Tiago B. Rodrigues

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.

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation

The glutamate–glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate–glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo 13C nuclear magnetic resonance labeling with 13C2acetate or (1-13C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.

Magnetic resonance analysis of the effects of acute ammonia intoxication on rat brain. Role of NMDA receptors

Acute ammonia intoxication leads to rapid death, which is prevented by blocking N‐methyl‐d‐aspartate (NMDA) receptors. The subsequent mechanisms leading to death remain unclear. Brain edema seems an important step. The aim of this work was to study the effects of acute ammonia intoxication on different cerebral parameters in vivo using magnetic resonance and to assess which effects are mediated by NMDA receptors activation. To assess edema induction, we injected rats with ammonium acetate and measured apparent diffusion coefficient (ADC) in 16 brain areas. We also analyzed the effects on T1, T2, and T2* maps and whether these effects are prevented by blocking NMDA receptors. The effects of acute ammonia intoxication are different in different brain areas. T1 relaxation time is reduced in eight areas. T2 relaxation time is reduced only in ventral thalamus and globus pallidus. ADC values increased in hippocampus, caudate‐putamen, substantia nigra and cerebellar cortex, reflecting vasogenic edema. ADC decreased in hypothalamus, reflecting cytotoxic edema. Myo‐inositol increased in cerebellum and substantia nigra, reflecting vasogenic edema. N‐acetyl‐aspartate decreased in cerebellum, reflecting neuronal damage. Changes in N‐acetyl‐aspartate, T1 and T2 are prevented by blocking NMDA receptors with MK‐801 while changes in ADC or myo‐inositol (induction of edema) are not.

Metabolic interactions between glutamatergic and dopaminergic neurotransmitter systems are mediated through D1 dopamine receptors

Interactions between the dopaminergic and glutamatergic neurotransmission systems were investigated in the adult brain of wild‐type (WT) and transgenic mice lacking the dopamine D1 or D2 receptor subtypes. Activity of the glutamine cycle was evaluated by using 13C NMR spectroscopy, and striatal activity was assessed by c‐Fos expression and motor coordination. Brain extracts from (1,2‐13C2) acetate‐infused mice were prepared and analyzed by 13C NMR to determine the incorporation of the label into the C4 and C5 carbons of glutamate and glutamine. D1R−/− mice showed a significantly higher concentration of cerebral (4,5‐13C2) glutamine, consistent with an increased activity of the glutamate‐glutamine cycle and of glutamatergic neurotransmission. Conversely, D2R−/− mice did not show any significant changes in (4,5‐13C2) glutamate or (4,5‐13C2) glutamine, suggesting that alterations in glutamine metabolism are mediated through D1 receptors. This was confirmed with D1R−/− and WT mice treated with reserpine, a dopamine‐depleting drug, or with reserpine followed by L‐DOPA, a dopamine precursor. Exposure to reserpine increased (4,5‐13C2) glutamine in WT to levels similar to those found in untreated D1R−/− mice. These values were the same as those reached in the reserpine‐treated D1R−/− mice. Treatment of WT animals with L‐DOPA returned (4,5‐13C2) glutamine levels to normal, but this was not verified in D1R−/− animals. Reserpine impaired motor coordination and decreased c‐Fos expression, whereas L‐DOPA restored both variables to normal values in WT but not in D1R−/−. Together, our results reveal novel neurometabolic interactions between glutamatergic and dopaminergic systems that are mediated through the D1, but not the D2, dopamine receptor subtype.

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.

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.

Methylglyoxal causes structural and functional alterations in adipose tissue independently of obesity.

