Lactate Production In Astrocytes Research Articles

Octopamine metabolically reprograms astrocytes to confer neuroprotection against α-synuclein

Octopamine is a well-established invertebrate neurotransmitter involved in fight or flight responses. In mammals, its function was replaced by epinephrine. Nevertheless, it is present at trace amounts and can modulate the release of monoamine neurotransmitters by a yet unidentified mechanism. Here, through a multidisciplinary approach utilizing invitro andin vivomodels of α-synucleinopathy, we uncovered an unprecedented role for octopamine in driving the conversion from toxic to neuroprotective astrocytes in the cerebral cortex by fostering aerobic glycolysis. Physiological levels of neuron-derived octopamine act on astrocytes via a trace amine-associated receptor 1-Orai1-Ca2+-calcineurin-mediated signaling pathway to stimulate lactate secretion. Lactate uptake in neurons via the monocarboxylase transporter2-calcineurin-dependent pathway increases ATP and prevents neurodegeneration. Pathological increases of octopamine caused by α-synuclein halt lactate production in astrocytes and short-circuits the metabolic communication to neurons. Our work provides a unique function of octopamine as a modulator of astrocyte metabolism and subsequent neuroprotection with implications to α-synucleinopathies.

Apolipoprotein E ε4 modulates astrocyte neuronal support functions in the presence of amyloid-β.

Apolipoprotein E (APOE) is a lipid transporter produced predominantly by astrocytes in the brain. The ε4 variant of APOE (APOE4) is the strongest and most common genetic risk factor for Alzheimer's disease (AD). Although the molecular mechanisms of this increased risk are unclear, APOE4 is known to alter immune signaling and lipid and glucose metabolism. Astrocytes provide various forms of support to neurons, including regulating neuronal metabolism and immune responses through cytokine signaling. Changes in astrocyte function because of APOE4 may therefore decrease neuronal support, leaving neurons more vulnerable to stress and disease insults. To determine whether APOE4 alters astrocyte neuronal support functions, we measured glycolytic and oxidative metabolism of neurons treated with conditioned media from APOE4 or APOE3 (the common, risk-neutral variant) primary astrocyte cultures. We found that APOE4 neurons treated with conditioned media from resting APOE4 astrocytes had similar metabolism to APOE3 neurons treated with media from resting APOE3 astrocytes, but treatment with astrocytic conditioned media from astrocytes challenged with amyloid-β (Aβ), a key pathological protein in AD, caused APOE4 neurons to increase their basal mitochondrial and glycolytic metabolic rates more than APOE3 neurons. These changes were not because of differences in astrocytic lactate production or glucose utilization, but instead correlated with increased glycolytic ATP production and a lack of cytokine secretion in response to Aβ. Additionally, we identified that astrocytic cytokine signatures could predict basal metabolism of neurons treated with the astrocytic conditioned media. Together, these findings suggest that in the presence of Aβ, APOE4 astrocytes alter immune and metabolic functions that result in a compensatory increase in neuronal metabolic stress.

Potential use of lactate for the treatment of neonatal hypoxic-ischemic encephalopathy.

Potential use of lactate for the treatment of neonatal hypoxic-ischemic encephalopathy.

Juvenile social isolation leads to schizophrenia-like behaviors via excess lactate production by astrocytes

Repeated early environmental deprivation is regarded as a typical paradigm to mimic the behavioral abnormalities and brain dysfunction that occur in psychiatric disorders. Previously, we reported that social isolation could disrupt prepulse inhibition (PPI) in Sprague-Dawley (SD) rats, producing the typical characteristics of a schizophrenia animal model. Based on further analysis of previous proteomic and transcriptomic data, a disrupted balance of glucose metabolism was found in the prefrontal cortex (PFC) of isolated rats. Subsequently, in the first experiment of this study, we investigated the effects of juvenile social isolation (postnatal days (PND) 21–34) on PPI and lactate levels in PND56 rats. Compared with the social rearing group, rats in the isolated rearing group showed disrupted PPI and increased lactate levels in the PFC. In the second experiment, at PND55, the model rats were acutely injected with a glycogen phosphorylase inhibitor (4-dideoxy-1,4-imino-darabinitol, DAB) or control saline in the bilateral PFC. Our data showed that acute DAB administration (50 pmol, 0.5 μl) significantly improved the disrupted PPI and decreased the levels of oxidative phosphorylation (OXPHOS)-related mRNAs as well as lactate. In summary, our results suggested that excess astrocytic lactate production was involved in the impairment of auditory sensory gating of isolated rats, which may contribute to the metabolic pathogenesis of schizophrenia.

