Carbohydrate deprivation reduces NADPH-production in fish liver but not in adipose tissue

Little is known about the way in which carnivorous fish such as salmonids mobilise and metabolise dietary carbohydrates, which are essential to lipid metabolism. Thus we have studied changes caused by the absence of dietary carbohydrates to the kinetics and molecular behaviour of the four cellular NADPH-production systems [glucose 6-phosphate dehydrogenase (G6PDH); 6-phosphogluconate dehydrogenase (6PGDH); malic enzyme (ME); and isocitrate dehydrogenase NADP-dependent (NADP-IDH)] in the liver and adipose tissue of rainbow trout (Oncorhynchus mykiss). We used spectrophotometry to study enzyme kinetics and nucleic acid concentrations, and immunoblot analysis to determine specific protein concentrations. The absence of carbohydrate reduced specific enzyme activity, maximum rate and catalytic efficiency by almost 65% in G6PDH and 6PGDH, by more than 50% in ME, and by almost 25% in NADP-IDH but caused no significant changes in the K(m) values or activity ratios in any of these hepatic enzymes. Molecular analysis clearly showed that this kinetic behaviour reflected concomitant changes in intracellular enzyme concentrations, produced by protein-induction/repression processes rather than changes in the activity of pre-existing enzymes. We conclude that the absence of carbohydrates significantly reduces intracellular concentrations of G6PDH, ME and NADP-IDH in trout liver in percentages similar to those recorded for enzyme activity. We found no such variations in the concentrations of any of these enzymes in adipose tissue and no change in the levels of their activity, suggesting that the liver and adipose tissues are subject to different regulation systems with regard to carbohydrates and play distinct roles in lipid metabolism.     

Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp

Glucokinase (GK) plays a central role in glucose homeostasis in mammals. The absence of an inducible GK has been suggested to explain the poor utilization of dietary carbohydrates in rainbow trout. In this context, we analyzed GK expression in three fish species (rainbow trout, gilthead seabream, and common carp) known to differ in regard to their dietary carbohydrate tolerance. Fish were fed for 10 wk with either a diet containing a high level of digestible starch (>20%) or a diet totally deprived of starch. Our data demonstrate an induction of GK gene expression and GK activity by dietary carbohydrates in all three species. These studies strongly suggest that low dietary carbohydrate utilization in rainbow trout is not due to the absence of inducible hepatic GK as previously suggested. Interestingly, we also observed a significantly lower GK expression in common carp (a glucose-tolerant fish) than in rainbow trout and gilthead seabream, which are generally considered as glucose intolerant. These data suggest that other biochemical mechanisms are implicated in the inability of rainbow trout and gilthead seabream to control blood glucose closely.     

The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.     

A Holistic View of Dietary Carbohydrate Utilization in Lobster: Digestion,Postprandial Nutrient Flux,and Metabolism

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2–6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6–12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.     

Cholecystokinin impact on rainbow trout glucose homeostasis: possible involvement of central glucosensors

Although the role of cholecystokinin (CCK) on fish appetite regulation has been widely studied, its involvement in the regulation of glucose metabolism had been little explored to date. In the present study we have carried out different experimental approaches to study CCK effects in rainbow trout (a so-called 'glucose intolerant' fish species) glucose homeostasis. We have found that for the first time in a vertebrate species, systemic or central CCK administration causes hyperglycemia, which is at least in part related to the presence of an ancestral gut-brain axis in which CCK is involved. By using capsaicin we have found that part of the action of CCK on glucose homeostasis is mediated by vagal and splanchnic afferents. Changes in hepatic metabolism after systemic CCK administration suggest that the effects are not directly taking place on the liver, but probably in other tissues, while after the central CCK administration, the glycogenolytic response observed in liver could be mediated by the activation of the sympathetic system. In hypothalamus and hindbrain changes elicited by CCK-8 treatment are likely related to the glucosensor response to the increased glycemia and/or vagal/splanchnic afferences whereas in hindbrain a possible action through specific CCK-1 receptors cannot be excluded. All these processes result in changes in metabolic parameters related with glucose homeostasis control. Further studies are needed to fully understand the role of this peptide on glucose homeostasis control in fish.     

