Glucogenic blood sugar formation in an insect Manduca sexta L.: asymmetric synthesis of trehalose from 13C enriched pyruvate.

Gluconeogenesis and blood sugar formation were examined in Manduca sexta, fed carbohydrate- and fat-free diets with varying levels of casein. De novo carbohydrate synthesis was examined by nuclear magnetic resonance spectroscopy of the 13C enrichment in blood trehalose and alanine derived from (2-(13)C)pyruvate and (2,3-(13)C(2))pyruvate administered to 5th instar larvae. Gluconeogenic flux and blood trehalose concentration were positively correlated with protein consumption. On all diets, the 13C distribution in trehalose was asymmetric, with C6 more highly enriched than C1. The C6/C1 13C enrichment ratio, however, decreased with increased protein consumption and gluconeogenic flux. Although the asymmetric 13C enrichment pattern in trehalose is consistent with pentose cycling via the pentose phosphate pathway following de novo synthesis, experiments employing [2,3-(13)C(2)]pyruvate demonstrated that pentose cycling is not detected in insects under these nutritional conditions. Analysis of the multiplet NMR signal structure in trehalose due to spin-spin coupling between adjacent 13C enriched carbons showed the absence of uncoupling expected by pentose phosphate pathway activity. Here we suggest that the asymmetric 13C distribution in trehalose results from a disequilibrium of the triose phosphate isomerase-catalyzed reaction.

Dietary nutrient levels regulate protein and carbohydrate intake, gluconeogenic/glycolytic flux and blood trehalose level in the insect Manduca sexta L.

This study examined the effects of dietary casein and sucrose levels on nutrient intake, and distinguished the effects of carbohydrate and protein consumption on growth, fat content, pyruvate metabolism and blood trehalose level of 5th instar Manduca sexta larvae. Growth increased with increasing casein consumption but was unaffected by carbohydrate intake. Fat content also increased with carbohydrate consumption, but on carbohydrate-free diets fat content increased with increased protein consumption. Blood trehalose level and pyruvate metabolism were examined by nuclear magnetic resonance spectroscopy analysis of blood following administration of (3-(13)C)pyruvate. On diets containing sucrose, blood trehalose increased with increasing carbohydrate intake, and on most diets trehalose was synthesized entirely from dietary sucrose. Pyruvate cycling, indicated by the alanine C2/C3 (13)C enrichment ratio, increased with carbohydrate consumption reflecting increased glycolysis, and pyruvate decarboxylation exceeded carboxylation on all sucrose diets. Larvae that consumed <75 mg/day sucrose were gluconeogenic, based on the [2 (trehalose C6)(Glx C3/C2)]/alanine C2] (13)C enrichment ratio. On carbohydrate-free diets, blood trehalose levels were low and maintained entirely by gluconeogenesis. Blood trehalose level increased with increasing protein intake. Pyruvate cycling was very low, although many insects displayed higher levels of pyruvate decarboxylation than carboxylation. All gluconeogenic larvae displayed alanine (13)C enrichment ratios <0.35 and had blood trehalose levels <50 mM.

The glucogenic response of a parasitized insect Manduca sexta L. is partially mediated by differential nutrient intake.

Induction of gluconeogenesis is accelerated in larvae of the insect Manduca sexta L. parasitized by Cotesia congregata (Say), maintaining the concentration of the blood sugar trehalose, an important nutrient for parasite development. Investigation has demonstrated that when host larvae are offered a choice of diets with varying levels of sucrose and casein, parasitized insects consume a different balance of these nutrients, principally due to a decrease in protein consumption. The result is metabolic homeostasis, with normal unparasitized and parasitized larvae exhibiting similar levels of gluconeogenesis and blood sugar level. In the present study, normal unparasitized and parasitized larvae were maintained on individual chemically defined diets having the balance of protein and carbohydrate consumed by each when offered a dietary choice. Total dietary nutrient, the sum of carbohydrate and protein, was provided at six levels, composed of three pairs of diets. Each diet pair consisting of diets having equivalent overall nutrient ratios of 2:1 and 1:1 casein/sucrose. Host growth and diet consumption were significantly affected by dietary nutrient level and the magnitude of these effects was influenced by parasitism. Due to the effects of dietary nutrient level on diet consumption, none of the unparasitized and parasitized larvae within any of the three diet pairs consumed protein and carbohydrate at the levels predicted by the earlier choice experiments. Among insects on all of the diets, however, two groups of unparasitized and parasitized larvae consumed the expected levels of protein and carbohydrate. In each case, gluconeogenesis, as measured by 13C nuclear magnetic resonance spectroscopy (NMR) analysis of pyruvate cycling and trehalose synthesis from [2-13C]pyruvate, was evident in unparasitized and parasitized insects, confirming the conclusions of the earlier experiments. Generally, all larvae that consumed less than approximately 250 mg of sucrose over the 3-day feeding period, were gluconeogenic, regardless of diet. Differential carbohydrate consumption, therefore, was an important factor in inducing gluconeogenesis in both unparasitized and parasitized insects. The selective 13C enrichment in trehalose displayed by non-gluconeogenic larvae on some diets demonstrated trehalose formation from [2]pyruvate. The absence of net carbohydrate synthesis in these insects was likely due to an elevation of glycolysis. There was no significant effect of diet consumption or parasitism on blood trehalose level. Parasitized larvae displayed higher levels of gluconeogenesis than did unparasitized insects, a finding consistent with the conclusion that blood sugar is rapidly sequestered by developing parasites. The parasite burden, the total number of parasites developing within host larvae, as well as the number of parasites emerging from host larvae to complete development, was significantly less at the lowest dietary nutrient level, but was otherwise similar at all dietary nutrient levels. Moreover, the number of parasites that emerged increased with increasing diet consumption as reflected by host final weight.

