as mean ± SD. A P value < 0.05 is considered statistically significant

Fig. 3

Expression levels of PPARα in bats determined by Western blotting. (A) Western blotting results of PPARα. (B) Relative levels of PPARα. The expression level of the PPARα protein in mice (M) was set as 2, and that of PPARα in each bat species was relative to it. RF, HA, and MR represent hibernating R. ferrumequinum, H. armiger, and M. ricketti bats, respectively. RL represents non-hibernating R. leschenaultii bats. T and A indicate bats in torpid and active states, respectively. Arrowheads in (A) indicate molecular weights (kDa) of protein markers. Relative levels of the PPARα protein in mice (gray), torpid (light blue) and active (dark blue) bats, and non-hibernating bats (pink) are shown in (B). Results are presented as mean ± SD. A P value < 0.05 is considered significant

Fig. 4

Bioinformatic analyses of PPARα in mammals. The y-axis in (A) represents the ratios of the number of PPARα potential target genes to the number of all genes of different animals. The small open rectangle indicates the threshold setting (5 x 10⁻⁷) for the matrix scan. The scores in (B) indicate the binding affinity of PPARα to its target genes in different animals. Blue and pink boxes indicate data calculated from 16 hibernating species (H) and 48 non-hibernating species (N) of mammals. Each dot represents the mean value obtained from each mammalian species. The box plot shows the median, 25/75 percentiles (box), and 10/90 percentiles (bars). A P value < 0.05 is considered significant