Hepatic Glucose Metabolism and Its Disorders in Fish.

Carbohydrate, which is the most abundant nutrient in plant-sourced feedstuffs, is an economically indispensable component in commercial compound feeds for fish. This nutrient can enhance the physical quality of diets and allow for pellet expansion during extrusion. There is compelling evidence that an excess dietary intake of starch causes hepatic disorders, thereby further reducing the overall food consumption and growth performance of fish species. Among the severe metabolic disturbances are glycogenic hepatopathy (hepatomegaly caused by the excessive accumulation of glycogen in hepatocytes) and hepatic steatosis (the accumulation of large vacuoles of triacylglycerols in hepatocytes). The development of those disorders is mainly due to the limited ability of fish to oxidize glucose and control blood glucose concentration. The prolonged elevations of blood glucose increase glucose intake by the liver, and excess glucose is stored either as glycogen through glycogenesis in hepatocytes or as triglycerides via lipogenesis in tissues, depending on the species. In some fish species (e.g., largemouth bass), the liver has a low ability to regulate glycolysis, gluconeogenesis, and glycogen breakdown in response to high starch intake. For most species of fish, the liver size increases with lipid or glycogen accumulation when they have a high starch intake. It is a challenge to develop the same set of diagnostic criteria for all fish species as their physiology or metabolic patterns differ. Although glycogenic hepatopathy appears to be a common disease in carnivorous fish, it has been under-recognized in many studies. As a result, understanding these diseases and their pathogeneses in different fish species is crucial for manufacturing cost-effective pellet diets to promote the health, growth, survival, and feed efficiency of fish in future.

Late/post-term decidual basalis-derived mesenchymal stem/stromal cells show evidence of advanced ageing and downregulation of microRNA-516b-5p.

INTRODUCTION: The placenta is a short-lived organ, yet it shows signs of progressive ageing in the third trimester. Studies of ageing chorionic placental tissue have recently flourished, providing evidence of advanced ageing of tissues in the late/post-term (L/PT) period of gestation. However, ageing of the maternal aspect of the maternal-fetal interface, specifically the decidua basalis, is poorly understood. Here, we investigated whether the L/PT period was associated with advanced ageing and exhaustion of important decidua basalis mesenchymal stem/stromal cells (DMSCs) functions. METHODS: In this study, DMSCs were isolated and characterised from early term (ET) and L/PT placental tissue and they were then investigated by employing various MSC potency and ageing assays. RNA sequencing was also performed to screen for specific microRNAs that are associated with stem cell exhaustion and ageing between ET- and L/PT-DMSCs. RESULTS: L/PT-DMSCs, when compared to ET-DMSCs, showed significantly lower cell proliferation and a significant higher level of cell apoptosis. L/PT-DMSCs showed significantly lower resistance to oxidative stress and a significant decrease in antioxidant capacity compared with ET-DMSCs. Western blot analysis revealed increased expression of the stress-mediated P-p38MAPK protein in L/PT-DMSCs. RNA Sequencing showed microRNA (miR) miR-516b-5p, was present at significantly lower levels in L/PT-DMSCs. Inhibition of miR-516b-5p in ET-DMSCs revealed a decline in the ability of the inhibited cells to survive in extended cell culture. DISCUSSION: These data provide the first evidence of advanced ageing and exhaustion of important stem cell functions in L/PT-DMSCs, and the involvement of specific miRs in the DMSC ageing process.

Nutrition and Functions of Amino Acids in Fish.

