Fish Physiol Biochem DOI 10.1007/s10695-017-0383-y
Effect of blood glucose level on acute stress response of grass carp Ctenopharyngodon idella Danli Jiang & Yubo Wu & Di Huang & Xing Ren & Yan Wang
Received: 12 October 2016 / Accepted: 8 May 2017 # Springer Science+Business Media Dordrecht 2017
Abstract Stress has a considerable impact on welfare and productivity of fish, and blood glucose level of fish may be a factor modulating stress response. This study evaluated the effect of blood glucose level and handling on acute stress response of grass carp Ctenopharyngodon idella. Fish were intraperitoneally injected with glucose at 0, 0.2, 0.5, and 1.0 mg g−1 body mass (BM) and then were exposed to handling for 5 min. Glucose injection resulted in increase of plasma glucose level and liver glycogen content and decrease of plasma lactate level. Handling resulted in increase of plasma levels of cortisol, glucose, and lactate and plasma lactic dehydrogenase (LDH) activity and decrease of liver glycogen content. At 1 h post-stress, the plasma cortisol level was lower in the stressed fish injected with glucose at 0.5 mg g−1 BM than the stressed fish injected with glucose at 0, 0.2, and 1.0 mg g−1 BM. No significant differences were found in the activities of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase (PK) in the liver between the stressed and unstressed fish, regardless of the dose of glucose injection. At 1 h post-stress, the liver glucose-6phosphatase (G6Pase) activity was higher in the fish without glucose injection than in the fish injected with glucose. This study reveals that blood glucose level can D. Jiang : Y. Wu : D. Huang College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang Province 310058, China X. Ren : Y. Wang (*) Ocean College, Zhejiang University, Zhoushan, Zhejiang Province 316021, China e-mail: [email protected]
affect stress response of grass carp by modulating cortisol release and glucose homeostasis through glycogen metabolism and gluconeogenesis in the liver. Keywords Acute stress . Plasma glucose level . Cortisol . Glycemia . Grass carp
Introduction Fish are usually subjected to stress in commercial farming practices since stressors, including handling, transportation, crowding, and noise from aerator, cannot be avoided in intensive aquaculture (Wendelaar Bonga 1997). Stress can reduce growth performance and feed utilization efficiency and impair immune system and reproduction of farmed fish (Mazur and Iwama 1993; Montero et al. 1999). Reducing stress is of considerable significance to the improvement of both welfare and productivity in fish aquaculture (Ashley 2007; Lupatsch et al. 2010). Blood glucose level of fish rapidly increases within a short period after stress primarily due to the activation of hepatic β-adrenoceptors and glycogenolysis (Wendelaar Bonga 1997; Fabbri et al. 1998). Fish subjected to stress challenges increase energy metabolism to cope with stress response, and glucose is used as the main energy fuel (Wendelaar Bonga 1997; Lupatsch et al. 2010). In human, preoperative carbohydrate administration could attenuate cortisol release (Warner et al. 2000; Ljungqvist 2009; Ian et al. 2011; Viganò et al. 2012), post-operative insulin resistance (Ljungqvist et al. 1994; Nygren et al. 1998;
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Henriksen et al. 2003), endogenous glucose release (Soop et al. 2004), and organ dysfunction-associated risk factors (Hoorn et al. 2005). It is conceivable that blood glucose level of fish may affect their stress response. Holloway et al. (1994) reported that plasma cortisol level of rainbow trout Oncorhynchus mykiss increased under hyperglycemic condition after feeding. Powers et al. (2010) reported that hyperglycemia-induced stress resulted in change of cortisol level in embryos (48 h post-fertilization) of zebrafish Danio rerio. Conde-Sieira et al. (2013) reported that cortisol release of rainbow trout was modulated by glucose level [adrenocorticotropic hormone (ACTH)-regulated)] in vitro head kidney tissue and indicated that high glucose level could not regulate cortisol synthesis without ACTH secretion by activating hypothalamic-pituitaryinterrenal (HPI) axis. Gesto et al. (2014) reported that exogenous glucose could decline the activity of glucose6-phosphatase (G6Pase) in the liver of stressed rainbow trout but did not affect cortisol synthesis in head kidney. To our knowledge, the role of blood glucose level in modulating energy generation, cortisol release, and glucose metabolism of farmed fish subjected to acute stress is not well known. It is hypothesized that an appropriate blood glucose level could reduce stress response of fish by inhibiting the release of cortisol and endogenous glucose. This hypothesis remains to be tested. Grass carp (Ctenopharyngodon idella) is one of the Chinese native carps with commercial importance for freshwater aquaculture. Grass carp grow fast but are susceptible to various stressors and diseases. As a herbivore, glucose tolerance of grass carp is higher than that of carnivorous black carp Mylopharyngodon piceus (Huang et al. 2005). In the present study, we evaluated the effect of blood glucose level on plasma levels of cortisol, glucose, and lactate; liver glycogen content; and activities of several key enzymes involved in glucose metabolism in grass carp subjected to handling. The purposes of our study aimed at clarifying the relationship between blood glucose level and stress response in grass carp and identifying if exogenous glucose supplementation could reduce stress response of grass carp.
Materials and methods Fish Grass carp were purchased from a freshwater fish hatchery in Deqing, Huzhou, China. Upon arrival, the fish
were reared in 4000-L outdoor polyethylene tanks for 2 weeks. Prior to the experiment, 256 fish were acclimated in 16 fiberglass tanks (80-cm diameter, 70-cm depth, 300-L volume) of an indoor freshwater circulation system at 16 fish tank−1. The acclimation lasted for 4 weeks, during which the fish were fed to satiation with a formulated feed twice daily. The feed contained 32.6% crude protein, 3.0% crude fat, and 10.8% ash. Water in the tanks circulated at 5 L min−1 and was continuously aerated. Water temperature was 25 ± 2 °C (mean ± SD), and photoperiod was controlled at 12-h light/12-h darkness. Dissolved oxygen (DO) was measured with an 85 DO meter (YSI Scientific Inc., Yellow Springs, OH, USA) and fluctuated from 4.3 to 5.2 mg L−1. Experimental design and sampling procedure A 2 × 4 layout, including two stress treatments (stressed or unstressed) and four levels of glucose injection [0, 0.2, 0.5, and 1.0 mg g −1 body mass (BM)], was designed. Prior to the stress treatment, fish were injected with glucose at 0, 0.2, 0.5, or 1.0 mg g−1 BM (abbreviated as 0G, 0.2G, 0.5G, and 1G), respectively. At 1 h after the injection, the fish accepted acute handling (stressed treatment, abbreviated as S) or were undisturbed (unstressed treatment, abbreviated as NS). Therefore, total eight treatments, abbreviated as 0G-S, 0.2GS, 0.5G-S, 1G-S, 0G-NS, 0.2G-NS, 0.5G-NS, and 1GNS, were established. At the start of the experiment, the acclimated fish were deprived of food for 24 h to achieve the basal hormone and metabolite levels and then were anesthetized with clove oil (60 mg L−1). After the loss of equilibrium, the fish were captured with a dip net, weighed, and injected with either PBS solution (45 mM NaCl, 3 mM Na2HPO4, and 0.6 mM NaH2PO4, pH 8.0) or glucose that was dissolved in PBS solution. All the fish were injected with equal volume (5 mL kg−1 body weight) of PBS or glucose. Initial fish mass was 82.9 ± 18.2 g (mean ± SD). After glucose injection, the fish were distributed in the original tanks and remained undisturbed. Total eight tanks were used as the stressed treatments (0G-S, 0.2G-S, 0.5G-S, and 1G-S), with two tanks (eight fish) for each treatment. At 1 h after glucose injection, 12 fish were captured from one tank with a dip net and exposed in air for 5 min. The stressed fish were randomly distributed into other three tanks at four fish tank−1 for monitoring physiological changes at 1, 2, and 4 h after the handling. Four fish
