Effects of seasonal temperature acclimatization on muscle metabolism in rainbow trout, Oncorhynchus mykiss

Maximal enzyme activities in cardiac and swimming muscles of rainbow trout (Oncorhynchus mykiss) were estimated during the natural progression of environmental temperature, between seasonal extremes of 4 and 18°C. Specific activity of marker enzymes were measured at a common temperature of 11°C to determine the potential for metabolic acclimatization. The results confirm an increased oxidative capacity in the cold, with little or no expansion in glycolytic capacity of slow muscle: citrate synthase (CS) activity was 23, 15 and 3 µmoles min^-1 g wet wt^-1 and pyruvate kinase (PK) 15, 23 and 17 µmoles min^-1 g wet wt^-1 from 4, 11 and 18°C acclimatized groups, respectively. Expansion of lipid metabolism was limited, with increased carnitine palmitoleoyltrasferase (CPT) seen only in the coldest group. Cardiac and fast muscle showed a similar response for CS but not PK or CPT which were greatest at 11 and 18°C, respectively. When compared with previous laboratory acclimation studies, these data suggest that environmental factors other than temperature are likely to influence the nature of metabolic acclimatization.     

Seasonal variation of muscle metabolic organization in rainbow trout (Oncorhynchus mykiss)

This study examined how muscle metabolic organization varied during an annual cycle in which rainbow trout (Oncorhynchus mykiss) were held in outdoor holding ponds in which they were exposed to natural changes in temperature (range 0.2 to 15.6°C) and photoperiod. We examined the activities of glycolytic and mitochondrial enzymes in red and white muscle to evaluate whether trout enhance their capacity for lipid and carbohydrate oxidation during cold-acclimization. When assayed at habitat temperature, the enzyme activities generally increased in spring to reach a maximum in summer followed by a decrease in the fall. This led to significantly higher activities at warm than cold periods for all enzymes measured in red muscle and all but one in white muscle. The activities at 10°C provided little evidence for compensatory adjustments of aerobic capacity. Particularly in red muscle, enzyme levels at 10°C were generally lower during cold than warm periods. The variation of enzyme activities throughout the cycle was not due to changes in protein concentration, as the same responses were observed when activities were expressed per g wet mass or per mg protein. Although the aerobic capacity did not increase with cold-acclimatization, the relative capacity for lipid oxidation was higher in winter than in summer trout. In contrast, the relative capacity for aerobic glycolysis was higher in summer than in winter trout. Thus, the metabolic capacities of trout muscle undergo seasonal reorganization.     

Myofibrillar proteins in white muscle of the developing African catfish Heterobranchus longifilis (Siluriforms, Clariidae)

Developmental changes in myofibrillar protein composition were investigated in the myotomal muscle of the African catfish, Heterobranchus longifilis (Clariidae), by several electrophoretic techniques. The main muscle fibres of larvae and the fast-white muscle fibres of juvenile and adult fish were found to express distinct myosin heavy chain and myosin light chain 2 (LC2) isoforms. Three myosin LC2 chains were successively detected, differing by their isoelectric points. In contrast, the alkali light chains remained qualitatively and quantitatively unchanged during fish growth. Actin, -tropomyosin, and troponin-C (TN-C) were also similar in larval, juvenile, and adult white muscle, but an additional larval tropomyosin isoform was found in the first developmental stages. Two isoforms of troponin-T (TN-T) and troponin-I (TN-I) were synthesised in the course of fish growth. Transition from the larval to the adult isoform was much faster for TN-T than for TN-I. Slow-red muscle myofibrils from adult H. longifilis showed no common component (except actin) with larval, juvenile, or adult fast-white muscle myofibrils. Red myofibrils displayed a single TN-T and a single TN-I isoform, but two isoforms of TN-C. The myofibrillar protein isoforms synthesised at any given developmental stage almost certainly reflect changes in the functional requirements of swimming muscles in the course of fish development.     

