Characterization of glucose-related metabolic pathways in differentiated rat oligodendrocyte lineage cells.

Although oligodendrocytes constitute a significant proportion of cells in the central nervous system (CNS), little is known about their intermediary metabolism. We have, therefore, characterized metabolic functions of primary oligodendrocyte precursor cell cultures at late stages of differentiation using isotope-labelled metabolites. We report that differentiated oligodendrocyte lineage cells avidly metabolize glucose in the cytosol and pyruvate derived from glucose in the mitochondria. The labelling patterns of metabolites obtained after incubation with [1,2-(13)C]glucose demonstrated that the pentose phosphate pathway (PPP) is highly active in oligodendrocytes (approximately 10% of glucose is metabolized via the PPP as indicated by labelling patterns in phosphoenolpyruvate). Mass spectrometry and magnetic resonance spectroscopy analyses of metabolites after incubation of cells with [1-(13)C]lactate or [1,2-(13)C]glucose, respectively, demonstrated that anaplerotic pyruvate carboxylation, which was thought to be exclusive to astrocytes, is also active in oligodendrocytes. Using [1,2-(13)C]acetate, we show that oligodendrocytes convert acetate into acetyl CoA which is metabolized in the tricarboxylic acid cycle. Analysis of labelling patterns of alanine after incubation of cells with [1,2-(13)C]acetate and [1,2-(13)C]glucose showed catabolic oxidation of malate or oxaloacetate. In conclusion, we report that oligodendrocyte lineage cells at late differentiation stages are metabolically highly active cells that are likely to contribute considerably to the metabolic activity of the CNS.

Effects of hypoglycaemia on neuronal metabolism in the adult brain: role of alternative substrates to glucose.

Hypoglycaemia is characterized by decreased blood glucose levels and is associated with different pathologies (e.g. diabetes, inborn errors of metabolism). Depending on its severity, it might affect cognitive functions, including impaired judgment and decreased memory capacity, which have been linked to alterations of brain energy metabolism. Glucose is the major cerebral energy substrate in the adult brain and supports the complex metabolic interactions between neurons and astrocytes, which are essential for synaptic activity. Therefore, hypoglycaemia disturbs cerebral metabolism and, consequently, neuronal function. Despite the high vulnerability of neurons to hypoglycaemia, important neurochemical changes enabling these cells to prolong their resistance to hypoglycaemia have been described. This review aims at providing an overview over the main metabolic effects of hypoglycaemia on neurons, covering in vitro and in vivo findings. Recent studies provided evidence that non-glucose substrates including pyruvate, glycogen, ketone bodies, glutamate, glutamine, and aspartate, are metabolized by neurons in the absence of glucose and contribute to prolong neuronal function and delay ATP depletion during hypoglycaemia. One of the pathways likely implicated in the process is the pyruvate recycling pathway, which allows for the full oxidation of glutamate and glutamine. The operation of this pathway in neurons, particularly after hypoglycaemia, has been re-confirmed recently using metabolic modelling tools (i.e. Metabolic Flux Analysis), which allow for a detailed investigation of cellular metabolism in cultured cells. Overall, the knowledge summarized herein might be used for the development of potential therapies targeting neuronal protection in patients vulnerable to hypoglycaemic episodes.

Estimation of intracellular fluxes in cerebellar neurons after hypoglycemia: importance of the pyruvate recycling pathway and glutamine oxidation.

Although glucose is the primary cerebral fuel, the brain is able to metabolize other substrates in hypoglycemia. Nevertheless, the metabolic consequences of this pathology at the cellular level remain largely unknown. Taking advantage of the metabolic flux analysis (MFA) methodology, this work was aimed at investigating and quantifying the effects of hypoglycemia on cerebellar neurons. After 12 hr without glucose, primary cultures were incubated with medium containing [1,6-(13)C]glucose and unlabeled glutamine, and metabolism was monitored for 30 hr. Metabolic rates of glucose, lactate, and amino acids were determined based on cell supernatant analysis and used to estimate metabolic fluxes with MFA. Percent (13)C enrichment time profiles of different keto and amino acids were measured by mass spectrometry in cell extracts and compared with the MFA results. Hypoglycemia decreased the glucose uptake rate and glycolytic metabolism by 35% whereas glutamine uptake was increased fourfold. Flux estimations fit well with data from (13)C labeling dynamics, indicating a significant activation of the pyruvate recycling (PR) pathway, accounting for 43% of the total pyruvate synthesized under control conditions and up to 71% after hypoglycemia. Increased PR appeared to be due mainly to increased glutamine oxidation given the higher label dilution observed in the hypoglycemia group. In summary, this work provides new evidence for PR as an important pathway for glutamine oxidation in cerebellar neurons, particularly after glucose deprivation.

Metabolic alterations induced by ischemia in primary cultures of astrocytes: merging 13C NMR spectroscopy and metabolic flux analysis.