3-LB: Glutamate Metabolism Is Coupled to Lactate Production from Astrocytes under Low-Glucose Conditions

Recurrent hypoglycemia (RH) results in elevated lactate levels in the ventromedial hypothalamus that impair the counter-regulatory responses to hypoglycemia. While the mechanisms that enhance lactate production are not clear, our previous data suggests that astrocytic glycogen is the major source of lactate resulting from RH. A number of different inputs regulate glycogen turnover in astrocytes and the interaction between metabolic and neurotransmitter signals may alter glycogenolytic capacity and ultimately, lactate production. The current study investigates how the interaction between norepinephrine and glutamate influence lactate production from astrocytes in the setting of hypoglycemia. We cultured primary rat astrocytes in normal (2.5mM) and low (0.1mM) glucose medium in the presence and absence of norepinephrine and/or glutamate for 3 hours and quantified lactate in the culture media. We also examined the contribution of glutamate oxidation using the glutamate dehydrogenase inhibitor, hexachlorophene. Our data indicates that under low glucose conditions, glutamate alone dramatically enhances lactate production in astrocytes compared to controls (P<0.0001). Of note, the inhibition of glutamate oxidation also increased lactate secretion, but not to the same extent as glutamate alone (P<0.01). In contrast, the addition of norepinephrine and glutamate significantly reduced lactate secretion (P<0.05). Our data suggests that glutamate uptake during acute hypoglycemia likely plays a large role in driving astrocytic lactate production, a metabolic deficit that is offset by the oxidation of glutamate and by norepinephrine. In conclusion, our data suggests that under low glucose conditions, the metabolism of glutamate is tightly coupled to lactate production in astrocytes. Disclosure D. A. Appadurai: None. O. Chan: None. Funding National Institutes of Health (DK099315)

AMPK-Regulated Astrocytic Lactate Shuttle Plays a Non-Cell-Autonomous Role in Neuronal Survival

SUMMARYLactate is used as an energy source by producer cells or shuttled to neighboring cells and tissues. Both glucose and lactate fulfill the bioenergetic demand of neurons, the latter imported from astrocytes. The contribution of astrocytic lactate to neuronal bioenergetics and the mechanisms of astrocytic lactate production are incompletely understood. Through in vivo 1H magnetic resonance spectroscopy, 13C glucose mass spectroscopy, and electroencephalographic and molecular studies, here we show that the energy sensor AMP activated protein kinase (AMPK) regulates neuronal survival in a non-cell-autonomous manner. Ampk-null mice are deficient in brain lactate and are seizure prone. Ampk deletion in astroglia, but not neurons, causes neuronal loss in both mammalian and fly brains. Mechanistically, astrocytic AMPK phosphorylated and destabilized thioredoxin-interacting protein (TXNIP), enabling expression and surface translocation of the glucose transporter GLUT1, glucose uptake, and lactate production. Ampk loss in astrocytes causes TXNIP hyperstability, GLUT1 misregulation, inadequate glucose metabolism, and neuronal loss.