Fillet texture and muscle structure in brown trout (Salmo trutta) subjected to long-term exercise

Brown trout (Salmo trutta) (2.7 g initial mean weight) were reared in freshwater for 8 months at water velocities of <0.1 (control group), 1 or 2 body lengths per second (BL s^?1) (exercise groups). Growth (body weight, body length and body width), muscle structure (muscle fibre diameter and width of myosepta) and flesh quality parameters (dry matter, muscle pH, collagen content and solubility, instrumental evaluation of texture) were measured at the end of the experiment. The body weight of fish at 1 BL s^?1 was 22% higher than the control group. Muscle development was stimulated at 1 and 2 BL s^?1, leading to a higher condition factor, greater body height and width, and muscle fibre hypertrophy (55 vs. 59.5 μm fibre diameter in the control and 2 BL s^?1 groups respectively). Connective tissue and collagen were only slightly affected by exercise (no difference in collagen solubility, but a greater proportion of γ trimer and fewer α chains in the control compared with the exercised group). Flesh quality was affected, with greater dry matter content and lower post‐mortem pH in the 1 BL s^?1 group compared with the control and 2 BL s^?1 groups. The mechanical resistance of the raw fillets was slightly but significantly increased by exercise. The exercise‐related changes in muscle structure and texture are discussed.     

Molecular adaptations of glucose and lipid metabolism to different levels of dietary carbohydrates in juvenile Japanese flounder Paralichthys olivaceus

Japanese flounder juveniles (initial body weight: 7.12 ± 0.02 g) were fed three diets containing 0, 120 and 200 g/kg of carbohydrates (C0, C120 and C200, respectively) for 10 weeks. Results showed that higher dietary carbohydrate intake enabled further deposition of glycogen and lipids in liver and muscle. The mRNA levels of glucokinase (gck), phosphofructokinase (pfkl) and hexokinase (HK) activity involved in glycolysis were significantly up‐regulated in C120 (p < .05) but showed no further up‐regulation except for gck in C200. Besides, the gluconeogenic phosphoenolpyruvate carboxykinase 1 (pck1) mRNA levels were down‐regulated significantly in fish fed the C120 (p < .05). However, further increase in dietary carbohydrate levels increased the mRNA levels and activities of enzymes involved in gluconeogenesis and lipolysis (p < .05). Additionally, plasma glucose remained unchanged in C120 (p > .05) but significantly increased in C200 group (p < .05). In conclusion, Japanese flounder was able to use carbohydrates efficiently through regulation of glucose and lipid metabolism when dietary carbohydrate was not higher than 120 g/kg, while 200 g/kg of dietary carbohydrate caused the deregulation of glucose homoeostasis.     

Effects of food deprivation on white muscle energy reserves in rainbow trout (Oncorhynchus mykiss): the relationships with body size and temperature

The relationship between body size and the depletion of white muscle metabolites (e.g., PCr, ATP, glucose and glycogen) was examined in two sizes (30 or 700 g) of rainbow trout deprived of food for one, four or seven days at either 5 or 15 °C. Following 7 days of food deprivation at 15 °C, the levels of muscle glycogen decreased by approximately 50% in small fish relative to control values (i.e., day 1). In comparison, small fish acclimated to cold temperatures did not exhibit a significant reduction of muscle glycogen over the seven day fasting regime. In contrast to small fish, the levels of white muscle glycogen in large fish remained unchanged after food deprivation, regardless of acclimation temperature. A seven day deprivation of food also resulted in a 50% depletion of white muscle glucose concentrations in small and large fish acclimated to warm temperatures, but there were no significant changes in this variable in fish acclimated to cold temperatures. In contrast to the negative effects of food deprivation on white muscle glycogen and glucose levels, the concentrations of white muscle PCr and ATP were not greatly affected by food deprivation under any of the experimental conditions. Taken together, these results clearly show that food deprivation can have an important influence on the storage of energy metabolites for anaerobic energy production, particularly in small fish at warm temperatures. In the future, it may be very important to consider the physiological effects of short-term food deprivation when interpreting results from studies in which fish have been fasted prior to treatments such as exercise.     

