Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons

Abstract

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca2+ during activation cause influx of Ca2+ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate-aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca2+ (i.e. independent of synaptic activity) on neuronal energy metabolism employing 13C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca2+ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca2+ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca2+-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca2+ signalling.

Figure 1. The CiMASH mechanism in glutamatergic neurons as originally described in Bak et al. (2009)

(A) Schematic depiction of a neuron showing possible mechanisms involved in regulation of glucose (glc) and lactate (lac) metabolism during neuronal depolarization and (B) the dynamics of glucose and lactate metabolism during re-polarization and at resting membrane potential. (C) A schematic depiction of cytosolic Ca²⁺ dynamics during neuronal spiking; arrows indicate when the CiMASH mechanism is operating. During neuronal depolarization (A), [Ca²⁺]_i is increased via flux of Ca²⁺ through NMDA receptors and voltage-gated Ca²⁺ channels (VGCC) subsequently inducing release from endoplasmic reticulum (ER, 1). This triggers accumulation of Ca²⁺ in the mitochondrial matrix (2) and activation of α-ketoglutarate dehydrogenase (α-KGDH, 3), which competes with the malate/α-ketoglutarate carrier (MKC) for substrate thus limiting efflux of α-ketoglutarate (α-KG, 4). This leads to less α-ketoglutarate being available for the cytosolic aspartate aminotransferase (AATc) reaction (5) in turn limiting activity of cytosolic malate dehydrogenase (MDHc) and thus re-oxidation of cytosolic NADH (6). The increased cytosolic [NADH] together with decreased [ATP] will activate anaerobic glycolysis leading to lactate synthesis via the LDH reaction and re-oxidation of NADH (7). During this period, oxidation of lactate will be limited because of the increased [NADH] (8, 9). (B) During neuronal re-polarization and in the period between depolarizations where [Ca²⁺]_i is low and MASH activity is restored, cytosolic [NAD⁺] will increase again. In conjunction with the increased [lactate] formed by anaerobic glycolysis, this will limit formation of lactate via the LDH reaction (10) whereas the opposite reaction is now favoured (11). The net effect at this point is formation of pyruvate (pyr) from glucose-derived lactate as well as extracellular lactate. Thus, extracellular lactate is only consumed during rest whereas glucose fuels the energy needed during neuronal depolarization. This latter part is now revised in the present paper (see Figure 8). c, cytosolic; m, mitochondrial; AGC, aspartate/glutamate carrier; Asp, aspartate; Glu, glutamate; GLUT, glucose transporter; Mal, malate; MCT, monocarboxylate transporter; OAA, oxaloacetate; TCA, tricarboxylic acid. Reproduced with permission from: LK Bak, AB Walls, A Schousboe, A Ring, U Sonnewald and HS Waagepetersen, Neuronal glucose but not lactate utilization is positively correlated with NMDA-induced neurotransmission and fluctuations in cytosolic Ca²⁺ levels, J Neurochem, 2009 Wiley-Blackwell. copyright 2009 The Authors Journal Compilation copyright 2009 International Society for Neurochemistry.

Figure 2. Effect of ionomycin on the intracellular Ca²⁺ concentration

Cultures of cerebellar neurons were exposed to increasing concentrations of ionomycin for 30 min and the resulting effect on the intracellular Ca²⁺ concentration was measured employing the ratiometric Ca²⁺ sensitive dye fura-2. The results are displayed as the ratio of emitted light at 550 nm after λ_ex at 340 and 380 nm and each bar represents the means±S.E.M. Data were obtained from two individual batches of cerebellar neurons with 14–15 repetitions in total for each condition. a, b, c, d and e denotes differences from 0, 0.2, 0.4, 0.6 and 0.8 μM ionomycin respectively (one-way ANOVA followed by Tukey–Kramer post hoc test). Ionomycin dose-dependently increases the intracellular Ca²⁺ concentration.

