Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii. In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.

Proteomic analysis of kidney in rats chronically exposed to fluoride

Two-dimensional gel electrophoresis (2-DE) was used to better understand alterations in renal metabolism induced by fluoride (F). Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F for 60 days (n=6/group). Kidneys were collected for proteomic and histological (HE) analysis. After protein isolation, renal proteome profiles were examined using 2-DE and Colloidal Coomassie Blue staining. Protein spots with a 2-fold significant difference as detected by quantitative intensity analysis (image Master Platinum software) and t-test (p < 0.05) were excised and analyzed by MALDI-TOF MS (matrix assisted laser desorption ionization-time-of-flight mass spectrometry). The histological analysis revealed no damage in kidneys induced by F, except for a vascular congestion in the 50 ppm F group. Between control vs 50 ppm F, and control vs 5 ppm F groups, 12 and 6 differentially expressed proteins were detected, respectively. Six proteins, mainly related with metabolism, detoxification and housekeeping, were successfully identified. At the high F group, pyruvate carboxylase, a protein involved in the formation of oxaloacetate was found to be downregulated, while enoyl coenzyme A hydratase, involved in fatty acids oxidation, was found to be upregulated. Thus, proteomic analysis can provide new insights into the alterations in renal metabolism after F exposure, even in low doses. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Reciprocal changes in forebrain contents of glycogen and of glutamate/glutamine during early memory consolidation in the day-old chick

Passive avoidance learning is with advantage studied in day-old chicks trained to distinguish between beads of two different colors, of which one at training was associated with aversive taste. During the first 30-min post-training, two periods of glutamate release occur in the forebrain. One period is immediately after the aversive experience, when glutamate release is confined to the left hemisphere. A second release, 30 min later, may be bilateral, perhaps with preponderance of the right hemisphere. The present study showed increased pool sizes of glutamate and glutamine, specifically in the left hemisphere, at the time when the first glutamate release occurs, indicating de novo synthesis of glutamate/glutamine from glucose or glycogen, which are the only possible substrates. Behavioral evidence that memory is extinguished by intracranial administration at this time of iodoacetate, an inhibitor of glycolysis and glycogenolysis, and that the extinction of memory is counteracted by injection of glutamine, supports this concept. A decrease in forebrain glycogen of similar magnitude and coinciding with the increase in glutamate and glutamine suggests that glycogen rather than glucose is the main source of newly synthesized glutamate/glutamine. The second activation of glutamatergic activity 30 min after training, when memory is consolidated into stable, long-term memory, is associated with a bilateral increase in pool size of glutamate/glutamine. No glycogenolysis was observed at this time, but again there is a temporal correlation with sensitivity to inhibition by iodoacetate and rescue by glutamine, indicating the importance of de novo synthesis of glutamate/glutamine from glucose or glycogen. (C) 2003 Elsevier B.V All rights reserved.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

In vivo quantification of neuro-glial metabolism and glial glutamate concentration using 1H-[13C] MRS at 14.1T.

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo (13) C NMR spectroscopy coupled with the infusion labeled glial-specific substrate, such as acetate. In this study, we infused alpha-chloralose anesthetized rats with [2-(13) C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using (1) H-[(13) C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two-compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (Vg ) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT ) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC ) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vx (g) and Vx (n) ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations. In this (13) C-acetate labeling study, we propose a refined two-compartment analysis of brain energy metabolism based on (13) C turnover curves of acetate, glutamate and glutamine measured with state of the art in vivo dynamic MRS at high magnetic field in rats, enabling a deeper understanding of the specific role of glial cells in brain oxidative metabolism. In addition, the robustness of the metabolic fluxes determination relative to MRS data quality was carefully studied.

Small RNA-dependent expression of secondary metabolism is controlled by Krebs cycle function in Pseudomonas fluorescens.

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.

Brain energy metabolism measured by (13) C magnetic resonance spectroscopy in vivo upon infusion of [3-(13) C]lactate.