Fish Physiol Biochem
remained in the original tank were anesthetized and sampled immediately and served as the reference (0 h). Eight tanks were used as the unstressed treatments (0G-NS, 0.2G-NS, 0.5G-NS, and 1G-NS). In each unstressed tank, 16 fish were anesthetized, were captured and randomly distributed into four tanks at four fish tank−1, and then were sampled at 1, 2, and 4 h after the handling. Prior to sample collection, water in the tanks was drawn off slowly until water depth was reduced to 10 cm. Clove oil was added into the tanks at 60 mg L−1 to ensure that all fish lost equilibrium within 2 to 3 min. Blood sample was collected with 1-mL heparinized syringe from the caudal artery of each fish, and each sample was collected within 1 min. The blood samples were centrifuged at 9000 rpm for 5 min, and then, plasma samples were collected. After the collection of plasma samples, the fish were dissected and liver samples were collected. The plasma and liver samples were frozen in liquid nitrogen and stored at −80 °C until assays. Assessment of metabolites and enzyme activities Prior to assay of plasma cortisol, 100 μL plasma was added to 1 mL diethyl ether, mixed using a vortex mixture for several seconds, and then left to rest to separate the organic phase. The extra diethyl ether was evaporated under a gentle stream of nitrogen. Cortisol content in plasma was measured with a commercial ELISA kit (Oxford Biomedical Research, Inc., Oxford, MI, USA). In addition, plasma glucose was measured with a commercial kit (Rsbio, Shanghai, China). Lactate and lactic dehydrogenase (LDH) activity in plasma was measured with commercial kits (Institute of Biological Engineering of Nanjing Jianchen, Nanjing, China). Glycogen content in the liver was measured with the method described by Breer and Rahmann (1974). Prior to assay of activities of the enzymes in the liver, the liver samples were homogenized (1:10 w/v) in icecold buffer (80 mM Tris, 5 mM EDTA, 1 mM KH2PO4, 2 mM NaHCO3, 1.4 mM dithiothreitol, and pH 7.5). The homogenates were centrifuged either at 900×g for 10 min for hexokinase (HK) assay or at 10,000×g for 20 min for assay of pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), and G6Pase. The supernatants were used as crude extracts for enzyme assay. Activities of the enzymes were assayed at previously established saturating concentrations of substrates
using a UV759 ultraviolet spectrophotometer (Shanghai Precision Scientific Instruments Inc., Shanghai, China) at 340 nm and 25 °C. HK (EC 2.7.1.1) activity was measured as described by Tranulis et al. (1996), and PK (EC 2.7.1.40) activity was measured as described by Moon et al. (1985). PEPCK activity was measured using a buffered salt medium (5 mM MnCl2, 0.1 mol L−1 TrisHCl, pH 7.4) in which 2 mM IDP, 1.1 U mL−1 MDH, 2.5 mM PEP, and 0.12 mM NADH were added. G6Pase activity was measured using a buffered salt medium (1.8 mM EDTA, 100 mM imidazole-HCl, pH 7.4) in which 26.5 mM glucose-6-phosphate, 2 mM NAD+, 0.7 U mL−1 mutarotase, and 6 U mL−1 glucose dehydrogenase were added. Protein content was measured as described by Bradford (1976) with bovine serum albumin as a standard. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 μmol of substrate min−1, and activities of the enzymes were expressed as unit per milligram protein. The biochemical reagents, including substrates, coenzymes, and purified enzymes, were purchased from Roche (Mannheim, Germany) and Sigma Chemical Co. (St. Louis, MO, USA). Statistics The data were expressed as means ± standard error (SEM). At the same sampling time, the differences in plasma levels of cortisol, glucose, and lactate; liver glycogen content; LDH activity in plasma; and activities of HK, PK, PEPCK, and G6Pase in the liver between the treatments were examined by a two-way ANOVA, with stress treatment and injected dose of glucose (0, 0.2, 0.5, 1.0 mg g−1 BM) as main factors. When the ANOVA showed a significant effect, post hoc comparisons were carried by using a Bonferroni’s test. The significance level was set at P < 0.05. The statistics was performed with the SPSS software (version 11.5).
Results Plasma levels of cortisol, glucose, and lactate Plasma cortisol level was dependent on stress treatment at 1 h (ANOVA, F[1, 49] = 132.0, P < 0.0001), 2 h (ANOVA, F [1, 49] = 47.8, P < 0.0001), and 4 h (ANOVA, F[1, 51] = 5.4, P = 0.024) post-stress and was dependent on the injected dose of glucose at 0 h
Fish Physiol Biochem
(ANOVA, F[3, 48] = 6.2, P = 0.001), 1 h (ANOVA, F[3, 49] = 7.8, P < 0.0001), and 2 h post-stress (ANOVA, F[3, 49] = 6.9, P = 0.001). Plasma cortisol level was dependent on the interactive effect of stress treatment and injected dose of glucose at 1 h (ANOVA, F[3, 49] = 5.9, P = 0.002) and 2 h (ANOVA, F[3, 49] = 4.1, P = 0.012) post-stress. At 0 h, the plasma cortisol level was higher in 1G-S group than in 0G-S, 0.2G-S, and 0.5G-S groups, while the plasma cortisol level was higher in 1G-NS group than in 0G-NS, 0.2G-NS, and 0.5G-NS groups (P < 0.05; Fig. 1a). At 1 h post-stress, the plasma cortisol level was lower in the 0.5G-S group than in 0GS and 0.2G-S groups and was higher in the 1G-NS group than in 0.2G-NS and 0.5G-NS groups (P < 0.05). At 2 h post-stress, the plasma cortisol level was lower in the 1G-S group than in 0G-S and 0.2G-S groups (P < 0.05). At 1 h post-stress, the plasma cortisol level was higher in the stressed groups than in the unstressed counterparts injected with the same dose of glucose (P < 0.05). At 2 h post-stress, the plasma cortisol level was higher in the stressed groups than in the unstressed counterparts (0G-S vs 0G-NS, 0.2G-S vs 0.2G-NS, and 0.5G-S vs 0.5G-NS, P < 0.05) except 1G-S and 1G-NS groups (P > 0.05). Plasma glucose level was dependent on stress treatment at 1 h (ANOVA, F[1, 53] = 22.3, P < 0.0001), 2 h (ANOVA, F [1, 54] = 61.6, P < 0.0001), and 4 h (ANOVA, F[1, 55] = 54.9, P < 0.0001) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 48] = 135.8, P < 0.0001), 1 h (ANOVA, F[3, 53] = 75.9, P < 0.0001), 2 h (ANOVA, F[3, 54] = 14.8, P < 0.0001), and 4 h (ANOVA, F [3, 55] = 10.4, P < 0.0001) post-stress. Plasma glucose level was dependent on the interactive effect of stress treatment and injected dose of glucose at 2 h (ANOVA, F[3, 54] = 6.8, P = 0.001) and 4 h (ANOVA, F[3, 55] = 6.6, P = 0.001) post-stress. The plasma glucose level increased with the increase of injected dose of glucose at 0 and 1 h poststress (P < 0.05; Fig. 1b). At 1 h post-stress, the plasma glucose level was higher in the stressed groups than in the unstressed counterparts injected with the same dose of glucose (0G-S vs 0G-NS and 0.2G-S vs 0.2G-NS, P < 0.05) except 0.5G-S and 0.5G-NS groups and 1G-S and 1G-NS groups (P > 0.05). At 2 h post-stress, the plasma glucose level was higher in the stressed groups than in the unstressed counterparts (0G-S vs 0G-NS, 0.2G-S vs 0.2G-NS, and 1G-S vs 1G-NS, P < 0.05) except 0.5G-S and 0.5G-NS groups (P > 0.05). At 4 h post-stress, the plasma glucose level was higher in the