Arginine kinase and phosphoarginine, a functional phosphagen, in the rhabditoid nematode steinernema carpocapsae.

Moderate activity of arginine kinase was found in Steinernema carpocapsae, an entomopathogenic nematode. In the forward reaction, 4.60 and 3.12 micromol ATP/min/mg protein was produced in infectious third-stage juveniles (J3s) and adult nematodes, respectively. For the reverse reaction, 3.20 and 2.27 micromol phosphoarginine/min/mg protein was produced by J3s and adults, respectively. The K(m)s for phosphoarginine and ADP were 0.73 and 0.42 mM, respectively, in the forward reaction, whereas in the reverse reaction, the K(m)s were 0.37 and 2.35 mM for arginine and ATP, respectively, for the enzyme from J3s. The pH optimum for the forward reaction was 7.2 and 7.3 in J3s and adults, respectively. The pH optimum was elevated for the reverse reaction, 7.8 and 7.9-8.5 in J3s and adults, respectively. In the J3s, the in vitro optima for arginine kinase activity was correlated with the in vivo tissue pH in hypoxic (6.9) and aerobic (7.5) J3s estimated by in vivo flow 31P-NMR.

Effects of histone and diolein on the structure of phosphatidylcholine/phosphatidylserine or phosphatidylcholine/phosphatidylglycerol bilayers.

The effects of the PKC substrate histone 1 and the PKC activator diolein (Ole2Gro) on the structure of phosphatidylcholine (PtdCho)/phosphatidylserine (PtdSer), or PtdCho/phosphatidylglycerol (PtdGro) bilayers were studied using 2H-NMR. The results showed that in PtdCho/PtdSer bilayers, histone preferentially increased order parameters of the acyl chains of the PtdSer, but not the PtdCho lipid component. This effect was additive with the effect of Ole2Gro, which equally increased the ordering of the acyl chains of both PtdCho and PtdSer. The histone-induced change in the conformation of the PtdCho headgroups in PtdCho/PtdSer bilayers indicated that positively charged residues of the bound histone are located above the lipid-water interface and their location was altered by the presence of Ole2Gro. A different picture was observed in the case of PtdCho/PtdGro bilayers; although the effect of Ole2Gro on both the PtdCho or the PtdGro components was similar to the case of the PtdCho/PtdSer bilayers, histone did not significantly affect the order parameters of PtdCho or PtdGro in either the absence or presence of Ole2Gro. The results indicate that histone 1 induces clustering of PtdSer in PtdCho bilayers which may contribute to PKC activation. Moreover, the observed differences in the interactions of histone with PtdCho/PtdSer compared with PtdCho/PtdGro bilayers may explain the higher efficiency of PtdSer in activating PKC.

Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.

High energy phosphate metabolites observed by NMR in infective larvae of Haemonchus contortus.

Phosphorus resonances consistent with phosphoarginine and ATP were observed in the in vivo 31P-nuclear magnetic resonance (NMR) spectra of infective larvae of Haemonchus contortus. The level of phosphoarginine quickly declined when nematode suspensions were purged with nitrogen and was restored upon return to aerobic conditions. Saturation transfer NMR demonstrated forward and reverse exchange of phosphorus between phosphoarginine and ATP.

Effects of diacylglycerols and Ca2+ on structure of phosphatidylcholine/phosphatidylserine bilayers.

