HSD causes insulin resistance in glia.

(A) Mean fold change in G6P expression in ND and HSD-fed flies. N = 3 technical replicates of cDNA collected from 100 fly heads/treatment. Student t test with Welch’s correction. (B) Mean fold change in Chico expression in ND and HSD-fed flies. N = 3 technical replicates of cDNA collected from 100 fly heads/treatment. Student t test with Welch’s correction. (C) Representative z-stack summation projections of the IPCs immunostained with anti-Dilp5 of ND and HSD-fed flies. (D) Mean fluorescent intensity of anti-Dilp5 in the IPCs of ND and HSD-fed flies, obtained from a Z-stack summation projection that spans the entire depth of the IPCs. Student t test with Welch’s correction. N = each circle represents an individual fly. (E) Mean fold change in Dilp 5 expression in ND and HSD-fed flies. N = 3 technical replicates of cDNA collected from 100 fly heads/treatment. Student t test with Welch’s correction. (F) Schematic of the insulin resistance model. Increased circulating insulin desensitizes the IRs leading to reduced Pi3k activity. (G) Schematic for experimental design and summarized results of (H and I). Flies fed an HSD exhibit reduced ensheathing glia Pi3k signaling despite increased Dilp release. (H) Representative confocal images of the antennal lobe region of flies expressing the Pi3k (tGPH) activity sensor that were fed either an ND or an HSD. (I) Mean fluorescent intensity of tGPH-GFP measured within a region of interest (white box) that coincides with the location of ensheathing glia in ND and HSD-fed flies. Measurements were obtained from a Z-stack summation projection that spans the entire depth of the antennal lobe. Student t test with Welch’s correction. N = each circle represents an individual fly. (J) Schematic for experimental design and summarized results of (K and L). Flies expressing TrpA1 specifically in their IPCs show reduced ensheathing glia Pi3k signaling similar to flies fed an HSD. (K) Confocal images of tGPH levels in the antennal lobe region of control flies and flies expressing TrpA1 in the IPCs that were fed an ND. TrpA1 expression force activates the IPCs to release Dilps. (L) Mean fluorescent intensity of GFP measured within a region of interest (white box) that coincides with the location of ensheathing glia in control and flies with Dilp2-driven TrpA1 expression. Measurements were obtained from a Z-stack summation projection that spans the entire depth of the antennal lobe. Student t test with Welch’s correction. N = each circle represents an individual fly. (M) Schematic for experimental design and summarized results of (N and O). HSD-fed flies expressing EKO specifically in their IPCs show increased ensheathing glia Pi3k signaling compared to control flies. (N) Confocal images of tGPH levels in the antennal lobe region of control flies and flies expressing EKO in the IPCs that were fed an HSD. EKO expression attenuates the release of Dilps from the IPCs. (O) Mean fluorescent intensity of GFP measured within a region of interest (white box) that coincides with the location of ensheathing glia in control and flies with Dilp2-driven EKO expression. Measurements were obtained from a Z-stack summation projection that spans the entire depth of the antennal lobe. Student’s t test with Welch’s correction. N = each circle represents an individual fly. The data underlying this figure can be found in the supporting information file S2 Data. Dilp5, Drosophila insulin-like peptide 5; G6P, glucose-6-phosphatase; HSD, high-sugar diet; IPC, insulin-producing cell; IR, insulin receptor; Pi3k, phosphoinositide 3-kinase.