Aquaculture is increasingly important for providing humans with high-quality animal protein to improve growth, development and health. Farm-raised fish and shellfish now exceed captured fisheries for foods. More than 70% of the production cost is dependent on the supply of compound feeds. A public debate or concern over aquaculture is its environmental sustainability as many fish species have high requirements for dietary protein and fishmeal. Protein or amino acids (AAs), which are the major component of tissue growth, are generally the most expensive nutrients in animal production and, therefore, are crucial for aquatic feed development. There is compelling evidence that an adequate supply of both traditionally classified nutritionally essential amino acids (EAAs) and non-essential amino acids (NEAAs) in diets improve the growth, development and production performance of aquatic animals (e.g., larval metamorphosis). The processes for the utilization of dietary AAs or protein utilization by animals include digestion, absorption and metabolism. The digestibility and bioavailability of AAs should be carefully evaluated because feed production processes and AA degradation in the gut affect the amounts of dietary AAs that enter the blood circulation. Absorbed AAs are utilized for the syntheses of protein, peptides, AAs, and other metabolites (including nucleotides); biological oxidation and ATP production; gluconeogenesis and lipogenesis; and the regulation of acid-base balance, anti-oxidative reactions, and immune responses. Fish producers usually focus on the content or digestibility of dietary crude protein without considering the supply of AAs in the diet. In experiments involving dietary supplementation with AAs, inappropriate AAs (e.g., glycine and glutamate) are often used as the isonitrogenous control. At present, limited knowledge is available about either the cell- and tissue-specific metabolism of AAs or the effects of feed processing methods on the digestion and utilization of AAs in different fish species. These issues should be addressed to develop environment-friendly aquafeeds and reduce feed costs to sustain the global aquaculture.

Nutrition and Functions of Amino Acids in Aquatic Crustaceans.

Crustaceans (e.g., shrimp and crabs) are a good source of protein-rich foods for human consumption. They are the second largest aquaculture species worldwide. Understanding the digestion of dietary protein, as well as the absorption, metabolism and functions of amino acids (AAs) and small peptides is essential to produce cost-effective and sustainable aquafeeds. Hepatopancreas (the midgut gland) is the main site for the digestion of dietary protein as well as the absorption of small peptides and AAs into the hemolymph. Besides serving as the building blocks of protein, AAs (particularly aspartate, glutamate, glutamine and alanine) are the primary metabolic fuels for the gut and extra-hepatopancreas tissues (e.g., kidneys and skeletal muscle) of crustaceans. In addition, AAs are precursors for the syntheses of glucose, lipids, H2S, and low-molecular-weight molecules (e.g., nitric oxide, glutathione, polyamines, histamine, and hormones) with enormous biological importance, such as physical barrier, immunological and antioxidant defenses. Therefore, both nutritionally essential and nonessential AAs are needed in diets to improve the growth, development, molt rate, survival, and reproduction of crustaceans. There are technical difficulties and challenges in the use of crystalline AAs for research and practical production due to the loss of free AAs during feed processing, the leaching of in-feed free AAs to the surrounding water environment, and asynchronous absorption with peptide-bounded AAs. At present, much knowledge about AA metabolism and functions in crustaceans is based on studies of mammals and fish species. Basic research in this area is necessary to lay a solid foundation for improving the balances and bioavailability of AAs in the diets for optimum growth, health and wellbeing of crustaceans, while preventing and treating their metabolic diseases. This review highlights recent advances in AA nutrition and metabolism in aquatic crustacean species at their different life stages. The new knowledge is expected to guide the development of the next generation of their improved diets.

Use of alternative protein sources for fishmeal replacement in the diet of largemouth bass (Micropterus salmoides). Part II: effects of supplementation with methionine or taurine on growth, feed utilization, and health.

