Magnetic resonance spectroscopy (MRS) of hyperpolarized 13C-labeled substrates provides unique noninvasive measurements of critical dynamic metabolic processes, with hyperpolarized [1-13C]pyruvate (Pyr) being the first (and currently only) 13C-compound to reach the clinic. Occupying a key nodal point in energy metabolism, among the fates of Pyr are reduction to lactate (Lac) as the end product of glycolysis (GLY), amidation to produce alanine (Ala), or conversion in mitochondria to form acetyl CoA and CO2 (measured with 13C-MRS as a 13C-bicarbonate [Bic] peak) as the first step towards oxidative phosphorylation (OXPHOS) via the tricarboxylic acid (TCA) cycle. To date, the most prominent applications of hyperpolarized 13C-Pyr metabolic imaging are studies of cancer and heart disease. With a growing interest into cancer metabolic reprogramming fueling new studies into Warburg's 1965 observation that less energy-efficient aerobic GLY is favored in neoplasms over more efficient OXPHOS, MRS of hyperpolarized 13C-Pyr and the subsequent labeling of 13C-Lac provides an unprecedented noninvasive measurement of GLY. While 13C-Lac changes have now been well documented in multiple brain tumors and other neoplasms, these cancer studies have been largely silent on other downstream Pyr metabolic products, which can provide important information beyond glycolytic capacity. A more complete understanding of cancer metabolism requires measurements reflecting the complex interplay between both GLY and OXPHOS, and our preliminary findings with a glioblastoma multiforme model demonstrate that imaging Pyr products beyond Lac can provide this information. Energy regulation and mitochondrial function are also critically important for understanding brain function, and current data suggest a complex relationship involving the exchange of metabolic intermediates between glia and neurons. Conventional 13C MRS is well established as the primary noninvasive tool to study neuroenergetics and linked neurotransmitter cycling, but hyperpolarized 13C-MRS, with its high spatial and temporal resolution, has the potential to provide new insights. Although hyperpolarized [1-13C]Pyr provides little information on the TCA cycle, in vivo measurements downstream metabolic products of [2-13C]Pyr, for which the 13C-label is retained with the conversion of Pyr to acetyl-CoA, potentially add significant insights ino both OXPHOS and mitochondrial function. However, outside of the heart, few results concerning [2-13C]Pyr products beyond Lac (and Ala) have been reported. We believe the dearth of brain research on downstream Pyr products beyond Lac is due to the low signal- to-noise ratio (SNR) and other imaging difficulties associated with these resonances, rather than a lack of biochemical significance. The overall goal of this 4-year technical development grant is to develop optimized methods for imaging brain energy metabolism using hyperpolarized 13C-pyruvate in order to assess GLY, OXPHOS, and mitochondrial function. Many of to-be-developed methods will also be applicable to other organs and diseases. The Specific Aims are to develop improved acquisition and quantification methods for use with [1-13C]Pyr (Aim 1) and [2-13C]Pyr (Aim 2), assess Pyr metabolism and anti-VEGF treatment response in a rat C6-glioma model (Aim 3), and optimize sequences and hardware for future clinical hyperpolarized 13C- Pyr studies (Aim 4).