β1-adrenoceptor-stimulated lactate production in cultured astrocytes is predominantly glycogen-independent

Noradrenaline (NA) promotes breakdown of the glucose-polymer, glycogen, and hence enhances glycolytic production of lactate in astrocytes. Here, in cultured rat cerebrocortical astrocytes, we examined the contributions of different adrenoceptor subtypes to NA-modulated glucose metabolism, and the relationship of NA-induced glycogenolysis to lactate production. Stimulation of astrocytic glucose metabolism by NA was mediated predominantly via β1-adrenoceptors and cAMP. Constitutive β 1-adrenoceptor activity – in the absence of exogenous NA - contributed to the basal rate of glycogen turnover. Although mRNAs encoding both β 1- and β 2-adrenoceptors were detected in these astrocytes, β 2-adrenoceptors contributed little to NA-induced modulation of glucose metabolism. Activation of α2- and α 1-adrenoceptors in these cells decreased cAMP and increased cytosolic Ca2+, respectively, but did not modulate NA-induced glycogenolysis: α 2-adrenoceptors because glycogenolysis was induced maximally by NA concentrations that only began to inhibit cAMP production; and α 1-adrenoceptors possibly because of desensitisation and depletion of Ca2+ stores. Under basal conditions, astrocytes converted glucose to extracellular lactate in near stoichiometric manner. When glucose-starved astrocytes were given fresh glucose-containing medium, lactate accumulation displayed a brief lag period before attaining a steady-state rate. During this lag period NA, acting at β 1-adrenoceptors, increased the rate of lactate accumulation both in the absence and presence of an inhibitor of glycogen turnover. At the steady-state, the rate of glucose incorporation into accumulated glycogen was ~ 5% of that into lactate, but NA enhanced lactate output by 20–50%: this further indicates that NA, via β 1-adrenoceptors and cAMP, can enhance astrocytic lactate production independently of its effect on glycogen turnover.

Arousal-induced cortical activity triggers lactate release from astrocytes.

It has been suggested that, in states of arousal, release of noradrenaline and β-adrenergic signalling affect long-term memory formation by stimulating astrocytic lactate production from glycogen. However, the temporal relationship between cortical activity and cellular lactate fluctuations upon changes in arousal remains to be fully established. Also, the role of β-adrenergic signalling and brain glycogen metabolism on neural lactate dynamics in vivo is still unknown. Here, we show that an arousal-induced increase in cortical activity triggers lactate release into the extracellular space, and this correlates with a fast and prominent lactate dip in astrocytes. The immediate drop in astrocytic lactate concentration and the parallel increase in extracellular lactate levels underline an activity-dependent lactate release from astrocytes. Moreover, when β-adrenergic signalling is blocked or the brain is depleted of glycogen, the arousal-evoked cellular lactate surges are significantly reduced. We provide in vivo evidence that cortical activation upon arousal triggers lactate release from astrocytes, a rise in intracellular lactate levels mediated by β-adrenergic signalling and the mobilization of lactate from glycogen stores.

The Response to Stimulation in Neurons and Astrocytes.

The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this high oxidative index, contrasts with the metabolic traits of the major cell types in the brain studied in culture, neurons and astrocytes, including the selective use of the malate-aspartate shuttle (MAS) in neurons and the glycerol-phosphate shuttle in astrocytes. Metabolic interactions among these cell types may partly explain the high oxidative index of the brain. In vivo, neuronal activation results in a decrease in the oxygen glucose index, which has been attributed to a stimulation of glycolysis and lactate production in astrocytes in response to glutamate uptake (astrocyte-neuron lactate shuttle, ANLS). Recent findings indicate that this is accompanied with a stimulation of pyruvate formation and astrocyte respiration, indicating that lactate formation is not the only astrocytic response to neuronal activation. ANLS proposes that neurons utilize lactate produced by neighboring astrocytes. Indeed, neurons can use lactate to support an increase in respiration with different workloads, and this depends on the Ca2+ activation of MAS. However, whether this activation operates in the brain, particularly at high stimulation conditions, remains to be established.

FRET-based imaging of intracellular ATP in organotypic brain slices.

