NMR Spectroscopic Studies of <sup>13</sup>C Acetate and <sup>13</sup>C Glucose Metabolism in Neocortical Astrocytes: Evidence for Mitochondrial Heterogeneity

TLDR

Glucose labeling is expected to reveal pyruvate carboxylation in glutamine C‑2 and citrate C‑4 positions. The study aimed to identify metabolic pathways in neocortical astrocytes by incubating them with 13C‑labeled substrates. 13C NMR spectroscopy was employed to track incorporation of 13C into glutamine and citrate from [1‑13C]glucose or [2‑13C]acetate. The data showed extensive pyruvate‑carboxylation in glutamine but not citrate, lower acetate labeling of lactate, and suggested that glutamine and citrate arise from distinct astrocyte subtypes or mitochondrial TCA cycles with differential pyruvate carboxylase expression, indicating mitochondrial heterogeneity.

Abstract

Neocortical astrocytes were incubated with 13C-labeled substrates to determine metabolic pathways. 13C NMR spectroscopy was used to analyze 13C incorporation into glutamine and citrate from the different precursors – [1-13C]glucose or [2-13C]acetate. When glucose was the labeling substrate, incorporation due to pyruvate carboxylation should be observed in the C-2 position in glutamine and the C-4 position in citrate. A large incorporation due to pyruvate carboxylation was observed in glutamine in the C-2 and C-3 positions, but not in citrate. When acetate was the precursor, 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. Moreover, acetate labeled lactate in the C-2 position much less than did glucose. Altogether, these observations led to the conclusion that glutamine precursors and citrate are either produced in different types of astrocytes or in different tricarboxylic acid cycles, situated in functionally different mitochondria in the same cell, and that in all likelihood pyruvate carboxylase is expressed differently in these mitochondria.