Effects of dietary fatty acids on mitochondrial phospholipid compositions, oxidative status and mitochondrial gene expression of zebrafish at different ages.

Mitochondrial decay is generally associated with impairment in the organelle bioenergetics function and increased oxidative stress, and it appears that deterioration of mitochondrial inner membrane phospholipids (PL) and accumulation of mitochondrial DNA (mtDNA) mutations are among the main mechanisms involved in this process. In the present study, mitochondrial membrane PL compositions, oxidative status (TBARS content and SOD activity) and mtDNA gene expression of muscle and liver were analyzed in zebrafish fed two diets with lipid supplied either by rapeseed oil (RO) or a blend 60:40 of RO and DHA500 TG oil (DHA). Two feeding trials were performed using zebrafish from the same population of two ages (8 and 21 months). Dietary FA composition affected fish growth in 8-month-old animals, which could be related to an increase in stress promoted by diet composition. Lipid peroxidation was considerably higher in mitochondria of 8-month-old zebrafish fed the DHA diet than in animals fed the RO diet. This could indicate higher oxidative damage to mitochondrial lipids, very likely due to increased incorporation of DHA in PL of mitochondrial membranes. Lipids would be among the first molecules affected by mitochondrial reactive oxygen species, and lipid peroxidation could propagate oxidative reactions that would damage other molecules, including mtDNA. Mitochondrial lipid peroxidation and gene expression of 21-month-old fish showed lower responsiveness to diet composition than those of younger fish. Differences found in the effect of diet composition on mitochondrial lipids between the two age groups could be indicating age-related changes in the ability to maintain structural homeostasis of mitochondrial membranes.

Roles of selenoprotein antioxidant protection in zebrafish, Danio rerio, subjected to dietary oxidative stress.

In vertebrates, selenium (Se) is an essential micronutrient for vertebrates that is involved in antioxidant protection and thyroid hormone regulation among other roles and functions through its incorporation into proteins, the selenoproteins. Long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are essential nutrients for fish although high dietary levels may lead to increased oxidative stress due to the high degree of unsaturation. The present study investigated the effects of Se supplementation on zebrafish, Danio rerio, oxidative status together with selenoprotein expression profiles when subjected to a high-DHA diet. Fish were fed for 8 weeks with one of the four experimental diets, containing high or low DHA in combination with or without organic Se (7 mg/kg). Fish performance, Se content, fatty acid composition and TBARS of zebrafish were determined, as well as gene expression of selected selenoproteins in liver and muscle. The Se levels in whole fish reflected dietary content. High dietary DHA increased oxidative stress as indicated by reduced growth and high TBARS content, although Se supplementation reduced oxidation. The expression patterns of selenoproteins varied between liver and muscle with only deiodinase type II displaying a transcriptional response when high dietary Se was supplied. High dietary DHA decreased selenoprotein W expression in muscle and sps2 expression in liver regardless of the dietary Se content. These data suggest that oxidative stress protection associated with a high dietary intake of Se may not be solely mediated by transcriptional changes in teleost selenoprotein expression.

Energy intake and macronutrient selection in sharpsnout seabream (Diplodus puntazzo) challenged with fat dilution and fat deprivation using encapsulated diets.

Sharpsnout seabream fed pure macronutrient capsules were challenged to fat dilution and fat deprivation in order to investigate the effects of fat level on energy intake regulation and macronutrient selection by fish, as they lack oropharyngeal chemosensory information from the diet. During the control phase, the fish were fed three individually encapsulated macronutrients, from which they composed a diet containing 67.36% protein (P), 19.08% carbohydrates (CH) and 13.57% fat (F), in terms of macronutrient weight intake percentage. During the second phase of the experiment, a lipid content reduction in F capsules from 55.0% to 13.4% did not significantly modify this selection pattern, energy ingestion or the number of capsules ingested of each macronutrient. During the third phase, in which they were subjected to fat deprivation, starting on almost the first day, the fish increased their total energy intake and total ingested number of capsules. These results reveal that fish are capable of distinguishing and selecting each of the three macronutrients contained in gelatine capsules, and that fish selection of a balanced diet from pure macronutrients is remarkably stable. Fish are capable of sustaining their macronutrient selection pattern and energy intake with very low amounts of fat in their diets (Phase 2). A certain instability in the initial P, CH and energy intake was only observed when fat was totally deprived (Phase 3), which resulted in higher values than those observed in Phase 1. In order to examine any possible effects of diet encapsulation, digestibility assays were performed in a second experiment. The fish were divided into two experimental groups and fed the same complete commercial diet, the only difference being the way it was presented to each group (pelleted or encapsulated). No statistical differences between the experimental groups were found with regards to both apparent digestibility coefficients and fish growth.