stressed groups than in the unstressed counterparts (0.2G-S vs 0.2G-NS, 0.5G-S vs 0.5G-NS, and 1G-S vs 1G-NS, P < 0.05) except 0G-S and 0G-NS groups (P > 0.05). Plasma lactate level was dependent on stress treatment at 1 h (ANOVA, F[1, 55] = 807.3, P < 0.0001), 2 h (ANOVA, F[1, 52] = 532.7, P < 0.0001), and 4 h (ANOVA, F[1, 51] = 29.4, P < 0.0001) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 49] = 31.0, P < 0.0001), 1 h (ANOVA, F[3, 55] = 13.0, P < 0.0001), 2 h (ANOVA, F[3, 52] = 10.1, P < 0.0001), and 4 h (ANOVA, F [3, 51] = 14.9, P < 0.0001) post-stress. Plasma lactate level was dependent on the interactive effect of stress treatment and injected dose of glucose at 1 h (ANOVA, F[3, 55] = 4.9, P = 0.004), 2 h (ANOVA, F[3, 52] = 3.6, P = 0.019), and 4 h (ANOVA, F[3, 51] = 5.9, P = 0.002) post-stress. The plasma lactate level was decreased with the increase of injected dose of glucose at 0 h (P < 0.05; Fig. 1c). At 1 h post-stress, the plasma lactate level was higher in the 1G-NS group than 0G-NS, 0.2G-NS, and 0.5G-NS groups and was higher in the 0G-S group than in the 0.5G-S group (P < 0.05). At 2 h post-stress, the plasma lactate level was lower in 0.5G-S and 1G-S groups than in the 0.2G-S group and lower in the 0.5G-NS group than in the 0.2G-NS group (P < 0.05). At 4 h post-stress, the plasma lactate level was higher in the 1G-S group than in 0G-S, 0.2G-S, and 0.5G-S groups and higher in the 0.5G-NS group than in the 0G-NS group (P < 0.05). At 1 and 2 h post-stress, the plasma lactate level was higher in all stressed groups than in the unstressed counterparts injected with the same dose of glucose (P < 0.05). At 4 h post-stress, the plasma lactate level was higher in the stressed groups than in the unstressed counterparts (0.2G-S vs 0.2G-NS and 1G-S vs 1G-NS, P < 0.05) except 0G-S and 0G-NS groups and 0.5G-S and 0.5G-NS groups (P > 0.05). Liver glycogen content Liver glycogen content was dependent on stress treatment at 2 h (ANOVA, F[1, 56] = 10.8, P = 0.002) and 4 h (ANOVA, F[1, 50] = 22.8, P < 0.0001) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 45] = 8.7, P < 0.0001), 1 h (ANOVA, F[3, 54] = 9.7, P < 0.0001), 2 h (ANOVA, F[3, 56] = 16.8, P < 0.0001), and 4 h (ANOVA, F [3, 50] = 27.5, P < 0.0001) post-stress. Liver glycogen content was dependent on the interactive effect of stress treatment
Fish Physiol Biochem
Fig. 1 Plasma levels of cortisol (a), glucose (b), and lactate (c) in grass carp prior to (0 h) and after handling (1, 2, and 4 h poststress). Fish were injected with glucose at 0 (0G), 0.2 (0.2G), 0.5 (0.5G), or 1.0 mg g−1 (1G) BM. At 1 h after injection, fish were stressed (S) or unstressed (NS). Bars represent mean ± SEM (n = 6–8). The different letters indicate significant differences
between the fish injected with different doses of glucose within the same stress treatment (the capital letter for stressed fish and the small letter for unstressed fish; Bonferroni’s test, P < 0.05). The asterisks indicate significant differences between the stressed and unstressed fish injected with the same injected dose of glucose (Bonferroni’s test, P < 0.05)
and injected dose of glucose at 2 h (ANOVA, F[3, 56] = 6.8, P = 0.001) and 4 h (ANOVA, F[3, 50] = 7.2, P < 0.0001) post-stress. The liver glycogen content was higher in 0.2G-NS, 0.5G-NS, and 1G-NS groups than in the 0G-NS group at 0 h (P < 0.05; Fig. 2). At 1 h post-
stress, the liver glycogen content was higher in 0.5G-S and 1G-S groups than in the 0G-S group and higher in the 1G-NS group than in the 0G-NS group (P < 0.05). At 2 and 4 h post-stress, the liver glycogen content in the unstressed fish was increased with the increase of
Fish Physiol Biochem
Fig. 2 Liver glycogen content of grass carp prior to (0 h) and after handling (1, 2, and 4 h post-stress). Fish were injected with glucose at 0 (0G), 0.2 (0.2G), 0.5 (0.5G), or 1.0 mg g−1 (1G) BM. At 1 h after injection, fish were stressed (S) or unstressed (NS). Bars represent mean ± SEM (n = 6–8). The different letters indicate significant differences between the fish injected with different
doses of glucose within the same stress treatment (the capital letter for stressed fish and the small letter for unstressed fish; Bonferroni’s test, P < 0.05). The asterisks indicate significant differences between the stressed and unstressed fish injected with the same injected dose of glucose (Bonferroni’s test, P < 0.05)
injected dose of glucose (P < 0.05), while the liver glycogen content was lower in the 1G-S group than in the 1G-NS group (P < 0.05). At 4 h post-stress, the liver glycogen content was higher in the 1G-S group than in the 0G-S group (P < 0.05).
with the increase of injected dose of glucose (P < 0.05), and the plasma LDH activity was higher in 0.2G-NS, 0.5G-NS, and 1G-NS groups than in the 0G-NS group (P < 0.05). Liver G6Pase activity was dependent on the injected dose of glucose at 1 h (ANOVA, F [3, 54] = 2.9, P = 0.041) post-stress and was dependent on the interactive effect of stress treatment and injected dose of glucose at 1 h (ANOVA, F[3, 54] = 3.3, P = 0.027) and 2 h (ANOVA, F[3, 56] = 3.4, P = 0.023) post-stress. At 1 h post-stress, the liver G6Pase activity was lower in 0.2G-S, 0.5G-S, and 1G-S groups than in the 0G-S group (P < 0.05; Fig. 3c). At 2 h post-stress, the liver G6Pase activity was lower in the 1G-S group than in the 0G-S group (P < 0.05). At 1 and 2 h post-stress, the liver G6Pase activity was higher in the 0G-S group than in the 0G-NS group (P < 0.05). The liver HK activity was dependent on stress treatment at 1 h post-stress (ANOVA, F[1, 48] = 9.0, P = 0.005). At 1 h post-stress,
Activities of the enzymes Plasma LDH activity was dependent on stress treatment at 2 h (ANOVA, F[1, 48] = 14.2, P = 0.001) and 4 h (ANOVA, F[1, 49] = 4.2, P = 0.047) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 49] = 9.9, P < 0.0001), 1 h (ANOVA, F[3, 54] = 5.7, P = 0.002), 2 h (ANOVA, F[3, 48] = 5.4, P = 0.003), and 4 h (ANOVA, F [3, 49] = 14.8, P < 0.0001) post-stress. At 0 h, the plasma LDH activity was higher in the 0.5G-S group than in 0G-S and 0.2G-S groups, while the plasma LDH activity was higher in the 0.5G-NS group than in 0G-NS and 0.2G-NS groups (P < 0.05; Fig. 3a). At 1 h post-stress, the plasma LDH activity was higher in 0.2G-S and 1G-S groups than in the 0G-S group (P < 0.05). At 2 h post-stress, the plasma LDH activity was higher in the 0.2G-S group than in the 0G-S group (P < 0.05), while the plasma LDH activity was higher in the stressed groups than in the unstressed groups injected with the same dose of glucose (0.2G-S vs 0.2G-NS and 1G-S vs 1G-NS, P < 0.05) except 0G-S and 0G-NS groups and 0.5G-S and 0.5G-NS groups (P > 0.05). At 4 h post-stress, the plasma LDH activity was increased in the stressed fish
Fig. 3 Activity of LDH in plasma (a) and activities of PEPCK (b), G6Pase (c), HK (d), and PK (e) in liver of grass carp prior to (0 h) and after handling (1, 2, and 4 h post-stress). Fish were injected with glucose at 0 (0G), 0.2 (0.2G), 0.5 (0.5G), or 1.0 mg g−1 (1G) BM. At 1 h after injection, fish were stressed (S) or unstressed (NS). Bars represent mean ± SEM (n = 6–8). The different letters indicate significant differences between the fish injected with different doses of glucose within the same stress treatment (the capital letter for stressed fish and the small letter for unstressed fish; Bonferroni’s test, P < 0.05). The asterisks indicate significant differences between the stressed and unstressed fish injected with the same injected dose of glucose (Bonferroni’s test, P < 0.05)
Fish Physiol Biochem
Fish Physiol Biochem
the liver HK activity was higher in the 0.2G-S group than in the 0.2G-NS group, and the liver HK activity was higher in the 1G-S group than in the 1G-NS group (P < 0.05; Fig. 3d). No significant differences were found in the activities of PEPCK and PK in the liver between the stressed and unstressed groups injected with the same dose of glucose or between the groups injected with different doses of glucose within each stress treatment (P > 0.05; Fig. 3b, e).