Influence of Plant Growth at High CO2 Concentrations on Leaf Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Intracellular Distribution of Soluble Carbohydrates in Tobacco,Snapdragon, and Parsley

We have examined the possible role of leaf cytosolic hexoses and the expression of mannitol metabolism as mechanisms that may affect the repression of photosynthetic capacity when plants are grown at 1000 versus 380 [mu]L L-1 CO2. In plants grown at high CO2, leaf ribulose-1,5-bisphosphate carboxylase/oxygenase content declined by [greater than or equal to]20% in tobacco (Nicotiana sylvestris) but was not affected in the mannitol-producing species snapdragon (Antirrhinum majus) and parsley (Petroselinum hortense). In the three species mesophyll glucose and fructose at midday occurred almost entirely in the vacuole (>99%), irrespective of growth CO2 levels. The estimated cytosolic concentrations of glucose and fructose were [less than or equal to]100 [mu]M. In the three species grown at high CO2, total leaf carbohydrates increased 60 to 100%, but mannitol metabolism did not function as an overflow mechanism for the increased accumulation of carbohydrate. In both snapdragon and parsley grown at ambient or high CO2, mannitol occurred in the chloroplast and cytosol at estimated midday concentrations of 0.1 M or more each. The compartmentation of leaf hexoses and the metabolism of alternate carbohydrates are further considered in relation to photosynthetic acclimation to high levels of CO2.     

Current theories on the mechanism of the effect of glucagon on carbohydrate metabolism

Glucagon is considered for mechanisms of its action on the carbon metabolism, its significance in the complex polyhormonal regulation of the glucose synthesis and metabolism being studied. Glucagon exerts its effect on cells through specific receptors arranged on the plasma membrane, while its effect on the carbohydrate metabolism is mediated, mainly, by cAMP-dependent phosphorylation os some proteins participating in regulation of carbohydrate synthesis and metabolism including the proteins controlling expression of many genes. The glucagon effect on the level and efficiency of the action of insulin and some other hormones is an important link in the glucagon action on the carbohydrate metabolism.     

Some insights into energy metabolism for osmoregulation in fish

A sufficient and timely energy supply is a prerequisite for the operation of iono- and osmoregulatory mechanisms in fish. Measurements of whole-fish or isolated-gill (or other organs) oxygen consumption have demonstrated regulation of the energy supply during acclimation to different osmotic environments, and such regulation is dependent on species, the situation of acclimation or acclimatization, and life habits. Carbohydrate metabolism appears to play a major role in the energy supply for iono- and osmoregulation, and the liver is the major source supplying carbohydrate metabolites to osmoregulatory organs. Compared with carbohydrates, the roles of lipids and proteins remain largely unclear. Energy metabolite translocation was recently found to occur between fish gill ionocytes and neighboring glycogen-rich (GR) cells, indicating the physiological significance of a local energy supply for gill ion regulatory mechanisms. Spatial and temporal relationships between the liver and other osmoregulatory and non-osmoregulatory organs in partitioning the energy supply for ion regulatory mechanisms during salinity challenges were also proposed. A novel glucose transporter was found to specifically be expressed and function in gill ionocytes, providing the first cue for investigating energy translocation among gill cells. Advanced molecular physiological approaches can be used to examine energy metabolism relevant to a particular cell type (e.g., gill ionocytes), and functional genomics may also provide another powerful approach to explore new metabolic pathways related to fish ion regulation.     