Fishmeal has long been a staple protein feedstuff for fish, but its global shortage and high price have prompted its replacement with alternative sustainable sources. In this experiment involving largemouth bass (a carnivorous fish), a new mixture of feedstuffs (45% poultry byproduct meal, 30% soybean meal, 15% blood meal, and 10% krill shrimp meal) was added to low (14.5%) fishmeal diets along with 0.0%, 0.5% taurine, 0.5% methionine, or 0.5% taurine plus 0.5% methionine (dry matter basis). The positive control diet [65.3% fishmeal (46% crude protein on dry matter basis)] and all low-fishmeal diets contained 40% true protein and 10% lipids. There were 3 tanks per treatment group (20 fish/tank). Fish with the mean initial body weight of 16.6 g were fed to satiety twice daily. Compared with the unsupplemented low-fishmeal group, supplementing either 0.5% methionine or 0.5% methionine plus 0.5% taurine to the low-fishmeal diet improved (P < 0.05) the growth, feed utilization, retention of dietary protein and lipids, and health of largemouth bass, reduced (P < 0.05) the occurrence of black skin syndrome from ~ 40 to ~ 10%. Histological sections of tissues from the fish with black skin syndrome showed retina degeneration, liver damage, and enteritis in the intestine. Compared with methionine supplementation, supplementing 0.5% taurine alone to the low-fishmeal diet did not affect the growth or feed efficiency of fish and had less beneficial effects (P < 0.05) on ameliorating the black skin syndrome. These results indicated that: (a) the basal low-fishmeal diet was inadequate in methionine or taurine; and (b) dietary supplementation with methionine was an effective method to improve the growth performance, feed efficiency, and health of largemouth bass. Further studies are warranted to understand the pathogenesis of the black skin syndrome in largemouth bass.

Use of alternative protein sources for fishmeal replacement in the diet of largemouth bass (Micropterus salmoides). Part I: effects of poultry by-product meal and soybean meal on growth, feed utilization, and health.

Five isonitrogenous and isocaloric diets [containing 54, 30, 15, 10, and 5% fishmeal crude-protein (CP), dry matter (DM) basis] were prepared by replacing fishmeal with poultry by-product meal plus soybean meal to feed juvenile largemouth bass (LMB, with an initial mean body weight of 4.9 g) for 8 weeks. All diets contained 54% CP and 13% lipids. There were four tanks of fish per treatment group (15 fish/tank). The fish were fed twice daily with the same feed intake (g/fish) in all the dietary groups. Results indicated that the inclusion of 15% fishmeal protein in the diet is sufficient for LMB growth. However, some of the fish that were fed diets containing ≤ 15% fishmeal CP had black skin syndrome (characterized by skin darkening and retinal degeneration, as well as intestinal and liver atrophies and structural abnormalities). The concentrations of taurine, methionine, threonine and histidine in serum were reduced (P < 0.05) in fish fed the diets containing 5, 10 and 15% fishmeal CP, compared with the 30 and 54% fishmeal CP diets. Interestingly, the concentrations of tyrosine and tryptophan in serum were higher in fish fed diets with ≤ 15% fishmeal CP than those in the 54% fishmeal CP group. These results indicated that 15% fishmeal CP in the diet containing poultry by-product meal and soybean meal was sufficient for the maximum growth and feed efficiency in LMB but inadequate for their intestinal, skin, eye, and liver health. A reduction in dietary methionine and taurine content and the possible presence of antinutritional factors in the fishmeal replacements diets containing high inclusion levels of soybean meal may contribute to black skin syndrome in LMB. We recommend that the diets of juvenile LMB contain 30% fishmeal CP (DM basis).

Effects of dietary protein intake on the oxidation of glutamate, glutamine, glucose and palmitate in tissues of largemouth bass (Micropterus salmoides).

Largemouth bass (Micropterus salmoides, a carnivorous fish native to North America) prefers to utilize amino acids as energy sources rather than glucose and fatty acids. However, little is known about the nutritional regulation of substrate oxidation in the fish. Therefore, this study was conducted to determine whether the oxidation of glutamate, glutamine, glucose and palmitate in its tissues might be influenced by dietary protein intake. Juvenile largemouth bass (initial weight 18.3 ± 0.1 g) were fed three isocaloric diets containing 40%, 45% and 50% protein for 8 weeks. The growth performance, energy retention, and lipid retention of juvenile fish increased with increasing dietary protein levels. The rate of oxidation of glutamate by the intestine was much greater than that of glutamine, explaining why increasing the dietary protein content from 40% to 50% had no effect on the serum concentration of glutamate but increased that of glutamine in the fish. The liver of fish fed the 50% protein diet had a higher (P < 0.05) rate of glutamine oxidation than that in the 40% and 45% protein groups. In contrast, augmenting dietary protein content from 40% to 45% increased (P < 0.05) both glutamine and glutamate oxidation in the proximal intestine of the fish and renal glutamine oxidation, without changes in intestinal or renal AA oxidation between the 45% and 50% protein groups. Furthermore, the rates of glucose oxidation in the liver, kidney, and intestine of largemouth bass were decreased in response to an  increase in dietary  protein content   from 40% to 45% and a concomitant decrease in dietary starch content from 22.3% to 15.78%, but did not differ between the 45% and 50% protein groups.   The rates of oxidation of glucose in skeletal muscle and those of palmitate in all tissues (except for the  kidney) were not affected by the diets. Collectively, these results indicate that the largemouth bass can regulate substrate metabolism in a  tissue-specific manner to favor protein and lipid gains as dietary protein content increases from 40% to 50% and have a lower ability to oxidize fatty acids and glucose than amino acids regardless of the dietary protein intake.