The brain uses lactate produced by glycolysis as an energy source. How lactate originated from the blood stream is used to fuel brain metabolism is not clear. The current study measures brain metabolic fluxes and estimates the amount of pyruvate that becomes labeled in glial and neuronal compartments upon infusion of [3-(13) C]lactate. For that, labeling incorporation into carbons of glutamate and glutamine was measured by (13) C magnetic resonance spectroscopy at 14.1 T and analyzed with a two-compartment model of brain metabolism to estimate rates of mitochondrial oxidation, glial pyruvate carboxylation, and the glutamate-glutamine cycle as well as pyruvate fractional enrichments. Extracerebral lactate at supraphysiological levels contributes at least two-fold more to replenish the neuronal than the glial pyruvate pools. The rates of mitochondrial oxidation in neurons and glia, pyruvate carboxylase, and glutamate-glutamine cycles were similar to those estimated by administration of (13) C-enriched glucose, the main fuel of brain energy metabolism. These results are in agreement with primary utilization of exogenous lactate in neurons rather than astrocytes. © 2014 Wiley Periodicals, Inc.

Exercise training enhances rat pancreatic islets anaplerotic enzymes content despite reduced insulin secretion

Endurance exercise has been shown to reduce pancreatic islets glucose-stimulated insulin secretion (GSIS). Anaplerotic/cataplerotic pathways are directly related to GSIS signaling. However, the effect of endurance training upon pancreatic islets anaplerotic enzymes is still unknown. In this sense, we tested the hypothesis that endurance exercise decreases GSIS by reducing anaplerotic/cataplerotic enzymes content. Male Wistar rats were randomly assigned to one of the four experimental groups as follows: control sedentary group (CTL), trained 1 day per week (TRE1x), trained 3 days per week (TRE3x) and trained 5 days per week (TRE5x) and submitted to an 8 weeks endurance-training protocol. After the training protocol, pancreatic islets were isolated and incubated with basal (2.8 mM) and stimulating (16.7 mM) glucose concentrations for GSIS measurement by radioimmunoassay. In addition, pyruvate carboxylase (PYC), pyruvate dehydrogenase (PDH), pyruvate dehydrogenase kinase 4 (PDK4), ATP-citrate lyase (ACL) and glutamate dehydrogenase (GDH) content were quantified by western blotting. Our data showed that 8 weeks of chronic endurance exercise reduced GSIS by 50% in a dose-response manner according to weekly exercise frequency. PYC showed significant twofold increase in TRE3x. PYC enhancement was even higher in TRE5x (p < 0.0001). PDH and PDK4 reached significant 25 and 50% enhancement, respectively compared with CTL. ACL and GDH also reported significant 50 and 75% increase, respectively. The absence of exercise-induced correlations among GSIS and anaplerotic/cataplerotic enzymes suggests that exercise may control insulin release by activating other signaling pathways. The observed anaplerotic and cataplerotic enzymes enhancement might be related to beta-cell surviving rather than insulin secretion.

Proteomic analysis of kidney in rats chronically exposed to fluoride

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Maturation of endogenous glucose production in preterm and term calves

Glucose disposability is often impaired in neonatal calves and even more in preterm calves. The objective of this study was to investigate ontogenic maturation of endogenous glucose production (eGP) in calves and its effects on postnatal glucose homeostasis. Calves (n = 7 per group) were born preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4 d (TC). Blood samples were taken immediately after birth and before and 2h after feeding at 24h after birth (PT; T) or on d 4 of life (TC) to determine metabolic and endocrine changes. After birth (PT and T) or on d 3 of life (TC), fasted calves were gavaged with deuterium-labeled water to determine gluconeogenesis (GNG) and intravenously infused with [U(13)C]-glucose to measure eGP and glucose oxidation (GOx) in blood plasma. After slaughter at 26h after birth (PT, T) or on d 4 of life (TC), glycogen concentrations in liver and hepatic mRNA concentrations and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase were measured. Preterm calves had the lowest plasma concentrations of cortisol and 3,5,3'-triiodothyronine at birth. Plasma glucose concentrations from d 1 to 2 decreased more, but plasma concentrations of lactate and urea and glucagon:insulin ratio were higher in PT than in T and TC calves. The eGP, GNG, GOx, as well as hepatic glycogen concentrations and PEPCK activities, were lowest in PT calves. Results indicate impaired glucose homeostasis due to decreased eGP in PT calves and maturation of eGP with ontogenic development.