Effect of abandonment and plant classification on carbohydrate reserves of meadow plants

We studied the effect of cessation of management on carbohydrate reserves of plants in meadows with different environmental characteristics and plant composition. We recorded storage carbohydrates and seasonal changes for 40 plant species. We asked whether there are differences in responses of carbohydrate reserves in forbs versus graminoids and in plants storing starch versus plants storing osmotically active carbohydrates. We analysed belowground organs before the meadows were mown and at the end of the vegetation season in mown versus recently abandoned plots. Whereas starch and fructans were widely distributed, raffinose family oligosaccharides were the main carbohydrate reserves of the Lamiaceae and Plantago lanceolata. Properties of carbohydrate reserves differed between forbs and graminoids but no difference was found between plants storing starch versus osmotically active carbohydrates. Graminoids had lower carbohydrate concentrations than forbs. We observed a positive effect of mowing on carbohydrate concentrations of graminoids in the dry, calcium-rich meadow and higher seasonal fluctuations of these values in the acid, wet meadow, suggesting that local factors and/or the species pool affect carbohydrate reserves. Despite local conditions, graminoids represent a distinct functional group in meadows from the point of view of their storage economy. We suggest that as well as growth, storage processes should also be considered for understanding the functioning of meadow plant communities.     

Dietary carbohydrates induce changes in glucosensing capacity and food intake of rainbow trout

We hypothesize that variations in dietary carbohydrate levels produce changes in glucosensor parameters in previously characterized glucosensing areas (hypothalamus and hindbrain) related with the regulation of food intake of a carnivorous fish species like rainbow trout. Therefore, we fed trout with standard, carbohydrate-free (CF) or high-carbohydrate (HC) diets for 10 days to assess changes in glucosensing system and food intake. Fish fed CF diet displayed hypoglycemia and increased food intake. Fish fed a HC diet displayed hyperglycemia and decreased food intake. Changes in food intake due to dietary carbohydrates were accompanied in hypothalamus and hindbrain of fish fed with HC diet by changes in parameters involved in glucosensing, such as increased glucose, glucose 6-phosphate, and glycogen levels and increased glucokinase (GK), glycogen synthase, and pyruvate kinase activities as well as increased GK and GLUT2 expression. All those results address for the first time in fish, despite the relative intolerance to glucose of carnivorous species, that dietary carbohydrates are important regulators of the glucosensing system in carnivorous fish, suggesting that the information generated by this system can be associated with the changes observed in food intake.     

Differentiation of Lactobacillus helveticus,Lactobacillus delbrueckii subsp bulgaricus,subsp lactis and subsp delbrueckii using physiological and genetic tools and reclassification of some strains from the ATCC collection

Several physiological tests of glucose metabolism and genetic tools including species specific probes and 16S rDNA sequences were used to identify strains of L. helveticus and the group of L. delbrueckii with its three subspecies lactis, bulgaricus, and delbrueckii. These species are important for the milk industry as fermenting lactic acid bacteria. The identification procedure was applied to the different strains of these species available from the ATCC collection and allowed to reclassify part of them.     

Derivatization of carbohydrates for chromatographic,electrophoretic and mass spectrometric structure analysis

Carbohydrates, either alone or as constituents of glycoproteins, proteoglycans and glycolipids, are mediators of several cellular events and (patho)physiological processes. Progress in the "glycome" project is closely related to the analytical tools used to define carbohydrate structure and correlate structure with function. Chromatography, electrophoresis and mass spectrometry are the indispensable analytical tools of the on-going research. Carbohydrate derivatization is required for most of these analytical procedures. This review article gives an overview of derivatization methods of carbohydrates for their liquid chromatographic and electrophoretic separation, as well as the mass spectrometric characterization. Pre-column and on-capillary derivatization methods are presented with special emphasis on the derivatization of large carbohydrates.     

Glucose and lipid metabolism in the pancreas of rainbow trout is regulated at the molecular level by nutritional status and carbohydrate intake