Responses to prolonged hypoxia by rainbow trout (Salmo gairdneri) I. Free amino acids and proteins in plasma,liver and white muscle

In order to estimate the mobilization of nitrogen compounds for energetic purposes in trout under hypoxic conditions, commercial-size rainbow trout, acclimated to 15°C, were maintained for 10 weeks at an oxygen level of 5.3 ± 0.5 mg/l (hypoxic group) or 8.4 ± 0.4 mg/I (control group), and the changes in tissue concentrations of free amino acids and proteins studied. In animals subjected to hypoxia, there was a decrease in plasma free amino acids involved in gluconeogenesis, liver alanine and aspartic acid, plasma and liver protein concentrations, and muscle free histidine. These results suggest a trend of rainbow trout metabolic activity towards energy production at the expense of anabolism when oxygen availability in water is limited over a long period of time.     

Myosin expression levels in trout muscle: a new method for monitoring specific growth rates for rainbow trout Oncorhynchus mykiss (Walbaum) on varied planes of nutrition

Fish growth is manifested by a number of measurable physical changes. We have developed a sensitive method for monitoring the growth rate of fish fed at four different planes of nutrition. This technique consists in measuring expression levels of myosin RNA isolated from the muscle of experimental animals. Using PCR (polymerase chain reaction) primers, and a fluorescence-labelled single-stranded DNA probe that hybridizes specifically to a region within the myosin mRNA of rainbow trout Oncorhynchus mykiss (Walbaum), we were able to detect differences in the relative level of myosin expression between groups of fish. This method also allows for the determination of absolute expression levels when reactions are performed with standards consisting of known levels of in vitro-transcribed myosin RNA. With the proper equipment, this novel procedure can be performed rapidly on large numbers of individuals, and with the procurement of non-invasive muscle biopsies the same experimental animal could theoretically be sampled multiple times throughout the course of the study. This new method could be used to measure differences in muscle synthesis in fish associated with various nutrient intake levels, environmental parameters, life-history stages and health status.     

Changes in smooth muscle contractility of rainbow trout (Oncorhynchus mykiss Walbaum) intestine during acclimation to altered temperature

The effects of altered water temperature in vivo on in vitro smooth muscle contractility of rainbow trout intestine were investigated. Temperature has a significant effect on receptor-mediated intestinal smooth muscle contractility in the rainbow trout. The efficacy of 5-HT, carbachol, and transmural stimulation increased with temperatures above 10°C, with an optimal increase at 15°C. There was also a modest increase in the potency of 5-HT and carbachol within 2 days of establishing trout at 20°C. By day 8, most of these changes had either stabilized or were returning to control values, suggesting that acclimation changes in membranes and enzyme activities were taking effect. However, the contractile responses to carbachol and transmural stimulation were still increasing at this time. This may imply that the muscarinic receptors are more resistant to membrane acclimation changes and may take longer to adapt. Because these experiments were controlled for handling stress and seasonal changes that affect contractility, we have been able to demonstrate some early changes in smooth muscle contractility that occur during acclimation to altered temperature.     

达里湖瓦氏雅罗鱼生殖洄游过程中能量代谢和消化酶活性的变化

为了解达里湖瓦氏雅罗鱼(Leuciscus waleckii Cyprinidae)生殖洄游过程中能量代谢和消化酶活性的变化,本研究对比了捕于达里湖和贡格尔河的瓦氏雅罗鱼血清生化参数、肝脏和肌肉组织中糖原含量、肝脏组织中转氨酶和己糖激酶活性以及肝脏和肠道组织中消化酶活性的差异;并利用实验生态学方法,研究达里湖中瓦氏雅罗鱼转入贡格尔河河水中 24 h 后其血清生化参数的响应。研究结果显示,与达里湖中瓦氏雅罗鱼相比,洄游到贡格尔河中后雅罗鱼血清葡萄糖含量显著降低(P<0.05),胆固醇含量显著升高(P<0.05),总蛋白和甘油三酯含量无显著变化;肝糖原含量极显著升高(P<0.01),肌糖原含量无显著变化;肝脏组织中谷草转氨酶活性显著降低(P<0.05),谷丙转氨酶和己糖激酶活性无显著差异;肝脏和肠道组织中纤维素酶、胃蛋白酶、胰蛋白酶和淀粉酶活性均显著升高(P<0.05)。将达里湖中瓦氏雅罗鱼转入河水中 24 h 后,其血清葡萄糖和总蛋白含量显著降低(P<0.05),而甘油三酯和胆固醇含量未见明显变化。本研究结果表明,达里湖瓦氏雅罗鱼洄游过程中以肝糖原的形式为其生殖活动储备能量;肝脏和肠道组织中消化酶活性的升高可提高鱼体对营养物质的消化吸收能力。     