Active neurons require a substantial amount of adenosine triphosphate (ATP) to re-establish ion gradients degraded by ion flux across their plasma membranes. Despite this fact, neurons, in contrast to astrocytes, do not contain any significant stores of energy substrates. Recent work has provided evidence for a neuro-metabolic coupling between both cell types, in which increased glycolysis and lactate production in astrocytes support neuronal metabolism. Here, we established the cell type-specific expression of the Förster resonance energy transfer (FRET) based nanosensor ATeam1.03YEMK ("Ateam") for dynamic measurement of changes in intracellular ATP levels in organotypic brain tissue slices. To this end, adeno-associated viral vectors coding for Ateam, driven by either the synapsin- or glial fibrillary acidic protein (GFAP) promoter were employed for specific transduction of neurons or astrocytes, respectively. Chemical ischemia, induced by perfusion of tissue slices with metabolic inhibitors of cellular glycolysis and mitochondrial respiration, resulted in a rapid decrease in the cellular Ateam signal to a new, low level, indicating nominal depletion of intracellular ATP. Increasing the extracellular potassium concentration to 8mM, thereby mimicking the release of potassium from active neurons, did not alter ATP levels in neurons. It, however, caused in an increase in ATP levels in astrocytes, a result which was confirmed in acutely isolated tissue slices. In summary, our results demonstrate that organotypic cultured slices are a reliable tool for FRET-based dynamic imaging of ATP in neurons and astrocytes. They moreover provide evidence for an increased ATP synthesis in astrocytes, but not neurons, during periods of elevated extracellular potassium concentrations.

DISC1 regulates lactate metabolism in astrocytes: implications for psychiatric disorders

Our knowledge of how genetic risk variants contribute to psychiatric disease is mainly limited to neurons. However, the mechanisms whereby the same genetic risk factors could affect the physiology of glial cells remain poorly understood. We studied the role of a psychiatric genetic risk factor, Disrupted-In-Schizophrenia-1 (DISC1), in metabolic functions of astrocytes. We evaluated the effects of knockdown of mouse endogenous DISC1 (DISC1-KD) and expression of a dominant-negative, C-terminus truncated human DISC1 (DN-DISC1) on the markers of energy metabolism, including glucose uptake and lactate production, in primary astrocytes and in mice with selective expression of DN-DISC1 in astrocytes. We also assessed the effects of lactate treatment on altered affective behaviors and impaired spatial memory in DN-DISC1 mice. Both DISC1-KD and DN-DISC1 comparably decreased mRNA and protein levels of glucose transporter 4 and glucose uptake by primary astrocytes. Decreased glucose uptake was associated with reduced oxidative phosphorylation and glycolysis as well as diminished lactate production in vitro and in vivo. No significant effects of DISC1 manipulations in astrocytes were observed on expression of the subunits of the electron transport chain complexes or mitofilin, a neuronal DISC1 partner. Lactate treatment rescued the abnormal behaviors in DN-DISC1 male and female mice. Our results suggest that DISC1 may be involved in the regulation of lactate production in astrocytes to support neuronal activity and associated behaviors. Abnormal expression of DISC1 in astrocytes and resulting abnormalities in energy supply may be responsible for aspects of mood and cognitive disorders observed in patients with major psychiatric illnesses.

Lanthanum chloride reduces lactate production in primary culture rat cortical astrocytes and suppresses primary co-culture rat cortical astrocyte-neuron lactate transport.

