Brain And Liver Proteome Studies On Hibernating Bats

Hibernation is a phenotype that hibernators reduce body temperature, metabolic rate, and other physiological processes in response to harsh environments. Bats are the only flying mammals comprised20%of the total mammal species. Taxonomically, bats belong to the order Chiroptera that is subdivided into suborders Yinpterochiroptera and Yangochiroptera. Hibernating bats are found in both suborders and non-hibernating bats are mostly found in the suborder Yinpterochiroptera. The molecular mechanisms underlying regulations of bat hibernation remain largely unknown. In this study, brain and liver proteomes were separatively compared between torpid and active bats (Myotis ricketti) to further understand bat hibernation. Moreover, we choosed target proteins and used multidisciplinary approaches, including western blots, molecular evolution, activity assay, structure simulation and immunohistochemistry to further explore molecular regulatory mechanisms of hibernation. Three main findings of this study are listed as follows:(1) Brain and liver proteins of torpid and active Myotis ricketti bats were compared using a proteomic approach. Results showed that1)21%(23proteins) of identified proteins were associated with amino acid metabolism and proteostasis in the brain. The expression levels of proteins involved in energy metabolism (15proteins), cytoskeletal structure (18proteins), and stress response (13proteins) were also significantly altered in torpid bats. Over30%(34proteins) of differentially expressed proteins were associated with mitochondrial functions. Post-translational modifications (PTMs) on PDHB, DLD, and ARG1were detected, suggesting that bats use PTMs to regulate protein functions during torpor. Antioxidation and stress responses in torpid bats were similar to those of hibernated squirrels, indicating a common strategy adopted by small hibernators against brain dysfunction. Because many amino acids that metabolize in mitochondria modulate neuronal transmissions, results of this study also reveal pivotal roles of mitochondria in neural communication, metabolic regulation, and brain cell survival during bat hibernation.2) Differential expression of proteins related to carbohydrate metabolism, lipid metabolism and amino acid metabolism was found in the liver. Proteins involved in carbohydrate metabolism were decreased and those in lipid metabolism were increased in the torpid Myotis ricketti bats. These suggest that the main way of energy supply is from glycolysis to lipolysis during bat hibernation. The expression of most of the proteins involved in synthesis and folding were up-regulated. The expression level of some structure proteins was down-regulated and those proteins involved in metabolisms of amino acids (such as Phe and Tyr) were increased, indicating that bat hibernators may consume amino acids for energy supply during torpor. In addition, the transcription factors (such as PPARs and HNFs) play an important role in the regulation of fuel utilization during hibernation.(2) We found that the expression level of betaine-homocysteine S-methyltransferase (BHMT) is increased in the proteomic analysis of brain, thus the biological significance of BHMT expression was further studied. The main function of the coenzyme-independent BHMT is involved in Hcy metabolism. Elevated homocysteine increases cerebrovascular and neurodegenerative disease morbidity. It suggests that elevated BHMT in the torpid Myotis ricketti bats is an important factor which protect brain tissues during hibernation. BHMT expression was observed in the amygdala of basal ganglia and the cerebral cortex where BHMT levels were clearly elevated during torpor. In mammals, B vitamin supplementation can reduce homocysteine levels. We found that homocysteine does not elevate in torpid brains, despite declining vitamin B levels. At low levels of vitamin B6and B12, we found no change in total expression level of the two main enzymes involved in homocysteine metabolism (methionine synthase and cystathionine β-synthase). This is the first report of BHMT protein expression in the mammalian brain and suggests that BHMT may have a neuroprotective role in the brains of hibernating bats. Further research on this system could expand our biomedical understanding of certain cerebrovascular and neurodegenerative disease processes.(3) In the liver proteomic analysis, we further found that three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate1,2-dioxygenase (HGD), fumarylacetoacetase (FAH), involved in phenylalanine and tyrosine catabolism were co-upregulated during hibernation in two distantly related species of bats, Myotis ricketti and Rhinolophus ferrumequinum. In addition, the levels of phenylalanine in the livers of these bats were significantly decreased during hibernation. Because phenylalanine and tyrosine are both glucogenic and ketogenic, these results indicate the role of this catabolic pathway in energy supply. Since any deficiency in the catabolism of these two amino acids can cause accumulations of toxic metabolites, these results also suggest the detoxification role of these enzymes during bat hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and the conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation.In conclusion, brain and liver proteome were compared separatively between torpid and active bats in this study, and the differentially expressed proteins were involved in carbohydrate metabolism, lipid metabolism, nitrogen metabolism, antioxidation defense, stress response and cytoskeletal plasticity. We found for the first time that BHMT is expressed in the brains of mammals, and discussed functions and medical significances of BHMT in brain protection during bat hibernation. In the catabolic pathway, the results showed the crucial roles of PAH, HPD, HGD and FAH in phenylalanine and tyrosine metabolism, and reveals this catabolic pathway is important in bat hibernation. This research expands our understanding of mammalian hibernation and may provide theoretical basis for the application of hibernation in translational medicine.