Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise

In this study, we investigated the effect of the supplementation with the dipeptide L-alanyl-L-glutamine (DIP) and a solution containing L-glutamine and L-alanine on plasma levels markers of muscle damage and levels of pro-inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty-one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg(-1)), a solution of free L-glutamine (1 g.kg(-1)) and free L-alanine (0.61 g.kg(-1)) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE-2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations Of glutamine and glutamate were measured. The concentrations of plasma TNF-alpha, PGE2 and the activity of CK were lower in the G&A-R and DIP-R groups, compared to the CON-R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP-R and G&A-R groups relative to the CON-R group. G&A-PE and DIP-PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF-alpha (p < 0.05), and higher concert I rations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON-PE group. We concluded that supplementation with free L-glutamine and the dipeptide LL-alanyl-LL-glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. Copyright (C) 2009 John Wiley & Sons, Ltd.

Homocysteine Thiolactone Induces Cardiac Dysfunction: Role of Oxidative Stress

This study investigates the cardiac functioning in male Wistar rats after treatments with methionine and homocysteine thiolactone (HcyT). The rats were distributed into 3 groups and treated for 8 weeks. Group I was the control (CO) group, given water, group II was treated with methionine, and group III with HcyT (100 mg/kg). Morphometric and functional cardiac parameters were evaluated by echocardiography. Superoxide dismutase (SOD), catalase, and glutathione S-transferase activities, chemiluminescence, thiobarbituric acid reactive substances, and immunocontent were measured in the myocardium. Hyperhomocysteinemia was observed in rats submitted to the both treatments. The results showed diastolic function was compromised in HcyT group, seen by the increase of E/A (peak velocity of early (E) and late (A) diastolic filling) ratio, decrease in deceleration time of E wave and left ventricular isovolumic relaxation time. Myocardial performance index was increased in HcyT group and was found associated with increased SOD immunocontent. HcyT group demonstrated an increase in SOD, catalase, and glutatione S-transferase activity, and chemiluminescence and thiobarbituric acid reactive substances. Overall, these results indicated that HcyT induces a cardiac dysfunction and could be associated with oxidative stress increase in the myocardium.

Nitrogen Metabolism in leaves of a tank epiphytic bromeliad: Characterization of a spatial and functional division

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO(3)(-)/NH(4)(+) or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary. GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH(4)(+) assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad`s leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Genomic Analysis of Wild Tomato Introgressions Determining Metabolism- and Yield-Associated Traits

With the aim of determining the genetic basis of metabolic regulation in tomato fruit, we constructed a detailed physical map of genomic regions spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 colocated markers from five selected genomic regions, and a total of 614 bacterial artificial chromosome (BAC)/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of Solanum lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analysis of this information resulted in a genome-wide map of a nondomesticated plant species and covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely colinear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated, and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive microsynteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 million years ago. The combined results serve as a reference for comparative studies both at the macrosyntenic and microsyntenic levels. They also provide a valuable tool for fine-mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition.

Angiotensin II induces superoxide generation via NAD(P)H oxidase activation in isolated rat pancreatic islets

Angiotensin II (Ang II) controls blood pressure, electrolyte balance, cell growth and vascular remodeling. Ang II activates NAD(P)H oxidase in several tissues with important function in the control of insulin secretion. Considering the concomitant occurrence of hypertension, insulin resistance and pancreatic B cell secretion impairment in the development of type II diabetes the aim of the present study was to evaluate the effect of ANG II on NAD(P)H oxidase activation in isolated pancreatic islets. We found that ANGII-induced superoxide generation via NAD(P)H oxidase activation and increased protein and mRNA levels of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)). (C) 2008 Elsevier B.V. All rights reserved.

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (170, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats (similar to 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats (similar to 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages. Copyright (C) 2008 John Wiley & Sons, Ltd.

Liver glycogen metabolism during short-term insulin-induced hypoglycemia in fed rats

The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was their infused with glucagon (1 nM), isoproterenot (2 mu M), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase. Copyright (c) 2008 John Wiley & Sons, Ltd.