Lanthanum (La) can impair learning memory and induce behavioral abnormalities in animals. However, the mechanism underlying these adverse effects of La is still elusive. It has been demonstrated that lactate derived from astrocytes is the major energy source for neurons during long-term memory (LTM) formation and the deficiency of lactate supply can result in LTM damage. However, little work has been done with respect to the impact of La on the lactate production in astrocytes and astrocyte-neuron lactate transport (ANLT). Herein, experiments were undertaken to explore if there was such an adverse effect of La. Primary culture rat cortical astrocytes and primary co-culture rat cortical astrocyte-neuron were treated with (0.125, 0.25 and 0.5mM) lanthanum chloride (LaCl3) for 24h. The results showed that LaCl3 treatment significantly downregulated the mRNA and protein expression of glucose transporter 1 (GLUT1), glycogen synthase (GS), glycogen phosphorylase (GP), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1, 2 and 4 (MCT 1 2 and 4); upregulated the mRNA and protein expression of lactate dehydrogenase B (LDHB); and decreased the glycogen level, total LDH and GP activity, GS/p-GS ratio and lactate contents. Moreover, rolipram (20, 40μM) or forskolin (20, 40μM) could increase the lactate content by upregulating GP expression and the GS/p-GS ratio, as well as antagonize the effects of La. These results suggested that La-induced learning-memory damage was probably related to its suppression of lactate production in astrocytes and ANLT. This study provides some novel clues for clarifying the mechanism underlying the neurotoxicity of La.

The Protein Tyrosine Kinase Inhibitor Tyrphostin 23 Strongly Accelerates Glycolytic Lactate Production in Cultured Primary Astrocytes.

Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases. To investigate potential acute effects of T23 on the viability and the glucose metabolism of brain cells, we exposed cultured primary rat astrocytes to T23 for up to 4h. While the viability and the morphology of the cultured astrocytes were not acutely affected by the presence of T23 in concentrations of up to 300µM, this compound caused a rapid, time- and concentration-dependent increase in glucose consumption and lactate release. Maximal effects on glycolytic flux were found for incubations with 100µM T23 for 2h which doubled both glucose consumption and lactate production. The stimulation of glycolytic flux by T23 was reversible, completely abolished upon removal of the compound and not found in presence of other known inhibitors of endocytosis. Structurally related compounds such as tyrphostin 25 and catechol or modulators of AMP kinase activity did neither affect the basal nor the T23-stimulated lactate production by astrocytes. In contrast, the presence of the phosphatase inhibitor vanadate completely abolished the stimulation by T23 of astrocytic lactate production in a concentration-dependent manner. These data suggest that T23-sensitive phosphorylation/dephosphorylation events are involved in the regulation of astrocytic glycolysis.

The Antidiabetic Drug Metformin Stimulates Glycolytic Lactate Production in Cultured Primary Rat Astrocytes.

Metformin is the most frequently used drug for the treatment of type 2 diabetes in humans. However, only little is known about effects of metformin on brain metabolism. To investigate potential metabolic consequences of an exposure of brain cells to metformin, we incubated rat astrocyte-rich primary cultures with this compound. Metformin in concentrations of up to 30mM did not acutely compromise the viability of astrocytes, but caused a time- and concentration-dependent increase in cellular glucose consumption and lactate production. For acute incubations in the hour range, the presence of 10mM metformin doubled the glycolytic flux, while already 1mM metformin doubled glycolytic flux during incubation for 24h. In addition to metformin, also other guanidino compounds increased astrocytic lactate production. After 4h of incubation, half-maximal stimulation of glycolysis was observed for metformin, guanidine and phenformin at concentrations of around 3mM, 3mM and 30µM, respectively. The acute stimulation of glycolytic lactate production by metformin was persistent after removal of extracellular metformin and was also observed, if glucose was absent from the incubation medium or replaced by other hexoses. The metformin-induced stimulation of glycolytic flux was not prevented by compound C, an inhibitor of AMP-dependent protein kinase, nor was it additive to the stimulation of glycolytic flux caused by respiratory chain inhibitors. These data demonstrate that the antidiabetic drug metformin has the potential to strongly activate glycolytic lactate production in brain astrocytes.

