Astrocytes expressing PyronicSF or Pyronic in mitochondria or cytosol were first incubated in a buffer containing physiological concentrations of glucose (2 mM), lactate (2 mM) and pyruvate (0.2 mM), followed by removal of pyruvate by accelerated-exchange with 10 mM lactate and exposure to 10 mM pyruvate. (A) Representative trace from a single astrocyte expressing mito-PyronicSF. Data are shown as percentage of the maximum change (left) and pyruvate concentration (right), with reference to the response of the sensor obtained in vitro (Figure 1C). (B) Representative trace from a single astrocyte expressing mito-Pyronic. Data are shown as percentage of the maximum change (left) and pyruvate concentration (right), with reference to the response of the sensor obtained in vitro (San Martín et al., 2014a). (C) Steady-state mitochondrial and cytosolic pyruvate concentrations measured with PyronicSF or Pyronic at physiological concentrations of glucose, lactate and pyruvate, as illustrated in panels A and B. Data are from 131 cells in ten experiments (mito-PyronicSF), 59 cells in four experiments (cytosolic PyronicSF), 20 cells in seven experiments (mito-Pyronic), and 50 cells in five experiments (cytosolic Pyronic). (D) Mitochondrial pyruvate dynamics. Pyruvate enters mitochondria (blue compartment) and is metabolized by PDH and the tricarboxylic acid cycle (TCA), or is carboxylated by PC. (E) Simulation of pyruvate dynamics in response to PDH and MPC modulation. The effects of activating PDH and MPC by 100% on mitochondrial pyruvate concentration (top panel) and on the fluxes of PDH and PC (bottom panel) are shown. Steady-state cytosolic and mitochondrial pyruvate were 33 μM and 21 μM. Cytosolic and mitochondrial pH were 7.2 and 7.8. Steady-state PDH and PC fluxes were 0.91 and 0.29 μM/s. (F) Ultrasensitive modulation of PC flux by the MPC. The curves show the degree of flux increase at PC and PDH relative to the degree of MPC activation. The shaded area under the PC curve indicates the range of pyruvate concentrations at which PC flux increases more than 1% when MPC is activated by 1%. MPC dosage was normalized at 3.24 μM. (G) Synergic effect of cytosolic pyruvate and MPC activity on PC flux. The effects of increasing cytosolic pyruvate and MPC activity by 100% on mitochondrial pyruvate concentration (top panel) and on the fluxes of PDH and PC (bottom panel) are shown. The sums of the independent effects are indicated by interrupted lines.