Cytosolic reducing power preserves glutamate in retina

Glutamate in neurons is an important excitatory neurotransmitter, but it also is a key metabolite. We investigated how glutamate in a neural tissue is protected from catabolism. Flux analysis using ¹³C-labeled fuels revealed that retinas use activities of the malate aspartate shuttle to protect >98% of their glutamate from oxidation in mitochondria. Isolation of glutamate from the oxidative pathway relies on cytosolic NADH/NAD⁺, which is influenced by extracellular glucose, lactate, and pyruvate.Glutamate is especially important as a metabolite because it is required for the synthesis of glutathione, other amino acids, and proteins. Glutamate also is a key intermediate in glutamine-dependent anaplerosis, now known to be a principal source of citric acid cycle intermediates in cancer cells (1).When it is released as a neurotransmitter at brain synapses, glutamate that escapes from the synapse is taken up by astrocytes. There it is converted to glutamine and is delivered back to neurons in a process called the “glutamate/glutamine cycle” (2). Uptake of glutamate and conversion to glutamine within astrocytes stimulates glycolysis and synthesis of lactate. Astrocytes export the lactate to neurons as fuel in a process called the “astrocyte neuron lactate shuttle” (ANLS) (3).Synaptic terminals of rod and cone photoreceptors have characteristics that appear incompatible with the ANLS. The photoreceptor terminal is enriched with transporters for reuptake of glutamate (4), and it encapsulates the synapse. It is unlikely that much glutamate can escape the synapse before being sequestered back into the photoreceptor. We initiated a study to evaluate the role of ANLS in retina. However, the unusual metabolic features of retina revealed a surprising feature of neuronal metabolism, that >98% of glutamate is protected from catabolism. We investigated this protection and show here that the protection is provided by activities associated with the metabolic pathway known as the “malate aspartate shuttle” (MAS) (shown schematically in Fig. 1).Open in a separate windowFig. 1.How the malate aspartate shuttle isolates glutamate. The glutamate/α-ketoglutarate cycle in retina isolates the carbon atoms of glutamate from the oxidative pathway inside mitochondria. In this report we demonstrate the influence that cytosolic reducing power and Aralar/AGC1 activity have on this pathway. ASP, aspartate; OAA, oxaloacetate; MAL, malate; GLU, glutamate; AKG, α-ketoglutarate; OGC, oxoglutarate carrier, IPM, interphotoreceptor matrix.MAS activity regenerates cytosolic NAD⁺ that is needed to support glycolysis. To do so, it uses two important transporters to trap the reducing power from cytosolic NADH and shuttle it into the mitochondrial matrix. One transporter is the neuronal aspartate/glutamate carrier (AGC1 or Aralar) (Fig. 1, orange circle); the other transporter is the oxoglutarate carrier (OGC) (Fig. 1, light blue circle). AGC1 transports glutamate from the cytoplasm into the mitochondrial matrix in exchange for aspartate from the matrix (Fig. 1). OGC transports α-ketoglutarate from the matrix into the cytoplasm in exchange for malate from the cytoplasm (Fig. 1) (5). An important consequence of MAS activity is that it diverts metabolic flux in mitochondria away from succinyl CoA, succinate, and fumarate (Fig. 1). Most importantly, glutamate that completes a MAS cycle functions as a catalyst for the importation of reducing power into the mitochondria. The carbon atoms of glutamate are isolated from the oxidative pathway in the mitochondrial matrix. To determine the extent of that isolation in a neuronal tissue, we used ¹³C-labeled fuels to identify metabolic networks in mouse retinas and quantify their metabolic flux.