Juan Peragón

Influence of temperature and dietary-protein supplementation either with free or coated lysine on the fractional protein-turnover rates in the white muscle of carp

The effect of protein quality and of supplementation of corn-glutenprotein with lysine on the growth, feed conversion and protein turnoverrates in white muscle was investigated in carp (Cyprinus carpio) acclimatedto either 18 or 25 °C. Fish fed the lysine-deficient diet showed asignificantly lower food intake, weight gain and feed-conversion efficiencythan animals fed the lysine-sufficient diets, regardless of environmentaltemperature. Coated lysine, compared with free lysine, proved to be asignificantly better way of supplementing dietary protein, as shown byfeed-conversion indices at 18 and 25 °C. White-muscle protein, RNA andDNA contents were not altered by dietary treatment or water temperature.Supplementation with coated lysine, but not with free lysine, significantlyincreased the protein-synthesis rate (KS) at 18 or 25 °Cin comparison to lysine deficiency, although not to control values. Theeffiiency of protein synthesis (KRNA) and retention (PRE)obtained for fish fed the coated-lysine diet, at 18 °C, reached controlvalues. At 25 °C PRE and protein accumulation rate (KG)showed the following significant differences: lysine-deficient diet <free-lysine supplemented diet < coated-lysine supplemented diet <control diet. Significant correlations were observed for Ks in relation withKD, KRNA or protein-related growth, at bothtemperatures.

Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout (Oncorhynchus mykiss)

Abstract We have investigated the effect of dietary carbohydrate on different parameters of protein-turnover rate, nature of growth, and nucleic acid content in the muscle of rainbow trout in order to better understand the molecular nature of these growth parameters in the absence of this dietary component. For this, we used a methodology based on the incorporation rate of tritium labelled phenylalanine in muscle protein. Juvenile rainbow trout of an initial body weight of 110 g were fed near to satiety with a control or a non-carbohydrate diet during 7 weeks. The absence of dietary carbohydrate significantly depressed fish growth, as well as daily body weight gain, as a consequence of muscular hypotrophy (the cell size diminished by almost 50%) and not by a reduction of number of cells (hypoplasia). This nutritional situation also significantly slowed (by almost 11%) muscle-protein accumulation rate ( K G ) as a result of a significant increase (eight-fold) in muscle-protein degradation rate ( K D ), without changing the other protein-turnover rates, protein synthesis rate ( K S ), protein synthesis capacity ( C S ), protein synthesis efficiency ( K RNA ), protein synthesis rate per cell unit ( K DNA ), or protein retention efficiency (PRE). These results, together with the nucleic acid content, clearly indicate that the absence of carbohydrate significantly exacerbates the muscular-protein degradation without affecting protein synthesis. In conclusion, carbohydrates are needed to prevent amino acids released during protein degradation from being used to synthesize carbohydrates and/or to be used for energy and not for growth.

Impact of starvation-refeeding on kinetics and protein expression of trout liver NADPH-production systems

Herein we report on the kinetic and protein expression of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase, and malic enzyme (ME) in the liver of the trout (Oncorhynchus mykiss) during a long-term starvation-refeeding cycle. Starvation significantly depressed the activity of these enzymes by almost 60%, without changing the Michaelis constant. The time response to this nutritional stimulus increased with fish weight. The sharp decline in G6PDH and ME activities was due to a specific protein-repression phenomenon, as demonstrated by molecular and immunohistochemical analyses. Also, the dimeric banding pattern of liver G6PDH shifted from the fully reduced and partially oxidized forms, predominant in control, to a fully oxidized form, more sensitive to proteolytic inactivation. Refeeding caused opposite effects in both protein concentration and enzyme activities of about twice the control values in the first stages, later reaching the normal enzyme activity levels. Additionally, the partially oxidized form of G6PDH increased. The kinetics of these enzymes were examined in relation to the various metabolic roles of NADPH. These results clearly indicate that trout liver undergoes protein repression-induction processes under these two contrasting nutritional conditions.

