Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the [U-13C6]-d-glucose tracer in mice

Previous genetic and proteomic studies identified altered activity of various enzymes such as those of fatty acid metabolism and glycogen synthesis after a single toxic dose of valproic acid (VPA) in rats. In this study, we demonstrate the effect of VPA on metabolite synthesis flux rates and the possible use of abnormal ¹³C labeled glucose-derived metabolites in plasma or urine as early markers of toxicity. Female CD-1 mice were injected subcutaneously with saline or 600 mg/kg) VPA. Twelve hours later, the mice were injected with an intraperitoneal load of 1 g/kg [U-¹³C]-d-glucose. ¹³C isotopomers of glycogen glucose and RNA ribose in liver, kidney and brain tissue, as well as glucose disposal via cholesterol and glucose in the plasma and urine were determined. The levels of all of the positional ¹³C isotopomers of glucose were similar in plasma, suggesting that a single VPA dose does not disturb glucose absorption, uptake or hepatic glucose metabolism. Three-hour urine samples showed an increase in the injected tracer indicating a decreased glucose re-absorption via kidney tubules. ¹³C labeled glucose deposited as liver glycogen or as ribose of RNA were decreased by VPA treatment; incorporation of ¹³C via acetyl-CoA into plasma cholesterol was significantly lower at 60 min. The severe decreases in glucose-derived carbon flux into plasma and kidney-bound cholesterol, liver glycogen and RNA ribose synthesis, as well as decreased glucose re-absorption and an increased disposal via urine all serve as early flux markers of VPA-induced adverse metabolic effects in the host.     

Diapause development and acclimation regulating enzymes associated with glycerol synthesis in the Shonai ecotype of the rice stem borer larva,Chilo suppressalis walker

Overwintering larvae of the Shonai ecotype of the rice stem borer, Chilo suppressalis, enter diapause in early September and terminate diapause at the end of October. Cold acclimation at 0°C did not influence glycerol, trehalose or glycogen content in larvae collected on 22 September. Acclimation at 0°C increased the glycerol content and reduced the glycogen content significantly in larvae collected on 2 October and 22 November compared with acclimation at 15°C. These results indicate that overwintering larvae at different phases of diapause development respond differently to the low temperature stimulus for glycerol synthesis. Thus, we evaluated the metabolic rearrangements associated with glycerol synthesis during diapause development and after temperature acclimation. Larvae collected on 2 October were acclimated at 15°C for 15 and 60 days. Some of those acclimated at 15°C were then moved to 0°C for 15 days. The larvae acclimated at 15°C for 15 days were in deep diapause and accumulated little glycerol, while larvae acclimated at 15°C for 60 days were nearly ready to emerge from diapause and accumulated glycerol at 155.5 μmol/g. When larvae acclimated to 15°C for 15 days were transferred to 0°C, glycerol accumulation was stimulated to the same extent (ca 140 μmol/g) as it was in larvae that were acclimated to 15°C for 60 days and then transferred to 0°C. These results indicate that low temperature has a cumulative effect on glycerol production in larvae at different phases of diapause development. Glycerol accumulation was accomplished by activation of glycogen phosphorylase and inhibition of fructose-1,6-bisphosphatase, and activation of enzymes associated with glycerol synthesis, mainly glyceraldehyde-3-phosphatase and polyol dehydrogenase with glyceraldehyde activity.     

Evidence for Transaldolase Activity in the Isolated Heart Supplied with [U-13C3]Glycerol

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-¹³C₃]glycerol, and ²H₂O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, ¹³C from [U-¹³C₃]glycerol appeared in glycogen in carbon positions 4–6 of glucosyl units but none in positions 1–3. The extent of [4,5,6-¹³C₃]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H₂O₂. Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked ¹³C asymmetry in glycogen can only be attributed to conversion of [U-¹³C₃]glycerol to [U-¹³C₃]dihydroxyacetone phosphate and [U-¹³C₃]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this ¹³C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.     