Intestinal glucose absorption but not endogenous glucose production differs between colostrum- and formula-fed neonatal calves

Glucose supply markedly changes during the transition to extrauterine life. In this study, we investigated diet effects on glucose metabolism in neonatal calves. Calves were fed colostrum (C; n = 7) or milk-based formula (F; n = 7) with similar nutrient content up to d 4 of life. Blood plasma samples were taken daily before feeding and 2 h after feeding on d 4 to measure glucose, lactate, nonesterified fatty acids, protein, urea, insulin, glucagon, and cortisol concentrations. On d 2, additional blood samples were taken to measure glucose first-pass uptake (FPU) and turnover by oral [U-(13)C]-glucose and i.v. [6,6-(2)H(2)]-glucose infusion. On d 3, endogenous glucose production and gluconeogenesis were determined by i.v. [U-(13)C]-glucose and oral deuterated water administration after overnight feed deprivation. Liver tissue was obtained 2 h after feeding on d 4 and glycogen concentration and activities and mRNA abundance of gluconeogenic enzymes were measured. Plasma glucose and protein concentrations and hepatic glycogen concentration were higher (P < 0.05), whereas plasma urea, glucagon, and cortisol (d 2) concentrations as well as hepatic pyruvate carboxylase mRNA level and activity were lower (P < 0.05) in group C than in group F. Orally administered [U-(13)C]-glucose in blood was higher (P < 0.05) but FPU tended to be lower (P < 0.1) in group C than in group F. The improved glucose status in group C resulted from enhanced oral glucose absorption. Metabolic and endocrine changes pointed to elevated amino acid degradation in group F, presumably to provide substrates to meet energy requirements and to compensate for impaired oral glucose uptake.

Differences in milk production, glucose metabolism, and carcass composition of 2 Charolais x Holstein F2 families derived from reciprocal paternal and maternal grandsire crosses

Two F(2) Charolais x German Holstein families comprising full and half sibs share identical but reciprocal paternal and maternal Charolais grandfathers differ in milk production. We hypothesized that differences in milk production were related to differences in nutritional partitioning revealed by glucose metabolism and carcass composition. In 18F(2) cows originating from mating Charolais bulls to German Holstein cows and a following intercross of the F(1) individuals (n=9 each for family Ab and Ba; capital letters indicate the paternal and lowercase letter the maternal grandsire), glucose tolerance tests were performed at 10 d before calving and 30 and 93 d in milk (DIM) during second lactation. Glucose half-time as well as areas under the concentration curve for plasma glucose and insulin were calculated. At 94 DIM cows were infused intravenously with 18.3 micromol of d-[U-(13)C(6)]glucose/kg(0.75) of BW, and blood samples were taken to measure rate of glucose appearance and glucose oxidation as well as plasma concentrations of metabolites and hormones. Cows were slaughtered at 100 DIM and carcass size and composition was evaluated. Liver samples were taken to measure glycogen and fat content, gene expression levels, and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase as well as gene expression of glucose transporter 2. Milk yield was higher and milk protein content at 30 DIM was lower in Ba than in Ab cows. Glucose half-life was higher but insulin secretion after glucose challenge was lower in Ba than in Ab cows. Cows of Ab showed higher glucose oxidation, and plasma concentrations at 94 DIM were lower for glucose and insulin, whereas beta-hydroxybutyrate was higher in Ba cows. Hepatic gene expression of pyruvate carboxylase, glucose 6-phosphatase, and glucose transporter 2 were higher whereas phosphoenolpyruvate carboxykinase activities were lower in Ba than in Ab cows. Carcass weight as well as fat content of the carcass were higher in Ab than in Ba cows, whereas mammary gland mass was lower in Ab than in Ba cows. Fat classification indicated leaner carcass composition in Ba than in Ab cows. In conclusion, the 2 families showed remarkable differences in milk production that were accompanied by changes in glucose metabolism and body composition, indicating capacity for milk production as main metabolic driving force. Sex chromosomal effects provide an important regulatory mechanism for milk performance and nutrient partitioning that requires further investigation.