Liver and white muscle protein turnover rates in the / European eel Anguilla anguilla: effects of dietary protein quality

Abstract The influence of the quality of dietary protein source on growth and protein synthesis and degradation rates was studied in the liver and white muscle of the European eel. Fish were fed isonitrogenous diets differing in protein source: one (control) contained fish meal, three others incorporating meat meal (MM) or sunflower meal (SFM) as the only protein source, and SFM supplemented with some essential amino acids (EAAs) were also tested. Fish fed diets containing unsupplemented MM or SFM exhibited dietary utilization and growth indices poorer than those fed the control, while EAA supplementation greatly improved the performance of the SFM-diet. Liver showed higher rates of protein synthesis ( k s ) and degradation ( k d ) associated with a higher capacity for protein synthesis per unit of DNA but a lower protein deposition efficiency (PDE), compared to muscle. Low quality dietary protein increased the protein turnover rate, with a higher protein synthesis rate per unit of DNA and RNA but a decrease of PDE. In white muscle, MM and unsupplemented SFM diets decreased k s without changing k d . The MM diet reduced the efficiency of protein synthesis and deposition. EAA supplementation of the SFM diet raised the protein synthesis rate and capacity as well as protein deposition compared to control values.

Growth, protein-turnover rates and nucleic-acid concentrations in the white muscle of rainbow trout during development.

We have studied the growth rate, nucleic-acid concentration, protein-accumulation rate (K(G)), and several other parameters relating to protein turnover, such as the protein-synthesis (K(S)), and protein-degradation rates (K(D)), protein-synthesis capacity (C(S)), protein-synthesis efficiency (K(RNA)), protein-synthesis rate per DNA unit (K(DNA)) and protein-retention efficiency (PRE), in the white muscle of rainbow trout during development. Both growth rate and relative food intake decreased significantly with age and weight, as did the food-efficiency ratio (FER) and protein-efficiency ratio (PER). Although absolute RNA and DNA contents increased with age, their relative concentrations decreased. The RNA/DNA ratio increased sharply from 14 to 28 weeks but afterwards decreased towards initial values. Hypertrophy increased rapidly to the 28-week stage but henceforth increased much more slowly. Hyperplasia, on the other hand, continued to increase linearly, resulting in a significant four- to fivefold predominance in this type of growth at the end of the 96-week experimental period. K(G) decreased significantly with age, as did K(S), and C(S), whereas at the 14-week stage, K(D) was significantly lower than at other ages. K(RNA) increased until 28 weeks. K(DNA) increased significantly in juvenile fish compared to both fingerlings and adults, where it showed similar lower values. PRE remained high at all ages.

Kinetic properties of hexose-monophosphate dehydrogenases. I. Isolation and partial purification of glucose-6-phosphate dehydrogenase from rat liver and kidney cortex☆

Glucose-6-phosphate dehydrogenase (G6PDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely separated from 6-phosphogluconate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 40-55% ammonium sulphate fractionation, by which both enzyme activities were separated, and finally, the application of the protein fraction to a column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of isolated liver and renal G6PDH were examined. Both enzymes showed a typical Michaelis-Menten kinetic saturation curve with no evidence of co-operativity. The optimum pH of both liver and kidney cortex G6PDH was 9.4. The Km values for glucose-6-phosphate (G6P) and for NADP were 3.29 x 10(-4) M and 1.00 x 10(-4) M respectively. The specific activity measured at 37 degrees C and optimum pH was 327.1 mU/ mg of protein. NADPH caused a competitive inhibition with a Ki of 10 microM. The Km values for the G6P and NADP of kidney-cortex G6PDH were 2.06 x 10(-4) and 0.25 x 10(-4) M respectively. The specific activity at pH 9.4 and 37 degrees C was 76.55 mU/mg of protein. The Ki value for NADPH inhibition was 4 microM. This work describes an easy, rapid and reliable method for the separation of the two dehydrogenases involved in the hexose-monophosphate shunt in animal tissues.