Discussion Most fish species exhibit impaired glucose tolerance, and prolonged hyperglycemia is commonly observed in fish after an acute glucose load (Moon 2001). Intraperitoneal injection of glucose has been used to increase blood glucose level in fishes, such as rainbow trout (Harmon et al. 1991), white sea bream Diplodus sargus (Enes et al. 2012), European sea bass Dicentrarchus labrax (Peres et al. 1999), and turbot Scophthalmus maximus (GarciaRiera and Hemre 2006). In the present study, the interval time between glucose injection and handling challenge was designed based on reports of earlier studies to ensure that the effect of exogenous glucose supplementation on stress response of grass carp could be detected. The plasma glucose level increased with the increase of injected dose of glucose at 0 h, suggesting that intraperitoneal injection of glucose can significantly increase plasma glucose level of grass carp. The effect of glucose level on cortisol release in fish remains to be clarified. Earlier studies reported that high glucose levels resulted in more cortisol generation (ACTH-dependent) in head kidney of rainbow trout in vitro (Holloway et al. 1994; Conde-Sieira et al. 2013). These results reveal that blood glucose level could modulate cortisol release in fish, and high glucose levels could result in high cortisol levels. Gesto et al. (2014) reported that plasma cortisol level in rainbow trout injected with glucose at 0.2 mg g−1 BM did not change in parallel with the changes in glycemia. In the present study, the plasma cortisol level was higher in the fish injected with glucose at 1.0 mg g−1 BM than in the fish injected with glucose at 0, 0.2, and 0.5 mg g−1 BM at 0 h, suggesting that high glucose injection dose could increase plasma cortisol level of grass carp. The plasma cortisol level steeply climbed in the stressed fish at 1 h post-stress, and then gradually declined. At 1 h post-stress, the plasma cortisol level was lower in the
stressed fish injected with 0.5 mg g−1 BM glucose than in the stressed fish injected with glucose at 0 and 0.2 mg g−1 BM. This result suggests that 0.5 mg g−1 BM glucose injection might benefit to suppress cortisol release of grass carp subjected to handling. At 4 h poststress, no significant difference was found in the plasma cortisol level between the stressed fish and unstressed fish injected with same dose of glucose, suggesting that the effect of handling on plasma cortisol of grass carp might not exceed 4 h. According to results of the previous and present study, it is concluded that the effect of blood glucose level on plasma cortisol of grass carp subjected to handling is dose dependent. Appropriate blood glucose level (0.5 mg g−1 BM) could inhibit cortisol release in grass carp subjected to acute stress, but abnormally high blood glucose level (1.0 mg g−1 BM) might stimulate cortisol release. The mechanisms by which blood glucose regulates cortisol release in fish warrant more studies. It has been demonstrated that the plasma lactate level increased in fish subjected to stress due to energy was exhausted in short time and fish need to generate energy through anaerobic metabolism (Pottinger 1998; De Boeck et al. 2001). In the present study, the fish injected with glucose at 0.5 and 1.0 mg g−1 BM showed lower plasma lactate level but higher LDH activity at 0 h, suggesting that high blood glucose level might result in increase of lactate generation. At 1 and 2 h post-stress, the plasma lactate level significantly increased in the stressed fish relative to the unstressed fish. At 4 h poststress, the plasma lactate level was still higher in the stressed fish injected with glucose at 1.0 mg g−1 BM than in the unstressed counterpart, while no significant difference was found in the plasma lactate level between the stressed and unstressed fish injected with glucose at 0 and 0.5 mg g−1 BM. These results reveal that hyperglycemia might enhance anaerobic metabolism in the stressed grass carp. In mammal, a major fraction of glycogen deposited in the liver is converted from exogenous carbohydrate (Newgard et al. 1983). The effect of exogenous carbohydrate on liver glycogen varied among fish species. Intraperitoneal injection of glucose did not significantly affect liver glycogen content in European sea bass (Enes et al. 2011), but resulted in increase of liver glycogen content in white sea bream (Enes et al. 2012). In the present study, liver glycogen content was higher in fish injected with glucose than in fish without glucose injection, suggesting that the exogenous glucose
Fish Physiol Biochem
supplementation could be partly converted into liver glycogen to maintain glucose homeostasis in grass carp under a hyperglycemic condition. At 2 and 4 h poststress, liver glycogen content was dramatically reduced in the fish injected with glucose at 1.0 mg g−1 BM, suggesting that grass carp could utilize liver glycogen as energy fuel in priority when subjected to stress challenge. This result is consistent with the reports that liver glycogen content significantly decreased in the stressed fish for glucose generation (Wendelaar Bonga 1997; López-Patiño et al. 2014). It has been demonstrated that acute stress could activate gluconeogenesis through cortisol modulation (Mommsen et al. 1999; Vijayan et al. 2003). In the present study, liver G6Pase activity was higher in the stressed than unstressed fish without glucose injection, which was not detected in fish with glucose injection. Moreover, the activity of liver G6Pase was lower in the stressed fish with glucose injection compared to the stressed fish without glucose injection at 1 and 2 h post-stress. These results suggest that hyperglycemic situation might attenuate gluconeogenesis in the liver during stress challenge. Guignot and Mithieux (1999) reported that hyperglycemia could suppress hepatic glucose generation in rats by inhibiting G6Pase activity. The response of gluconeogenesis in the liver to hyperglycemic and stress challenge might be a mechanism for maintaining glucose homeostasis in fishes. The attenuation of gluconeogenesis in the liver of grass carp under a hyperglycemic and stressed condition should be attributed to the complex regulation of cortisol, catecholamine, and pancreatic hormones. Earlier studies reported that activities of glycolytic enzymes did not significantly increase in rainbow trout subjected to stress (Morales et al. 1990; López-Patiño et al. 2014). In the present study, the liver HK activity was higher in the stressed fish injected with glucose at 0.2 and 1.0 mg g−1 BM than in unstressed counterpart at 1 h post-stress, suggesting that plasma glucose level might stimulate the liver glycolysis in grass carp after handling. Oppositely, no significant differences were found in the PEPCK and PK activities in the liver among the fish injected with different doses of glucose, regardless of the stressed and unstressed fish. These results indicate that glycemia and acute handling stress could not significantly change the activity of glycolytic enzymes in grass carp. In conclusion, blood glucose level could affect the plasma levels of cortisol, glucose, and lactate; liver
glycogen content; LDH activity in plasma; and G6Pase activity in the liver of stressed grass carp, but had limited effect on activity of HK, PK, and PEPCK in the liver. Glucose supplementation at 0.5 mg g−1 BM can benefit to reduce stress response to handing in grass carp by modulating glycogen metabolism and cortisol release, and hyperglycemic might attenuate gluconeogenesis in the liver. Acknowledgements This research was funded by the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303056-2). The authors thank Dr. Jinyu Tang (Zhejiang University) and Dr. Xiafei Zheng (Zhejiang University) for their assistance in preparing fish feed and sampling blood samples.