Amino Acid Metabolism in the Kidneys: Nutritional and Physiological Significance.

The kidneys are developed from the intermediate mesoderm of the embryo. They are important for osmoregulation, regulation of acid-base balance, reabsorption of nutrients, and excretion of metabolites. In fish, the kidneys also serve as a hematopoietic, lymphoid and endocrine organ for the generation of red blood cells, the development of lymphocytes, and the production of hormones (e.g., glucocorticoids, catecholamines, and thyroid hormones). In humans and all animals, kidneys play a vital role in the metabolism and reabsorption of amino acids (AAs) and glucose. Specifically, this organ contributes to glucose synthesis from AAs, lactate and pyruvate via the gluconeogenesis pathway; regulates acid-base balance via inter-organ metabolism of glutamine; and synthesizes arginine, tyrosine, and glycine, respectively, from citrulline, phenylalanine, and 4-hydroxyproline. In mammals and birds, kidneys participate in creatine synthesis. Renal dysfunction adversely alters the concentrations of AAs in blood, while promoting muscle protein breakdown, inflammation, mitochondrial abnormalities, defects in the immune response, and cardiovascular diseases. Moderation of dietary AA intake has a protective and therapeutic effect on chronic kidney disease. Understanding the functions and metabolism of AAs in kidneys is essential for maintaining whole-body homeostasis, improving health and well-being, and preventing or treating renal metabolic diseases in humans and farm animals (including swine, poultry, ruminants, fish and shrimp).

Decidual mesenchymal stem/stromal cell-derived extracellular vesicles ameliorate endothelial cell proliferation, inflammation, and oxidative stress in a cell culture model of preeclampsia.

Oxidative stress and endothelial dysfunction contribute substantially to the pathogenesis of preeclampsia (PE). Decidual mesenchymal stem/stromal cells (DMSC), reportedly reduce endothelial cell dysfunction and alleviate PE-like symptoms in a murine model. However, as a therapeutic strategy, the use of whole DMSC presents significant technical limitations, which may be overcome by employing DMSC-secreted extracellular vesicles (DMSC_EV). DMSC_EV restoration of endothelial dysfunction through a paracrine effect may alleviate the clinical features of PE. OBJECTIVE: To determine whether DMSC-secreted, extracellular vesicles (DMSC_EV) restore endothelial cell function and reduce oxidative stress. METHODS: DMSC were isolated from the placentae of uncomplicated term pregnancies and DMSC_EV prepared by ultracentrifugation. Human umbilical vein endothelial cells (HUVEC) were treated with bacterial lipopolysaccharide (LPS), or with serum from PE patients, to model the effects of PE. DMSC_EV were then added to treated HUVEC and their growth profiles, inflammatory state, and oxidative stress levels measured. RESULTS: DMSC_EV displayed characteristic features of extracellular vesicles. In both LPS- and PE serum-treatment models, addition of DMSC_EV significantly increased HUVEC cell attachment and proliferation, and significantly reduced production of pro-inflammatory cytokine IL-6. The addition of DMSC_EV to LPS-treated HUVEC had no significant effect on total antioxidant capacity, superoxide dismutase levels or on lipid peroxidation levels. In contrast, the addition of DMSC_EV to PE serum-treated HUVEC resulted in a significant reduction in levels of lipid peroxidation. CONCLUSION: Addition of DMSC_EV had beneficial effects in both LPS- and PE serum- treated HUVEC but the two treatment models to induce endothelial cell dysfunction showed differences. The LPS treatment of HUVEC model may not accurately model the endothelial cell dysfunction characteristic of PE. Human cell culture models of PE show that DMSC_EV improve endothelial cell dysfunction in PE, but testing in in vivo models of PE is required.