Nitric oxide reduces astrocytic lactate production and induces neuronal vulnerability in stroke‐prone spontaneously hypertensive rats

Nitric oxide (NO) leads to neuronal death in ischemia/reperfusion (I/R), including stroke. Here, we examined the NO-induced vulnerability of neurons and lactate production by astrocytes in stroke-prone spontaneously hypertensive rats (SHRSP) in vitro. Neuronal cell death induced by the NO donor sodium nitroprusside (SNP) was significantly increased in SHRSP compared with Wistar kyoto rats (WKY). Furthermore, levels of lactate production by astrocytes were significantly reduced in SHRSP compared with WKY. At the same time, expressions of the lactate dehydrogenase (LDH) and monocarboxylate transporter 1 (MCT1) genes were significantly decreased by SNP in SHRSP compared with WKY. Moreover, in astrocytes isolated from SHRSP, the gene expression of isoforms of 6-phosphofracto-2-kinase (PFK2), a master regulator of glycolysis, namely PFK2.1, PFK2.2, PFK2.3, and PFK2.4, had deteriorated significantly. Notably, the SNP-evoked gene expression of PFK2.4 was lower in astrocytes of SHRSP than those of WKY. These results indicated that the neurons and astrocytes of SHRSP differed in responsiveness to SNP from those of WKY. This difference might explain the deficiency of energy and vulnerability to SNP of the neurons of SHRSP.

Evidence for time-dependent glutamate-mediated glycolysis in head-injured patients: a microdialysis study.

In the brain, lactate is not only a marker of anaerobic glycolysis due to hypoxia/ischemia, but also a neuronal energy source which is provided by glutamate-induced astrocytic glycolysis. In the present study we wanted to investigate the relationship between glutamate release and lactate production during the entire time-course and during three time periods of microdialytic monitoring in 54 severely head injured patients. Within-subject Spearman rank correlations were calculated in each period for glutamate and lactate, for each patient and the mean of all correlation coefficients were analyzed for difference from zero by a one-sample t-test. The results show a strong overall positive relationship between glutamate and lactate. However, during the first 12 hours after injury, there was no significant correlation. Thereafter, good correlation was seen. The splitting of patients into groups with good (Glasgow Outcome Scale; GOS 0-2) and poor outcome (GOS 3-4) showed a similar strong correlation for patients with good outcome, but this was lost for patients with poor outcome. The results clearly indicate that glutamate "drives" astrocytic lactate production in head-injured patients. The contribution of glutamate to overall lactate release is thus time-dependent. During the first 12 hours after injury, factors such as hypoxia, ischemia or edema overshadowed glutamate-induced glycolysis in astrocytes. In addition, the effect of glutamate is more pronounced in patients with good outcome.

Astrocytic mitochondrial respiratory chain damage: effect on cellular ATP levels.

72 h The primary function of the mitochondrial respiratory chain is to generate energy in the form of ATP via the four complexes of the electron transport chain and the ATP synthetase. Damage to one or more of the respiratory chain complexes may therefore result in depletion of cellular energy reserves and cell death. There is increasing evidence that defects in mitochondrial energy metabolism may underlie the pathology of some neurodegenerative diseases [ I ] . Furthermore the mitochondrial respiratory chain is susceptible to oxidative damage, and nitric oxide (NO) has been implicated in the development of mitochondrial dysfunction [ I ] . Exposure of astrocytes, a major source of NO within the brain, to interferon (1FN)-y k lipopolysaccharide (LPS) results in induction of nitric oxide synthase (NOS) activity and a corresponding increase in NO release. We have previously shown that induction of NOS in cultured astrocytes by IFN-y k LPS is accompanied by damage to the mitochondrial respiratory chain [2, 31. However this damage occurs in the absence of cell death. We hypothesised that this was due to an up-regulation of glycolysis resulting in maintenance of astrocytic energy homeostasis. To test this hypothesis, astrocytes were exposed to IFN-y+ LPS for up to 72 h. The activities of the mitochondria1 complexes NADH-CoQ, (complex I), succinate-cytochrome c reductase (complex IVIII) and cytochrome c oxidase (complex IV) and citrate synthase (a mitochondrial marker) were determined in cell homogenates; cell death was assessed by lactate dehydrogenase (LDH) release; and ATP was extracted from astrocytes and measured using the luciferin-luciferase method. The exposure of astrocytes to IFN-y + LPS produced a significant time-dependent loss of both complex IMII and complex IV, which was prevented by the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) (Table 1). Both complex I and citrate synthase were unaffected. There was no significant increase in LDH release, indicating that IFN-y + LPS treatment did not cause cell death. In addition, astrocytic ATP levels were also unaffected by IFN-y + LPS treatment (Table 2). We have previously demonstrated that exposure to IFh-y + LPS causes a significant increase in both astrocytic lactate production and glucose consumption, which is prevented by a NOS inhibitor [2]. These findings indicate that although IFN-y + LPS-mediated NOS induction in astrocytes causes marked inhibition of mitochondrial respiratory chain complexes, these events do not affect astrocyte survival due to the ability of these cells to switch to glycolysis and consequently maintain their ATP levels and energy homeostasis. However it has previously been demonstrated that complex activity can be decreased by approximately 70% before major changes in mitochondrial respiration and ATP synthesis occurs [4]. Therefore it is possible that despite the mitochondrial respiratory chain damage observed here, the threshold of decreased complex activity was not reached. y+LPS 10.4+3.2* 0.43M. 19*