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Fish Physiol Biochem Henriksen MG, Hessov I, Dela F, Hansen HV, Haraldsted V, Rodt SÅ (2003) Effects of preoperative oral carbohydrates and peptides on postoperative endocrine response, mobilization, nutrition and muscle function in abdominal surgery. Acta Anaesth Scand 47:191–199 Holloway AC, Reddy PK, Sheridan MA, Leatherland JF (1994) Diurnal rhythms of plasma growth hormone, somatostatin, thyroid hormones, cortisol and glucose concentrations in rainbow trout, Oncorhynchus mykiss, during progressive food deprivation. Biol Rhythm Res 25:415–432 Hoorn ECV et al (2005) Preoperative supplementation with a carbohydrate mixture decreases organ dysfunctionassociated risk factors. Clin Nutr 24:114–123 Huang HZ, Ding L, Song XH, Wang YL, Yang CG (2005) Comparative research on glucose tolerance betweem black carp Mylopharyngodon piceus and grass carp Ctenopharyngodon idellus. J Fish Sci China 12:496–500 Ian S et al (2011) Perioperative fasting in adults and children: guidelines from the European Society of Anaesthesiology. Eur J Anaesthesiol 28:556–569 López-Patiño MA, Hernández-Pérez J, Gesto M, Librán-Pérez M, Míguez JM, Soengas JL (2014) Short-term time course of liver metabolic response to acute handling stress in rainbow trout, Oncorhynchus mykiss. Comp Biochem Physiol A 168: 40–49 Ljungqvist O (2009) Modulating postoperative insulin resistance by preoperative carbohydrate loading. Best Pract Res Cl An 23:401–409 Ljungqvist O, Thorell A, Gutniak M, Häggmark T, Efendic S (1994) Glucose infusion instead of preoperative fasting reduces postoperative insulin resistance. J Am Coll Surgeons 178:329–336 Lupatsch I, Santos GA, Schrama JW, Verreth JAJ (2010) Effect of stocking density and feeding level on energy expenditure and stress responsiveness in European sea bass Dicentrarchus labrax. Aquaculture 298:245–250 Mazur CF, Iwama GK (1993) Handling and crowding stress reduces number of plaque-forming cells in Atlantic salmon. J Aquat Anim Health 5:98–101 Mommsen TP, Vijayan MM, Moon TW (1999) Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Rev Fish Biol Fisher 9:211–268 Montero D, Marrero M, Izquierdo M, Robaina L, Vergara J, Tort L (1999) Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture 171:269–278
Moon TW (2001) Glucose intolerance in teleost fish: fact or fiction? Comp Biochem Physiol B 129:243–249 Moon TW, Walsh PJ, Mommsen TP (1985) Fish hepatocytes: a model metabolic system. Can J Fish Aquat Sci 42:1772– 1782 Morales AE, García-Rejón L, Higuera L (1990) Influence of handling and/or anaesthesia on stress response in rainbow trout. Effects on liver primary metabolism. Comp Biochem Physiol A 95:87–93 Newgard CB, Hirsch LJ, Foster DW, Mcgarry JD (1983) Studies on the mechanism by which exogenous glucose is converted to liver glycogen in the rat: a direct or an indirect pathway. J Biol Chem 258:8046–8052 Nygren J, Soop M, Thorell A, Efendic S, Nair KS, Ljungqvist O (1998) Preoperative oral carbohydrate administration reduces postoperative insulin resistance. Clin Nutr 17:65–71 Peres H, Gonçalves P, Oliva-Teles A (1999) Glucose tolerance in gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax). Aquaculture 179:415–423 Pottinger TG (1998) Changes in blood cortisol, glucose and lactate in carp retained in anglers’ keepnets. J Fish Biol 53:728–742 Powers JW, Mazilu JK, Lin S, McCabe ERB (2010) The effects of hyperglycemia on adrenal cortex function and steroidogenesis in the zebrafish. Mol Genet Metab 101:421–422 Soop M, Nygren J, Thorell A, Weidenhielm L, Lundberg M, Hammarqvist F, Ljungqvist O (2004) Preoperative oral carbohydrate treatment attenuates endogenous glucose release 3 days after surgery. Clin Nutr 23:733–741 Tranulis MA, Dregni O, Christophersen B, Krogdahl Å, Borrebaek B (1996) A glucokinase-like enzyme in the liver of Atlantic salmon (Salmo salar). Comp Biochem Physiol B 114:35–39 Viganò J, Cereda E, Caccialanza R, Carini R, Cameletti B, Spampinato M, Dionigi P (2012) Effects of preoperative oral carbohydrate supplementation on postoperative metabolic stress response of patients undergoing elective abdominal surgery. World J Surg 36:1738–1743 Vijayan MM, Raptis S, Sathiyaa R (2003) Cortisol treatment affects glucocorticoid receptor and glucocorticoidresponsive genes in the liver of rainbow trout. Gen Comp Endocr 132:256–263 Warner MA et al (2000) Practice guidelines for preoperative fasting and the use of pharmacologic agents to reduce the risk of pulmonary aspiration: application to healthy patients undergoing elective procedures. Surv Anesthesiol 44:47–48 Wendelaar Bonga SE (1997) The stress response in fish. Physiol Rev 77:591–625
Effect of blood glucose level on acute stress response of grass carp Ctenopharyngodon idella Danli Jiang & Yubo Wu & Di Huang & Xing Ren & Yan Wang
Received: 12 October 2016 / Accepted: 8 May 2017 # Springer Science+Business Media Dordrecht 2017
Abstract Stress has a considerable impact on welfare and productivity of fish, and blood glucose level of fish may be a factor modulating stress response. This study evaluated the effect of blood glucose level and handling on acute stress response of grass carp Ctenopharyngodon idella. Fish were intraperitoneally injected with glucose at 0, 0.2, 0.5, and 1.0 mg g−1 body mass (BM) and then were exposed to handling for 5 min. Glucose injection resulted in increase of plasma glucose level and liver glycogen content and decrease of plasma lactate level. Handling resulted in increase of plasma levels of cortisol, glucose, and lactate and plasma lactic dehydrogenase (LDH) activity and decrease of liver glycogen content. At 1 h post-stress, the plasma cortisol level was lower in the stressed fish injected with glucose at 0.5 mg g−1 BM than the stressed fish injected with glucose at 0, 0.2, and 1.0 mg g−1 BM. No significant differences were found in the activities of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase (PK) in the liver between the stressed and unstressed fish, regardless of the dose of glucose injection. At 1 h post-stress, the liver glucose-6phosphatase (G6Pase) activity was higher in the fish without glucose injection than in the fish injected with glucose. This study reveals that blood glucose level can D. Jiang : Y. Wu : D. Huang College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang Province 310058, China X. Ren : Y. Wang (*) Ocean College, Zhejiang University, Zhoushan, Zhejiang Province 316021, China e-mail: [email protected]
affect stress response of grass carp by modulating cortisol release and glucose homeostasis through glycogen metabolism and gluconeogenesis in the liver. Keywords Acute stress . Plasma glucose level . Cortisol . Glycemia . Grass carp
Introduction Fish are usually subjected to stress in commercial farming practices since stressors, including handling, transportation, crowding, and noise from aerator, cannot be avoided in intensive aquaculture (Wendelaar Bonga 1997). Stress can reduce growth performance and feed utilization efficiency and impair immune system and reproduction of farmed fish (Mazur and Iwama 1993; Montero et al. 1999). Reducing stress is of considerable significance to the improvement of both welfare and productivity in fish aquaculture (Ashley 2007; Lupatsch et al. 2010). Blood glucose level of fish rapidly increases within a short period after stress primarily due to the activation of hepatic β-adrenoceptors and glycogenolysis (Wendelaar Bonga 1997; Fabbri et al. 1998). Fish subjected to stress challenges increase energy metabolism to cope with stress response, and glucose is used as the main energy fuel (Wendelaar Bonga 1997; Lupatsch et al. 2010). In human, preoperative carbohydrate administration could attenuate cortisol release (Warner et al. 2000; Ljungqvist 2009; Ian et al. 2011; Viganò et al. 2012), post-operative insulin resistance (Ljungqvist et al. 1994; Nygren et al. 1998;
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Henriksen et al. 2003), endogenous glucose release (Soop et al. 2004), and organ dysfunction-associated risk factors (Hoorn et al. 2005). It is conceivable that blood glucose level of fish may affect their stress response. Holloway et al. (1994) reported that plasma cortisol level of rainbow trout Oncorhynchus mykiss increased under hyperglycemic condition after feeding. Powers et al. (2010) reported that hyperglycemia-induced stress resulted in change of cortisol level in embryos (48 h post-fertilization) of zebrafish Danio rerio. Conde-Sieira et al. (2013) reported that cortisol release of rainbow trout was modulated by glucose level [adrenocorticotropic hormone (ACTH)-regulated)] in vitro head kidney tissue and indicated that high glucose level could not regulate cortisol synthesis without ACTH secretion by activating hypothalamic-pituitaryinterrenal (HPI) axis. Gesto et al. (2014) reported that exogenous glucose could decline the activity of glucose6-phosphatase (G6Pase) in the liver of stressed rainbow trout but did not affect cortisol synthesis in head kidney. To our knowledge, the role of blood glucose level in modulating energy generation, cortisol release, and glucose metabolism of farmed fish subjected to acute stress is not well known. It is hypothesized that an appropriate blood glucose level could reduce stress response of fish by inhibiting the release of cortisol and endogenous glucose. This hypothesis remains to be tested. Grass carp (Ctenopharyngodon idella) is one of the Chinese native carps with commercial importance for freshwater aquaculture. Grass carp grow fast but are susceptible to various stressors and diseases. As a herbivore, glucose tolerance of grass carp is higher than that of carnivorous black carp Mylopharyngodon piceus (Huang et al. 2005). In the present study, we evaluated the effect of blood glucose level on plasma levels of cortisol, glucose, and lactate; liver glycogen content; and activities of several key enzymes involved in glucose metabolism in grass carp subjected to handling. The purposes of our study aimed at clarifying the relationship between blood glucose level and stress response in grass carp and identifying if exogenous glucose supplementation could reduce stress response of grass carp.
