Hepatocellular adaptation to extreme nutritional conditions in ide,Leuciscus idus melanotus L. (Cyprinidae). A morphofunctional analysis

The adaptive response of the omnivorous ide,Leuciscus idus melanotus, to drastic metabolic conditions was analyzed on different levels of organisation investigating a variety of parameters: Organism (condition factor, liver-somatic index), organ (liver structure), cellular and subcellular level (hepatocyte structure, glycogen and lipid storage, contents and distribution of nucleic acids, enzyme alterations).During starvation, ide were able to maintain liver integrity in a biphasic process: after an initial phase of disturbance, ide established a new structural and metabolic homeostasis. Recovery from starvation was possible only with a complete diet but not with a sucrose diet. Carbohydrate overload, as evoked by sucrose refeeding, did not result in liver or carcass fattening as known from mammals.To the best of our knowledge, the present study is the first to use enzyme histochemistry in fish nutrition research. In mammals, histochemistry is particularly helpful for understanding processes of hepatic metabolic adaptation. In fish, however, on the basis of our results, enzyme histochemical studies appear to be of limited value, as long as no further data are available on a zonal distribution of enzyme activities in teleost liver parenchyma. Instead, the histochemical detection of the distribution of hepatic storage products and RNA-positive material yielded important information on liver adaptive processes.Abbreviations H starvation - SU sucrose - AD artificial diet - AcP acid phosphatase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GLU -glucuronidase - ME malic enzyme - PHO glycogen phosphorylase - UE unspecific esterase - RER rough endoplasmic reticulum     

Interacting effects of diet and environmental temperature on biochemical parameters in the liver ofLeuciscus idus melanotus (Cyprinidae: Teleostei)

One year old golden ide (Leuciscus idus melanotus) were starved for 2 weeks at 20°C (time zero) and then they were fed either freeze-dried mosquito larvae (natural diet) or a commercial fish chow (artificial diet) at an ambient temperature of 14°C and 20°C, respectively. Growth parameters and biochemical data in the liver were measured at time zero and after 3 and 7 weeks of specific regimen. If compared to natural food, the artificial diet caused transient increase in anabolic activity of the liver, but prevented long-lasting hepatocyte proliferation (increase of total liver DNA). After 3 weeks on artificial diet, the body weight was significantly higher and the liver mass doubled compared to ide kept on the natural regimen; tissue DNA content indicated that the hepatocyte volume increased mainly by increased protein content and corresponding uptake of water; lipid and glycogen contents were increased by a factor of 3–6, and the RNADNA ratio was increased accordingly. The growth of the ide, as reflected by the condition factor and hepatosomatic index (HSI), was virtually stopped when the artificial regimen was maintained for 7 weeks, whereas on natural food the condition factor was increased and liver weight and DNA were doubled. Growth of the fish maintained at 14°C was significantly lower than that of fish maintained at 20°C, though the total liver DNA was still increased on the natural regimen during the experiment. Less lipid was stored by fish maintained at 14°C, whereas protein and glycogen deposits were enlarged, if compared to fish maintained at 20°C. The specific activity of cytochrome oxidase in liver mitochondria and of NADPH cytochrome c reductase in the microsomal fraction were found independent of diet and ambient temperature. Oxidative capacity of hepatocytes (mitochondrial protein/mg DNA) remained unchanged, and microsomal protein/cell appeared reduced in response to the artificial food. However, reduction of cell number on this diet resulted in less total mitochondrial and microsomal protein in the organ. Golden ide liver is found to be a sensitive experimental model to characterize the mutual influence of diet and temperature on fish; the results are discussed with regard to the usage of golden ide as test fish for water pollution.     

棘头梅童鱼肝型脂肪酸结合蛋白基因克隆及组织表达分析

为进一步了解肝型脂肪酸结合蛋白基因(L-FABP基因)在棘头梅童鱼(Collichthys lucidus)营养调控、脂肪酸代谢和免疫应答等方面的功能作用,以棘头梅童鱼全基因组组装数据为基础,获得L-FABP基因的含开放阅读框的cDNA序列,利用生物信息学方法分析编码的蛋白质结构及进化关系,采用荧光定量(qRT-PCR)技术研究了不同组织中L-FABP基因的表达情况。结果表明,L-FABP基因cDNA序列为475 bp,其中5’非翻译区为51 bp,3’非翻译区为40 bp,开放阅读框为384 bp,编码127个氨基酸。其结构由10个β-折叠链和2个α-螺旋链组成,在氨基酸3~126位点间具有保守的脂质/胞质性脂肪酸结合结构域。棘头梅童鱼L-FABP基因与大黄鱼、条纹狼鲈同源性较近,与绿河鲀、尼罗罗非鱼和斑马鱼同源性较远。L-FABP基因在棘头梅童鱼肝脏、肠道、眼、大脑、心脏、肌肉、胃、鳃、脾脏、头肾和中肾中均有表达,其中肝脏中的L-FABP相对表达量最高,显著高于其他组织(P<0.05)。     