Figure 3. Effect of ionomycin on ¹³C-labelling from [U-¹³C]glucose and [U-¹³C]lactate into intracellular glutamate

Cultures of cerebellar neurons were incubated for 1 h with either 2.5 mM [U-¹³C]glucose in the presence or absence of 1 mM unlabelled lactate (A and C respectively) or 1 mM [U-¹³C]lactate in the presence or absence of 2.5 mM unlabelled glucose (B and D respectively) as substrates. In order to titrate the intracellular Ca²⁺ level, increasing concentrations of ionomycin (0, 0.25, 0.50 and 0.75 μM) were present during the last 30 min of incubation in combination with 10 μM MK-801 and 30 μM verapamil. The resulting ¹³C-enrichment in intracellular glutamate was determined by GC-MS. The results are displayed as percentage MCL, which is a measure of the average ¹³C-labelling of a given metabolite. The data were obtained from 2 to 4 individual batches of cerebellar neurons with 5–14 repetitions in total for each condition and the bars represent means±S.E.M. Significant differences (P<0.05, one-way ANOVA followed by Bonferroni's post hoc test) from 0 and 0.25 μM ionomycin are indicated by a and b, respectively. ¹³C-Labelling from glucose into glutamate is enhanced by ionomycin-induced increased intracellular Ca²⁺ concentration both in the presence and absence of lactate (A and C respectively). This signifies increased glycolysis and tricarboxylic acid cycle activity since glutamate is in rapid equilibrium with the tricarboxylic acid cycle intermediate α-ketoglutarate. In contrast, ¹³C-labelling from lactate is decreased by ionomycin in the presence but not in the absence of glucose (B and D respectively). Collectively these results indicate that extracellular lactate is metabolized independently of Ca²⁺-induced effects on glycolysis and tricarboxylic acid cycle activity, the latter causing dilution of ¹³C-labelling from increased metabolism of unlabelled glucose when present.

Figure 4. Effect of ionomycin on ¹³C-labelling from [U-¹³C]glucose and [U-¹³C]lactate into intracellular alanine

Cultures of cerebellar neurons were incubated for 1 h with either 2.5 mM [U-¹³C]glucose in the presence or absence of 1 mM unlabelled lactate (A and C respectively) or 1 mM [U-¹³C]lactate in the presence or absence of 2.5 mM unlabelled glucose (B and D respectively) as substrates. In order to titrate the intracellular Ca²⁺ level, increasing concentrations of ionomycin (0, 0.25, 0.50 and 0.75 μM) were present during the last 30 min of incubation in combination with 10 μM MK-801 and 30 μM verapamil. The resulting ¹³C-enrichment in intracellular alanine was determined by GC-MS. The results are displayed as the ¹³C-enrichment of the M+3 isotopomer, i.e. alanine labelled in all three carbon atoms that originate directly from [U-¹³C]glucose- or [U-¹³C]lactate-derived pyruvate via transamination. The data were obtained from 3 to 4 individual batches of cerebellar neurons with 5–14 repetitions in total for each condition and the bars represent means±S.E.M. Significant differences (P<0.05, one-way ANOVA followed by Bonferroni's post hoc test) from 0 and 0.25 μM ionomycin are indicated by a and b, respectively. ¹³C-labelling from glucose into alanine is enhanced by ionomycin-induced increased intracellular Ca²⁺ concentration in the presence but not the absence of lactate (A and C respectively). Moreover, the ¹³C-labelling from glucose is substantially higher in the absence than in the presence of unlabelled lactate. Since ¹³C-labelling in alanine reflects that of pyruvate, these findings indicate not only Ca²⁺-induced up-regulation of glycolytic activity but also that lactate is extensively metabolized even in unstimulated neurons. When lactate is the labelled substrate, ionomycin brings about a decrease in ¹³C-labelling in alanine in the presence of glucose (B). This might be explained by dilution of labelling due to metabolism of unlabelled glucose, although the corresponding decrease observed in the absence of glucose (D) argues against this.

Figure 5. Effect of ionomycin on ¹³C-labelling from [U-¹³C]glucose and [U-¹³C]lactate into intracellular lactate

