Fig. 2: Brain-derived AKH is responsible for the ICWs in the fat body.

A Quantification of Ca²⁺ activities triggered by different synthesized fly neuropeptides (0.1 ug/mL) in cultured larval fat body expressing GCaMP5G (N = 3 fat bodies per group). B Ca²⁺ activities were monitored in the cultured fat body. AKH (0.1 ug/mL) was added to the dissected fat body, incubated for 15 min, and then washed away. Representative trace of Ca²⁺ dynamics was collected from a single fat cell. C Relationship between the period of Ca²⁺ oscillation and concentration of applied AKH. D, E Ca²⁺ activity triggered by brain-conditioned medium was blocked by the addition of either a control rabbit serum or rabbit serum with anti-AKH antibody (1:100 dilution). Representative Ca²⁺ activity from a selected single fat body cell were plotted in D. N = 3, 3, 3 fat bodies (E). F AKH was efficiently knocked-down in APCs by Akh>Akh-RNAi. The result was repeated in 3 independent experiments. G Conditional medium derived from brains with APCs silenced by Kir2.1 or with Akh knockdown showed significantly reduced ability to trigger Ca²⁺ activity in the fat body. N = 3, 3, 3 fat bodies. H, I Cultured fat body with AkhR mutant or Gαq knockdown failed to respond to AKH. The dynamic Ca²⁺ activities are highlighted in the lower panel by subtracting the constant background signal and showing the organ outline. N = 3, 3, 3 fat bodies (I). J, K Ca²⁺ waves in free-behaving larvae were significantly reduced in AkhR mutant larvae. N = 6, 6 independent biological replicates (K). Ca²⁺ intensity was expressed as arbitrary units (a.u.) (B, D). Ca²⁺ intensity was presented using a linear colour scale (minimum = 0, maximum = 255) (H, J). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test was used in (E, G, I), and unpaired two-tailed Student’s t-test was used in (K). Data were plotted as mean ± SD. Scale bars, 200 μm (F), 500 μm (H), 1000 μm (J). Source data are provided as a Source Data file.