Glucose and lipid metabolism in pancreatic islet organs is poorly characterized. In the present study, using as a model the carnivorous rainbow trout, a glucose-intolerant fish, we assessed mRNA expression levels of several genes involved in glucose and lipid metabolism (including ATP-citrate lyase; carnitine palmitoyltransferase-1 isoforms, CPT; the mitochondrial isoform of the phosphoenolpyrutave carboxykinase, mPEPCK and pyruvate kinase, PK) and glucosensing (glucose transporter type 2, Glut2; glucokinase, GK and the potassium channel, K_ATP) in Brockmann bodies. We evaluated the response of these parameters to changes in feeding status (food deprived vs. fed fish) as well as to changes in the amount of carbohydrate (dextrin) in the diet. A general inhibition of the glycolytic (including the glucosensing marker GK) and β-oxidation pathways was found when comparing fed versus food-deprived fish. When comparing fish feeding on either low- or high-carbohydrate diets, we found that some genes related to lipid metabolism were more controlled by the feeding status than by the carbohydrate content (fatty acid synthase, CPTs). Findings are discussed in the context of pancreatic regulation of glucose and lipid metabolism in fish, and show that while trout pancreatic metabolism can partially adapt to a high-carbohydrate diet, some of the molecular actors studied seem to be poorly regulated (K_ATP) and may contribute to the glucose intolerance observed in this species when fed high-carbohydrate diets.     

Glucose homeostasis in rainbow trout fed a high-carbohydrate diet: metformin and insulin interact in a tissue-dependent manner

Carnivorous fish species such as the rainbow trout (Oncorhynchus mykiss) are considered to be "glucose intolerant" because of the prolonged hyperglycemia experienced after intake of a carbohydrate-enriched meal. In the present study, we use this species to study glucose homeostasis in fish chronically infused with the hypoglycemic agents, insulin, and metformin, and fed with a high proportion of carbohydrates (30%). We analyzed liver, skeletal muscle, and white adipose tissue (WAT), which are insulin- and metformin-specific targets at both the biochemical and molecular levels. Trout infused with the combination of insulin and metformin can effectively utilize dietary glucose at the liver, resulting in lowered glycemia, increased insulin sensitivity, and glucose storage capacity, combined with reduced glucose output. However, in both WAT and skeletal muscle, we observed decreased insulin sensitivity with the combined insulin + metformin treatment, resulting in the absence of changes at the metabolic level in the skeletal muscle and an increased potential for glucose uptake and storage in the WAT. Thus, the poor utilization by rainbow trout of a diet with a high proportion of carbohydrate can at least be partially improved by a combined treatment with insulin and metformin, and the glucose intolerance observed in this species could be, in part, due to some of the downstream components of the insulin and metformin signaling pathways. However, the predominant effects of metformin treatment on the action of insulin in these three tissues thought to be involved in glucose homeostasis remain exclusive in this species.     

Species‐specific regulation of herbivory‐induced defoliation tolerance is associated with jasmonate inducibility

Induced changes in root carbohydrate pools are commonly assumed to determine plant defoliation tolerance to herbivores. However, the regulation and species specificity of these two traits are not well understood. We determined herbivory‐induced changes in root carbohydrates and defoliation tolerance in seven different solanaceous plant species and correlated the induced changes in root carbohydrates and defoliation tolerance with jasmonate inducibility. Across species, we observed strong species‐specific variation for all measured traits. Closer inspection revealed that the different species fell into two distinct groups: Species with a strong induced jasmonic acid (JA) burst suffered from a reduction in root carbohydrate pools and reduced defoliation tolerance, while species with a weak induced JA burst maintained root carbohydrate pools and tolerated defoliation. Induced JA levels predicted carbohydrate and regrowth responses better than jasmonoyl‐L‐isoleucine (JA‐Ile) levels. Our study shows that induced JA signaling, root carbohydrate responses, and defoliation tolerance are closely linked, but highly species specific, even among closely related species. We propose that defoliation tolerance may evolve rapidly via changes in the plant's defense signaling network.     

