| In this study,we selected four lukewarm water fish species(grass carp Ctenopharyngodon idella,gibel carp Carassius auratus gibelio,black carp Mylophayngodon piceus and largemouth bass Micropterus salmoides,with different feeding habits,to study the effects of stress on hypothalamic-pituitary-interrenal axis(HPI)axis,plasma and liver physiology,and liver enzymes involved in glucose metabolism,as well as elucidate the effects of species,blood glucose concentration and nutritional history on stress response.The main results are shown as follows:1.The effects of handling stress on the HPI axis activity in grass carp Ctenopharyngodon idella:We recovered partial cDNA of corticotropin releasing factor(CRF),steroidogenic acute regulatory protein(StAR),11?-hydroxylase(CYP11C1)and glucocorticoid receptor(GR)genes.CRF is 209 bp in partial length and encoding a polypeptide of 69 amino acids;StAR is 314 bp in partial length and encoding a polypeptide of 104 amino acids;CYP11C1 is 347 bp in partial length and encoding a polypeptide of 115 amino acids;GR is 259 bp in partial length and encoding a polypeptide of 85 amino acids.The deduced amino acid sequences of CRF,StAR,CYP11C1 and GR in C.idella have 69%-99%,73%-86%,73%-89%and 86%-98%identify with the corresponding sequences of other species respectively.We also investigated the effects of handling stress on the HPI axis activity in grass carp,and mRNA expressions of hypothalamic CRP,kidney StAR and CYP11C1,pituitary proopiomelanocortin(POMC)and liver GR were analyzed 1 h post-stress.The primary phase occurring HPI axis was characterized by enhanced transcripts of hypothalamic CRF,kidney StAR and liver GR(P<0.05).2.The effects of handling stress on plasma physiological parameters(cortisol,glucose and lactate),liver glycogen and activities of enzymes[phosphoenolpyruvate carboxykinase(PEPCK),glucose-6-phosphatase(G6Pase),hexoxinase(HK)and pyruvate kinase(PK)]involved in glucose metabolism in liver of grass carp throughout a 24 h(5 min,30 min,1 h,4 h and 24 h)cycle:The results showed plasma cortisol concentration in fish exposed to acute handling stress rose rapidly within 5 min(P<0.05),peaking within 1 h(P<0.05).Plasma glucose and lactate concentrations rose rapidly within 30 min(P<0.05),peaking within 1 h(P<0.05).Liver glycogen content was decreased with 1 h(P<0.05),and returned to concentrations indistinguishable from those of the unstressed fish within 24 h(P>0.05).Besides,liver phosphoenolpyruvate carboxykinase(PEPCK)activity and glucose-6-phosphatase(G6Pase)activities were enhanced within 1 h and 30 min post-stress(P<0.05).3.1 The influence of handling on concentrations of cortisol,glucose and lactate in plasma,glycogen content in liver and activities of two enzymes(HK and PK)responsible to glycolysis in liver of three cyprinidaes including herbivorous grass carp,omnivorous gibel carp and carnivorous black carp:The concentrations of cortisol,glucose and lactate in plasma of grass carp,gibel carp and black carp significantly increased after handling(P<0.05).The contents of liver glycogen of grass carp and black carp declined at 2 h after handling(P<0.05),while no significant difference was found in content of liver glycogen of gibel carp before and after handling(P>0.05).The glucose concentration in plasma was higher in black carp than in grass carp and gibel carp(P<0.05),while the content of liver glycogen was higher in gibel carp than in grass carp and black carp(P<0.05).A slight increase in concentrations of lactate and glucose in plasma was observed in gibel carp.No significant difference was found in the activities of HK and PK in liver of grass carp and gibel carp before and after handling(P>0.05),while the activity of HK in liver of black carp decreased at 2 h after handling(P<0.05).3.2 The effects of plasma glucose level on physiological and liver glucose metabolic stress response in grass carp:We evaluated the parameters involving the physiology in plasma[(cort:isol,glucose and lactate concentrations and lactic dehydrogenase(LDH)activity]and brain(glucose,glycogen and lactate concentrations),and glucose metabolism in liver(glycogen content and activities of PEPCK,G6Pase,HK and PK).Fish were intraperitoneal injected with 0,0.2,0.5 and 1.0 mg/g body mass(BM),which were named as 0G-S,0.2G-S,0.5G-S and 1G-S.After 1 h injection,the fish were handled and exposed to air,or unstressed prior to sampling at 0 h(pre-stress),1 h,2 h and 4 h post-stress.They were abbreviated as OG-S,0.2G-S,0.5G-S and 1G-S for stressed groups or OG-NS,0.2G-NS,0.5G-NS and 1G-NS for unstressed groups,respectively.The results showed as follows.1)Plasma physiology:Before stress,the plasma cortisol concentration in 1G-S group was significantly higher than OG-S,0.2G-S and 0.5G-S groups,and the plasma cortisol concentration in 1G-NS group was also significantly higher than OG-NS,0.2G-NS and 0.5G-NS groups(P<0.05).At 1 h post-stress,the plasma cortisol concentration was significantly lower in 0.5G-S group than other stressed