Regulación nutricional de enzimas clave en la glucolisis-gluconeogénesis: expresión del gen 6-PF 2-K/FRU 2,6-P(2)ASA en hígado de Sparus aurata

[spa] A través de estudios de ayuno y realimentación, y de tipo y cantidad de dieta suministrada, hemos comprobado cómo afecta el estado nutricional a enzimas clave implicadas en glucólisis-gluconeogénesis, vía de las pentosas fosfato y metabolismo de aminoácidos en hígado del pez teleósteo Sparus aurata. La investigacion se ha centrado en el control de la expresión génica de la enzima bifuncional 6-PF 2-K/FRU 2,6-P2ASA, responsable de la sintesis y degradación de FRU 2,6-P2, principal activador de la glucólisis e inhibidor de la gluconeogénesis. Flujo glucolítico, ruta de las pentosas, así como actividad quinasa, cantidad de proteína y niveles de mRNA de 6-PF 2-K/FRU 2,6-P2ASA, parámetros todos disminuídos por el ayuno, correlacionaron con la cantidad de dieta suministrada y con el nivel de carbohidratos aportado por la dieta. Esta enzima ha mostrado ser regulada a corto plazo fundamentalmente por alosterismo y/o modificación covalente, y a largo plazo por el nivel de proteína y mensajero. El análisis de los niveles de mRNA por Northern Blot se ha realizado con una sonda homóloga obtenida por RT-PCR, cuya secuencia es la primera descrita para el mensajero de 6-PF 2-K/FRU, 2,6-P2ASA en peces

Dietary cobalt supplementation improves growth and body composition and induces the expression of growth and stress response genes in Tor putitora

A 90-day randomized feeding experiment was performed to assess the effects of dietary cobalt (Co) supplementation on the growth performance, muscle composition, status of iron and manganese in the muscle as well as the expression of growth-related genes in the muscle (myoblast determination protein 1 homolog (MyoD) and myogenin) and the stress-related gene heat shock protein 70 KDa (Hsp-70) in the liver of mahseer (Tor putitora). Feeding trial was conducted in triplicate under controlled semi-static conditions, and graded levels of dietary cobalt (0.5-3 mg/kg) were fed to six groups of advanced fry of T. putitora. The results obtained indicated a curvilinear relationship of dietary Co levels with body crude protein content and weight gain (%). A positive correlation was observed with up to 2 mg Co/kg diet. However, a decreasing trend was found with values over 2 mg Co/kg diet. The expression of muscle growth biomarkers MyoD and myogenin showed a similar response, upregulation up to 2 mg Co/kg diet and decreased expression at 3 mg Co/kg diet. Indeed, the highest dietary Co supplementation increased the expression of Hsp-70, a key gene expressed in response to stress. Moreover, the muscle content of iron and manganese showed an inverse relationship with the dietary Co supplementation. Our findings suggest that 2 mg/kg Co dietary supplementation stimulates myogenesis and optimize muscle growth and body composition, while higher levels enhanced the expression of stress response genes and impaired growth of T. putitora

Chitosan-Based Drug Delivery System: Applications in Fish Biotechnology

Chitosan is increasingly used for safe nucleic acid delivery in gene therapy studies, due to well-known properties such as bioadhesion, low toxicity, biodegradability and biocompatibility. Furthermore, chitosan derivatization can be easily performed to improve the solubility and stability of chitosan-nucleic acid polyplexes, and enhance e cient target cell drug delivery, cell uptake, intracellular endosomal escape, unpacking and nuclear import of expression plasmids. As in other fields, chitosan is a promising drug delivery vector with great potential for the fish farming industry. This review highlights state-of-the-art assays using chitosan-based methodologies for delivering nucleic acids into cells, and focuses attention on recent advances in chitosan-mediated gene delivery for fish biotechnology applications. The e ciency of chitosan for gene therapy studies in fish biotechnology is discussed in fields such as fish vaccination against bacterial and viral infection, control of gonadal development and gene overexpression and silencing for overcoming metabolic limitations, such as dependence on protein-rich diets and the low glucose tolerance of farmed fish. Finally, challenges and perspectives on the future developments of chitosan-based gene delivery in fish are also discussed

Effects of Pecan Nut (Carya illinoiensis) and Roselle Flower (Hibiscus sabdariffa) as Antioxidant and Antimicrobial Agents for Sardines (Sardina pilchardus)

