Variable impacts of L-arginine or L-NAME during early life on molecular and cellular markers of muscle growth mechanisms in rainbow trout.

Two experiments were conducted to test if manipulations of the Arginine-Nitric oxide pathway during the early life of rainbow trout would act on its early myogenic process. In experiment 1, trout embryos were immersed at 72° days post-fertilization (°dpf) or 104°dpf in water alone (control treatment, C) or containing 2 mM/L L-Arg (treatment A) or 1 mM/L of L-NAME, a NOS inhibitor (treatment N). We observed the beginning of expression of myf5 and fmhc genes at 72°dpf and 96°dpf, respectively. "A" treatment doubled the free Arg content of eggs but did not affect either the pattern of expression of myf5 and fmhc, nor white muscle cross-sectional area and number of white muscle fibres at hatching, nor embryo survival and fry growth. "N" treatment also did not affect these markers. In experiment 2, trout fry were fed from first feeding onwards and during 20 days either a control diet (C) or the same diet supplemented with L-NAME (0.1 g/100 g diet, N-diet). In C-fed fry, distribution of a single meal after overnight fasting induced changes in pcna, myod1, myog, fmhc, inos, nnos and ctsd gene expressions. N-feeding decreased fry growth but did not change their growth trajectory or survival. Twenty days of N-feeding led, compared to C-feeding, to changes in kinetics of transcription of pcna, myod1, myog, fmhc, inos, nnos, ctsd genes and to decreased white muscle cross-sectional area, total number of white muscle fibres, and number of large muscle fibres. L-NAME feeding thus decreased fry muscle growth by altering both hyperplasia and hypertrophy.

The effect of choline and cystine on the utilisation of methionine for protein accretion, remethylation and trans-sulfuration in juvenile shrimp Penaeus monodon.

This 35-d feeding experiment examined in juvenile shrimp Penaeus monodon (3·3 g initial body weight) the effects of methionine (Met), choline and cystine on protein accretion and the activity of two key enzymes of remethylation (betaine-homocysteine methyltransferase; BHMT) and trans-sulfuration (cystathionine ß-synthase; CBS). The interaction between Met and choline was tested using semi-purified diets either adequate or limiting (30 or 50 %) in total sulphur amino acid (SAA) content with a constant cystine:Met ratio. The diets contained either basal or excess choline (3 v. 7 g/kg feed). Cystine was added to two other 30 and 50 % Met-limiting diets to adjust the SAA supply to that of the control diet in order to evaluate the interaction between Met and cystine. As expected, N accretion was significantly lower with the SAA-limiting diets but increased back to control levels by the extra choline or cystine, demonstrating their sparing effect on Met utilisation for protein accretion. We show, for the first time, the activities of BHMT and CBS in shrimp hepatopancreas. Only BHMT responded to the SAA deficiencies, whereas the extra choline and cystine did not stimulate remethylation or down-regulate trans-sulfuration. Our data also suggest the capacity of P. monodon to synthesise taurine, being significantly affected by the cystine level in the 30 % SAA-limiting diets. Further research is warranted to better understand the metabolic regulation of taurine synthesis in shrimp and of the observed Met-sparing effects.

Feeding status regulates the polyubiquitination step of the ubiquitin-proteasome-dependent proteolysis in rainbow trout (Oncorhynchus mykiss) muscle.

