Anne-Karine Bouzier-Sore

Activity-dependent regulation of energy metabolism by astrocytes: an update.

Astrocytes play a critical role in the regulation of brain metabolic responses to activity. One detailed mechanism proposed to describe the role of astrocytes in some of these responses has come to be known as the astrocyte‐neuron lactate shuttle hypothesis (ANLSH). Although controversial, the original concept of a coupling mechanism between neuronal activity and glucose utilization that involves an activation of aerobic glycolysis in astrocytes and lactate consumption by neurons provides a heuristically valid framework for experimental studies. In this context, it is necessary to provide a survey of recent developments and data pertaining to this model. Thus, here, we review very recent experimental evidence as well as theoretical arguments strongly supporting the original model and in some cases extending it. Aspects revisited include the existence of glutamate‐induced glycolysis in astrocytes in vitro, ex vivo, and in vivo, lactate as a preferential oxidative substrate for neurons, and the notion of net lactate transfer between astrocytes and neurons in vivo. Inclusion of a role for glycogen in the ANLSH is discussed in the light of a possible extension of the astrocyte‐neuron lactate shuttle (ANLS) concept rather than as a competing hypothesis. New perspectives offered by the application of this concept include a better understanding of the basis of signals used in functional brain imaging, a role for neuron‐glia metabolic interactions in glucose sensing and diabetes, as well as novel strategies to develop therapies against neurodegenerative diseases based upon improving astrocyte‐neuron coupled energetics.

Lactate is a Preferential Oxidative Energy Substrate over Glucose for Neurons in Culture

The authors investigated concomitant lactate and glucose metabolism in primary neuronal cultures using 13C- and 1H-NMR spectroscopy. Neurons were incubated in a medium containing either [1-13C]glucose and different unlabeled lactate concentrations, or unlabeled glucose and different [3-13C]lactate concentrations. Overall, 13C-NMR spectra of cellular extracts showed that more 13C was incorporated into glutamate when lactate was the enriched substrate. Glutamate 13C-enrichment was also found to be much higher in lactate-labeled than in glucose-labeled conditions. When glucose and lactate concentrations were identical (5.5 mmol/L), relative contributions of glucose and lactate to neuronal oxidative metabolism amounted to 21% and 79%, respectively. Results clearly indicate that when neurons are in the presence of both glucose and lactate, they preferentially use lactate as their main oxidative substrate.

Competition between glucose and lactate as oxidative energy substrates in both neurons and astrocytes: a comparative NMR study.

Competition between glucose and lactate as oxidative energy substrates was investigated in both primary cultures of astrocytes and neurons using physiological concentrations (1.1 mm for each). Glucose metabolism was distinguished from lactate metabolism by using alternatively labelled substrates in the medium ([1‐13C]glucose + lactate or glucose + [3‐13C]lactate). After 4 h of incubation, 1H and 13C‐NMR spectra were realized on perchloric acid extracts of both cells and culture media. For astrocytic cultures, spectra showed that amino acids (glutamine and alanine) were more labelled in the glucose‐labelled condition, indicating that glucose is a better substrate to support oxidative metabolism in these cells. The opposite was observed on spectra from neuronal cultures, glutamate being much more labelled in the lactate‐labelled condition, confirming that neurons consume lactate preferentially as an oxidative energy substrate. Analysis of glutamine and glutamate peaks (singlets or multiplets) also suggests that astrocytes have a less active oxidative metabolism than neurons. In contrast, they exhibit a stronger glycolytic metabolism than neurons as indicated by their high lactate production yield. Using a mathematical model, we have estimated the relative contribution of exogenous glucose and lactate to neuronal oxidative metabolism. Under the aforementioned conditions, it represents 25% for glucose and 75% for lactate. Altogether, these results obtained on separate astrocytic and neuronal cultures support the idea that lactate, predominantly produced by astrocytes, is used as a supplementary fuel by neurons in vivo already under resting physiological conditions.