Changes in cytoplasmic carbohydrate content during Helleborus pollen presentation

Pollen grains of Helleborus foetidus and H. bocconei were exposed to low temperature treatments to simulate the natural events in pollen presentation of these two winter flowering species and to analyze the pollen carbohydrate content (glucose, fructose, sucrose and starch). In both species, cytoplasmic polysaccharides, monosaccharides and sucrose were found, while only Helleborus foetidus contained starch. Polysaccharide, sucrose and monosaccharide content varied as low temperature exposure time varied, a decrease in temperature decreases polysaccharide content and increases sucrose and monosaccharides. The relative quantities of the various types of carbohydrates were not affected by variations in the naturally occurring thermal cycles. Treatments did not greatly affect pollen viability. Although the occurrence of carbohydrates in pollen is known since many years, their function is still unclear. The findings of this research suggest a role of cytoplasmic pollen carbohydrates in resistance to low temperature exposure. The inter-conversion of carbohydrate type may be an adaptation for sustaining viability during pollen presentation that is particularly important for a winter flowering species such as Helleborus foetidus and H. bocconei .     

Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition

A C-type lectin was previously isolated from the blood of healthy Atlantic salmon (Salmo salar) and this salmon serum lectin (SSL) was found to opsonise bacteria. Selective binding to bacteria in vivo requires that the lectin be able to recognise a carbohydrate pattern on the bacterial surface distinguishable from that of the host. In order to investigate this selectivity in the lectin, a phage-display antibody was prepared and then used for detection of lectin by Western blotting. A carbohydrate binding-inhibition assay with Western blot detection of the lectin showed mannose to be the primary ligand and related sugars including glucose, N-acetylglucosamine and methyl alpha-D-mannopyranoside to be additional ligands of this lectin. The SSL in serum detected by Western blotting was shown to form a complex oligomer. These results show that the salmon serum lectin is oligomeric in blood and that it recognizes a broad spectrum of carbohydrates with optimal binding to mannose. The lectin might therefore be an ideal opsonin for multiple salmon pathogens with carbohydrate arrays on their surfaces. No similar lectins were identified in the sera of other fish by Western blotting using the phage-display antibody. Molecular analysis will be required in order to determine whether homologous lectins are expressed in related fish species. It is anticipated that similar lectins might have related pathogen recognition roles in divergent fish species.     

Modeling protein recognition of carbohydrates

We have a limited understanding of the details of molecular recognition of carbohydrates by proteins, which is critical to a multitude of biological processes. Furthermore, carbohydrate-modifying proteins such as glycosyl hydrolases and phosphorylases are of growing importance as potential drug targets. Interactions between proteins and carbohydrates have complex thermodynamics, and in general the specific positioning of only a few hydroxyl groups determines their binding affinities. A thorough understanding of both carbohydrate and protein structures is thus essential to predict these interactions. An atomic-level view of carbohydrate recognition through structures of carbohydrate-active enzymes complexed with transition-state inhibitors reveals some of the distinctive molecular features unique to protein-carbohydrate complexes. However, the inherent flexibility of carbohydrates and their often water-mediated hydrogen bonding to proteins makes simulation of their complexes difficult. Nonetheless, recent developments such as the parameterization of specific force fields and docking scoring functions have greatly improved our ability to predict protein-carbohydrate interactions. We review protein-carbohydrate complexes having defined molecular requirements for specific carbohydrate recognition by proteins, providing an overview of the different computational techniques available to model them.     

Competition for dietary carbohydrates between streptococci in dental plaque

Abstract The predominant microorganisms in dental plaque, i.e., streptococci and actinomycetes, are carbohydrate fermenters. In the natural environment these organisms experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. Recently, we have studied the competition between pairs of oral Streptococcus species in the chemostat under continuous glucose or sucrose limitation and under glucose pulsed conditions.
In the present study we have investigated the competition for dietary carbohydrates in dental plaque of gnotobiotic rats infected with the same combinations of streptococci. The rats were exposed to similar regimes of carbohydrate administration as in the chemostat by incorporation of low molecular carbohydrates in the diet and drinking water. Addition of glucose to the diet favoured the organism with the highest q _max for glucose. This result parallels the outcome of earlier competition experiments in glucose-pulsed chemostats, which showed that the organism with the highest q _max glucose became dominant in the cultures. Previous observations that bacteriocin production and extracellular glucan synthesis are major ecological factors for oral streptococci were also confirmed in this experiment.