Disruption of brain energy metabolism is the hallmark of cerebral ischemia, a major cause of death worldwide. Astrocytes play a key role in the regulation of brain metabolism and their vulnerability to ischemia has been described. Aiming to quantify the effects of an ischemic insult in astrocytic metabolism, primary cultures of astrocytes were subjected to 5 h of oxygen and glucose deprivation in a bioreactor. Flux distributions, before and after ischemia, were estimated by metabolic flux analysis using isotopic information and the consumption/secretion rates of relevant extracellular metabolites as constraints. During ischemia and early recovery, 30% of cell death was observed; several metabolic alterations were also identified reflecting a metabolic response by the surviving cells. In the early recovery ( approximately 10 h), astrocytes up-regulated glucose utilization by 30% and increased the pentose phosphate pathway and tricarboxylic acid cycle fluxes by three and twofold, respectively. Additionally, a two to fivefold enhancement in branched-chain amino acids catabolism suggested the importance of anaplerotic molecules to the fast recovery of the energetic state, which was corroborated by measured cellular ATP levels. Glycolytic metabolism was predominant in the late recovery. In summary, this work demonstrates that changes in fluxes of key metabolic pathways are implicated in the recovery from ischemia in astrocytes.

Improving retroviral vectors production: role of carbon sources in lipid biosynthesis.

This work investigates to which extent different carbon sources are metabolized and used for lipid biosynthesis in retrovirus producer cells, with the ultimate goal of understanding its importance regarding the stability/productivity of the vectors. For that purpose, isotopically labeled substrates (U-(13)C glucose, U-(13)C galactose, U-(13)C fructose, and U-(13)C glutamine) were used in combination with (13)C nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Our results show that glucose plays the major role in lipid biosynthesis, whereas glutamine, fructose and galactose are not significantly incorporated into lipids. Moreover, a correlation between medium osmolality (imposed by the presence of sorbitol) and virus stability and productivity was verified, apparently due to an enhancement in sugar metabolism. Since low stability and short half-life constitute the major bottleneck in process development for retrovirus and other enveloped viral vectors, this work presents useful knowledge for improved process robustness for these essential gene therapy vectors.

Succinate supplementation improves metabolic performance of mixed glial cell cultures with mitochondrial dysfunction.

Mitochondrial dysfunction, the inability to efficiently utilise metabolic fuels and oxygen, contributes to pathological changes following traumatic spinal cord or traumatic brain injury (TBI). In the present study, we tested the hypothesis that succinate supplementation can improve cellular energy state under metabolically stressed conditions in a robust, reductionist in vitro model of mitochondrial dysfunction in which primary mixed glial cultures (astrocytes, microglia and oligodendrocytes) were exposed to the mitochondrial complex I inhibitor rotenone. Cellular response was determined by measuring intracellular ATP, extracellular metabolites (glucose, lactate, pyruvate), and oxygen consumption rate (OCR). Rotenone produced no significant changes in glial ATP levels. However, it induced metabolic deficits as evidenced by lactate/pyruvate ratio (LPR) elevation (a clinically-established biomarker for poor outcome in TBI) and decrease in OCR. Succinate addition partially ameliorated these metabolic deficits. We conclude that succinate can improve glial oxidative metabolism, consistent our previous findings in TBI patients' brains. The mixed glial cellular model may be useful in developing therapeutic strategies for conditions involving mitochondrial dysfunction, such as TBI.

Oligodendrocytes: Development, Physiology and Glucose Metabolism.

The glutamate-glutamine cycle is an outstanding example of how essential neuronal-glial interactions are for brain function. For several decades, this and other metabolic cycles in the brain have only included neurons and astrocytes but not oligodendrocytes, the myelinating cells of the central nervous system (CNS). Recent data revealed that oligodendrocytes are highly metabolically active cells in the brain and, therefore, should not be ignored. Using 13C-labelled glucose in combination with nuclear magnetic resonance spectroscopy (MRS) and/or mass spectrometry (MS) it is possible to characterize metabolic functions in primary oligodendrocyte cultures. Mature rat oligodendrocytes avidly metabolize glucose in the cytosol and pyruvate derived from glucose in mitochondria. Moreover, they seem to have the ability of performing anaplerosis from pyruvate, which might enable them to synthesize metabolites de novo and transfer them to neighbouring cells. All these original findings highlight the importance of investigating oligodendrocyte metabolism separately from that of astrocytes and neurons to be able to discern the roles played by the individual partners. This is of particular importance in the white matter where the number of oligodendrocytes is considerable. The present book chapter provides some background on oligodendrocyte biology and physiology and summarizes the not very extensive information published on glucose metabolism in oligodendrocytes.

Tamoxifen accelerates the repair of demyelinated lesions in the central nervous system.