Rearing of pike-perch larvae using formulated diets – first success with starter feeds

The present study evaluated the performance of two commercial diets: AgloNorse (AN) and BioKyowa (BK), and two experimental, formulated diets based on casein (C) or casein plus casein hydrolysates (CH) in rearing of pike‐perch larvae (Sander lucioperca L.). All fish were 5 day old and control group was fed live Artemia nauplii. Fish were sampled periodically for histological comparison of morphological changes in the digestive tracts. Survival of fish fed Artemia nauplii, BK and AN was similar: 54.4%, 50.8% and 52.4%, respectively, while the fish fed formulated diets C and CH showed considerably lower survival: 28.4% and 21.6% respectively. After 5 weeks of rearing, the average body mass of fish ranged from 212±32 mg in Artemia fed group to 53.8±6.8 mg in C diet fed group. A considerable vacuolization of supranuclear zone in enterocytes of posterior intestine was observed in the larvae fed commercial diets. No anomalies in liver development were found. Hepatocytes of fish fed BK diet showed larger glycogen storage areas, compared with those occupied by lipids. The highest zymogen accumulation of pancreatic cytoplasm was observed in fish fed Artemia. In fish fed C and CH diets, anomalies in digestive system development were indicated by lower and less numerous intestinal folds, smaller hepatocytes, retarded development of gastric glands, and in CH group – also local fatty degeneration of liver.     

Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [¹²⁵I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [¹²⁵I]-insulin binding reached a steady state within 20–30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K_d1 = 5.5 pM) and low capacity (B_max1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K_d2 = 2.4 nM) and high capacity (B_max2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [¹²⁵I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC₅₀ values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis. dedicated to Prof. Dr. W. Hanke on the occasion of his 65^th birthday.     

The cytochrome P450 system of Atlantic salmon (Salmo salar): II. Variations in hepatic catalytic activities and isozyme patterns during an annual reproductive cycle

A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5–7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17 was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b₅, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b₅, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17 levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17 as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17 independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goks\/oyr 1989).     

饥饿和再投喂对美国红鱼消化器官组织学的影响

用石蜡切片方法观察和分析饥饿和再投喂对美国红鱼(Sciaenopsocellatus)消化器官形态结构和组织学的影响。鱼样体重(8.25±0.5)g,体长(8.84±0.27)cm。从形态结构看,饥饿与再投喂前后食道无明显变化,而饥饿10、15d的实验鱼胃壁变薄,幽门盲囊变小,肠管收缩,呈透明状,肝胰腺萎缩,呈姜黄色,胆管呈深绿色。从组织结构看,食道无明显变化;饥饿时间不同,各组织受损害和恢复程度不同。饥饿5d的实验鱼基本同对照组;而饥饿10、15d的实验鱼组织变化较明显:皱襞和上皮细胞高度均减少,分泌颗粒减少。胃腺细胞收缩,结构不完整;幽门盲囊长度和直径变小;肠直径变小,微绒毛退化;肝组织致密,肝细胞内脂滴减少,体积缩小;胰腺泡缩小,排列不规则。再投喂各主要结构均有所恢复,有些恢复到饥饿前水平,但大部分未能达到。     

Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout (Oncorhynchus mykiss). II. Adaptive response of glucose 6-phosphate dehydrogenase activity to high-carbohydrate/low-protein and high-fat/non-carbohydrate diets

The influence of long-term administration of high-carbohydrate/low-protein and high-fat/non-carbohydrate diets were studied in relation to kinetic behaviour of glucose 6-phosphate dehydrogenase in liver and kidney of rainbow trout. In all cases studied, the saturation curves of these enzyme showed typical hyperbolic kinetics without evidence of sigmoidicity. After 30 days of feeding with a high-fat diet (170 g kg^?1), there was a significant decrease in V_max and specific activity (45%) as well as catalytic efficiency (39%) without changes in K_m or activity ratio of hepatic glucose 6-phosphate dehydrogenase. These changes agree more with a clearly decreased cell concentration than with an inhibition of the pre-existing enzyme. The administration of a high-carbohydrate diet (60 g kg^?1), contrary to what was previously thought, decreased V_max by 21% and specific activity and catalytic efficiency by 30%, without significant changes in the other kinetic parameters of the hepatic enzyme. The kinetic behaviour under these nutritional conditons was due to the rejection of this diet by the fish and thus could be considered a low-feeding situation. On the other hand, no variations in the kinetics of renal glucose 6-phosphate dehydrogenase were found, clearly demonstrating that in this organ, the pentosephosphate pathway showed no adaptive response related to fattyacid and other lipid synthesis. The activity of the renal enzyme was consistently half that of the hepatic enzyme.     

