Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons

Abstract

The brain is highly susceptible to oxidative injury, and the pentose phosphate pathway (PPP) has been shown to be affected by pathological conditions, such as Alzheimer's disease and traumatic brain injury. While this pathway has been investigated in the intact brain and in astrocytes, little is known about the PPP in neurons. The activity of the PPP was quantified in cultured cerebral cortical and cerebellar neurons after incubation in the presence of [2-(13)C]glucose or [3-(13)C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism of (13)C-labeled glucose via the PPP does not appear to contribute to the production of releasable lactate, it contributes to labeling of tricarboxylic acid (TCA) cycle intermediates and related amino acids. Based on glutamate isotopomers, it was calculated that PPP activity accounts for ~6% of glucose metabolism in cortical neurons and ~4% in cerebellar neurons. This is the first demonstration that pyruvate generated from glucose via the PPP contributes to the synthesis of acetyl CoA for oxidation in the TCA cycle. Moreover, the fact that (13)C labeling from glucose is incorporated into glutamate proves that both the oxidative and the nonoxidative stages of the PPP are active in neurons.

Figure 1

Simplified presentation of ¹³C-labeling patterns of metabolites from incubation of cells with [2-¹³C]glucose representing the pentose phosphate pathway (PPP), glycolysis, and tricarboxylic acid (TCA) cycle activity. The circles symbolize the carbon backbone of the molecules. Dark grey circles mark the position of the label resulting from the PPP. Filled circles indicate that all the molecules are labeled in the position indicated. Half-filled circles indicate that 50% of the molecules formed are labeled in that position. Grey crosses mark the position of the label resulting from glycolysis. Complete crosses indicate that all the molecules are labeled in the position indicated. Demi-crosses indicate that half of the molecules are labeled in that position. Panel A displays glycolysis (right) and the PPP (left). Panel B displays labeling from acetyl CoA derived from either PPP or glycolysis in TCA cycle metabolites and glutamate and γ-amino butyric acid (GABA). Small circles indicate the molecules that are formed from the second turn of the TCA cycle. For simplicity, glyco-phosphates are not included, and only condensation of oxaloacetate with unlabeled acetyl CoA is shown for the second turn. α-KG, α-ketoglutarate; G-3-P, glyceraldehyde-3-phosphate.

Figure 2

Labeling patterns from [3-¹³C]glucose resulting from the pentose phosphate pathway (PPP), glycolysis, and tricarboxylic acid (TCA) cycle activity. The circles symbolize the carbon backbone of the molecules. Dark grey circles mark the position of the label resulting from the PPP. Filled circles indicate that all the molecules are labeled in the position indicated. Half-filled circles indicate that 50% of the molecules formed are labeled in that position. Grey crosses mark the position of the label resulting from glycolysis. Panel A displays glycolysis (right) and the PPP (left). Panel B displays labeling from acetyl CoA derived from either PPP or glycolysis in TCA cycle metabolites and glutamate and γ-amino butyric acid (GABA). Small circles indicate the molecules that are formed from the second turn of the TCA cycle. For simplicity, glyco-phosphates are not included, and only condensation of oxaloacetate with unlabeled acetyl CoA is shown for the second turn. α-KG, α-ketoglutarate; G-3-P, glyceraldehydes-3-phosphate.

Figure 3

Cortical (open bars) and cerebellar (filled bars) neurons were incubated for 4 hours. Cell extracts were analyzed by high-performance liquid chromatography (HPLC). For more details, see Materials and methods. Results are presented as mean±95% confidence interval (CI) (n=6). Unpaired Student's t-test was used to compare cerebellar and cortical neurons. Asterisks indicate statistical difference from the same metabolite in cortex. P values: ^*=<0.05, ^**=<0.001. GABA, γ-amino butyric acid.

Figure 4

¹³C Magnetic resonance (MR) spectra of the cell culture extracts from incubation with [2-¹³C]glucose. (A) Neocortical neurons and (B) cerebellar neurons. The horizontal axis has been truncated. The detectable peaks of glutamate and γ-amino butyric acid (GABA) are marked. GLU, glutamate; p.p.m., parts per million.