Feeding active neurons: (re)emergence of a nursing role for astrocytes

Despite unquestionable evidence that glucose is the major energy substrate for the brain, data collected over several decades with different approaches suggest that lactate may represent a supplementary metabolic substrate for neurons. Starting with the pioneering work of McIlwain in the early 1950s which showed that lactate can sustain the respiratory rate of small brain tissue pieces, this idea receives confirmation with more recent studies using nuclear magnetic resonance spectroscopy undoubtedly demonstrating that lactate is efficiently oxidized by neurons, both in vitro and in vivo. Not only is lactate able to maintain ATP levels and promote neuronal survival but it was also found to support neuronal activity, at least if low levels of glucose are present. Despite the early suggestion for a role of astrocytes in metabolic supply to neurons, it is only recently however that they have been considered as a potential source of lactate for neurons. Moreover, it has been proposed that astrocytes might provide lactate to neurons in response to enhanced synaptic activity by a well-characterized mechanism involving glutamate uptake. The description of specific transporters for lactate on both astrocytes and neurons further suggest that there exist a coordinated mechanism of lactate exchange between the two cell types. Thus it is proposed that astrocytes play a nursing role toward neurons by providing lactate as an additional energy substrate especially during periods of enhanced synaptic activity. The importance of this metabolic cooperation within the central nervous system, although not unique if compared to other organs, still remains to be explored.

MRI of inducible P‐selectin expression in human activated platelets involved in the early stages of atherosclerosis

The noninvasive imaging of atherosclerotic plaques at an early stage of atherogenesis remains a major challenge for the evaluation of the pathologic state of patients at high risk of acute coronary syndromes. Recent studies have emphasized the importance of platelet–endothelial cell interactions in atherosclerosis‐prone arteries at early stages, and the prominent role of P‐selectin in the initial loose contact between platelets and diseased vessel walls. A specific MR contrast agent was developed here for the targeting, with high affinity, of P‐selectin expressed in large amounts on activated platelets and endothelial cells. For this purpose, PEGylated dextran/iron oxide nanoparticles [PEG, poly(ethylene glycol)], named versatile ultrasmall superparamagnetic iron oxide (VUSPIO) particles, labeled with rhodamine were coupled to an anti‐human P‐selectin antibody (VH10). Flow cytometry and microscopy experiments on human activated platelets were highly correlated with MRI (performed at 4.7 and 0.2 T), with a 50% signal decrease in T2 and T1 values corresponding to the strong labeling of activated vs resting platelets. The number of 1000 VH10–VUSPIO nanoparticles attained per activated platelet appeared to be optimal for the detection of hypo‐ and hyper‐signals in the platelet pellet on T2‐ and T1‐weighted MRI. Furthermore, in vivo imaging of atherosclerotic plaques in ApoE mice at 4.7 T showed a spatial resolution adapted to the imaging of intimal thickening and a hypo‐signal at 4.7 T, as a result of the accumulation of VH10–VUSPIO nanoparticles in the plaque. Our work provides support for the further assessment of the use of VH10–VUSPIO nanoparticles as a promising imaging modality able to identify the early stages of atherosclerosis with regard to the pertinence of both the target and the antibody‐conjugated contrast agent used. Copyright

Time-dependence of the contribution of pyruvate carboxylase versus pyruvate dehydrogenase to rat brain glutamine labelling from [1–13C]glucose metabolism