Thyroidal compensation in rainbow trout (Salmo gairdneri) fed canola meal

Rainbow trout (Salmo gairdneri Richardson) were fed either a soybean mealbased (SM) or canola meal-based (CM) diet for up to 20 weeks. Plasma thyroxine (T₄) and triiodothryonine (T₃) levels were significantly lower in the CM-fed fish sampled after 12 weeks. However, there appeared to be some compensation after 12 and 20 weeks in that the thyroid hormone levels in trout fed the CM were not significantly different from those of the SM-fed fish. Nevertheless, there was marked thyroid hyperplasia and hypertrophy in the CM-fed fish sampled at 12, 16 and 20 weeks after commencement of the experiment. Moreover, the growth rate was significantly lower in the CM-fed fish in comparison to the SM-fed fish throughout the 20 week study period.Plasma T₄ levels were similar in SM-fed fish sampled 12, 16 and 20 weeks after commencement of the experiment, but plasma T₃ levels progressively increased over this period, as did the apparent activity of the thyroid tissue based on histological criteria.Fasting for up to 8 weeks resulted in the arrested growth of the SM-fed fish, and a loss in body weight of the CM-fed animals over the 8 week period of the fast. In addition, the plasma thyroid hormone levels in the fasted fish tended to be lower than in fish fed both the SM and CM diets prior to fasting, and there was histological evidence indicating a reduced activity of the pituitary-thyroid axis. However, thyroid hyperplasia and hypertrophy were still evident in the fasted fish previously fed the CM diet indicating that the adverse affects of CM diets are not completely reversible after 8 weeks.In fish fed the CM diet for 12 weeks and then the SM diet for up to a further 8 weeks (diet C-S) there was a compensatory increase in plasma thyroid hormone levels evident within 4 weeks after the change in diet, but no apparent decrease in thyroid hyperplasia or hypertrophy. In addition, in the fish fed the C-S diet there was a marked compensatory growth rate, and an increased feed: gain ratio; body weights of this group of fish were not significantly different from those of the SM-fed animals after 20 weeks of study, indicating a considerably higher growth rate over the last 8 week period.     

Role of molecular species catabolism in the temperature-induced restructuring of phosphatidylcholines in liver microsomes of thermally-acclimated rainbow trout (Oncorhynchus mykiss)

Most previous studies of the temperature-induced restructuring of phospholipid molecular species composition have examined steps in the biosynthesis of phospholipids to explain the accumulation of unsaturated fatty acids in membranes of cold-acclimated poikilotherms. In contrast, the present study explores the role of phospholipases in this restructuring process by determining the rates of degradation of specific molecular species of phosphatidylcholine, using enzymes (microsomes) freshly isolated from the liver of rainbow trout. (Oncorhynchus mykiss) acclimated to either 5° or 20°C. The substrate preparation employed to assay phospholipase activity possessed a range of molecular species, all radiolabeled with 1-¹⁴C-palmitic acid at thesn-1 position, similar to that present in native trout liver microsomes. After defined periods of incubation (120 and 240 min at 5°C; 60 and 120 min at 20°C), phospholipids were extracted from the reaction mixture and the distribution of radioactivity among the molecular species of phosphatidylcholine was determined by HPLC/liquid scintillation counting. In general, molecular species catabolism was not significantly influenced by either assay or acclimation temperature. Only in 20°C-acclimated fish did a reduction in assay temperature (from to 20 to 5°C) result in significantly increased proportions of radioactivity being recovered in one polyunsaturated fatty acid-containing species (16:0/22:6-PC). It is concluded: 1) that phospholipase specificity, assayed under conditions approximating thosein situ, is not significantly influenced by temperature; and 2), that the increased proportions of unsaturated fatty acid-containing molecular species of phosphatidylcholine observed at low temperatures must reflect the specificity of biosynthetic rather than degradative processes.