Cultures of cerebellar neurons were incubated for 1 h with either 2.5 mM of [U-¹³C]glucose in the presence or absence of 1 mM unlabelled lactate (A and C respectively) or 1 mM [U-¹³C]lactate in the presence or absence of 2.5 mM unlabelled glucose (B and D respectively) as substrates. In order to titrate the intracellular Ca²⁺ level, increasing concentrations of ionomycin (0, 0.25, 0.50 and 0.75 μM) were present during the last 30 min of incubation in combination with 10 μM MK-801 and 30 μM verapamil. The resulting ¹³C-enrichment in intracellular lactate was determined by GC-MS. The results are displayed as the ¹³C-enrichment of the M+3 isotopomer, i.e. lactate labelled in all three carbon atoms that originate directly from [U-¹³C]glucose- or [U-¹³C]lactate-derived pyruvate via the action of LDH. In addition, the data are expressed as percentage of control within each batch of neurons. The data were obtained from 2 individual batches of cerebellar neurons with 4–8 repetitions in total for each condition and the bars represent means±S.E.M. Significant differences (P<0.05, one-way ANOVA followed by Bonferroni's post hoc test) from 0 and 0.25 μM ionomycin are indicated by a and b, respectively. ¹³C-Labelling from glucose into lactate is enhanced by ionomycin-induced increased intracellular Ca²⁺ concentration both in the presence and absence of extracellular lactate (A and C respectively). This signifies increased glycolytic activity since labelling in lactate reflects that of pyruvate. In contrast, ionomycin has no effect on ¹³C-labelling of intracellular lactate from the extracellular pool of the same metabolite (B and D).

Figure 6. Effect of ionomycin on tricarboxylic acid cycle activity

Cultures of cerebellar neurons were incubated for 1 h with either 2.5 mM [U-¹³C]glucose in the presence or absence of 1 mM unlabelled lactate (A and C respectively) or 1 mM [U-¹³C]lactate in the presence or absence of 2.5 mM unlabelled glucose (B and D respectively) as substrates. In order to titrate the intracellular Ca²⁺ level, increasing concentrations of ionomycin (0, 0.25, 0.50 and 0.75 μM) were present during the last 30 min of incubation in combination with 10 μM MK-801 and 30 μM verapamil. The resulting ¹³C-enrichment in intracellular glutamate was determined by GC-MS. Based on the isotopomeric ¹³C-labelling of glutamate, the CR, which is a measure of the activity of the tricarboxylic acid cycle, was calculated as described in the Materials and Methods section. The data were obtained from 2 to 4 individual batches of cerebellar neurons with 5–14 repetitions in total for each condition and the bars represent means±S.E.M. Significant differences (P<0.05, one-way ANOVA followed by Bonferroni's post hoc test) from 0 to 0.25 μM ionomycin are indicated by a and b, respectively. Irrespective of the presence of extracellular lactate, the rate of [U-¹³C]glucose metabolism in the tricarboxylic acid cycle increases with increasing ionomycin-induced intracellular Ca²⁺ levels (A and C). However, with the exception of 0.25 μM ionomycin in the presence of glucose, the metabolism of lactate through the tricarboxylic acid cycle is unaffected by ionomycin (B and D).

Figure 7. NMDA-induced release of lactate from CCNs during superfusion in the presence of 2.5 mM glucose

CCNs were superfused (2 ml/min) in a Hepes-buffered medium containing glucose (2.5 mM) as substrate and subjected to 30-s pulses consisting of NMDA (100 μM) and glycine (10 μM). The rate of lactate release was quantified as described in the Materials and methods section and expressed as nmol of lactate released per min per mg of protein. a; significantly different from the control (i.e. no NMDA pulses) as determined employing Student's t test (P<0.05).

Figure 8. A compartmentalized CiMASH model for glutamatergic neurons

In the compartmentalized model, the CiMASH mechanism is working in the postsynaptic compartment in which NMDA-receptor-mediated Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum (ER) directly signals to a subset of postsynaptic mitochondria (type B) that increase their tricarboxylic acid cycle activity driven by breakdown of glucose-derived pyruvate (pyr). Notice that glucose-derived pyruvate is only in partial equilibrium with lactate (Lac)-generated pyruvate. When the CiMASH mechanism is activated, glucose-derived lactate is produced and released to the extracellular space for oxidation at a later time point. Pyruvate derived from extracellular lactate is metabolized in a mitochondrial compartment that is not affected by postsynaptic Ca²⁺ signalling (type A). At the presynaptic compartment, the voltage-gated Ca²⁺ channel (VGCC)-activated ER-to-mitochondria Ca²⁺-signalling will affect mitochondrial tricarboxylic acid cycle metabolism (type C) of glucose-derived pyruvate and activate the CiMASH mechanism to some extent; the lactate generated in this compartment is not able to leave the cell due to lack of transporters. It should be noted that substrate-level phosphorylation in the glycolytic pathway probably plays a functionally important role at the presynaptic terminal (not shown here).