Intensive insulin treatment induces insulin resistance in diabetic rats by impairing glucose metabolism-related mechanisms in muscle and liver

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM. Journal of Endocrinology (2011) 211, 55-64

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells. (C) 2010 Elsevier Inc. All rights reserved.

Low doses of hydrogen peroxide impair glucose-stimulated insulin secretion via inhibition of glucose metabolism and intracellular calcium oscillations

The inhibitory effect of hydrogen peroxide (H(2)O(2)) on glucose-stimulated insulin secretion was previously reported. However, the precise mechanism involved was not systematically investigated. In this study, the effects of low concentrations of H(2)O(2) (5-10 mu mol/L) on glucose metabolism, intracellular calcium ([Ca(2+)](i)) oscillations, and dynamic insulin secretion in rat pancreatic islets were investigated. Low concentrations of H(2)O(2) impaired insulin secretion in the presence of high glucose levels (16.7 mmol/L). This phenomenon was observed already after 2 minutes of exposure to H(2)O(2). Glucose oxidation and the amplitude of [Ca(2+)](i); oscillations were dose-dependently suppressed by H(2)O(2). These findings indicate that low concentrations of H(2)O(2) reduce insulin secretion in the presence of high glucose levels via inhibition of glucose metabolism and consequent impairment in [Ca(2+)](i); handling. (C) 2010 Elsevier Inc. All rights reserved.

Effect of exercise on glutamine metabolism in macrophages of trained rats

This study investigated the effect of exercise on glutamine metabolism in macrophages of trained rats. Rats were divided into three groups: sedentary (SED); moderately trained (MOD) rats that were swim trained 1 h/day, 5 days/week for 6 weeks; and exhaustively trained (EXT) rats that were similarly trained as MOD for 5 weeks and, in the 6th week, trained in three 1-h sessions/day with 150 min of rest between sessions. The animals swam with a load equivalent to 5.5% of their body weight and were killed 1 h after the last exercise session. Cells were collected, and glutamine metabolism in macrophage and function were assayed. Exercise increased phagocytosis in MOD when compared to SED (34.48 +/- 1.79 vs 15.21 +/- 2.91%, P < 0.05); however, H(2)O(2) production was higher in MOD (75.40 +/- 3.48 nmol h x 10(5) cell(-1)) and EXT (79.20 +/- 1.18 nmol h x 10(5) cell(-1)) in relation to SED (32.60 +/- 2.51 nmol h x 10(5) cell(-1), P < 0.05). Glutamine consumption increased in MOD and EXT (26.53 +/- 3.62 and 19.82 +/- 2.62 nmol h x 10(5) cell(-1), respectively) relative to SED (6.72 +/- 0.57 nmol h x 10(5) cell(-1), P < 0.05). Aspartate increased in EXT (9.72 +/- 1.14 nmol h x 10(5) cell(-1)) as compared to SED (1.10 +/- 0.19 nmol h x 10(5) cell(-1), P < 0.05). Glutamine decarboxylation was increased in MOD (12.10 +/- 0.27 nmol h x 10(5) cell(-1)) and EXT (16.40 +/-\ 2.17 nmol h x 10(5) cell(-1)) relative to SED (1.10 +/- 0.06 nmol h x 10(5) cell(-1), P < 0.05). This study suggests an increase in macrophage function post-exercise, which was supported by enhanced glutamine consumption and metabolism, and highlights the importance for glutamine after exercise.

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. (C) 2009 Elsevier Masson SAS. All rights reserved.

Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved in oligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, and two are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalian immunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterial recombinant EP24.15 with oxidized glutathione concentration as low as 10 mu M. The in vitro S-glutathionylation of EP24.15 was responsible for its oxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showed increased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also show that EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the protein oligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization by interprotein thiol-disulfide exchange. (c) 2007 Elsevier Inc. All rights reserved.

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed.

Laterally transferred genomic islands in Xanthomonadales related to pathogenicity and primary metabolism

Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes.