Fig. 8: AKH secretion from APCs is regulated by amino acids.

A Brain-conditioned HL6 media with each amino acid (5 mM) were generated and transferred to the dissected 3rd instar larval fat body. B Representative Ca²⁺ images of fat bodies before and after the addition of brain-conditioned HL6 media with indicated amino acids. Ca²⁺ intensity (a.u.) was presented using a linear colour scale (minimum = 0, maximum = 255). The result was repeated in 3 independent experiments. C Ca²⁺ activities in the bodies treated by brain-conditioned HL6 medium with indicated amino acids. Percentage of fat body cells with positive Ca²⁺ signal was indicated: “++” means more than 10%, “+” means 10–5%, “-” means less than 5%. D AKH-producing CC cells are located at the “foot” region of the ring gland associated with the larval brain. Ca²⁺ activity in the CC cells was monitored using Akh-Gal4 > GCaMP5G. E–F Dissected larval brains together with ring glands were incubated in a HL6 medium with amino acids. Representative Ca²⁺ activities in APCs after the addition of the indicated amino acids (5 mM) are shown. A representative trace of Ca²⁺ dynamics from a single APC cluster was plotted in (F). Ca²⁺ intensity was plotted in arbitrary units (a.u.) G Quantification of Ca²⁺ activities in the APCs 15 mins after the addition of the indicated amino acids. N = 3, 3 brains. H 3rd instar larvae fed on indicated food for 36 h and then dissected. AKH levels in the APCs were stained with anti-AKH antibody. I–J Representative staining of AKH and quantifications of the fluorescent intensity in APCs of fly larvae fed on 2% sucrose, 2% sucrose + 40 mM Met, or 2% sucrose + 40 mM Leu. N = 14, 11, 15 brains (J). K TAG contents of adult flies fed on indicated foods for 48 h were measured. Diets containing 2% sucrose or 2% sucrose + 40 mM Met were used. N = 4, 4, 4, 4 independent biological replicates. L Sugar and amino acid sensing properties of AKH as well as its downstream Ca²⁺ waves are functionally conserved compared with mammalian glucagon. For (G, J, K), each dot represents an independent biological replicate. Unpaired two-tailed Student’s t-test was used in (G), ordinary one-way ANOVA with Dunnett’s multiple comparisons was used in (J), and ordinary two-way ANOVA with uncorrected Fisher’s LSD was used in (K). Data were plotted as mean ± SD. Scale bars, 50 μm (B), 25 μm (E, I). Source data are provided as a Source Data file.