In mammals, the ubiquitin-proteasome proteolytic pathway is a major route of protein degradation and has been shown to be regulated by the feeding status via the protein kinase B (PKB)-Forkehead box-O transcription factor signaling pathway-mediated transcription regulation of atrophy-related ubiquitin ligases, atrogin1 and muscle RING finger 1. In contrast, in rainbow trout (Oncorhynchus mykiss), the activity of the proteasome in muscle was not affected during starvation-induced muscle degradation. The aim of this study was therefore to explore the molecular basis for this lack of induction of this proteolytic route during starvation. In this study, rainbow trout were food deprived for 7 and 14 d, refed ad libitum, and the effect of the nutritional status was assessed on the different steps involved in the regulation of the ubiquitin-proteasome system in muscle. We observed that starvation reduced the phosphorylation of PKB and enhanced the expression of atrogin1 in muscle, whereas refeeding led to the opposite effects. The level of polyubiquitinated proteins in muscle increased to over 2 times the initial value on d 0 after 14 d of starvation and decreased significantly at 12 h after refeeding, but there were no major changes in the activity of the main proteasomal peptidases (chymotrypsin-like and trypsin-like). Altogether, these results indicate that in rainbow trout muscle, the polyubiquitination step of the ubiquitin-proteasome route is regulated by the feeding status similarly to what is observed in mammals.

Response of hexokinase enzymes and the insulin system to dietary carbohydrates in the common carp, Cyprinus carpio.

The response of the common carp to diets with varying amounts of digestible starch, provided either as pea meal (LP, HP, 30 and 46% peas, respectively) or as cereal (LW, HW, 30 and 46% wheat, respectively), was studied and compared with the response to a carbohydrate-free protein-rich diet (CF). Here we focused on the utilisation of dietary carbohydrates by examining the relationship between dietary starch intake, hepatic hexokinase activities, circulating insulin and muscle insulin receptor system. Plasma glucose concentration and hepatic high Km hexokinase (glucokinase, GK) activity were not affected by the content of digestible starch, but 6 h after feeding enzyme activity was higher in the fish fed carbohydrate diets. Similarly, low Km hexokinase (HK) activity was also higher in the fish 24 h after feeding. Fat gain and protein retention were significantly improved by increased digestible starch intake, especially in the HP group, which in turn, presented the highest plasma insulin levels. Glycogen stores were moderately increased by the ingestion of digestible starch. The number of insulin receptors was greater in the CF group than in fish on carbohydrates, except the HP group. Our results confirmed that the common carp uses dietary carbohydrates efficiently, especially when there are provided by peas. This efficiency might be related to the enhanced response of postprandial insulin observed in the HP group.

Muscle insulin binding and plasma levels in relation to liver glucokinase activity, glucose metabolism and dietary carbohydrates in rainbow trout.

Rainbow trout were fed for 10 weeks with either a carbohydrate-free diet (C-free) or with four experimental diets containing various levels (20 or 40%) and sources of starch (extruded wheat or peas) in order to examine metabolic utilisation of dietary vegetable carbohydrates and its endocrine control. The study was focused on the parameters described as limiting in glucose metabolism in fish. Feeding trials were conducted at 8 and 18 degrees C to establish whether carbohydrate-rich diets can be used in trout farming irrespective of water temperature. At both temperatures, pea diets (especially the highest level) resulted in a feed efficiency as high as the C-free diet. Fish had similar growth rates except when fed the low wheat content diet. Glycaemia values 6 h after feeding were significantly higher in trout fed carbohydrate diets than those given the C-free diet, whereas plasma insulin levels were similar independently of the levels of dietary starch. This study provides the first evidence that glucokinase (GK) activity and mRNA level in trout liver increase in proportion to the content of dietary starch. Nevertheless, these changes were not correlated with plasma insulin levels. Insulin-like growth factor-I (IGF-I) binding and number of receptors in skeletal muscle were consistently higher than those for insulin but no diet-induced differences were found for any of these parameters. Temperature clearly affected the postprandial profile of glucose and insulin, which both showed lower levels 6 h after feeding at 8 degrees C than at 18 degrees C, which was consistent with a lower feed intake. Glucose and insulin levels decreased markedly 24 h after feeding at 18 degrees C, while they were still high at 8 degrees C, an observation concordant with delayed transit rate. These findings indicate satisfactory adaptation of rainbow trout to diets with a relatively high vegetable starch content, especially when provided as extruded peas, and indicate that diets with increased levels of carbohydrates can be used in this species even when it is reared at low temperature.