The effects of pecan nut (Carya illinoinensis) and roselle flower (Hibiscus sabdariffa) as antioxidant and antimicrobial agents on shelf life extension of sardines (Sardina pilchardus) were evaluated over a period of 5 days at 7 ± 1 ºC. Treatments consisted of the addition of 5% and 10% w/w pecan nut, 5% w/w roselle flower and a combination of 5% of each. Physicochemical (lipid oxidation, fatty acids, hexanal and biogenic amines), sensory and microbiological characteristics of fish samples were periodically analyzed. All treatments effectively improved physicochemical quality parameters, with 10% w/w pecan nut having the highest effectiveness. The presence of roselle flower reduced microbial growth. Our findings suggest that addition of a natural preservative combining pecan nut and roselle flower may extend the shelf life of fresh sardines during chilled storage while maintaining quality indexes

Antibacterial and Antiproliferative Activities of Azadirachta indica Leaf Extract and Its Effect on Oil-in-Water Food Emulsion Stability

The present study aims to identify and quantify the phenolic compounds of Azadirachta indica leaf extract using HPLC-MS and to evaluate the antioxidant, antibacterial (against different Gram-positive and negative bacteria) and in vitro anti-proliferative activities of this extract (against breast, human liver and cervix adenocarcinoma-derived cells). The application of this extract as a natural antioxidant for food preservation was also tested on oil-in-water food emulsions for the first time in the present work in order to determine the use of Azadirachta indica leaves as a natural additive to preserve the food against lipid oxidation and rancidity. The results obtained revealed that 50%-aqueous ethanol leaf extract showed the best extraction yield (25.14%), which was characterized by a high content in phenolic compounds and strong antioxidant activity. Moreover, this leaf extract inhibited the growth of the bacterial strains tested (Staphylococcus aureus, Escherichia coli, Salmonella paratyphi and Micrococcus luteus) and showed better anti-proliferative activity against breast and cervix adenocarcinoma-derived cells than human liver cancer cells after 48 h of treatment. Additionally, Azadirachta indica leaf extract showed almost similar effects as gallic acid solutions (0.25% and 0.5%) in preserving the oxidation of oil-in-water food emulsions and prevented the formation of secondary oxidation products (malondialdehyde) as well. The results obtained suggested that extracts of Azadirachta indica leaves are a potential source of antioxidant and antibacterial compounds and pointed to the potential of these natural extracts as therapeutic agents. Keywords: antioxidant; Azadirachta indica; anti-bacterial; cancer cells; food emulsion; HPLC-M

Effect of diet composition on growth performance, hepatic metabolism and antioxidant activities in Siberian sturgeon (Acipenser baerii, Brandt, 1869) submitted to starvation and refeeding

Many fish species undergo natural starvation periods. Adaptation to starvation is possible through the activation of behavioral, biochemical and physiological mechanisms. Knowledge of the effect of dietary nutrients on the intermediary metabolism during starvation and refeeding can be useful to improve fish health and optimize aquaculture production. To analyze the effect of dietary nutrients on liver metabolism of Siberian sturgeon (Acipenser baerii) submitted to starvation and refeeding, four isoenergetic diets differing in nutrient composition were designed: LP-St (38 % protein, 12 % lipid, 36 % carbohydrate), HP-St (44 % protein, 10 % lipid, 30 % carbohydrate), LP-L (38 % protein, 18 % lipid, 25 % carbohydrate) and HP-L (44 % protein, 16 % lipid, 22 % carbohydrate). Four groups of fish were fed 3 weeks to satiety with the corresponding diet, starved for 2 weeks and then refed 5 weeks to satiety on the same diet. Starvation mobilized the hepatic lipid store to a greater extent than glycogen. Starvation increased superoxide dismutase activity irrespective of the diet, while low protein diets (LP-St and LP-L) increased catalase activity. The oxidative damage decreased after 5 weeks of refeeding. Refeeding the starved fish on the HP-St diet promoted the greatest growth performance. In addition to report for the first time the effect of diet composition on growth, liver composition and antioxidant activities in Siberian sturgeon submitted to starvation and refeeding, our findings suggest that refeeding on HP-St diet stimulated the use of dietary carbohydrates and allowed a protein sparing effect in Siberian sturgeon

Metformin counteracts glucose-dependent lipogenesis and impairs transdeamination in the liver of gilthead sea bream (Sparus aurata)