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein‐turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2‐DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish‐farm conditions and fed with a 100 mg/kg MA‐enriched diet (MA100). After the comparison of the protein profiles from MA100 fed fish and from control, 49 protein spots were found to be altered in abundance (≥2‐fold). Analysis by MALDI‐TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S‐adenosyl methionine‐dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6‐phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4‐hydroxyphenylpyruvic dioxygenase, methylmalonate‐semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat‐shock protein, 58 kDa glucose‐regulated protein, cytokeratin E7, type‐II keratin, intermediate filament proteins, 17‐β‐hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6‐phosphate dehydrogenase, elongation factor 2, 60 kDa heat‐shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein‐expression levels of these proteins, we proposed a cellular‐signalling pathway to explain the hepatic‐cell response to the intake of a diet containing MA.

Selective changes in the protein-turnover rates and nature of growth induced in trout liver by long-term starvation followed by re-feeding

We report upon the effects of a cycle of long-term starvation followed by re-feeding on the liver-protein turnover rates and nature of protein growth in the rainbow trout (Oncorhynchus mykiss). We determined the protein-turnover rate and its relationship with the nucleic-acid concentrations in the livers of juvenile trout starved for 70 days and then re-fed for 9 days. During starvation the total hepatic-protein and RNA contents decreased significantly and the absolute protein-synthesis rate (AS) also fell, whilst the fractional protein-synthesis rate (KS) remained unchanged and the fractional protein-degradation rate (KD) increased significantly. Total DNA content, an indicator of hyperplasia, and the protein:DNA ratio, an indicator of hypertrophy, both fell considerably. After re-feeding for 9 days the protein-accumulation rates (KG, AG) rose sharply, as did KS, AS, KD, protein-synthesis efficiency (KRNA) and the protein-synthesis rate/DNA unit (KDNA). The total hepatic protein and RNA contents increased but still remained below the control values. The protein:DNA and RNA:DNA ratios increased significantly compared to starved fish. These changes demonstrate the high response capacity of the protein-turnover rates in trout liver upon re-feeding after long-term starvation. Upon re-feeding hypertrophic growth increased considerably whilst hyperplasia remained at starvation levels.

Dietary protein effects on growth and fractional protein synthesis and degradation rates in liver and white muscle of rainbow trout (Oncorhynchus mykiss)

Abstract We have studied the effects of a decrease in dietary protein on the growth and proteinturnover parameters [fractional protein synthesis ( K S ), degradation ( K D ) and accumulation ( K G ) rates, synthesis capacity ( C S ), synthesis efficiency ( K RNA and K DNA ), and protein retention efficiency (PRE)]. The administration of a low-protein diet caused a significant decrease in the availability of protein precursors due to a decline in feed efficiency and thus a concomitant decrease in whole-body, white-muscle and liver growth capacity throughout the experimental period. The reduction in dietary protein caused a significant decrease in the K G value in the liver as a consequence of a decrease in protein retention efficiency (PRE). In white muscle, however, a considerable reduction in the K G value was due to a decrease in K S , C S , K DNA and K RNA rather than a decrease in PRE.

The influence of dietary protein on the kinetics of NADPH production systems in various tissues of rainbow trout (Oncorhynchus mykiss)

Abstract The effects of a decrease in dietary protein level on the kinetic behaviour of the four NADPH production systems [glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME), and NADP-isocitrate dehydrogenase (NADP-IDH)] were investigated in four different trout tissues. Typical hyperbolic saturation curves were always obtained for the activity of these enzymes. A low-protein diet caused a significant decrease in the maximum velocity values ( V max ) of all the hepatic enzyme systems studied. The decrease in hexose monophosphate dehydrogenases (G6PDH and 6PGDH) was about 35 and 50%, respectively, whereas for ME and NADP-IDH it was about 35 and 25%. No significant changes were found in the Michaelis constant. These kinetic characteristics are compatible with an increase in the catalytic efficiency of these enzymes without there being any changes in their activity ratio values. The kinetic parameters for these enzymes in the kidney, spleen, and gill tissue did not undergo any significant change.