[1–13C]glucose metabolism in the rat brain was investigated after intravenous infusion of the labelled substrate. Incorporation of the label into metabolites was analysed by NMR spectroscopy as a function of the infusion time: 10, 20, 30 or 60 min. Specific enrichments in purified mono‐ and dicarboxylic amino acids were determined from 1H‐observed/13C‐edited and 13C‐NMR spectroscopy. The relative contribution of pyruvate carboxylase versus pyruvate dehydrogenase (PC/PDH) to amino acid labelling was evaluated from the enrichment difference between either C2 and C3 for Glu and Gln, or C4 and C3 for GABA, respectively. No contribution of pyruvate carboxylase to aspartate, glutamate or GABA labelling was evidenced. The pyruvate carboxylase contribution to glutamine labelling varied with time. PC/PDH decreased from around 80% after 10 min to less than 30% between 20 and 60 min. This was interpreted as reflecting different labelling kinetics of the two glutamine precursor glutamate pools: the astrocytic glutamate and the neuronal glutamate taken up by astrocytes through the glutamate‐glutamine cycle. The results are discussed in the light of the possible occurrence of neuronal pyruvate carboxylation. The methods previously used to determine PC/PDH in brain were re‐evaluated as regards their capacity to discriminate between astrocytic (via pyruvate carboxylase) and neuronal (via malic enzyme) pyruvate carboxylation.

Microglia in Close Vicinity of Glioma Cells: Correlation Between Phenotype and Metabolic Alterations

Microglia are immune cells within the central nervous system. In brain-developing tumors, gliomas are able to silence the defense and immune functions of microglia, a phenomenon which strongly contributes to tumor progression and treatment resistance. Being activated and highly motile, microglia infiltrate tumors and secrete macrophagic chemoattractant factors. Thereafter, the tumor cells shut down their immune properties and stimulate the microglia to release tumor growth-promoting factors. The result of such modulation is that a kind of symbiosis occurs between microglia and tumor cells, in favor of tumor growth. However, little is known about microglial phenotype and metabolic modifications in a tumoral environment. Co-cultures were performed using CHME5 microglia cells grown on collagen beads or on coverslips and placed on monolayer of C6 cells, limiting cell/cell contacts. Phagocytic behavior and expression of macrophagic and cytoskeleton markers were monitored. Respiratory properties and energetic metabolism were also studied with regard to the activated phenotype of microglia. In co-cultures, transitory modifications of microglial morphology and metabolism were observed linked to a concomitant transitory increase of phagocytic properties. Therefore, after 1 h of co-culture, microglia were activated but when longer in contact with tumor cells, phagocytic properties appear silenced. Like the behavior of the phenotype, microglial respiration showed a transitory readjustment although the mitochondria maintained their perinuclear relocation. Nevertheless, the energetic metabolism of the microglia was altered, suggesting a new energetic steady state. The results clearly indicate that like the depressed immune properties, the macrophagic and metabolic status of the microglia is quickly driven by the glioma environment, despite short initial phagocytic activation. Such findings question the possible contribution of diffusible tumor factors to the microglial metabolism.

Short-Term Effect of Erythropoietin on Brain Lesions and Aquaporin-4 Expression in a Hypoxic-Ischemic Neonatal Rat Model Assessed by Magnetic Resonance Diffusion Weighted Imaging and Immunohistochemistry

Erythropoietin (Epo) is an endogenous cytokine that regulates hematopoiesis and is widely used to treat anemia. In addition, it has recently increased interest in the neurosciences since the new concept of Epo as a neuroprotective agent has emerged. The potential protective effect of human recombinant Epo (r-hu-Epo) on a hypoxic-ischemic (HI) pup rat model was studied. Cerebral HI was obtained by permanent left carotid artery ligature of pups followed by a 2-h hypoxia. Three hours after carotid occlusion, brain lesions were assessed by magnetic resonance diffusion weighted imaging. Intraperitoneal administration of r-hu-Epo (30,000 U/kg dose) limited both the HI-induced brain lesion area and the decrease in apparent diffusion coefficient (ADC) in the lesion. To identify potential mechanisms underlying the effects of Epo, immunohistochemical detection of caspase-3 and water channel protein aquaporin-4 (AQP4) were performed. No early apoptosis was detected, but up-regulation of AQP4 expression was observed in HI pups that received r-hu-Epo compared with HI animals without treatment. This study demonstrates an early neuroprotective effect of Epo with regard to brain lesion area and ADC values. One possible mechanism of Epo for decreasing brain edema and cellular swelling could be a better clearance of water excess in brain tissue, a process possibly mediated by AQP4.