Metformin is an anti-diabetic drug with a major impact on regulating blood glucose levels by decreasing hepatic gluconeogenesis but also affecting other pathways, including glucose transport and energy/lipid metabolism. Carnivorous fish are considered glucose intolerant, as they exhibit poor ability to using dietary carbohydrates. To increase the current knowledge about the molecular mechanisms by which metformin can improve glucose homeostasis in carnivorous fish, we addressed the effect of intraperitoneal administration of metformin, in the presence or absence of a glucose load, on metabolic rate-limiting enzymes and lipogenic factors in the liver of gilthead sea bream (Sparus aurata). Hyperglycemia markedly up-regulated the expression of glycolytic enzymes (glucokinase and 6-phosphofructo-1-kinase, PFK1) 5 h following glucose administration, while at 24 h post-treatment it increased isocitrate dehydrogenase (IDH) activity, a key enzyme of the tricarboxylic acid cycle, and the expression of lipogenic factors (PGC1b, Lpin1 and SREBP1). Metformin counteracted glucose-dependent effects, and down-regulated glutamate dehydrogenase, alanine aminotransferase and mTOR 5 h post-treatment in the absence of a glucose load, leading to decreased long-term activity of PFK1 and IDH. The results of the present study suggest that hyperglycemia enhances lipogenesis in the liver of S. aurata, and that metformin may exert specific metabolic effects in fish by decreasing hepatic transdeamination and supressing the use of amino acids as gluconeogenic substrates. Our findings highlight the role of amino acid metabolism in the glucose-intolerant carnivorous fish model. KEYWORDS: Glutamate dehydrogenase; Lipogenesis; Liver; Metformin; Sparus aurat

The Effects of Pecan Shell, Roselle Flower and Red Pepper on the Quality of Beef Patties during Chilled Storage

The antioxidant and antimicrobial effects of pecan shell (PSW), combined with roselle flower (RS) and red pepper (CA) were analyzed in beef patties by several methods during chilled storage for 13 days. Additionally, the antioxidant and antimicrobial activities of PSW, RS and CA extracts were determined. The PSW extract exhibited a higher radical scavenging activity (by the DPPH method) and more total phenolic compounds than RS and CA. RS presented the best antimicrobial capacity. Nine formulations of beef patties were prepared, including a control (CM), a synthetic preservative (CAMPA N.3 (A)) and different combinations of PSW, RS and CA. The bacterial counts of the beef patties with RS (4-5 log colony-forming units (CFU)/g meat) were significantly lower than those of the control sample (CM) (6-7 CFU/g meat) at day 6. The thiobarbituric acid-reactive substance (TBARS) values at day 7 of all treatments were similar to the values of samples containing the synthetic antioxidant and significantly lower than the CM group. The order of stability assessed by the TBARS values were in agreement with the hexanal content. Thus, these results support the hypothesis that the combination of PWS, RS and CA could represent a good natural food preservative

Disposition of a Glucose Load into Hepatic Glycogen by Direct and Indirect Pathways in Juvenile Seabass and Seabream

In carnivorous fish, conversion of a glucose load to hepatic glycogen is widely used to assess their metabolic flexibility towards carbohydrate utilization, but the activities of direct and indirect pathways in this setting are unclear. We assessed the conversion of an intraperitoneal glucose load (2 g.kg−1) enriched with [U-13C6]glucose to hepatic glycogen in juvenile seabass and seabream. 13C-NMR analysis of glycogen was used to determine the contribution of the load to glycogen synthesis via direct and indirect pathways at 48-hr post-injection. For seabass, [U-13C6]glucose was accompanied by deuterated water and 2H-NMR analysis of glycogen 2H-enrichment, allowing endogenous substrate contributions to be assessed as well. For fasted seabass and seabream, 47 ± 5% and 64 ± 10% of glycogen was synthesized from the load, respectively. Direct and indirect pathways contributed equally (25 ± 3% direct, 21 ± 1% indirect for seabass; 35 ± 7% direct, 29 ± 4% indirect for seabream). In fasted seabass, integration of 2H- and 13C-NMR analysis indicated that endogenous glycerol and anaplerotic substrates contributed an additional 7 ± 2% and 7 ± 1%, respectively. In fed seabass, glucose load contributions were residual and endogenous contributions were negligible. Concluding, direct and indirect pathways contributed equally and substantially to fasting hepatic glycogen repletion from a glucose load in juvenile seabream and seabass