Extended Data Figure 2. Characterization of feeding behaviors in STZ-treated diabetic mice; Additional analyses of ectopic activation of AgRP neurons and its pathologic contributions to STZ-induced hyperphagia and hyperglycemia.

a, Quantitative PCR results showing that Agrp and Npy mRNA levels are significantly upregulated in the mediobasal hypothalamus of STZ-treated animals, which is consistent with increased AgRP neuronal activity following STZ-injection (n=5 mice per group). b–f, Food intake during light cycle (7 a.m. to 7 p.m.) (b) and dark cycle (7 p.m. to 7 a.m.) (c), representative 1-hour heat map (10 to 11 a.m.) showing percent occupancy time in food zone (upper right corner) and nesting zone (lower right corner) (d), feeding duration (e), and 1-hour food intake (f) in saline- or STZ-treated C57BL/6 mice (n=8 mice per group). g, h, Representative sections and quantification of hrGFP immunostaining in the ARC of saline- or STZ-treated Npy-hrGFP transgenic mice, suggesting that STZ-treatment does not induce obvious cell loss of AgRP neurons (n=3 mice per group). i, Representative sections and quantification of hrGFP::pS6 co-immunostaining in the ARC of saline- or STZ-treated Npy-hrGFP transgenic mice (n=4 mice per group). j, Schematic diagram of chemogenetic inhibition of AgRP neurons in virus-transduced Agrp-IRES-Cre mice. k, 4-hour food intake measurement during dark cycle (8 p.m.–12 a.m.) following the administration of saline or CNO (n=6 mice per group). l, m, 4-hour food intake assay (10 a.m.–2 p.m.) (l) and blood glucose measurement (m, without food in the cage) in female Agrp-IRES-Cre littermates after AAV pSyn-FLEX-hM4D_i-mCherry virus injection into the ARC and upon saline/CNO treatment (n=8 mice per group). n–q, Schematic diagram of experiments to assess CNO’s effects with Cre-dependent AAV-FLEX-mCherry virus injected to into the ARC of Agrp-IRES-Cre mice (n). Representative brain sections (o) and quantification (p, n=3 mice per group) of mCherry::Fos co-immunostaining, and food intake assay (q, 10 a.m. to 2 p.m., n=8 mice per group) in STZ-treated mice following the i.p. injection of saline/CNO, demonstrating that CNO administration without the expression of hM₄Di in AgRP neurons induces null effects and the changes observed in Figure 1g–i are caused by the chemogenetic inhibition of AgRP neurons. Data are mean ± s.e.m. and representative of three independent experiments; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Student’s two-tailed, unpaired t-test (a–c, e, f, h, p), paired t-test (m) or two-way ANOVA analysis (l, q) with Šidák post hoc test (k).