Uncertainties in pentose-phosphate pathway flux assessment underestimate its contribution to neuronal glucose consumption: relevance for neurodegeneration and aging

The pentose-phosphate pathway (PPP) promotes the oxidative decarboxylation of glucose-6-phosphate (G6P) in two consecutive steps, catalyzed by glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), yielding ribulose-5-phosphate (Ru5P) (Figure 1) (Wamelink et al., 2008). These steps constitute the so-called oxidative PPP branch, where the redox energy of G6P is conserved as NADPH(H +). Together with other NADPH(H +) regenerating systems, such as NADP-dependent isocitrate dehydrogenase and malic enzyme (ME), the oxidative PPP branch represents the most important source of reducing equivalents for (i) antioxidant enzymes, such as glutathione peroxidases and thioredoxin reductases, and (ii) fatty acid synthase (Dringen et al., 2007). Ru5P is isomerized into ribose-5-phosphate (R5P), which serves either as the precursor for nucleotide biosynthesis, or it continues metabolism through the non-oxidative PPP branch. In the latter, R5P epimerize into xylulose-5-phosphate (Xu5P), with which it transketolases producing sedoheptulose-7-phosphate (S7P) plus glyceraldehyde-3-phosphate (G3P). In turn, S7P and G3P transaldolase to form fructose-6-phosphate (F6P) and erythrose-4-phosphate (E4P). E4P is then transketolated with Xu5P forming F6P and G3P. Thus, through the PPP, three moles of G6P yield three CO 2 , two F6P, and one G3P. Since PPP-derived F6P and G3P are glycolytic intermediates too, they can follow conversion into pyruvate. Thus, glycolysis and PPP are two different pathways that share common pools of F6P and G3P intermediates. Accordingly, G6P converted into pyruvate through the PPP conserves both the redox and the energetic values of glucose, highlighting a yet unrecognized high impact of PPP flux activity in redox/energy conservation.

Effect of exogenous lactate on rat glioma metabolism

Glioma‐bearing rats were infused intravenously with a solution containing either [3‐13C]lactate or both glucose and [3‐13C]lactate for 20 min or 1 hr. Perchloric acid extracts of healthy and tumoral brain tissues were prepared and analyzed by 13C‐ and 1H‐observed 13C‐edited nuclear magnetic resonance (NMR) spectroscopy to determine 13C‐label incorporation into brain tissue and glioma metabolites. Moreover, 13C enrichments in blood lactate and glucose were determined from 1H‐NMR spectra. In the nontumoral tissue, 13C labeling of amino acids indicated that [3‐13C]lactate entered the brain and was metabolized. There was no labeling difference between the contralateral and the ipsilateral hemispheres. Lactate metabolism appeared more specifically neuronal, in agreement with our previous results obtained with normal rat brain (Bouzier et al. [ 2000 ] J. Neurochem. 75:480–486). In the glioma tissue, comparison of Ala C3, Glu C4, and Gln C4 labeling indicated that the contributions of blood glutamine and tricarboxylic acid (TCA) cycle to glutamate labeling were about 80% and 20%, respectively, after 1 hr of [3‐13C]lactate infusion. In contrast, these contributions were about 10% and 90%, respectively, when [1‐13C]glucose was infused in the absence of lactate. This indicated a major effect of the exogenous lactate on glioma metabolism, which may be due to the following process: The high blood lactate level might hinder the drain of glycolytic lactate produced inside the glioma and thus generate a change in redox potential such that the tumor cells are unable to restore it with oxidative phosphorylation. Thereafter, the high NADH level might inhibit glycolysis and the TCA cycle, and glutamine could become the major carbon source for glutamate labeling. J. Neurosci. Res. 65:543–548, 2001.