Circadian monitoring as an aging predictor.

The ageing process is associated with sleep and circadian rhythm (SCR) frailty, as well as greater sensitivity to chronodisruption. This is essentially due to reduced day/night contrast, decreased sensitivity to light, napping and a more sedentary lifestyle. Thus, the aim of this study is to develop an algorithm to identify a SCR phenotype as belonging to young or aged subjects. To do this, 44 young and 44 aged subjects were recruited, and their distal skin temperature (DST), activity, body position, light, environmental temperature and the integrated variable TAP rhythms were recorded under free-living conditions for five consecutive workdays. Each variable yielded an individual decision tree to differentiate between young and elderly subjects (DST, activity, position, light, environmental temperature and TAP), with agreement rates of between 76.1% (light) and 92% (TAP). These decision trees were combined into a unique decision tree that reached an agreement rate of 95.3% (4 errors out of 88, all of them around the cut-off point). Age-related SCR changes were very significant, thus allowing to discriminate accurately between young and aged people when implemented in decision trees. This is useful to identify chronodisrupted populations that could benefit from chronoenhancement strategies.

Impact of a shift work-like lighting schedule on the functioning of the circadian system in the short-lived fish Nothobranchius furzeri.

Adult Nothobranchius furzeri of the MZM-04/10 strain were individually kept and subjected to a "5 + 2" shifting lighting schedule (SHIFT) for 8 weeks in order to evaluate the desynchronizing effects of a simulated human-like shift-work schedule on the functioning of the circadian system (CS). With this aim, sixteen 21-week-old N. furzeri were placed into a Morning, Night and Evening schedule (lights on from 08:00 to 16:00, 00:00 to 08:00 and 16:00 to 00:00 h, respectively) and fed once a day in the middle of the corresponding photophase (12:00, 04:00 and 20:00 h, respectively). Then, in the weekends (2 days), fish were always returned to the Morning shift. As controls, 16 fish were maintained under a non-shifting LD cycle condition (CONTROL) throughout the whole experiment, with lights on from 08:00 to 16:00 h. Rest-activity rhythm (RAR) of fish subjected to SHIFT showed several symptoms of chronodisruption, such as a decrease in the percentage of diurnal activity and a reduction of the relative amplitude and the circadian function index with time. When a periodogram analysis was performed, RAR of N. furzeri under SHIFT conditions showed up to three separate circadian components: one longer than 24 h (26.5 h) that followed the weekly 8 h delays; a short-period component (~23 h) that was related to the weekend's phase advances, and finally, a 24 h component. The shifting LD schedule also affected fish CS at a molecular level, with several significant differences in the expression of core genes of the molecular clock (bmal1, clock, rorα, rev-erbα) between SHIFT and CONTROL animals. RAR impairment along with changes in clock gene expression could be associated with high stress and accelerated aging in these fish.

Macronutrient selection through post-ingestive signals in sharpsnout seabream fed gelatine capsules and challenged with protein dilution.