Enhancing central nervous system (CNS) myelin regeneration is recognized as an important strategy to ameliorate the devastating consequences of demyelinating diseases such as multiple sclerosis. Previous findings have indicated that myelin proteins, which accumulate following demyelination, inhibit remyelination by blocking the differentiation of rat oligodendrocyte progenitor cells (OPCs) via modulation of PKCα. We therefore screened drugs for their potential to overcome this differentiation block. From our screening, tamoxifen emerges as a potent inducer of OPC differentiation in vitro. We show that the effects of tamoxifen rely on modulation of the estrogen receptors ERα, ERß, and GPR30. Furthermore, we demonstrate that administration of tamoxifen to demyelinated rats in vivo accelerates remyelination. Tamoxifen is a well-established drug and is thus a promising candidate for a drug to regenerate myelin, as it will not require extensive safety testing. In addition, Tamoxifen plays an important role in biomedical research as an activator of inducible genetic models. Our results highlight the importance of appropriate controls when using such models.

Metabolic aspects of neuron-oligodendrocyte-astrocyte interactions.

Whereas astrocytes have been in the limelight of scientific interest in brain energy metabolism for a while, oligodendrocytes are still waiting for a place on the metabolic stage. We propose to term the interaction of oligodendrocytes with astrocytes and neurons: NOA (neuron-oligodendrocyte-astrocyte) interactions. One of the reasons to find out more about metabolic interactions between oligodendrocytes, neurons, and astrocytes is to establish markers of healthy oligodendrocyte metabolism that could be used for the diagnosis and assessment of white matter disease. The vesicular release of glutamate in the white matter has received considerable attention in the past. Oligodendrocyte lineage cells express glutamate receptors and glutamate toxicity has been implicated in diseases affecting oligodendrocytes such as hypoxic-ischaemic encephalopathy, inflammatory diseases and trauma. As oligodendrocyte precursor cells vividly react to injury it is also important to establish whether cells recruited into damaged areas are able to regenerate lost myelin sheaths or whether astrocytic scarring occurs. It is therefore important to consider metabolic aspects of astrocytes and oligodendrocytes separately. The present review summarizes the limited evidence available on metabolic cycles in oligodendrocytes and so hopes to stimulate further research interests in this important field.

Manufacturing of retroviruses.

Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of viral vectors developed for gene therapy. They have been extensively used in clinical trials, particularly in ex vivo transduction of hematopoietic stem cells. Although there is a vast experience acquired with retroviruses, their manufacturing is still a difficult task due to the low cell productivities and inherent instability of the infective virus. These viral vectors are most commonly produced using stable producer cell lines in adherent monolayer culture systems. In order to obtain high transduction efficiencies and low toxicity in clinical applications, the viral preparations should be purified, concentrated, and well characterized to attain stringent quality specifications. This chapter describes currently used protocols for manufacturing retroviruses.

A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose.

Metabolic models have been used to elucidate important aspects of brain metabolism in recent years. This work applies for the first time the concept of isotopic transient 13C metabolic flux analysis (MFA) to estimate intracellular fluxes in primary cultures of astrocytes. This methodology comprehensively explores the information provided by 13C labeling time-courses of intracellular metabolites after administration of a 13C-labeled substrate. Cells were incubated with medium containing [1-13C]glucose for 24 h and samples of cell supernatant and extracts collected at different time points were then analyzed by mass spectrometry and/or high performance liquid chromatography. Metabolic fluxes were estimated by fitting a carbon labeling network model to isotopomer profiles experimentally determined. Both the fast isotopic equilibrium of glycolytic metabolite pools and the slow labeling dynamics of TCA cycle intermediates are described well by the model. The large pools of glutamate and aspartate which are linked to the TCA cycle via reversible aminotransferase reactions are likely to be responsible for the observed delay in equilibration of TCA cycle intermediates. Furthermore, it was estimated that 11% of the glucose taken up by astrocytes was diverted to the pentose phosphate pathway. In addition, considerable fluxes through pyruvate carboxylase [PC; PC/pyruvate dehydrogenase (PDH) ratio = 0.5], malic enzyme (5% of the total pyruvate production), and catabolism of branched-chained amino acids (contributing with ∼40% to total acetyl-CoA produced) confirmed the significance of these pathways to astrocytic metabolism. Consistent with the need of maintaining cytosolic redox potential, the fluxes through the malate-aspartate shuttle and the PDH pathway were comparable. Finally, the estimated glutamate/α-ketoglutarate exchange rate (∼0.7 µmol mg prot-1 h-1) was similar to the TCA cycle flux. In conclusion, this work demonstrates the potential of isotopic transient MFA for a comprehensive analysis of energy metabolism.

Production of retroviral vectors: review.

Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, genetic diseases or neurological disorders. This review provides an overview on the evolution of retroviral vector design and production for gene therapy applications, including state of the art developments in flexible producer cells and safe vectors. In addition, production and purification processes will be addressed, with a particular focus on the improvements undertaken to increase vector productivity and to reduce the rapid loss of infectivity, which presently represent the main challenges in retroviral vectors production for gene therapy.