Effect of extracellular calcium on the contractility of warm-and cold-acclimated crucian carp heart

Crucian carp (Carassius carassius L.) were acclimated for at least 4 weeks to 2°C or 22°C, and the consequences of thermal acclimation on force development, time-course of contraction and action potential duration of the ventricular myocardium were studied. In cold-acclimated fish contraction was activated at much lower external [Ca] than in warm-acclimated fish: [Ca] for half-maximal force was 0.9±0.15 and 3.1±0.92 mmol·l^-1 (P<0.05) for cold- and warm-acclimated fish, respectively. Durations of contraction and relaxation were significantly longer in fish acclimated to 2°C than in fish acclimated to 22°C, especially at [Ca] below 2 mmol·l^-1. In low-Ca solution ventricular action potential was prolonged both in cold- and warm-acclimated fish. In 0.5 mmol·l^-1 Ca action potential duration at zero voltage level was longer in cold- than warm-acclimated fish. Although lengthening of action potential was evident in both acclimation groups, a marked prolongation of contraction duration by low-Ca solutions occurred only in cold-acclimated fish. This suggests that a plateau component of contraction is present in cold-acclimated fish but less well developed in warm-acclimated fish hearts. Contractions were strongly inhibited by sarcolemmal Ca-channel blocker, cadmium (100 and 300 mol·l^-1), in both warm- and cold-acclimated crucian carp hearts. However, the sarcoplasmic reticulum Ca release channel blocker, ryanodine (10 mol·l^-1), had no effect on the force of contraction in either acclimation group. These results suggest that the contraction of crucian carp heart is controlled by sarcolemmal mechanisms without contribution by sarcoplasmic reticulum Ca release. Since the Ca sensitivity of myofilaments was not altered by thermal acclimation, the results indicate that thermal acclimation alters Ca activation of contraction of the crucian carp heart at the level of sarcolemma.Abbreviations AP action potential - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra-acetic acid - F _max maximum force - F _max maximum rate of contraction - F _min maximum rate of relaxation - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - pCa log [Ca] - Pl action potential plateau - SL sarcolemma - SR sarcoplasmic reticulum - TPF time to peak force - T1/2R time to half relaxation from the peak force     

Effect of freezing and dehydration on ion and cryoprotectant distribution and hemolymph volume in the goldenrod gall fly, Eurosta solidaginis

Extracellular freezing and dehydration concentrate hemolymph solutes, which can lead to cellular injury due to excessive water loss. Freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, may experience extreme cold and desiccation in winter. To determine whether larvae employ protective mechanisms against excessive cellular water loss we examined the effect of extracellular freezing and dehydration on hemolymph volume, and cryoprotectant and ion levels in the hemolymph. Dehydrated larvae or ones that had been frozen at −5 or −20 °C had a significantly smaller proportion of their body water as hemolymph (26.0-27.4%) compared to controls (30.5%). Even with this reduction in water content, hemolymph osmolality was similar or only slightly higher in frozen or dehydrated individuals than controls (908 mOsm kg⁻¹), indicating these stresses led to a reduction in hemolymph solutes. Hemolymph and intracellular content of ions remained largely unchanged between treatment groups; although levels of Mg⁺⁺ in the hemolymph were lower in larvae subjected to freezing (0.21 ± 0.01 μg mg⁻¹ dry mass) compared to controls (0.29 ± 0.01 μg mg⁻¹ dry mass), while intracellular levels of K⁺ were lower in groups exposed to low temperature (8.31 ± 0.21 μg mg⁻¹ dry mass). Whole body glycerol and sorbitol content was similar among all treatment groups, averaging 432 ± 25 mOsm kg⁻¹ and 549 ± 78 mOsm kg⁻¹ respectively. However, larvae subjected to dehydration and freezing at −20 °C had a much lower relative amount of cryoprotectants in their hemolymph (∼35%) compared to controls (54%) suggesting these solutes moved into intracellular compartments during these stresses. The correlation between reduced hemolymph volume (i.e. increased cellular water content) and intracellular movement of cryoprotectants may represent a link between tolerance of dehydration and cold in this species.     

Substrate regulation of ascorbate transport activity in astrocytes

Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na⁺-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[¹⁴C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V _max) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[¹⁴C]ascorbate. The increase inV _max was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[³H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.     

