A Study On The Seasonality Of Metabolism And The Mechanism Of Pre-hibernation Urea Accumulation In Frog Nanorana Pleskei

The frog Nanorana pleskei is an endemic species to Qinghai-Tibet Plateau,and is an excellent material for studying high-elevation adaptation of animals.The previous study of our lab showed that the frog could accumulate substantial amounts of urea as cryoprotectant during late fall or early winter to cope with the cold stress and freezing risk in winter.At present,there is a lack of research on seasonal metabolism,as well as the mechanism of urea accumulation in this species.In this paper,using N.pleskei as the research object,we determined the standard metabolic rate(SMR)of different season under the various temperature(5?15?25 ?),the main metabolic enzyme activities in the crucial metabolic pathways to explore the seasonal adaptation strategy of metabolism.The assayed enzymes include citrate synthase(CS),pyruvate kinase(PK),lactate dehydrogenase(LDH),?-hydroxyacyl coenzyme A dehydrogenase(HOAD),glutamate dehydrogenase(GDH).To reveal the mechanism of urea accumulation before hibernation from physiological and biochemical aspects,we also examined the content of some metabolites including urea,ammonia,and water content,total protein and the organ weight ratio from different tissues,as well as the activity of key regulatory enzymes of urea cycle such as carbamoyl phosphate synthase(CPS-1),N-acetyl-L-glutamate synthetase(NAGS),and the seasonal changes of urea and ammonia excretion rate,At the same time,the effects of low temperature acclimation(4 ?,dark,and wet conditions for 1 month)on metabolism and urea synthesis of summer frogs were also studied.The results showed that the metabolic rate of N.pleskei was seasonally varied and was affected by cold acclimation.The metabolic rate in spring was significantly higher than other seasons,and metabolic depression occurred in autumn.The key metabolic enzyme activities in the liver also showed seasonal variations.The activities of PK and LDH were significantly higher than other seasons indicating the intense anaerobic glycolysis activity in spring.This may be related to the reproduction,damage repair after hibernation,and increase of violent activity in a short period;However,PK activity significantly decreased in autumn,indicating that the oxidative metabolism of carbohydrates was strongly inhibited.The modulation of PK activity is important for the regulation of animal metabolism and the seasonal variation of PK activity may be the main cause of seasonal differences in metabolic rate.HOAD activity in summer and autumn was significantly higher than in spring,indicating that the lipid metabolism in the summer and autumn was more active.GDH activity was significantly increased in autumn indicating the high level of amino acid catabolism in this season.In addition,the significant increase in CS activity after cold acclimation may be a low temperature compensation strategy.We speculate that the changes of activities of certain enzyme account for the conversion of main fuel substances,which is an important strategy for seasonal adaptation of frogs.The enhancement of amino acid catabolism in autumn can provide sufficient nitrogen source for urea cycle,which may be an important basis for urea accumulation.At the same time,the total protein content in the liver and muscle tissues in autumn was significantly higher than summer and spring,yet the protein may act as both an energy reserve and a nitrogen reserve.The results of CPS-1 and NAGS activity assays showed that the activity of CPS-1 is strongly dependent upon the activation of N-acetyl-L-glutamic acid(NAG).The CPS-1 activity is extremely low in the absence of NAG,and the enzyme activity does not increase with temperature.Because the regulation of NAGS activity controls the activity of CPS-1 the changes of NAGA activity affect urea production capacity.Therefore,the significant increase of NAGS activity in autumn may be an important reason for urea accumulation.After one month of cold acclimation,the NAGS activity of the frogs collected in summer was significantly increased.The change of urea content in plasma and liver was consistent with the change of NAGS activity.The accumulation of urea may be in response to low temperature.The ammonia and urea excretion rate also showed seasonal changes.In autumn the ammonia and urea excretion rates were significantly reduced,indicating that the urea reabsorption was enhanced.It is worth noting that there is also a seasonal difference in the ratio of kidney weight to body weight,and it is significantly higher in autumn than in summer.The ratio was also significantly increased after cold acclimation,which may be related to the urea reabsorption while the relevant mechanism needs further investigation.In summary,the metabolism of N.pleskei showed significant seasonality,which probably is an important strategy for this frog to adapt to the environmental changes.The increase of urea production capacity and the enhancement of urea reabsorption in autumn may conjointly led to the urea accumulation before hibernation.This is considered as an active adaptation to its specific environment.The urea accumulation caused by cold acclimation also confirmed the relationship between urea accumulation and low temperature from another aspect.