Materials and methods Fish Grass carp were purchased from a freshwater fish hatchery in Deqing, Huzhou, China. Upon arrival, the fish
were reared in 4000-L outdoor polyethylene tanks for 2 weeks. Prior to the experiment, 256 fish were acclimated in 16 fiberglass tanks (80-cm diameter, 70-cm depth, 300-L volume) of an indoor freshwater circulation system at 16 fish tank−1. The acclimation lasted for 4 weeks, during which the fish were fed to satiation with a formulated feed twice daily. The feed contained 32.6% crude protein, 3.0% crude fat, and 10.8% ash. Water in the tanks circulated at 5 L min−1 and was continuously aerated. Water temperature was 25 ± 2 °C (mean ± SD), and photoperiod was controlled at 12-h light/12-h darkness. Dissolved oxygen (DO) was measured with an 85 DO meter (YSI Scientific Inc., Yellow Springs, OH, USA) and fluctuated from 4.3 to 5.2 mg L−1. Experimental design and sampling procedure A 2 × 4 layout, including two stress treatments (stressed or unstressed) and four levels of glucose injection [0, 0.2, 0.5, and 1.0 mg g −1 body mass (BM)], was designed. Prior to the stress treatment, fish were injected with glucose at 0, 0.2, 0.5, or 1.0 mg g−1 BM (abbreviated as 0G, 0.2G, 0.5G, and 1G), respectively. At 1 h after the injection, the fish accepted acute handling (stressed treatment, abbreviated as S) or were undisturbed (unstressed treatment, abbreviated as NS). Therefore, total eight treatments, abbreviated as 0G-S, 0.2GS, 0.5G-S, 1G-S, 0G-NS, 0.2G-NS, 0.5G-NS, and 1GNS, were established. At the start of the experiment, the acclimated fish were deprived of food for 24 h to achieve the basal hormone and metabolite levels and then were anesthetized with clove oil (60 mg L−1). After the loss of equilibrium, the fish were captured with a dip net, weighed, and injected with either PBS solution (45 mM NaCl, 3 mM Na2HPO4, and 0.6 mM NaH2PO4, pH 8.0) or glucose that was dissolved in PBS solution. All the fish were injected with equal volume (5 mL kg−1 body weight) of PBS or glucose. Initial fish mass was 82.9 ± 18.2 g (mean ± SD). After glucose injection, the fish were distributed in the original tanks and remained undisturbed. Total eight tanks were used as the stressed treatments (0G-S, 0.2G-S, 0.5G-S, and 1G-S), with two tanks (eight fish) for each treatment. At 1 h after glucose injection, 12 fish were captured from one tank with a dip net and exposed in air for 5 min. The stressed fish were randomly distributed into other three tanks at four fish tank−1 for monitoring physiological changes at 1, 2, and 4 h after the handling. Four fish
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remained in the original tank were anesthetized and sampled immediately and served as the reference (0 h). Eight tanks were used as the unstressed treatments (0G-NS, 0.2G-NS, 0.5G-NS, and 1G-NS). In each unstressed tank, 16 fish were anesthetized, were captured and randomly distributed into four tanks at four fish tank−1, and then were sampled at 1, 2, and 4 h after the handling. Prior to sample collection, water in the tanks was drawn off slowly until water depth was reduced to 10 cm. Clove oil was added into the tanks at 60 mg L−1 to ensure that all fish lost equilibrium within 2 to 3 min. Blood sample was collected with 1-mL heparinized syringe from the caudal artery of each fish, and each sample was collected within 1 min. The blood samples were centrifuged at 9000 rpm for 5 min, and then, plasma samples were collected. After the collection of plasma samples, the fish were dissected and liver samples were collected. The plasma and liver samples were frozen in liquid nitrogen and stored at −80 °C until assays. Assessment of metabolites and enzyme activities Prior to assay of plasma cortisol, 100 μL plasma was added to 1 mL diethyl ether, mixed using a vortex mixture for several seconds, and then left to rest to separate the organic phase. The extra diethyl ether was evaporated under a gentle stream of nitrogen. Cortisol content in plasma was measured with a commercial ELISA kit (Oxford Biomedical Research, Inc., Oxford, MI, USA). In addition, plasma glucose was measured with a commercial kit (Rsbio, Shanghai, China). Lactate and lactic dehydrogenase (LDH) activity in plasma was measured with commercial kits (Institute of Biological Engineering of Nanjing Jianchen, Nanjing, China). Glycogen content in the liver was measured with the method described by Breer and Rahmann (1974). Prior to assay of activities of the enzymes in the liver, the liver samples were homogenized (1:10 w/v) in icecold buffer (80 mM Tris, 5 mM EDTA, 1 mM KH2PO4, 2 mM NaHCO3, 1.4 mM dithiothreitol, and pH 7.5). The homogenates were centrifuged either at 900×g for 10 min for hexokinase (HK) assay or at 10,000×g for 20 min for assay of pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), and G6Pase. The supernatants were used as crude extracts for enzyme assay. Activities of the enzymes were assayed at previously established saturating concentrations of substrates
using a UV759 ultraviolet spectrophotometer (Shanghai Precision Scientific Instruments Inc., Shanghai, China) at 340 nm and 25 °C. HK (EC 2.7.1.1) activity was measured as described by Tranulis et al. (1996), and PK (EC 2.7.1.40) activity was measured as described by Moon et al. (1985). PEPCK activity was measured using a buffered salt medium (5 mM MnCl2, 0.1 mol L−1 TrisHCl, pH 7.4) in which 2 mM IDP, 1.1 U mL−1 MDH, 2.5 mM PEP, and 0.12 mM NADH were added. G6Pase activity was measured using a buffered salt medium (1.8 mM EDTA, 100 mM imidazole-HCl, pH 7.4) in which 26.5 mM glucose-6-phosphate, 2 mM NAD+, 0.7 U mL−1 mutarotase, and 6 U mL−1 glucose dehydrogenase were added. Protein content was measured as described by Bradford (1976) with bovine serum albumin as a standard. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 μmol of substrate min−1, and activities of the enzymes were expressed as unit per milligram protein. The biochemical reagents, including substrates, coenzymes, and purified enzymes, were purchased from Roche (Mannheim, Germany) and Sigma Chemical Co. (St. Louis, MO, USA). Statistics The data were expressed as means ± standard error (SEM). At the same sampling time, the differences in plasma levels of cortisol, glucose, and lactate; liver glycogen content; LDH activity in plasma; and activities of HK, PK, PEPCK, and G6Pase in the liver between the treatments were examined by a two-way ANOVA, with stress treatment and injected dose of glucose (0, 0.2, 0.5, 1.0 mg g−1 BM) as main factors. When the ANOVA showed a significant effect, post hoc comparisons were carried by using a Bonferroni’s test. The significance level was set at P < 0.05. The statistics was performed with the SPSS software (version 11.5).
Results Plasma levels of cortisol, glucose, and lactate Plasma cortisol level was dependent on stress treatment at 1 h (ANOVA, F[1, 49] = 132.0, P < 0.0001), 2 h (ANOVA, F [1, 49] = 47.8, P < 0.0001), and 4 h (ANOVA, F[1, 51] = 5.4, P = 0.024) post-stress and was dependent on the injected dose of glucose at 0 h
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(ANOVA, F[3, 48] = 6.2, P = 0.001), 1 h (ANOVA, F[3, 49] = 7.8, P < 0.0001), and 2 h post-stress (ANOVA, F[3, 49] = 6.9, P = 0.001). Plasma cortisol level was dependent on the interactive effect of stress treatment and injected dose of glucose at 1 h (ANOVA, F[3, 49] = 5.9, P = 0.002) and 2 h (ANOVA, F[3, 49] = 4.1, P = 0.012) post-stress. At 0 h, the plasma cortisol level was higher in 1G-S group than in 0G-S, 0.2G-S, and 0.5G-S groups, while the plasma cortisol level was higher in 1G-NS group than in 0G-NS, 0.2G-NS, and 0.5G-NS groups (P < 0.05; Fig. 1a). At 1 h post-stress, the plasma cortisol level was lower in the 0.5G-S group than in 0GS and 0.2G-S groups and was higher in the 1G-NS group than in 0.2G-NS and 0.5G-NS groups (P < 0.05). At 2 h post-stress, the plasma cortisol level was lower in the 1G-S group than in 0G-S and 0.2G-S groups (P < 0.05). At 1 h post-stress, the plasma cortisol level was higher in the stressed groups than in the unstressed counterparts injected with the same dose of glucose (P < 0.05). At 2 h post-stress, the plasma cortisol level was higher in the stressed groups than in the unstressed counterparts (0G-S vs 0G-NS, 0.2G-S vs 0.2G-NS, and 0.5G-S vs 0.5G-NS, P < 0.05) except 1G-S and 1G-NS groups (P > 0.05). Plasma glucose level was dependent on stress treatment at 1 h (ANOVA, F[1, 53] = 22.3, P < 0.0001), 2 h (ANOVA, F [1, 54] = 61.6, P < 0.0001), and 4 h (ANOVA, F[1, 55] = 54.9, P < 0.0001) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 48] = 135.8, P < 0.0001), 1 h (ANOVA, F[3, 53] = 75.9, P < 0.0001), 2 h (ANOVA, F[3, 54] = 14.8, P < 0.0001), and 4 h (ANOVA, F [3, 55] = 10.4, P < 0.0001) post-stress. Plasma glucose level was dependent on the interactive effect of stress treatment and injected dose of glucose at 2 h (ANOVA, F[3, 54] = 6.8, P = 0.001) and 4 h (ANOVA, F[3, 55] = 6.6, P = 0.001) post-stress. The plasma glucose level increased with the increase of injected dose of glucose at 0 and 1 h poststress (P < 0.05; Fig. 1b). At 1 h post-stress, the plasma glucose level was higher in the stressed groups than in the unstressed counterparts injected with the same dose of glucose (0G-S vs 0G-NS and 0.2G-S vs 0.2G-NS, P < 0.05) except 0.5G-S and 0.5G-NS groups and 1G-S and 1G-NS groups (P > 0.05). At 2 h post-stress, the plasma glucose level was higher in the stressed groups than in the unstressed counterparts (0G-S vs 0G-NS, 0.2G-S vs 0.2G-NS, and 1G-S vs 1G-NS, P < 0.05) except 0.5G-S and 0.5G-NS groups (P > 0.05). At 4 h post-stress, the plasma glucose level was higher in the