groups(P<0.05).Before stress and 1 h post-stress,the plasma glucose concentration of both stressed and unstressed groups was significantly increased with the increase of injected dose of glucose(P<0.05).Before stress,and 1h,2 h and 4 h post-stress,the plasma glucose concentration was significantly higher in 1G-S group than other three stressed groups(P<0.05).At 4 h post-stress,the plasma lactate and LDH activity was significantly higher in 1G-S group than other thress stressed groups.2)Brain physiology:Before stress,the brain glucose concentration in 0.5G-NS and 1G-NS was significantly higher than 0G-NS and 0.2G-NS groups,and the brain glucose concentration in 0.5G-S and 1G-N was also significantly higher than 0G-S and 0.2G-S groups(P<0.05).At 1 h and 2 h post-stress,the brain glucose concentration in 1G-S group was significantly higher than other three stressed groups(P<0.05).At 1 h post-stress,the brain glycogen content in 0.5G-S group was significantly higher than 0G-S and 0.2G-S groups(P<0.05).At 2 h post-stress,the brain lactate concentration in 0.5G-S group was significantly higher than 0.5G-NS group(P<0.05).3)Liver glucose metabolism:At 2 h and 4 h post-stress,the liver glycogen content in 1G-S group was significantly lower than 1G-NS group(P<0.05).The liver G6Pase activity in 0G-S group was significantly higher than other stressed groups at 1 h post-stress(P<0.05).At 2 h post-stress,the liver G6Pase activity in 0G-S group was significantly higher than 1G-S group(P<0.05).At 1 h and 2 h post-stress,the liver G6Pase activity in 0G-S group was significantly higher than their parallel unstressed groups(P<0.05).At 1 h post-stress,the activity of liver HK in 0.2G-S and 1G-S groups was significantly higher than their parallel unstressed groups(P<0.05).3.3 The effects of nutritional history on stress response and the liver enzymes activities related to glucose metabolism after stress,and made a comparison of the effects between two species:A 2 × 2 layout,including two fish species(gibel carp or largemouth bass)and two nutritional history(fasted or fed to satiation for four weeks),was used.After being fed to satiation or fasted for 28 days,gibel carp and largemouth bass were handled and exposed to air,and sampled at 0 h(before stress),1 h,2 h,4 h and 24 h post-stress.Following the feeding and fasting periods,hepatosomatic index(HSI)and body compositions was analyzed.Plasma biochemical parameters(cortisol,glucose and lactate),concentrations of brain energy substrates(glucose,glycogen and lactate),liver glycogen content and enzyme activities of liver glucose metabolism(PEPCK,G6Pase,HK and PK)were analyzed after stress during a 24 h cycle.The results showed as follows.1)HSI and body composition:Compared with the initial fish,gibel carp displayed an average of 61%decrease(P<0.05)in crude lipid and 34%increase(P<0.05)in ash after 28 days fasting.Fasted largemouth bass had an average of 77%decrease(P<0.05)in HSI compared with the initial fish.2)Plasma physiology:The plasma cortisol concentration in fed gibel carp increased significantly at 2 h,4 h and 24 h post-stress(P<0.05).Fed largemouth bass had an increased concentration of plasma cortisol at 4 h and 24 h,and the plasma cortisol concentration in fasted largemouth bass increase at 1 h post-stress(P<0.05).In general(0 h-24 h),the plasma cortisol concentration was 3.5 times higher in the fed gibel carp than in the fasted gibel carp,while the plasma cortisol concentration was 1.2 times higher in the fed largemouth bass than in the fasted largemouth bass.Plasma glucose concentration significantly increased in fed and fasted gibel carp at 2 h and 1 h post-stress respectively(P<0.05).In largemouth bass,only fed fish had an increased concentration of plasma glucose at 1 h,2h and 4 h post-stress(P<0.05).In general,the plasma glucose concentration was 1.4 times higher in the fed gibel carp than in the fasted gibel carp,while plasma glucose concentration was 2.1 times higher in the fed largemouth bass than in the fasted largemouth bass.The plasma lactate concentration increased in both gibel carp and largemouth bass after stress(P<0.05),and was not significantly influenced by nutritional history(P>0.05).3)Brain physiology:The brain glucose concentration in both fed and fasted gibel carp significantly increased at 2 h post-stress(P<0.05).Fed largemouth bass also had an increased concentration of brain glucose at 1 h,2 h and 4 h post-stress(P<0.05).In general,the glucose concentration in brain was 1.2 times higher in the fed gibel carp than in the fasted gibel carp,while the brain glucose concentration was 6.0 times higher in the fed largemouth bass than in the fasted largemouth bass.Handling stress did not induce any significant changes of brain glycogen concentration in both species(P>0.05).In general,the glycogen concentration in brain was 2.0 times higher in the fed gibel carp than in the fasted gibel carp,while the glycogen concentration in brain was 1.5 times higher in the fed largemouth bass than