Metabolic dynamics across prolonged diapause development in larvae of the sawfly,Cephalcia chuxiongica (Hymenoptera: Pamphiliidae)

Nutrient metabolism is crucial for the survival of insects through the diapause. However, little is known about the metabolic mechanism of prolonged diapause. The sawfly, Cephalcia chuxiongica (Hymenoptera: Pamphiliidae), is a notorious defoliator of pine trees in southwest China. One of the distinguishing biological characteristics of this pest is the prolonged diapause of about 1.5 years. In this study, the body lipid, carbohydrate (total body sugar, glycogen, trehalose, and glucose), protein, and glycerol contents were measured in diapausing larvae of C. chuxiongica. The results showed that the changes of biochemical composition in C. chuxiongica are associated with the diapause initiation, maintenance, and termination phases. During the initiation phase, trehalose, glucose, and glycerol increased significantly, but glycogen decreased sharply. In general, the lipid, carbohydrate, and glycerol levels decreased gradually across the maintenance phase. At termination phase, the contents of glycogen and lipid persistently decreased, while an increase of trehalose, glucose, and glycerol contents were detected. The protein level was significantly higher at maintenance phase than at initiation and termination phases. It was also found that elevation of trehalose, glucose, and glycerol contents occurred in winter. These implies that the metabolites with altered levels in diapausing larvae of C. chuxiongica are responsible for maintaining a prolonged development and overwintering.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

The fermentation of xylose: studies by carbon-13 nuclear magnetic resonance spectroscopy

Summary The fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by¹³C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-¹³C]xylose or [2-¹³C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-¹³C]xylose or [1-¹³C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-¹³C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions.     

Aberrant nutritional regulation of carbohydrate synthesis by parasitized Manduca sexta L

The present studies confirm that storage carbohydrate synthesis from [1-(13)C]glucose is elevated in Manduca sexta parasitized by Cotesia congregata, despite a decrease in the rate of metabolism of the labeled substrate. Further, the results demonstrate that a similar pattern of carbohydrate synthesis and glucose metabolism was induced in normal larvae by administration of the glycolytic inhibitor, iodoacetate. (13)C enrichment of C6 of trehalose and glycogen demonstrated randomization of the C1 label at the triose phosphate step of the glycolytic/gluconeogenic pathway and suggested that gluconeogenesis, that is, de novo carbohydrate formation, contributed to the synthesis of carbohydrate in both normal and parasitized insects. Accounting for differences in the (13)C enrichment in C1 of trehalose and glycogen due to direct labeling from [1-(13)C]glucose, the mean C6/C1 labeling ratios in trehalose and glycogen of parasitized larvae and insects treated with iodoacetate were greater than the mean ratio observed in normal larvae, suggesting a greater contribution of gluconeogenesis to trehalose labeling in parasitized insects. This conclusion was confirmed by additional investigations on the metabolism of [3-(13)C]alanine by normal and parasitized insects. The pattern of (13)C enrichment in hemolymph trehalose observed in normal larvae maintained on a low carbohydrate diet indicated a large contribution of gluconeogenesis, while gluconeogenesis contributed very little to trehalose labeling in normal insects maintained on a high carbohydrate diet. Parasitized insects maintained on a high or a low carbohydrate diet displayed a significantly greater contribution of gluconeogenesis to trehalose labeling than was observed in normal larvae maintained on the same diets. In conclusion, these investigations indicate that regulation over the utilization of dietary glucose for trehalose and glycogen synthesis as well as the dietary regulation of de novo carbohydrate synthesis were altered by parasitism.     