The combined effects of the diacylglycerols (DAGs) with the various acyl chains and Ca2+ on the structure of phosphatidylcholine/phosphatidylserine (4:1 mole/mole) bilayers were studied using 2H- and 31P NMR. The following DAG- and Ca(2+)-induced bilayer perturbations were identified. 1) Increased tendency to form nonbilayer lipid phases was induced by diolein or stearoylarachidonoylglycerol, and was synergistically enhanced by the addition of Ca2+. 2) "Transverse" bilayer perturbation was induced by dioctanoylglycerol. The addition of this DAG caused increased ordering of the phospholipid acyl side chains in the region adjacent to the headgroup, with the concomitant decrease of the order toward the bilayer interior. 3) Separation of the phosphatidylcholine and phosphatidylserine bilayer components was induced by combinations of relatively high (1:5 mole/mole to phosphatidylserine) Ca2+ and 25 mol% (to the phospholipids) of diolein, stearoylarachidonoylglycerol, or oleoylacetylglycerol. 4) Lateral phase separation of the bilayers on the regions of different fluidities was induced by dipalmitin. These physicochemical effects were correlated with the effects of these DAGs and Ca2+ on the activity of protein kinase C. The increased tendency to form nonbilayer lipid phases and the transverse bilayer perturbations correlated with the increased protein kinase C activity, whereas the actual presence of the nonbilayer lipid phases, as well as the separation of the phosphatidylcholine and phosphatidylserine components, was associated with the decrease in the protein kinase C activity. The lateral phase separation of the bilayer on gel-like and liquid crystalline regions did not have an effect on the activity of the enzyme. These results demonstrate the importance of the physicochemical properties of the membranes in the process of activation of protein kinase C.

Chloroquine stabilization of phospholipid membranes against diacylglycerol-induced perturbation.

The effects of 1-stearoyl,2-sn-arachidonoylglycerol (SAG) and the antimalarial drug chloroquine on lipid bilayer structure were studied by 2H-NMR spectroscopy. Model lipid systems were established with compositions similar to those of normal human erythrocytes, malaria-infected erythrocytes, or malaria parasite membranes. The 2H-NMR spectra of the membranes formed from the lipids extracted from normal human erythrocytes were similar to those obtained using the corresponding lipid mixtures. The order parameters of the model "infected" and model "parasite" membranes were reduced markedly relative to that of normal erythrocytes. Addition of SAG induced formation of non-bilayer lipid phases in all lipid systems. Only a small decrease in the order parameters of the acyl side chains of the phosphatidylserine, but not of the phosphatidylcholine component of the lipid membranes, was observed upon the addition of chloroquine. A dramatic effect was observed upon the addition of chloroquine to the SAG-containing membranes: this antimalarial almost totally abolished the formation of SAG-induced non-bilayer lipid phases. Since SAG, endogenously formed in erythrocyte membranes, is a potent activator of phospholipase A2, this membrane-stabilizing action of chloroquine may partially account for the phospholipase A2-inhibiting properties of this drug, and, consequently, for both its therapeutic and toxic modes of action.

Physiologic studies of snail-schistosome interactions and potential for improvement of in vitro culture of schistosomes.

Despite extensive efforts to develop suitable media for rearing the intramolluscan stages of schistosomes, successful in vitro culture of these parasites remains elusive. Recent 31P NMR studies demonstrated that the levels of free phospholipids, particularly phosphatidylcholine, in the digestive gland of the snail, Biomphalaria glabrata, were dramatically reduced when the host was infected with Schistosoma mansoni. It was speculated that absorption of host phosphatides may be an important source of membrane phospholipid precursors and fatty acids for developing sporocysts and cercariae. During the present investigations, B. glabrata was maintained on a high fat diet of egg yolk, and the lipid composition of control uninfected and infected snails examined by 31P and 13C NMR. In addition, the levels of host hemolymph metabolites, including glucose and urea, considered as indicators of parasite nutrient uptake, were monitored. The lipid level of snails fed egg yolk was greatly increased, and hosts developed patient infections in approximately half the time of infected snails maintained on lettuce. The composition of the free phospholipids accumulated in the tissues of B. glabrata fed egg yolk were the same as those previously reported in the cercarial stage of S. mansoni. Moreover, the fatty acids of S. mansoni and those reported here in the neutral lipids and free phosphatides in the host tissues were similar. Uninfected snails maintained on lettuce had higher hemolymph levels of glucose than those reared on egg yolk, and infected hosts on egg yolk had significantly lower levels of hemolymph urea. Beta-hydroxybutyrate was the principal hemolymph metabolite in snails fed egg yolk, but was not detected in snails maintained on lettuce.(ABSTRACT TRUNCATED AT 250 WORDS)