Biomedical subjects

Tiago B Rodrigues

Publications and source records attributed to Tiago B Rodrigues.

9 recordsLinked to original sources

PubMed2006

The redox switch/redox coupling hypothesis.

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD(+) limitation, under sufficiently reduced cytosolic NAD(+)/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD(+)/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD(+)/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD(+)/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.

Animals↗

PubMed2006

Functional genomics in Dictyostelium: MidA, a new conserved protein, is required for mitochondrial function and development.

Genomic sequencing has revealed a large number of evolutionary conserved genes of unknown function. In the absence of characterized functional domains, the discovery of the role of these genes must rely on experimental approaches. We have selected 30 Dictyostelium discoideum genes of unknown function that showed high similarity to uncharacterized human genes and were absent in the complete proteomes from Saccharomyces cerevisiae and S. pombe. No putative functional motifs were found in their predicted encoded proteins. Eighteen genes were successfully knocked-out and three of them showed obvious phenotypes. A detailed analysis of one of them, midA, is presented in this report. Disruption of midA in Dictyostelium leads to pleiotropic defects. Cell size, growth rate, phagocytosis and macropinocytosis were affected in the mutant. During development, midA- cells showed an enhanced tendency to remain at the slug stage, and spore viability was compromised. The expression of MidA fused to GFP in midA- strain rescued the phenotype and the fused protein was located in the mitochondria. Although cellular oxygen consumption, mitochondrial content and mitochondrial membrane potential were similar to wild type, the amount of ATP was significantly reduced in the mutant suggesting a mitochondrial dysfunction. Metabolomic analysis by natural-abundance 13C-nuclear magnetic resonance has shown the lack of glycogen accumulation during growth. During starvation, mutant cells accumulated higher levels of ammonia, which inhibited normal development. We hypothesize that the lack of MidA reduces mitochondrial ATP synthetic capacity and this has an impact in some but not all energy-dependent cellular processes. This work exemplifies the potential of Dictyostelium as a model system for functional genomic studies.

Amino Acid Sequence↗

PubMed2005

A fast and sensitive 1H NMR method to measure the turnover of the H2 hydrogen of lactate.

A fast and sensitive procedure to determine the turnover of the H2 hydrogen of lactate and quantify its (2)H-enrichment by (1)H NMR is illustrated using C6 cells metabolizing (3-(13)C) lactate in 50% (2)H(2)O (vol/vol). (2)H substitution of the lactate H2 hydrogen resulted in two easily detectable transformations of the vicinal H3 doublet resonance: 1) the formation of an H3 singlet due to the disappearance of the homonuclear coupling to H2 ((3)J(betaH-alphaH) = 7.0 Hz), and 2) an upfield isotopic shift derived from the vicinal (2)H2 substitution (Delta(3) = -0.007 ppm). Only those lactate molecules that have passed through the cell cytosol experience these effects, since H2 deuteration involves lactate dehydrogenase activity and NAD((2)H). Thus, analysis of the observed shifted and unshifted H3 lactate resonances from the incubation medium allows the discrimination of the perprotonated (3-(13)C) lactate added as substrate, and the (3-(13)C, 2-(2)H) lactate recycled to the incubation medium after passage through the cytosol.

Algorithms↗

PubMed2004

A (13)C isotopomer kinetic analysis of cardiac metabolism: influence of altered cytosolic redox and [Ca(2+)](o).

Rat hearts were perfused with mixtures of [3-(13)C]pyruvate and [3-(13)C]lactate (to alter cytosolic redox) at low (0.5 mM) or high (2.5 mM) Ca(2+) concentrations to alter contractility. Hearts were frozen at various times after exposure to these substrates, were extracted, and were then analyzed by (13)C NMR spectroscopy. The time-dependent multiplets observed in the (13)C NMR resonances of glutamate in all hearts and in malate and aspartate in hearts perfused with high-pyruvate/low-lactate concentrations were analyzed using a kinetic model of the tricarboxylic acid (TCA) cycle. The analysis showed that TCA cycle flux (V(TCA)) and exchange flux (V(X)) that involved cycle intermediates were both sensitive to cell redox and altered Ca(2+) concentration, and the ratio of these fluxes (V(X)/V(TCA)) varied >10-fold.

Animals↗

PubMed2003

Sources of glucose production in cirrhosis by 2H2O ingestion and 2H NMR analysis of plasma glucose.

Plasma glucose 2H enrichment was quantified by 2H NMR in patients with cirrhosis (n=6) and healthy subjects (n=5) fasted for 16 h and given 2H(2)O to approximately 0.5% body water. The percent contribution of glycogenolysis and gluconeogenesis to glucose production (GP) was estimated from the relative enrichments of hydrogen 5 and hydrogen 2 of plasma glucose. Fasting plasma glucose levels were normal in both groups (87+/-7 and 87+/-24 mg/dl for healthy and cirrhotic subjects, respectively). The percent contribution of glycogen to GP was smaller in cirrhotics than controls (22+/-7% versus 46+/-4%, P<0.001), while the contribution from gluconeogenesis was larger (78+/-7% versus 54+/-4%, P<0.001). In all subjects, glucose 6R and 6S hydrogens had similar enrichments, consistent with extensive exchange of 2H between body water and the hydrogens of gluconeogenic oxaloacetate (OAA). The difference in 2H-enrichment between hydrogen 5 and hydrogen 6S was significantly larger in cirrhotics, suggesting that the fractional contribution of glycerol to the glyceraldehyde-3-phosphate (G3P)-moiety of plasma glucose was higher compared to controls (19+/-6% versus 7+/-6%, P<0.01). In all subjects, hydrogens 4 and 5 of glucose had identical enrichments while hydrogen 3 enrichments were systematically lower. This reflects incomplete exchange between the hydrogen of water and that of 1-R-dihydroxyacetone phosphate (DHAP) or incomplete exchange of DHAP and G3P pools via triose phosphate isomerase.