Context:Adipose tissue is one of the first organs to develop insulin resistance even with moderate BMI. However, the contribution of developing hyperglycaemia and concomitant methylglyoxal increment to tissue dysfunction during type 2 diabetes progression was not addressed before. Methods:Young and aged Wistar and Goto-Kakizaki rats (non-obese model of type 2 diabetes) and a group of MG-treated W rats were used to investigate the chronic effects of hyperglycaemia and ageing and specifically MG-induced mechanisms. Results:Diabetic and aged rats showed decreased adipose tissue irrigation and interstitial hypoxia. Hyperglycaemia of diabetic rats leaded to fibrosis and accumulation of PAS-positive components, exacerbated in aged animals, which also showed decreased hipoadiponectinemia, increased MCP-1 expression and macrophage infiltration to glycated fibrotic regions. MG leaded to increased free fatty acids, hipoadiponectinemia, decreased irrigation, hypoxia and macrophage recruitment for glycated fibrotic regions. Conclusions:MG contributes to dysfunction of adipose tissue during type 2 diabetes progression.

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

We report a novel (13C, 2H) 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 [3‐13C]lactate in Krebs‐Henseleit Buffer containing 50% 2H2O (vol/vol) for up to 30 hr. 13C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at ∼20.85 parts per million (ppm; −0.119 μmol/mg protein/hr) derived from the methyl carbon of [3‐13C]lactate; and (2) an exponentially increasing shifted singlet at ∼20.74 ppm (0.227 μmol/ mg protein/hr) from the methyl carbon of [3‐13C, 2‐2H]lactate. The shifted singlet appears because during its transit through the cytosol, [3‐13C]lactate generates [3‐13C, 2‐2H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3‐13C, 2‐2H]lactate is shifted −0.11 ppm with respect to that of [3‐13C]lactate, making it possible to distinguish between both molecules by 13C NMR. During incubations with 2.5 mM [1‐13C]glucose and 3.98 mM [U‐13C3]lactate or with 2.5 mM [1‐13C]glucose and 3.93 mM [2‐13C]pyruvate, C2‐deuterated lactate was produced only from [1‐13C]glucose or [U‐13C3]lactate, revealing that this deuteration process is redox sensitive. When [1‐13C]glucose and [U‐13C3]lactate were used as substrates, no significant [3‐13C]lactate production from [1‐13C]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.

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

A simple 2H NMR method for quantifying absolute 2H‐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 2H‐enriched formate to the NMR sample. When the method was applied to [2‐2H]monoacetone glucose samples prepared from [2‐2H]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 [2‐2H]glucose precursors. The measurement was also applied to monoacetone glucose derived from human plasma glucose samples following administration of 2H2O 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 2H‐enrichment of body water measured by an independent method. Magn Reson Med 48:535–539, 2002.

Kinetic properties of the redox switch/redox coupling mechanism as determined in primary cultures of cortical neurons and astrocytes from rat brain

We investigate the mechanisms underlying the redox switch/redox coupling hypothesis by characterizing the competitive consumption of glucose or lactate and the kinetics of pyruvate production in primary cultures of cortical neurons and astrocytes from rat brain. Glucose consumption was determined in neuronal cultures incubated in Krebs ringer bicarbonate buffer (KRB) containing 0.25–5 mM glucose, in the presence and absence of 5 mM lactate as an alternative substrate. Lactate consumption was measured in neuronal cultures incubated with 1–15 mM lactate, in the presence and absence of 1 mM glucose. In both cases, the alternative substrate increased the Km (mM) values for glucose consumption (from 2.2 ± 0.2 to 3.6 ± 0.1) or lactate consumption (from 7.8 ± 0.1 to 8.5 ± 0.1) without significant changes on the corresponding Vmax. This is consistent with a competitive inhibition between the simultaneous consumption of glucose and lactate. When cultures of neurons or astrocytes were incubated with increasing lactate concentrations 1–20 mM, pyruvate production was observed with Km (mM) and Vmax (nmol/mg/h) values of 1.0 ± 0.1 and 109 ± 4 in neurons, or 0.28 ± 0.1 and 342 ± 54 in astrocytes. Thus, astrocytes or neurons are able to return to the incubation medium as pyruvate, a significant part of the lactate consumed. Present results support the reversible exchange of reducing equivalents between neurons and astrocytes in the form of lactate or pyruvate. Monocarboxylate exchange is envisioned to operate under near equilibrium, with the transcellular flux directed thermodynamically toward the more oxidized intracellular redox environment.