stressed groups than in the unstressed counterparts (0.2G-S vs 0.2G-NS, 0.5G-S vs 0.5G-NS, and 1G-S vs 1G-NS, P < 0.05) except 0G-S and 0G-NS groups (P > 0.05). Plasma lactate level was dependent on stress treatment at 1 h (ANOVA, F[1, 55] = 807.3, P < 0.0001), 2 h (ANOVA, F[1, 52] = 532.7, P < 0.0001), and 4 h (ANOVA, F[1, 51] = 29.4, P < 0.0001) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 49] = 31.0, P < 0.0001), 1 h (ANOVA, F[3, 55] = 13.0, P < 0.0001), 2 h (ANOVA, F[3, 52] = 10.1, P < 0.0001), and 4 h (ANOVA, F [3, 51] = 14.9, P < 0.0001) post-stress. Plasma lactate level was dependent on the interactive effect of stress treatment and injected dose of glucose at 1 h (ANOVA, F[3, 55] = 4.9, P = 0.004), 2 h (ANOVA, F[3, 52] = 3.6, P = 0.019), and 4 h (ANOVA, F[3, 51] = 5.9, P = 0.002) post-stress. The plasma lactate level was decreased with the increase of injected dose of glucose at 0 h (P < 0.05; Fig. 1c). At 1 h post-stress, the plasma lactate level was higher in the 1G-NS group than 0G-NS, 0.2G-NS, and 0.5G-NS groups and was higher in the 0G-S group than in the 0.5G-S group (P < 0.05). At 2 h post-stress, the plasma lactate level was lower in 0.5G-S and 1G-S groups than in the 0.2G-S group and lower in the 0.5G-NS group than in the 0.2G-NS group (P < 0.05). At 4 h post-stress, the plasma lactate level was higher in the 1G-S group than in 0G-S, 0.2G-S, and 0.5G-S groups and higher in the 0.5G-NS group than in the 0G-NS group (P < 0.05). At 1 and 2 h post-stress, the plasma lactate level was higher in all stressed groups than in the unstressed counterparts injected with the same dose of glucose (P < 0.05). At 4 h post-stress, the plasma lactate level was higher in the stressed groups than in the unstressed counterparts (0.2G-S vs 0.2G-NS and 1G-S vs 1G-NS, P < 0.05) except 0G-S and 0G-NS groups and 0.5G-S and 0.5G-NS groups (P > 0.05). Liver glycogen content Liver glycogen content was dependent on stress treatment at 2 h (ANOVA, F[1, 56] = 10.8, P = 0.002) and 4 h (ANOVA, F[1, 50] = 22.8, P < 0.0001) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 45] = 8.7, P < 0.0001), 1 h (ANOVA, F[3, 54] = 9.7, P < 0.0001), 2 h (ANOVA, F[3, 56] = 16.8, P < 0.0001), and 4 h (ANOVA, F [3, 50] = 27.5, P < 0.0001) post-stress. Liver glycogen content was dependent on the interactive effect of stress treatment
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Fig. 1 Plasma levels of cortisol (a), glucose (b), and lactate (c) in grass carp prior to (0 h) and after handling (1, 2, and 4 h poststress). Fish were injected with glucose at 0 (0G), 0.2 (0.2G), 0.5 (0.5G), or 1.0 mg g−1 (1G) BM. At 1 h after injection, fish were stressed (S) or unstressed (NS). Bars represent mean ± SEM (n = 6–8). The different letters indicate significant differences
between the fish injected with different doses of glucose within the same stress treatment (the capital letter for stressed fish and the small letter for unstressed fish; Bonferroni’s test, P < 0.05). The asterisks indicate significant differences between the stressed and unstressed fish injected with the same injected dose of glucose (Bonferroni’s test, P < 0.05)
and injected dose of glucose at 2 h (ANOVA, F[3, 56] = 6.8, P = 0.001) and 4 h (ANOVA, F[3, 50] = 7.2, P < 0.0001) post-stress. The liver glycogen content was higher in 0.2G-NS, 0.5G-NS, and 1G-NS groups than in the 0G-NS group at 0 h (P < 0.05; Fig. 2). At 1 h post-
stress, the liver glycogen content was higher in 0.5G-S and 1G-S groups than in the 0G-S group and higher in the 1G-NS group than in the 0G-NS group (P < 0.05). At 2 and 4 h post-stress, the liver glycogen content in the unstressed fish was increased with the increase of
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Fig. 2 Liver glycogen content of grass carp prior to (0 h) and after handling (1, 2, and 4 h post-stress). Fish were injected with glucose at 0 (0G), 0.2 (0.2G), 0.5 (0.5G), or 1.0 mg g−1 (1G) BM. At 1 h after injection, fish were stressed (S) or unstressed (NS). Bars represent mean ± SEM (n = 6–8). The different letters indicate significant differences between the fish injected with different
doses of glucose within the same stress treatment (the capital letter for stressed fish and the small letter for unstressed fish; Bonferroni’s test, P < 0.05). The asterisks indicate significant differences between the stressed and unstressed fish injected with the same injected dose of glucose (Bonferroni’s test, P < 0.05)
injected dose of glucose (P < 0.05), while the liver glycogen content was lower in the 1G-S group than in the 1G-NS group (P < 0.05). At 4 h post-stress, the liver glycogen content was higher in the 1G-S group than in the 0G-S group (P < 0.05).
with the increase of injected dose of glucose (P < 0.05), and the plasma LDH activity was higher in 0.2G-NS, 0.5G-NS, and 1G-NS groups than in the 0G-NS group (P < 0.05). Liver G6Pase activity was dependent on the injected dose of glucose at 1 h (ANOVA, F [3, 54] = 2.9, P = 0.041) post-stress and was dependent on the interactive effect of stress treatment and injected dose of glucose at 1 h (ANOVA, F[3, 54] = 3.3, P = 0.027) and 2 h (ANOVA, F[3, 56] = 3.4, P = 0.023) post-stress. At 1 h post-stress, the liver G6Pase activity was lower in 0.2G-S, 0.5G-S, and 1G-S groups than in the 0G-S group (P < 0.05; Fig. 3c). At 2 h post-stress, the liver G6Pase activity was lower in the 1G-S group than in the 0G-S group (P < 0.05). At 1 and 2 h post-stress, the liver G6Pase activity was higher in the 0G-S group than in the 0G-NS group (P < 0.05). The liver HK activity was dependent on stress treatment at 1 h post-stress (ANOVA, F[1, 48] = 9.0, P = 0.005). At 1 h post-stress,
Activities of the enzymes Plasma LDH activity was dependent on stress treatment at 2 h (ANOVA, F[1, 48] = 14.2, P = 0.001) and 4 h (ANOVA, F[1, 49] = 4.2, P = 0.047) post-stress and was dependent on the injected dose of glucose at 0 h (ANOVA, F[3, 49] = 9.9, P < 0.0001), 1 h (ANOVA, F[3, 54] = 5.7, P = 0.002), 2 h (ANOVA, F[3, 48] = 5.4, P = 0.003), and 4 h (ANOVA, F [3, 49] = 14.8, P < 0.0001) post-stress. At 0 h, the plasma LDH activity was higher in the 0.5G-S group than in 0G-S and 0.2G-S groups, while the plasma LDH activity was higher in the 0.5G-NS group than in 0G-NS and 0.2G-NS groups (P < 0.05; Fig. 3a). At 1 h post-stress, the plasma LDH activity was higher in 0.2G-S and 1G-S groups than in the 0G-S group (P < 0.05). At 2 h post-stress, the plasma LDH activity was higher in the 0.2G-S group than in the 0G-S group (P < 0.05), while the plasma LDH activity was higher in the stressed groups than in the unstressed groups injected with the same dose of glucose (0.2G-S vs 0.2G-NS and 1G-S vs 1G-NS, P < 0.05) except 0G-S and 0G-NS groups and 0.5G-S and 0.5G-NS groups (P > 0.05). At 4 h post-stress, the plasma LDH activity was increased in the stressed fish
Fig. 3 Activity of LDH in plasma (a) and activities of PEPCK (b), G6Pase (c), HK (d), and PK (e) in liver of grass carp prior to (0 h) and after handling (1, 2, and 4 h post-stress). Fish were injected with glucose at 0 (0G), 0.2 (0.2G), 0.5 (0.5G), or 1.0 mg g−1 (1G) BM. At 1 h after injection, fish were stressed (S) or unstressed (NS). Bars represent mean ± SEM (n = 6–8). The different letters indicate significant differences between the fish injected with different doses of glucose within the same stress treatment (the capital letter for stressed fish and the small letter for unstressed fish; Bonferroni’s test, P < 0.05). The asterisks indicate significant differences between the stressed and unstressed fish injected with the same injected dose of glucose (Bonferroni’s test, P < 0.05)
Fish Physiol Biochem
Fish Physiol Biochem
the liver HK activity was higher in the 0.2G-S group than in the 0.2G-NS group, and the liver HK activity was higher in the 1G-S group than in the 1G-NS group (P < 0.05; Fig. 3d). No significant differences were found in the activities of PEPCK and PK in the liver between the stressed and unstressed groups injected with the same dose of glucose or between the groups injected with different doses of glucose within each stress treatment (P > 0.05; Fig. 3b, e).