Adult↗

PubMed2003

Integration of [U-13C]glucose and 2H2O for quantification of hepatic glucose production and gluconeogenesis.

Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13C]glucose and 2H2O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13C-isotopomers derived from the incorporation of [U-13C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13C]glucose and provided reasonable estimates (1.90 +/- 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2H-enrichment from 2H2O by 2H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 +/- 0.14 and 1.03 +/- 0.10 mg/kg/min, respectively). Hepatic glutamine 13C-isotopomers representing multiply-enriched oxaloacetate and [U-13C]acetyl-CoA were identified as multiplets in the 13C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and approximately 0.05% to carbon 4 of glutamine.

Acetyl Coenzyme A↗

PubMed2002

Quantitation of absolute 2H enrichment of plasma glucose by 2H NMR analysis of its monoacetone derivative.

A simple (2)H NMR method for quantifying absolute (2)H-enrichments in all seven aliphatic positions of glucose following its derivatization to monoacetone glucose is presented. The method is based on the addition of a small quantity of (2)H-enriched formate to the NMR sample. When the method was applied to [2-(2)H]monoacetone glucose samples prepared from [2-(2)H]glucose standards of known enrichments in the range of 0.2-2.5%, enrichment estimates derived by the NMR method were in good agreement with the real enrichment values of the [2-(2)H]glucose precursors. The measurement was also applied to monoacetone glucose derived from human plasma glucose samples following administration of (2)H(2)O and attainment of isotopic steady state, where glucose H2 and body water enrichment are equivalent. In these studies, the absolute H2 enrichment of plasma glucose estimated by the formate method was in good agreement with the (2)H-enrichment of body water measured by an independent method.

Acetone↗

PubMedDate not supplied

Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR.

We report a novel ((13)C, (2)H) nuclear magnetic resonance (NMR) procedure to investigate lactate recycling through the monocarboxylate transporter of the plasma membrane of cells in culture. C6 glioma cells were incubated with [3-(13)C]lactate in Krebs-Henseleit Buffer containing 50% (2)H(2)O (vol/vol) for up to 30 hr. (13)C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at approximately 20.85 parts per million (ppm; -0.119 micromol/mg protein/hr) derived from the methyl carbon of [3-(13)C]lactate; and (2) an exponentially increasing shifted singlet at approximately 20.74 ppm (0.227 micromol/ mg protein/hr) from the methyl carbon of [3-(13)C, 2-(2)H]lactate. The shifted singlet appears because during its transit through the cytosol, [3-(13)C]lactate generates [3-(13)C, 2-(2)H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3-(13)C, 2-(2)H]lactate is shifted -0.11 ppm with respect to that of [3-(13)C]lactate, making it possible to distinguish between both molecules by (13)C NMR. During incubations with 2.5 mM [1-(13)C]glucose and 3.98 mM [U-(13)C(3)]lactate or with 2.5 mM [1-(13)C]glucose and 3.93 mM [2-(13)C]pyruvate, C2-deuterated lactate was produced only from [1-(13)C]glucose or [U-(13)C(3)]lactate, revealing that this deuteration process is redox sensitive. When [1-(13)C]glucose and [U-(13)C(3)]lactate were used as substrates, no significant [3-(13)C]lactate production from [1-(13)C]glucose was detected, suggesting that glycolytic lactate production may be stopped under the high lactate concentrations prevailing under mild hypoxic or ischemic episodes or during cerebral activation.

Animals↗

PubMedDate not supplied

Cerebral glucose metabolism and the glutamine cycle as detected by in vivo and in vitro 13C NMR spectroscopy.

We review briefly 13C NMR studies of cerebral glucose metabolism with an emphasis on the roles of glial energetics and the glutamine cycle. Mathematical modeling analysis of in vivo 13C turnover experiments from the C4 carbons of glutamate and glutamine are consistent with: (i) the glutamine cycle being the major cerebral metabolic route supporting glutamatergic neurotransmission, (ii) glial glutamine synthesis being stoichiometrically coupled to glycolytic ATP production, (iii) glutamine serving as the main precursor of neurotransmitter glutamate and (iv) glutamatergic neurotransmission being supported by lactate oxidation in the neurons in a process accounting for 60-80% of the energy derived from glucose catabolism. However, more recent experimental approaches using inhibitors of the glial tricarboxylic acid (TCA) cycle (trifluoroacetic acid, TFA) or of glutamine synthase (methionine sulfoximine, MSO) reveal that a considerable portion of the energy required to support glutamine synthesis is derived from the oxidative metabolism of glucose in the astroglia and that a significant amount of the neurotransmitter glutamate is produced from neuronal glucose or lactate rather than from glial glutamine. Moreover, a redox switch has been proposed that allows the neurons to use either glucose or lactate as substrates for oxidation, depending on the relative availability of these fuels under resting or activation conditions, respectively. Together, these results suggest that the coupling mechanisms between neuronal and glial metabolism are more complex than initially envisioned.

Adenosine Triphosphate↗