Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.

Acute stress alters the rates of degradation of cardiac muscle proteins.

Stressful experiences can have detrimental effects on many aspects of health and wellbeing. The zebrafish (Danio rerio) is a widely used model for stress research and a stress phenotype can be induced by manipulating the environmental conditions and social interactions. In this study we have combined a zebrafish stress model with the measurement of degradation rates of soluble cardiac muscle proteins. The results showed that the greater the stress response in the zebrafish the lower the level of overall protein degradation. On comparing the rates of degradation for individual proteins it was found that four main pathways were altered in response to stress conditions with decreased degradation for proteins involved in glucose metabolism, gluconeogenesis, the ubiquitin-proteasome system (UPS) and peroxisomal proliferator-activated receptor (PPAR) signalling pathways. Taken together, these data indicate that under stress conditions zebrafish preserve cardiac muscle proteins required for the 'fight or flight' response together with proteins that play a role in stress mitigation. SIGNIFICANCE: This study is the first to investigate the impact of stressful experiences on the dynamics of protein turnover in cardiac muscle. Using an established zebrafish model of human stress it has been possible to map key pathways at the protein level. The results show that the rates of degradation of cardiac proteins involved in glucose metabolism, UPS activity, hypoxia and PPAR signalling are decreased in stressed zebrafish. These findings indicate that proteins involved in the 'fight or flight' response to stress are conserved by the heart together with proteins that play a role in stress mitigation. This work provides the basis for more detailed investigations aimed at understanding the molecular effects of stress, which has implications for human health and disease.

Impact of the partial replacement of fish meal with a chloroplast rich fraction on the growth and selected nutrient profile of zebrafish (Danio rerio).

Increasing global aquaculture production, is putting pressure on fishmeal and fish oil supply. There is therefore a growing search for more sustainable sources of proteins and polyunsaturated fatty acids as fish feed ingredients. Chloroplasts are the organelles in the leaves of plants where many of the valuable nutrients, fatty acids (FAs), amino acids, vitamins and pigments, are synthesised. Chloroplasts could be incorporated into fish diets either retained in, or liberated from, plant cells. In this study zebrafish were fed with seven different diets individually; fish were fed with diets reducing fishmeal levels (10, 20 or 50%) using either spinach leaf powder (SLP) or a chloroplast rich fraction (CRF) prepared by an established method to recover chloroplasts. Both SLP and CRF had a positive impact on the growth, taste response, whole fish FA composition, and carotenoid profile. Fish fed with CRF diets showed significantly (P ≤ 0.05) greater α-linolenic (C18:3 n-3) and hexadecatrienoic (C16:3) acid contents than those of SLP and the control. Hexadecanoic acid (C16:3) is a unique FA in the galactolipids of the chloroplast; its presence in zebrafish tissues proves that zebrafish digest and absorb chloroplast galactolipids. The lutein profile of eggs produced by zebrafish fed with the CRF diet was significantly (P ≤ 0.05) higher than those of SLP and the control. Alterations in egg colour were also noted, warranting further investigations of the diet impact on fish fecundity, embryo fertility, hatch rate and larval survival.