Carbon balance and energetics of cyooprotectant synthesis in a freeze-tolerant insect: responses to perturbation by anoxia

Summary The capacity for polyol synthesis by larvae of Eurosta solidaginis was evaluated under aerobic versus anoxic (N₂ gas atmosphere) conditions. Glycerol production occurred readily in aerobic larvae at 13°C. Under anoxic conditions, however, net glycerol accumulation was only 57% of the aerobic value after 18 d, but the total hydroxyl equivalents available for cryoprotection were balanced by the additional synthesis of sorbitol. The efficiency of carbon conversion to polyols was much lower in anaerobic larvae. The ATP requirement of glycerol biosynthesis necessitated a 22% greater consumption of carbohydrate, when anaerobic and resulted in the accumulation of equimolar amounts of l-lactate and l-alanine as fermentative end products. The ratio of polyols produced to glycolytic end products formed was consistent with the use of the hexose monophosphate shunt to generate the reducing equivalents needed for cryoprotectant synthesis. A comparable experiment analyzed sorbitol synthesis at 3°C under aerobic versus anoxic conditions. Sorbitol synthesis was initiated more rapidly in anaerobic larvae, and the final sorbitol levels attained after 18 d were 60% higher than in aerobic larvae. The enhanced sorbitol output under anoxia may be due to an obligate channeling of a high percentage of total carbon flow through the hexose monophosphate shunt at 3°C. Carbon processed in this way generates NADPH which, along with the NADH output of glycolysis, must be reoxidized if anaerobic ATP synthesis is to continue. Redox balance within the hexose monophosphate shunt is maintained through NADPH consumption in the synthesis of sorbitol.     

The action of insulin of glycogen synthesis in rat diaphragm from intracellular and extracellular glucose

Rat hemidiaphragms were loaded with [U-¹⁴C] glucose at 2°C and subsequently incubated at 37°C with non-labeled glucose or [¹⁴C] glucose in the presence or absence of insulin. The incorporation of isotope into glycogen and lactate was determined. The results showed that insulin markedly stimulated the synthesis of glycogen from extracellular glucose while it had no effect on incorporation of isotope into glycogen from intracellular glucose. Lactate formation was not influenced by insulin. It was concluded that glucose transport in muscle is linked to glycogen sythesis and that insulin preferentially directs glucose entering the cell toward the formation of glycogen.     

Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of (³H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl₂ inhibited the [³H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [³H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the B_max of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK _D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [³H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO D-Ala²-(Me)Phe⁴-Gly-ol⁵-enkephalin - DALE d-Ala²-l-Leu⁵-enkephalin - DADLE d-Ala²-d-Leu⁵-enkephalin - EKC Ethylketocyclazocine - DHM Dihydromorphine - BIT 2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate - FIT Fentanyl isothiocyanate     

Characterization of S-layers from mesophilic bacillaceae and studies on their protective role towards muramidases

High resolution ¹³C NMR combined with chemical analysis were used to study the formation of metabolites from [1-¹³C]-labelled glucose by the salt-tolerant yeast Debaryomyces hansenii after transfer to media containing 8% NaCl. Time course spectroscopy of an aerobic cell suspension showed [1,3-¹³C]glycerol as the predominant end product. Perchloric acid extracts revealed additional less prominent incorporation of label into arabinitol, trehalose, glutamic acid, and alanine. The incorporation into trehalose and arabinitol showed a transient increase after shift to the high salinity medium. It is concluded that glycerol and arabinitol are the major organic solutes in D. hansenii, the production of glycerol being strongly induced by high salinity. Analysis of labelled extracts of D. hansenii after transfer to 8% NaCl media containing [1-¹³C]- or [6-¹³C]glucose, demonstrated that glucose is dissimilated via a combination of the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway, with the former playing a major role in glycerol formation and the latter in arabinitol production. The almost exclusive labelling of C5 of arabinitol from [6-¹³C]glucose indicates that the pathway to arabinitol proceeds via reduction of ribulose-5-phosphate.Abbreviations used NMR nuclear magnetic resonance - EMP Emden-Meyerhof-Parnas - PP pentose phosphate - GAP glyceraldehyde phosphate - DHAP dihydroxyacetone phosphate - ppm parts per million     

Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii

The conversion of glycerol to 1,3-propanediol by Citrobacter freundii DSM 30040 was optimized in single- and two-stage continuous cultures. The productivity of 1,3-propanediol formation was highest under glycerol limitation and increased with the dilution rate (D) to a maximum of 3.7 g·l^–1·h^–1. Glycerol dehydratase seemed to be the rate-limiting step in 1,3-propanediol formation. Conditions for the two-stage fermentation process were as follows: first stage, glycerol limitation (250mM), pH 7.2, D=0.1 h^–, 31° C; second stage, additional glycerol, pH 6.6, D=0.05 h^–1, 28° C. Under these conditions 875mM glycerol were consumed, the final 1,3-propanediol concentration was 545mM, and the overall productivity 1.38 g·1^–1·h^–1. Correspondence to: G. Gottschalk     

Interaction between the Pentose Phosphate Pathway and Gluconeogenesis from Glycerol in the Liver

After exposure to [U-¹³C₃]glycerol, the liver produces primarily [1,2,3-¹³C₃]- and [4,5,6-¹³C₃]glucose in equal proportions through gluconeogenesis from the level of trioses. Other ¹³C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex ¹³C-labeling patterns in exported glucose. Here, we investigated ¹³C labeling in plasma glucose in rats given [U-¹³C₃]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a ¹³C-¹³C bond within the trioses contributing to glucose, the appearance of [1,2-¹³C₂]-, [2,3-¹³C₂]-, [5,6-¹³C₂]-, and [4,5-¹³C₂]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-¹³C₂]glucose/[2,3-¹³C₂]glucose was significantly greater than [5,6-¹³C₂]glucose/[4,5-¹³C₂]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by ¹³C distribution in blood glucose after [U-¹³C₃]glycerol administration.     

13C nuclear magnetic resonance studies of anaerobic glycolysis inTrypanosoma brucei spp.

Anaerobic glycolysis inTrypanosoma brucei spp. has been studied by¹³C NMR at 50 and 75.5 MHz. The uptake of [U-¹³C]glucose by cell suspensions ofT. b. brucei was monitored by time-course spectroscopy, and while no anomeric specificity was found, the end -products of glycolysis were confirmed as glycerol and pyruvate together with alanine and dihydroxypropionat e. The intermediacy of L-glycerol-3-phosphate was also ascertained. The incorporation of C-I of [1-¹³C]glucose and of C-6 of [6-¹³C]glucose into glycerol and pyruvate inT. b. gambiense was quantified by measurement of the longitudinal relaxation times of the species involved. An incorporation to the extent of 66% of each substrate into equimolar amounts of glycerol and pyruvate indicate that Keq for the triosephosphate-isomerase-mediated reaction approaches unity.     

Overwintering strategy of endoparasitoids in Chilo suppressalis: a perspective from the cold hardiness of a host

Although parasitoids ultimately kill their host, koinobiont parasitoids must protect not only themselves but also their hosts against extreme environments. In this study, the parasitism rate of Chilo suppressalis Walker (Lepidoptera: Pyralidae) was investigated, and the average body weights, supercooling points, and concentrations of glycerol (acting as a cryoprotectant) in the hemolymph were compared between parasitized and non‐parasitized larvae. Five species of koinobiont endoparasitoids parasitized the overwintering C. suppressalis larvae and the total parasitism rate was 47.6% (n = 1 537). Average body weight of parasitized larvae was significantly lower than that of non‐parasitized larvae, and the parasitism rate of the lighter group (20–30 mg) was highest. The supercooling point of parasitized C. suppressalis larvae (?15.7 ± 0.3 °C) was significantly lower than that of the non‐parasitized larvae (?14.3 ± 0.2 °C). In addition, supercooling points were not correlated with body weights between parasitized and non‐parasitized larvae, indicating that cold hardiness of parasitized larvae was enhanced by endoparasitoids. Furthermore, the concentration of glycerol in the hemolymph was significantly higher in parasitized larvae (205.0 ± 7.1 μmol ml^?1) than in non‐parasitized larvae (169.8 ± 14.4 μmol ml^?1), which suggests that the mechanism that decreases the supercooling point of parasitized larvae was associated with glycerol. All these results indicated that the cold hardiness of parasitized C. suppressalis larvae was enhanced by their endoparasitoids, which benefitted overwintering endoparasitoids.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.