Gluconeogenesis in trout (Oncorhynchus mykiss) white muscle: purification and characterization of fructose-1,6-bisphosphatase activity in vitro

Studies of the enzyme fructose-1,6-bisphosphatase (FBPase) of rainbow trout (Oncorhynchus mykiss) have been undertaken in order to illuminate aspects of skeletal muscle gluconeogenesis in these animals. Maximal activities in crude homogenates of several organs suggest that the liver possesses the greatest FBPase activity on a unit g^–1 tissue basis but that the white muscle, owing to its bulk, contributes substantially to whole body FBPase activity. Studies of fructose-6-phosphate-1-kinase (PFK) and FBPase in crude homogenates of several organs suggests an important role for intracellular pH in regulating the relative carbon flux through the FBPase/PFK locus in vivo. Furthermore, a three-step purification scheme is described for trout white muscle FBPase by which a stable and homogeneous (by SDS PAGE) enzyme preparation (isoelectric point = 7.2; molecular weight = 37.6 kd) was obtained. Kinetic studies of the purified enzyme were undertaken at 20°C under conditions reflective of "rest" and "exercise/recovery" intramuscular pH in vivo. Affinity for substrate (F-1,6-P₂) was increased (Km = 6.88 versus 2.44 mol 1-^–1 as was enzyme activity when pH was lowered from 7.0 to 6.5. Various inhibitor metabolites are identified including F-2,6-P₂ (mixed-type inhibitor, K_i = 0.201 mol 1^–1, pH 7.0) and AMP (non-competitive inhibitor, K_i = 0.438 mol 1^–1, pH 7.0). Inhibition by F-2,6-P₂ was strongly alleviated by a reduction in pH from 7.0 to 6.5 (I₅₀ increased from 0.14 to 0.32 mol 1^–1). AMP on the other hand was a more potent inhibitor at pH 6.5 but this inhibition was totally reversed under conditions of citrate, NH₄ ⁺ and AMP typical of muscle during recovery from exercise in vivo. In purified white muscle enzyme preparations, FBPase demonstrated maximal activity at pH 6.5 whereas the optimal pH of PFK was 7.0 or greater. Indeed, it appears from these in vitro data that regulation by metabolite levels as well as pH are required for net FBPase flux in vivo. It is concluded, therefore that trout white muscle FBPase demonstrates the potential to play an important enzymatic role in the control of intramuscular gluconeogenesis in these animals. The results are discussed in relation to present knowledge regarding the metabolic responses of trout white muscle to, and its subsequent recovery from, exhaustive exercise.     

Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout, Oncorhynchus mykiss (Walbaum): adaptive response to a high-protein/non-carbohydrate diet and starvation of glucose 6-phosphate dehydrogenase activity

The adaptive response of the kinetics of glucose 6-phosphate dehydrogenase (G6PDH) was studied in trout liver and kidney after feeding (control or high-protein/non-carbohydrate diet) or starving for 30 days, as well as influences on growth and other nutritive parameters. Typical Michaelis-Menten kinetics were found for the hepatic catalysis of G6PDH under all conditions studied, without evidence of sigmoidicity. The administration of a high-protein/non-carbohydrate diet (61.0% protein, 7.9% lipids) produced no significant changes in specific activity or other kinetic parameters of the liver enzyme. This nutritional situation caused a significant daily increase in relative growth (20%) and feed efficiency (13.5%), as well as a 13% decrease in the protein-conversion efficiency, with respect to the control diet (46% protein, 8% lipids, 22% carbohydrates). On the other hand, long-term starvation (30 days), as a lipolytic condition, significantly decreased the activity and catalytic efficiency of hepatic G6PDH, by almost 45%, without significant changes in the K_m and activity-ratio values. These changes agree with a fall in the intracellular concentration of the enzyme as a consequence of a protein-repression process. The activity of the renal G6PDH was less than in the liver of control fish, and no variation in kinetics was found under the high-protein diet or starvation. This behaviour clearly demonstrates that the kidney pentose-phosphate pathway showed no adaptive response in relation to synthesis of fatty acids and other lipids.     

