Adaptation of Phenylalanine and Tyrosine Catabolic Pathway to Hibernation in Bats

et al. (2013) Adaptation of Phenylalanine and Tyrosine Catabolic Pathway to Hibernation in Bats. PLoS ONE 8(4): e62039. doi:10.1371/journal.pone.0062039 Adaptation of Phenylalanine and Tyrosine Catabolic Pathway to Hibernation in Bats Yi-Hsuan Pan 0 Yijian Zhang 0 Jie Cui 0 Yang Liu 0 Bronwyn M. McAllan 0 Chen-Chung Liao 0 Shuyi Zhang 0 Frank Seebacher, University of Sydney, Australia 0 1 Laboratory of Molecular Ecology and Evolution, Institute for Advanced Studies in Multidisciplinary Science and Technology, East China Normal University , Shanghai , China , 2 Discipline of Physiology and Bosch Institute, School of Medical Science, The University of Sydney , Sydney, New South Wales , Australia , 3 Proteomic Research Center, National Yang-Ming University , Taipei , Taiwan Some mammals hibernate in response to harsh environments. Although hibernating mammals may metabolize proteins, the nitrogen metabolic pathways commonly activated during hibernation are not fully characterized. In contrast to the hypothesis of amino acid preservation, we found evidence of amino acid metabolism as three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate 1,2-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 hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and hibernators had more conserved amino acid residues in each of these enzymes than non-hibernators. These conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Taken together, results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation. - Funding: This study was supported by grants of key construction program of the National 985 projects to SZ, National Natural Science Foundation of China (31100273/C030101-Pan), and the Science and Technology Commission, Shanghai Municipality (10R21412400-Pan). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Hibernation is a controlled reversible reduction of body temperature, metabolic rate, and many other physiological processes of some animals in order to survive in winter [14]. Understanding the mechanisms by which hibernators control their metabolism during hibernation to cope with stress conditions, such as hypothermia, ischemia, and muscle atrophy, may aid in the prevention or treatment of human diseases. A winter hibernation consists of a sequence of torpor bouts lasting for days or weeks interrupted by short normothermic arousal bouts [1,2]. In preparation for hibernation, the weight and fat mass of some hibernators (e.g., squirrels, bats, and black bears) are greatly increased in summer and autumn [1]. During hibernation, many hibernators do not feed and depend largely on fat reserves for energy by switching from glucose utilization to lipid consumption [1,3]. In large hibernators (e.g., bears), only a small loss of proteins occurs during hibernation [5,6]. Although small hibernators (e.g., bats and squirrels) lose a significant overall protein mass during hibernation [710], little loss of pectoral and biceps brachii muscles or liver proteins is observed [11,12]. Several mechanisms that may prevent muscle atrophy or loss of liver proteins have been proposed, including the production of proteolytic inhibitors [5,6], continual protein synthesis [12,13], maintenance of proper protein folding [11], and preservation of essential amino acids such as phenylalanine (Phe) and tyrosine (Tyr) [1214]. These two amino acids are both glucogenic and ketogenic in energy production. They are also precursors of thyroid hormones and catecholamines (e.g., adrenaline, noradrenaline, and dopamine) that can act on adipose tissues to modulate thermal and energy balance during hibernation [15,16]. In mammals, tyrosine is synthesized from phenylalanine. Conversion of phenylalanine to tyrosine is an irreversible step catalyzed by phenylalanine hydroxylase (PAH) [17,18]. Four enzymes including tyrosine aminotransferase (TAT), hydroxyphenylpyruvate dioxygenase (HPD) [19], homogentisate 1,2-dioxygenase (HGD) [20,21], and fumarylacetoacetase (FAH) are involved in tyrosine catabolism [2224]. Any deficiency in these enzymes may result in accumulations of toxic metabolites such as 4-hydroxyphenylpyruvate, homogentisate, 4-maleylacetoacetate, and fumarylacetate in blood, liver, or urine leading to DNA and tissue damage, depletion of glutathione levels, and activation of apoptosis [25,26]. Although hibernators undergo tremendous alterations in physiology with many genes differentially expressed during hibernation [1], there has been no evolutionary evidence for the adaptation of an entire metabolic pathway to hibernation. Bats represent approximately twenty percent of all mammalian species and are the only flying mammals that hibernate. Their physiological status during hibernation has been extensively studied [27 30]. Taxonomically, bats belong to the order Chiroptera that is subdivided into suborders Yinpterochiroptera and Yangochiroptera. Hibernating bats are found in both suborders (e.g., Rhinolophus in Yinpterochiroptera and Myotis in Yangochiroptera suborder), while most non-hibernating bats are in the Yinpterochiroptera suborder (e.g., Cynopterus) [27,31]. Bats offer the unique advantage over bears and squirrels for investigation of enzymes in adaptation to hibernation as there are at least 1,200 species of bats, and the phylogenetic relationships of most of these bats are well defined [31]. A careful comparison of the activities, expression levels, and structures of enzymes between different hibernating and non-hibernating species of bats will yield useful information in the regulation and evolution of the enzymes. The adaptation of liver proteins in 13-lined ground squirrels to hibernation has been investigated using proteomic approaches [12]. In this study, we used a combination of proteomic and evolutional approaches to investigate the adaptation of PAH, HPD, HGD, and FAH in the phenylalanine and tyrosine catabolic pathway to hibernation. Materials and Methods An (...truncated)