Regional mRNA expression of key gluconeogenic enzymes in the liver of dairy cows

Liver tissue was collected from eight random dairy cows at a slaughterhouse to test if gene expression of pyruvate carboxylase (PC), mitochondrial phosphoenolpyruvate carboxykinase (PEPCKm) and cytosolic phosphoenolpyruvate carboxykinase (PEPCKc) is different at different locations in the liver. Obtained liver samples were analysed for mRNA expression levels of PC, PEPCKc and PEPCKm and subjected to the MIXED procedure of SAS to test for the sampled locations with cow liver as repeated subject. Additionally, the general linear model procedure (GLM) for analysis of variance was applied to test for significant differences for mRNA abundance of PEPCKm, PEPCKc and bPC between the livers. In conclusion, this study demonstrated that mRNA abundance of PC, PEPCKc and PEPCKm is not different between locations in the liver but may differ between individual cows.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

Variation in hepatic regulation of metabolism during the dry period and in early lactation in dairy cows

The purpose of this study was to investigate variations in hepatic regulation of metabolism during the dry period, after parturition, and in early lactation in dairy cows. For this evaluation, cows were divided into 2 groups based on the plasma concentration of beta-hydroxybutyric acid (BHBA) in wk 4 postpartum (PP; group HB, BHBA >0.75 mmol/L; group LB, BHBA <0.75 mmol/L, respectively). Liver biopsies were obtained from 28 cows at drying off (mean 59 +/- 8 d antepartum), on d 1, and in wk 4 and 14 PP. Blood samples were collected every 2 wk during this entire period. Liver samples were analyzed for mRNA abundance of genes related to carbohydrate metabolism (pyruvate carboxylase, PC; phosphoenolpyruvate carboxykinase, PEPCK; citrate synthase, CS), fatty acid biosynthesis (ATP citrate lyase, ACLY) and oxidation (acyl-CoA synthetase long-chain, ACSL; carnitine palmitoyltransferase 1A, CPT 1A; carnitine palmitoyltransferase 2, CPT 2; acyl-coenzyme A dehydrogenase very long chain, ACADVL), cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, HMGCS1), ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2, HMGCS2), and of genes encoding the transcription factors peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferator-activated receptor gamma (PPARgamma), and sterol regulatory element binding factor 1 (SREBF1). Blood plasma was assayed for concentrations of glucose, BHBA, nonesterified fatty acids, cholesterol, triglycerides, insulin, insulin-like growth factor-I, and thyroid hormones. In both groups, plasma parameters followed a pattern usually observed in dairy cows. However, changes were moderate and the energy balance in cows turned positive in wk 7 PP for both groups. Additionally, the energy balance and milk yield were similar for both groups after parturition onwards. Significant group effects were found at drying off, when plasma concentrations of triglycerides were higher in LB than in HB, and in wk 4 PP, when plasma concentrations of glucose and IGF-I were lower in HB than in LB. Similarly, moderate changes in mRNA expression of hepatic genes between the different time points were observed, although HB cows showed more adaptive performance than LB cows based on changes in mRNA expression of PEPCKc, PEPCKm, CS, CPT 1A, CPT 2, and PPARalpha. Part of the variation measured in this study was explained by parity. Significant Spearman rank correlation coefficients between the variables were not similar at each time point and were not similar between the groups at each time point, suggesting that metabolic regulation differs between cows. In conclusion, metabolic regulation in dairy cows is a dynamic system, and differs obviously between cows at different metabolic stages related to parturition.