Discussion Most fish species exhibit impaired glucose tolerance, and prolonged hyperglycemia is commonly observed in fish after an acute glucose load (Moon 2001). Intraperitoneal injection of glucose has been used to increase blood glucose level in fishes, such as rainbow trout (Harmon et al. 1991), white sea bream Diplodus sargus (Enes et al. 2012), European sea bass Dicentrarchus labrax (Peres et al. 1999), and turbot Scophthalmus maximus (GarciaRiera and Hemre 2006). In the present study, the interval time between glucose injection and handling challenge was designed based on reports of earlier studies to ensure that the effect of exogenous glucose supplementation on stress response of grass carp could be detected. The plasma glucose level increased with the increase of injected dose of glucose at 0 h, suggesting that intraperitoneal injection of glucose can significantly increase plasma glucose level of grass carp. The effect of glucose level on cortisol release in fish remains to be clarified. Earlier studies reported that high glucose levels resulted in more cortisol generation (ACTH-dependent) in head kidney of rainbow trout in vitro (Holloway et al. 1994; Conde-Sieira et al. 2013). These results reveal that blood glucose level could modulate cortisol release in fish, and high glucose levels could result in high cortisol levels. Gesto et al. (2014) reported that plasma cortisol level in rainbow trout injected with glucose at 0.2 mg g−1 BM did not change in parallel with the changes in glycemia. In the present study, the plasma cortisol level was higher in the fish injected with glucose at 1.0 mg g−1 BM than in the fish injected with glucose at 0, 0.2, and 0.5 mg g−1 BM at 0 h, suggesting that high glucose injection dose could increase plasma cortisol level of grass carp. The plasma cortisol level steeply climbed in the stressed fish at 1 h post-stress, and then gradually declined. At 1 h post-stress, the plasma cortisol level was lower in the
stressed fish injected with 0.5 mg g−1 BM glucose than in the stressed fish injected with glucose at 0 and 0.2 mg g−1 BM. This result suggests that 0.5 mg g−1 BM glucose injection might benefit to suppress cortisol release of grass carp subjected to handling. At 4 h poststress, no significant difference was found in the plasma cortisol level between the stressed fish and unstressed fish injected with same dose of glucose, suggesting that the effect of handling on plasma cortisol of grass carp might not exceed 4 h. According to results of the previous and present study, it is concluded that the effect of blood glucose level on plasma cortisol of grass carp subjected to handling is dose dependent. Appropriate blood glucose level (0.5 mg g−1 BM) could inhibit cortisol release in grass carp subjected to acute stress, but abnormally high blood glucose level (1.0 mg g−1 BM) might stimulate cortisol release. The mechanisms by which blood glucose regulates cortisol release in fish warrant more studies. It has been demonstrated that the plasma lactate level increased in fish subjected to stress due to energy was exhausted in short time and fish need to generate energy through anaerobic metabolism (Pottinger 1998; De Boeck et al. 2001). In the present study, the fish injected with glucose at 0.5 and 1.0 mg g−1 BM showed lower plasma lactate level but higher LDH activity at 0 h, suggesting that high blood glucose level might result in increase of lactate generation. At 1 and 2 h post-stress, the plasma lactate level significantly increased in the stressed fish relative to the unstressed fish. At 4 h poststress, the plasma lactate level was still higher in the stressed fish injected with glucose at 1.0 mg g−1 BM than in the unstressed counterpart, while no significant difference was found in the plasma lactate level between the stressed and unstressed fish injected with glucose at 0 and 0.5 mg g−1 BM. These results reveal that hyperglycemia might enhance anaerobic metabolism in the stressed grass carp. In mammal, a major fraction of glycogen deposited in the liver is converted from exogenous carbohydrate (Newgard et al. 1983). The effect of exogenous carbohydrate on liver glycogen varied among fish species. Intraperitoneal injection of glucose did not significantly affect liver glycogen content in European sea bass (Enes et al. 2011), but resulted in increase of liver glycogen content in white sea bream (Enes et al. 2012). In the present study, liver glycogen content was higher in fish injected with glucose than in fish without glucose injection, suggesting that the exogenous glucose
Fish Physiol Biochem
supplementation could be partly converted into liver glycogen to maintain glucose homeostasis in grass carp under a hyperglycemic condition. At 2 and 4 h poststress, liver glycogen content was dramatically reduced in the fish injected with glucose at 1.0 mg g−1 BM, suggesting that grass carp could utilize liver glycogen as energy fuel in priority when subjected to stress challenge. This result is consistent with the reports that liver glycogen content significantly decreased in the stressed fish for glucose generation (Wendelaar Bonga 1997; López-Patiño et al. 2014). It has been demonstrated that acute stress could activate gluconeogenesis through cortisol modulation (Mommsen et al. 1999; Vijayan et al. 2003). In the present study, liver G6Pase activity was higher in the stressed than unstressed fish without glucose injection, which was not detected in fish with glucose injection. Moreover, the activity of liver G6Pase was lower in the stressed fish with glucose injection compared to the stressed fish without glucose injection at 1 and 2 h post-stress. These results suggest that hyperglycemic situation might attenuate gluconeogenesis in the liver during stress challenge. Guignot and Mithieux (1999) reported that hyperglycemia could suppress hepatic glucose generation in rats by inhibiting G6Pase activity. The response of gluconeogenesis in the liver to hyperglycemic and stress challenge might be a mechanism for maintaining glucose homeostasis in fishes. The attenuation of gluconeogenesis in the liver of grass carp under a hyperglycemic and stressed condition should be attributed to the complex regulation of cortisol, catecholamine, and pancreatic hormones. Earlier studies reported that activities of glycolytic enzymes did not significantly increase in rainbow trout subjected to stress (Morales et al. 1990; López-Patiño et al. 2014). In the present study, the liver HK activity was higher in the stressed fish injected with glucose at 0.2 and 1.0 mg g−1 BM than in unstressed counterpart at 1 h post-stress, suggesting that plasma glucose level might stimulate the liver glycolysis in grass carp after handling. Oppositely, no significant differences were found in the PEPCK and PK activities in the liver among the fish injected with different doses of glucose, regardless of the stressed and unstressed fish. These results indicate that glycemia and acute handling stress could not significantly change the activity of glycolytic enzymes in grass carp. In conclusion, blood glucose level could affect the plasma levels of cortisol, glucose, and lactate; liver
glycogen content; LDH activity in plasma; and G6Pase activity in the liver of stressed grass carp, but had limited effect on activity of HK, PK, and PEPCK in the liver. Glucose supplementation at 0.5 mg g−1 BM can benefit to reduce stress response to handing in grass carp by modulating glycogen metabolism and cortisol release, and hyperglycemic might attenuate gluconeogenesis in the liver. Acknowledgements This research was funded by the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303056-2). The authors thank Dr. Jinyu Tang (Zhejiang University) and Dr. Xiafei Zheng (Zhejiang University) for their assistance in preparing fish feed and sampling blood samples.
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