饲料中蛋白脂肪比对斑点叉尾鮰幼鱼生长、消化酶活性及肌肉成分的影响

探讨了不同的蛋白和脂肪水平对斑点叉尾生长与品质等的影响,试验设计以豆粕调节蛋白含量,混合油脂(鱼油∶豆油=1∶1)调节脂肪含量,并以α-淀粉和次粉为填充物,共配制3个蛋白(P)水平(28%,32%,36%),每一蛋白水平设3个脂肪(L)水平(5.0%,7.5%,10.0%),共9种饲料,分别为P28L10,P32L10,P36L10,P28L7.5,P32L7.5,P36L7.5,P28L5,P32L5和P36L5,每组4重复,每重复80尾鱼[(1.5±0.02)g]。饲养60d后,进行生产性能测定,并采集肌肉等组织样本,测定相关指标。结果表明:饲料蛋白脂肪比对斑点叉尾幼鱼生长有显著影响(P<0.05)。其中P36L7.5组增重率(WGR)、饲料效率(FCR)最高,与P36L10和P32L7.5组差异不显著(P>0.05),但显著高于其它组(P<0.05)。P36L10组试验鱼肥满度(CF)最高,与P32L7.5和P36L7.5组差异不显著(P>0.05),但显著高于其他组(P<0.05)。鱼体内脏比(VBR)、腹脂率(MSI)随脂肪水平降低而显著降低(P<0.05)。试验各组肌肉水分含量差异不显著(P>...     

Effect of recombinant crustacean hyperglycemic hormones rCHH‐B1 and rCHH‐B2 on lipid metabolism in the Pacific white shrimp Litopenaeus vannamei

The crustacean hyperglycemic hormones (CHH) are pleiotropic neuropeptides controlling diverse processes in the life cycle of crustaceans. In the Pacific white shrimp Litopenaeus vannamei, CHH‐B1 and CHH‐B2 are generated by alternative splicing of the same gene, and the recombinant versions of these peptides have shown differential effects on glucose metabolism and osmoregulation in this species. In this work, we explored the role of both recombinant variants, expressed in yeast Pichia pastoris, on lipid metabolism. The results showed that rCHH‐B1 and rCHH‐B2 injections into eyestalk‐ablated shrimp significantly increased the phospholipid and triglyceride levels in haemolymph and restored free fatty acid levels, suggesting that these CHH variants mobilize lipid reserves in shrimp. The effects on lipids were concomitant to its hyperglycemic activity, suggesting that both variants, in addition to their hyperglycemic activity, have hyperlipidemic functions.     

饲料中维生素A对青鱼幼鱼生长、血清生化指标和肝脏糖脂代谢酶活性及基因表达的影响

为研究饲料中维生素A(VA)对青鱼幼鱼生长、血清生化指标和肝脏糖脂代谢相关酶活性及基因表达的影响,实验选取360尾初始体质量为(6.10±0.10) g的青鱼幼鱼,随机分配至3个实验组中,每个实验组设置3个平行。采用单因素实验设计,以无维酪蛋白和明胶为蛋白源、菜籽油为脂肪源、糊精为糖源,同时添加矿物质混合物和维生素混合物(无VA添加)配制成3组实验饲料,分别以饲料1 (Diet1)、饲料2 (Diet2)和饲料3 (Diet3)表示。在饲料1、饲料2和饲料3中分别添加0、2 200和20 000 IU/kg VA醋酸酯(500 000IU/g),经高效液相色谱法(Agilent-1100, Agilent,美国)检测后实验饲料中VA的实际含量分别为178.2、2 058.9和18 436.2 IU/kg,养殖周期为8周。结果显示:饲料中VA缺乏会显著降低青鱼幼鱼的增重率(WGR)和特定生长率(SGR);VA缺乏会显著降低血清血糖(GLU)、甘油三酯(TG)和低密度脂蛋白(LDL)浓度,增加总胆固醇(TCH)浓度。饲料中添加2 058.9IU/kg VA能显著提高肝脏己糖激酶(HK)、磷...