Sharpsnout seabream ability for macronutrient self-selection was studied using gelatine capsules containing pure macronutrients. In particular, the existence of non-oropharyngeal chemosensory pathways involved on protein (P), fat (F) and carbohydrate (CH) selection, as well as sharpsnout seabream response to dietary protein dilution were investigated. In a sequence of experimental phases, sharpsnout seabream were fed a pelleted complete diet, an encapsulated complete diet or a combination of separately encapsulated pure macronutrients. In order to induce associative learning, capsules containing a given macronutrient were paired with a particular colour. The animals composed a diet containing 62.7% P, 21.3% CH and 16.0% F, in terms of macronutrient percentage intake, and this selection pattern was maintained throughout all experimental phases. In a second experiment, individually kept sharpsnout seabream were challenged with protein dilution. After protein capsules were diluted (from 91.9% P to 56.3% P) with cellulose, the animals increased their protein intake to compensate for dilution in such a way that their energy intake was not significantly modified (17.4 kJ/100 g BW vs. 17.6 kJ/100 g BW after dilution). These results show that sharpsnout seabream feeding on encapsulated diets are able to select and maintain a particular diet composition, as well as sustain their energy intake, without using the diet's oropharyngeal chemosensory properties. Moreover, they were also able to maintain their protein intake after dilution, which highlights the importance of this macronutrient in this omnivorous species.

Dietary fatty acids affect mitochondrial phospholipid compositions and mitochondrial gene expression of rainbow trout liver at different ages.

Mitochondria are among the first responders to various stressors that challenge the homeostasis of cells and organisms. Mitochondrial decay is generally associated with impairment in the organelle bioenergetics function and increased oxidative stress, and it appears that deterioration of mitochondrial inner membrane phospholipids (PL), particularly cardiolipin (CL), and accumulation of mitochondrial DNA (mtDNA) mutations are among the main mechanisms involved in this process. In the present study, liver mitochondrial membrane PL compositions, lipid peroxidation, and mtDNA gene expression were analyzed in rainbow trout fed three diets with the same base formulation but with lipid supplied either by fish oil (FO), rapeseed oil (RO), or high DHA oil (DHA) during 6 weeks. Specifically, two feeding trials were performed using fish from the same population of two ages (1 and 3 years), and PL class compositions of liver mitochondria, fatty acid composition of individual PL classes, TBARS content, and mtDNA expression were determined. Dietary fatty acid composition strongly affected mitochondrial membrane composition from trout liver but observed changes did not fully reflect the diet, particularly when it contained high DHA. The changes were PL specific, CL being particularly resistant to changes in DHA. Some significant differences observed in expression of mtDNA with diet may suggest long-term dietary effects in mitochondrial gene expression which could affect electron transport chain function. All the changes were influenced by fish age, which could be related to the different growth rates observed between 1- and 3-year-old trout but that could also indicate age-related changes in the ability to maintain structural homeostasis of mitochondrial membranes.

Age-related changes in fatty acid profile and locomotor activity rhythms in Nothobranchius korthausae.

The life cycle of Nothobranchius korthausae, a Cyprinodontiformes fish, was studied in our laboratory to characterise the ageing process. Some morphological changes, such as spine curvature, skin colour, and fin and eye appearance are described. Growth and survival curves reflected a fast life cycle with rapid initial growth until 4weeks of age, after which the fish grew more slowly before reaching their final size in week 40. Senescence onset was established at week 48 with a decrease in spawn size and viability and a general decline in the animal's appearance (weight and colouration losses, caudal fin degradation, and cataractogenesis). The fatty acid composition changed with age, with high unsaturation in the adult stage as reflected by a high peroxidation index, a condition that is associated with high susceptibility to oxidative damage if elevated reactive oxygen species (ROS) production occurs. Senescent fish had an increase in monounsaturated fatty acid proportions and a lower peroxidation index (226.5±19.7 in adults versus 120.2±19.1 in senescent fish, P<0.05). The circadian system, as reflected by locomotor activity rhythms, showed noticeable changes with age. Twenty-four-week-old fish (adults) had a robust diurnal rhythm that showed a decrease in total activity, an increase in rhythm fragmentation, and a fall in amplitude and regularity with age. Changes were clearly reflected in the Circadian Function Index variations (0.56, 0.47 and 0.25 at 24, 48 and 72weeks of age, respectively). In conclusion, N. korthausae appears to be a species with appropriate characteristics for ageing studies because it manifests clear signs of progressive ageing. Comparing species of Nothobranchius genus with different lifespans may be useful for increasing our understanding of the ageing process.