Extended Data Fig. 2: Control experiments for KB uptake by MB neurons.

(a) MCT switches to the other side of the membrane at a higher rate when a substrate is bound (MCT – L; k2) than when no substrate is bound (MCT; k1), with k1<k2. Trans-acceleration occurs because the conformational switch of MCT across the cell membrane is facilitated when an adequate substrate such as KB is bound. Lactate efflux can be monitored indirectly by an intracellular Laconic FRET sensor that is sensitive to lactate concentration. When KB is applied, the high concentration of KB in the extracellular media increases the rate of lactate transport in the opposite direction, corresponding here to lactate efflux. (b) In fed flies expressing the intracellular Laconic FRET sensor in MB neurons, application of 10 mM of L-lactate on the brain induced a strong increase in the Laconic FRET ratio showing that the intracellular lactate concentration subsequently increased in response to lactate uptake by MB neurons (t₅ = 2.627, P = 0.047). Thus, as previously shown by several other research groups, the Laconic FRET sensor can be used to monitor the intracellular lactate level. (c) To assess that Sln downregulation in MB neurons do not affect the lactate intracellular concentration to a level below the Laconic FRET sensor sensitivity threshold, thus preventing detection of any Laconic FRET ratio change in Fig. 2d, we applied a saturation treatment to reach a maximal plateau of Laconic FRET ratio (that is saturation of the sensor) that was used to normalize the different genotypic conditions. In fed flies expressing the Laconic FRET sensor in MB neurons, application of 5 mM NaAz, a strong inhibitor of mitochondrial respiration, induced a strong increase in the Laconic FRET ratio, reaching the saturation level of the sensor (mean trace ± s.e.m.). The last 100 s of recording (red bar), when the Laconic FRET sensor had reached saturation, were used to normalize; the initial Laconic ratio was measured during the 120-s baseline recording before NaAz application. In fed flies, the lactate basal concentration in MB neurons of flies expressing Sln RNAi was similar to the genotypic control (t₁₄ = 0.2091, p = 0.837). n represents the response of a single recorded fly. Data are expressed as mean ± s.e.m. with dots as individual values, and analyzed by one-sample two-sided t-test with theoretical mean = 0 (b) or unpaired two sided two-sample t-test (c). Asterisks refer to the P-value of the t-test comparison. *P<0.05, ns: not significant.