in the fasted largemouth bass.4)Liver glucose metabolism:No significant changes of liver glycogen content in both species were observed after handling stress(P>0.05).In general,the liver glycogen content was 1.3 times higher in the fed gibel carp than in the fasted gibel carp,while liver glycogen content was 5.0 times higher in the fed largemouth bass than in the fasted largemouth bass.Handling stress induced an increase of liver PEPCK activity in fed gibel carp at 2 h and 24 h post-stress(P<0.05).In general,the liver PEPCK activity was 1.4 times higher in the fed gibel carp than in the fasted gibel carp,while the liver PEPCK activity was 2.1 times higher in the fasted largemouth bass than in the fed largemouth bass.Fasted gibel carp had significantly lower activity of liver G6Pase than fed fish at 4 h post-stress(P<0.05).Liver HK activity in fasted largemouth bass was significantly higher than fed fish before stress,and at 2 h,4 h and 24 h post-stress(P<0.05).3.4 The effects of different nutritional history on stress response and the liver enzymes activities related to glucose metabolism after stress in gibel carp:After being fed to satiation(Fed),or fasted for 14 days(Fasted-14d)or 28 days(Fasted-28d),fish were handled and exposed to air,and sampled at 0 h(pre-stress),1 h,2 h,4 h and 24 h post-stress.The results showed the dynamic changes of plasma biochemical parameters(cortisol,glucose and lactate),concentrations of brain energy substrates(glucose,glycogen and lactate),liver glycogen content and enzyme activities of liver glucose metabolism(PEPCK,G6Pase,HK and PK).The results showed as follows.1)Plasma physiology:Only Fed group displayed increased plasma cortisol concentration at 1 h,2 h,4 h and 24 h post-stress(P<0.05).At 2 h,4 h and 24 h post-stress,the plasma cortisol concentration in Fed group was significantly higher than Fasted-14d and Fasted-28d groups(P<0.05).The plasma glucose and lactate concentrations in all treatments increased significantly at 1 h and 2 h post-stres(P<0.05).2)Brain physiology:Fed and Fasted-28d groups showed a significantly increase of brain glucose concentration at 2 h post-stress(P<0.05).At 2 h post-stress,the brain glucose concentration in Fed and Fasted-28d groups was significantly higher than Fasted-14d group(P<0.05).Before stress,and 1 h,2 h,4 h and 24 h post-stress,the brain glucose concentration in Fasted-28d groups was significantly lower than Fed group(P<0.05).3)Liver glucose metabolism:The liver glycogen content in Fasted-14d group was significantly higher than Fasted-28d group at 2 h post-stress(P<0.05).The liver PEPCK activity in Fed group increased significantly at 2 h,4 h and 24 h post-stress(P<0.05).To conclued,1)The key genes of HPI axis,including CRF,StAR and GR were activated in grass carp after stress.Meanwhile,the increase of exogenous glucose was derived from glycogenolysis and gluconeogenesis in grass carp.2)The stress response induced by handling was lower in gibel carp compared to grass carp and black carp.Besides,the increase of glucose concentration in plasma did not enhance the enzymes activities for glycolysis in liver of grass carp,gibel carp and black carp.3)The pre-injection of exogenous glucose with 1.0 mg/g BM glucose could stress the grass carp by a temporary increase of plasma cortisol concentration,however,the cortisol stress response was attenuated when the fish pre-injected with 0.5 mg/g BM glucose.Both stress and exogenous glucose increased the brain glucose concentration.Meanwhile,exogenous glucose attenuated endogenous glucose release,showing the low rising rate of plasma glucose,enhancing the generation of plasma lactate,and inhibiting liver G6Pase activity.4)Nutritional history affected physiological stress response that influenced plasma biochemistry,brain energy substrates and liver glucose metabolism,which exhibited different effects between gibel carp and largemouth bass.Compared with fed fish,fasted gibel carp had lower plasma cortisol and glucose concentrations,whereas fasted largemouth bass had lower plasma and brain glucose concentrations when subjected to handling,which elucidates that the stress response is less severe in fasted fish.Different influence of nutritional history on the liver glucose metabolic response to stress was also observed between the two fish species.Fasting could attenuate the liver gluconeogenesis response to stress in gibel carp,however,the effect was opposite in largemouth bass.5)The gibel carp during short-term or long-term fasting were all less sensitive to handling stress than fed individuals,showing lower plasma cortisol and glucose concentrations in fasted individuals after stress.Brain glycogen content was decreased due to short-term or long-term fasting,which suggests brain glycogenolysis can be enhanced to maintain brain glucose concentration.Besides,the enhancement of liver gluconeogenesis after stress would be attenuated due to short-term or long-term fasting. |
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