13C NMR study of IGF-I- and insulin-effects on mitochondrial function in cultured brain cells.

Development of differential cell culture systems has facilitated the study of cellular and physiological actions of growth factors. Here we report the novel effects of insulin and insulin-like growth factor (IGF)-I on energy metabolism in cerebral cortical neurones, astrocytes and co-cultures thereof. Incorporation of label from [1-13C]glucose into lactate has been shown to involve both glycolysis and the tricarboxylic acid cycle in astrocytes. Thus 13C nuclear magnetic resonance spectroscopy was used to monitor mitochondrial function by measuring 13C labelling of the C-2 position of lactate. It could be demonstrated that insulin and IGF-I have similar effects on mitochondria but individual effects on cytosolic glucose metabolism. Evidence is presented that IGF-I and insulin stimulate neurones to release factors affecting astrocytic lactate production. Furthermore, lactate released from neuronal-astrocytic co-cultures was mostly of astrocytic origin, implicating the importance of lactate for neuronal metabolism.

NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: Compartmentation of astrocyte metabolism

Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases). The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy. Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation. After 3 h of hypoxia full recovery was possible during reoxygenation. Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia. Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia. Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes. In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia. Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing [2- 13C]acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors. The media were subsequently analyzed by 13C NMR spectroscopy. After shorter periods of hypoxia (3 h) full recovery was possible. Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia. 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged. These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia. Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios. During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.

Keats and Anaesthetic Cyanosis

<h3>Abstract</h3> Apolipoprotein E (APOE) is a lipid transporter produced predominantly by astrocytes in the brain. The ε4 variant of APOE (APOE4) is the strongest and most common genetic risk factor for Alzheimer’s disease (AD). Although the molecular mechanisms of this increased risk are unclear, APOE4 is known to alter immune signaling and lipid and glucose metabolism. Astrocytes provide various forms of support to neurons, including regulating neuron metabolism and immune responses through cytokine signaling. Changes in astrocyte function due to APOE4 may therefore decrease neuronal support, leaving neurons more vulnerable to stress and disease insults. To determine whether APOE4 alters astrocyte neuronal support functions, we measured glycolytic and oxidative metabolism of neurons treated with conditioned media from APOE4 or APOE3 (the common, risk-neutral variant) primary astrocyte cultures. We found that APOE4 neurons treated with conditioned media from resting APOE4 astrocytes had similar metabolism to APOE3 astrocytes, but treatment with ACM from astrocytes challenged with amyloid-β (Aβ), a key pathological protein in AD, caused APOE4 neurons to increase their basal mitochondrial and glycolytic metabolic rates more than APOE3 neurons. These changes were not due to differences in astrocytic lactate production or glucose utilization, but instead correlated with increased glycolytic ATP production and a lack of cytokine secretion response to Aβ. Together, these findings suggest that in the presence of Aβ, APOE4 astrocytes alter immune and metabolic functions that result in a compensatory increase in neuronal metabolic stress.