Effects of dietary starch and lipid levels on the protein retention and growth of largemouth bass (Micropterus salmoides).

Protein accretion in some fish species is affected by dietary lipids, starch and their interactions, but this aspect of nutrition is largely unknown in largemouth bass (LMB). Therefore, we designed six experimental diets with three starch levels (5%, 10%, and 15%; dry matter  basis) and two lipid levels (10% and 12.5%; dry matter basis) to evaluate the effects of dietary starch and lipid levels on the protein retention, growth, feed utilization, and liver histology of LMB. There were three tanks (18 fish per tank, ~ 4.85 g per fish) per dietary treatment group and the trial lasted for 8 weeks. Fish were fed to apparent satiation twice daily. Results indicated that increasing the dietary starch level from 5 to 15% reduced (P < 0.05) absolute feed intake (AFI; - 9.0%, - 15% and - 14% on days 14-28, 28-42, and 42-56, respectively) and weight gains (- 4.4% and - 6.5% on days 42 and 56, respectively) of LMB. Increasing the dietary lipid level from 10 to 12.5% reduced (P < 0.05) AFI (- 9.7%, - 11.7% and - 11.9% on days 14-28, 28-42; and 42-56, respectively), weight gains (- 4.2%, - 5.9% and - 6.9% on days 28, 42 and 56, respectively), and survival rate (by a 5.6% unit) of LMB. The retention of dietary protein and some amino acids in the body was affected by dietary starch or lipid levels and their interactions. The viscerosomatic index (VSI), hepatosomatic index (HSI), and intraperitoneal fat ratio (IPFR) increased with increasing the dietary starch level from 5 to 15%. Compared with 10% lipids, 12.5% lipids in diets increased IPFR but had no effect on VSI or HSI. The concentrations of glucose in serum increased with increasing the dietary starch level from 5 to 15% at 4 to 24 h after feeding, with the effect of dietary lipids being time-dependent. Compared with a 5%-starch diet, fish fed a diet with 10%- or 15%-starch exhibited an enlarged and pale liver with excessive glycogen. Based on these findings, we recommend dietary lipid and starch levels to be 10% and < 10%, respectively, for juvenile LMB to maximize the retention of dietary protein in their bodies.

Effects of dietary protein and lipid levels on the growth performance, feed utilization, and liver histology of largemouth bass (Micropterus salmoides).

The reported requirements of largemouth bass (LMB, which is native to North America) for dietary protein and lipids varied substantially among previous studies, and this fish fed current formulated diets exhibit poor growth performance and pale liver syndrome. Because amino acids and lipids are known to affect hepatic metabolism and function in mammals, it is imperative to understand the impacts of these dietary macronutrients on the growth and liver morphology of LMB. In this study, we designed six isocaloric diets to determine the effects of different dietary crude protein (CP; 40%, 45%, and 50%; dry matter basis) and lipid levels (7.5% and 10%; dry matter basis) on fat and glycogen deposits, as well as hepatosis in LMB. There were four tanks (12 fish per tank, an average initial weight of 18.4 g/fish) per dietary treatment group and the trial lasted for 8 weeks. Fish were fed to apparent satiation three times daily. Results indicated that LMB fed the 45% or 50% CP diet grew faster (P < 0.05), had less (P < 0.05) glycogen in the liver and smaller (P < 0.05) hepatocyte sizes than fish fed the 40% CP diet, but there was no difference in weight gain or feed efficiency between the 45% and 50% CP diets. The hepatic lipid content did not differ between LMB fed the 40% and 45% CP diets, and the values for these two groups were 29% lower (P < 0.05) than those for LMB fed the 50% CP diet. Compared with the 40% CP group, LMB fed the 45% or 50% CP diet had 8-12% lower content of total minerals, phosphorus, and calcium in the body. Increasing the dietary lipid level from 7.5 to 10% enhanced the weight gains (+ 15%) and feed efficiency (+ 22%), as well as the retention of dietary protein (+ 18%), energy (+ 25%), and phosphorus (+ 7.6%) in the body. No fatty liver occurred in any group of LMB (with hepatic lipid concentrations being < 2%, wet weight basis). Based on these growth, metabolic and histologic data, we recommend dietary CP and lipids levels to be 45% and 10%, respectively, for juvenile LMB.

