The production of glycolytic end products, such as lactate, usually evokes a cellular shift from aerobic to anaerobic ATP generation and O₂ insufficiency. In the classical view, muscle lactate must be exported to the liver for clearance. However, lactate also forms under well-oxygenated conditions, and this has led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle¹ and the glycogen shunt^2,3 hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study reports the use of hyperpolarized [1-¹³C]lactate and [2-¹³C]pyruvate to measure the rapid pyruvate and lactate kinetics in rat skeletal muscle.Clinical GE 3T MR scanner and HyperSense DNP polarizer were used. Healthy male Sprague-Dawley rats (517–681g, N=12) were prepared for two studies. For the first study, metabolic kinetics was measured using the 40-mM hyperpolarized [1-¹³C]lactate before and 1hr after dichloroacetate (DCA), which stimulates pyruvate dehydrogenase (PDH) activity. To measure the spatial distribution of the metabolites in skeletal muscle, metabolite maps were acquired from one of the DCA-injected rats after an additional injection of hyperpolarized [1-¹³C]lactate. For the second study, metabolic kinetics was measured using 80-mM hyperpolarized [2-¹³C]pyruvate before and 1hr after DCA injection. A ¹³C surface coil (Ø=28mm) was placed on top of right rectus femoris of each animal. Dynamic free induction decay was measured for metabolic kinetics (10^o hard RF pulse, temporal resolution=3 s, spectral width/points=10 kHz/4096). A volumetric single time-point spiral CSI sequence (field of view=80×80×60mm³, matrix size=16×16×12, spectral bandwidth/points=972.7Hz/96, acquisition time=4s, 20^o hard pulse) with a circularly reduced k-space sampling scheme was used to acquire the CSI⁴. To obtain the maximum signal of ¹³C-labeled metabolic products, the CSI scan was started 25s after the start of the injection. Metabolites were quantified by integrating the respective peak in the absorption mode from time-averaged spectra (0–2min), followed by normalization to total carbon signal (tC). Temporal change of each metabolite signal was estimated from the peak integrals of the time-resolved spectra using two parameters⁵: τ and r. Time point (τ) where the half-maximum of each metabolite signal was achieved was compared to assess the metabolite production rate. The production rate of each metabolite was also estimated from the mean slope of the first four time points (r) following its appearance in the dynamic curve by linearly fitting the metabolite signal, which is then normalized to the initial mean slope of the injected substrate.Fig. 1 shows the time-averaged ¹³C spectra in rat leg muscle acquired after an injection of hyperpolarized [1-¹³C]lactate. In control muscle, the metabolic products such as pyruvate, alanine, and HCO₃⁻ were detected. DCA increased the HCO₃⁻/tC ratio ~11 times. The pyruvate/alanine ratio decreased by 18%, whereas the pyruvate/lactate ratio fell by 60%. The composite metabolism and exchange rates (r_x→y) relative to the time-dependent change of the lactate signal were also altered: r_Lac→HCO3- increased by a factor of ~10, r_Lac→Pyr decreased by a factor of 2 and r_Lac→Ala slightly decreased (Fig. 2). Fig. 3 shows the metabolic response to injected hyperpolarized [2-¹³C]pyruvate. In control muscle, only lactate and pyruvate had sufficient SNR to map the dynamic response confidently. With DCA, acetyl-carnitine, glutamate, and acetoacetate signals appeared prominently. The ¹³C label distribution of the metabolites and dynamic parameters are summarized in Fig. 4. 3D metabolite maps of lactate, alanine, pyruvate and HCO₃⁻ were reconstructed from the 2.5h post-DCA CSI data (Fig. 5). Normalized metabolite maps and a ratio map of products are shown in axial and coronal planes. Despite the large point spread function and partial volume effects, each metabolite map in both axial and coronal planes showed distinct distribution from the other metabolite maps. Both the intracellular lactate shuttle and glycogen shunt models require that muscle must mobilize and rapidly utilize lactate and can convert it quickly to acetyl CoA for oxidative metabolism in the mitochondria. Indeed, the present DNP studies have established that muscle can rapidly mobilize and use lactate. PDH poses no metabolic inertia and can compete readily with LDH to divert accumulated lactate to acetyl CoA. As a consequence, the results provide support for a critical underpinning of both the glycogen shunt model and the intracellular lactate shuttle hypothesis, and cautions against an overly simplistic view of glycolytic end products as merely hypoxia biomarkers. The findings have established a critical basis to conduct experiments underway that investigate muscle metabolism under pathophysiological conditions.