Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using C NMR spectroscopy on brain extracts following intravenous [1,2-C]-glucose (to probe glia and neuron metabolism), [2-C]-acetate (probing astrocyte-specific metabolites), or [3-C]-lactate. An increase in [4,5-C]-glutamine and [2,3-C]-lactate coupled with a decrease in [4,5-C]-glutamate was observed in the [1,2-C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-C]-lactate and decrease in [4,5-C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.