Nutrition and metabolism of glutamate and glutamine in fish.

Glutamate (Glu) and glutamine (Gln) comprise a large proportion of total amino acids (AAs) in fish in the free and protein-bound forms. Both Glu and Gln are synthesized de novo from other α-amino acids and ammonia. Although these two AAs had long been considered as nutritionally non-essential AAs for an aquatic animal, they must be included adequately in its diet to support optimal health (particularly intestinal health) and maximal growth. In research on fish nutrition, Glu has been used frequently as an isonitrogenous control on the basis of the assumption that this AA has no nutritional or physiological function. In addition, purified diets used for feeding fish generally lack glutamine. As functional AAs, Glu and Gln are major metabolic fuels for tissues of fish (including the intestine, liver, kidneys, and skeletal muscle), and play important roles not only in protein synthesis but also in glutathione synthesis and anti-oxidative reactions. The universality of Glu and Gln as abundant intracellular AAs depends on their enormous versatility in metabolism. Dietary supplementation with Glu and Gln to farmed fish can improve their growth performance, intestinal development, innate and adaptive immune responses, skeletal muscle development and fillet quality, ammonia removal, and the endocrine status. Glu (mainly as monosodium glutamate), glutamine, or AminoGut (a mixture of Glu and Gln) is a promising feed additive to reduce the use of fishmeal, while gaining the profitability of global aquaculture production. Thus, the concept of dietary requirements of fish for Glu and Gln is a paradigm shift in the nutrition of aquatic animals (including fish).

Amino acids are major energy substrates for tissues of hybrid striped bass and zebrafish.

Fish generally have much higher requirements for dietary protein than mammals, and this long-standing puzzle remains unsolved. The present study was conducted with zebrafish (omnivores) and hybrid striped bass (HSB, carnivores) to test the hypothesis that AAs are oxidized at a higher rate than carbohydrates (e.g., glucose) and fatty acids (e.g., palmitate) to provide ATP for their tissues. Liver, proximal intestine, kidney, and skeletal muscle isolated from zebrafish and HSB were incubated at 28.5 °C (zebrafish) or 26 °C (HSB) for 2 h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5 mM D-glucose) containing 2 mM L-[U-14C]glutamine, L-[U-14C]glutamate, L-[U-14C]leucine, or L-[U-14C]palmitate, or a trace amount of D-[U-14C]glucose. In parallel experiments, tissues were incubated with a tracer and  a mixture of unlabeled substrates [glutamine, glutamate, leucine, and palmitate (2 mM each) plus 5 mM D-glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In the presence of glucose or a mixture of substrates, the rates of oxidation of glutamate and ATP production from this AA by the proximal intestine, liver, and kidney of HSB   were much higher than those for glucose and palmitate. This was also true for glutamate in the skeletal muscle and glutamine in the liver of both species, glutamine in the HSB kidney, and leucine in the zebrafish muscle, in the presence of a mixture of substrates. We conclude that glutamate plus glutamine plus leucine contribute to ~80% of ATP production in the liver, proximal intestine, kidney, and skeletal muscle of zebrafish and HSB. Our findings provide the first direct evidence that the major tissues of fish use AAs (mainly glutamate and glutamine) as primary energy sources instead of carbohydrates or lipids.