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. 2023 Feb 15;133(4):e156722. doi: 10.1172/JCI156722

Figure 7. Slug directly suppresses LepRb expression and induces leptin resistance.

Figure 7

(A) Male overnight-fasted plasma leptin levels (8 weeks old, chow diet, n = 6 mice per group). (B) Male mice (7 weeks old, chow diet) were treated with leptin for 3 days. Body weight changes were measured (n = 5 mice per group). (C) AAV-hSyn-DIO-Slug (n = 8) or AAV-hSyn-DIO-mCherry (n = 7) vectors were bilaterally microinjected into the MBH of SlugΔLepRb male mice (Slugfl/fl; LepRb-Cre+/+) on a chow diet. Twelve weeks later, mice were treated with leptin and body weight changes were measured. (D and E) Slugfl/fl and SlugΔLepRb males (8 weeks old, chow diet) were fasted overnight and treated with leptin (i.p., 1 mg/kg, 45 minutes). Hypothalamic extracts were immunoblotted with anti–phospho-Stat3 (pTyr705) and anti-Stat3 antibodies. Stat3 phosphorylation was normalized to Stat3 levels (n = 3 mice per group). a.u., arbitrary units. (F) AAV-CAG-DIO-Slug or AAV-CAG-DIO-mCherry vectors were bilaterally microinjected into the MBH of SlugΔLepRb (Slugfl/fl;LepRb-Cre+/–) males (9 weeks old, on chow diet). Two weeks later, the mice were treated with leptin (i.p., 1.2 mg/kg, 45 minutes). Hypothalamic extracts were immunoblotted with anti–phospho-Stat3 and anti-Stat3 antibodies. Stat3 phosphorylation was normalized to Stat3 levels (E, n = 3 mice per group). (G) Male hypothalamic LepR mRNA levels (normalized to 36B4 levels, 8 weeks old, chow diet, n = 8 mice per group). (H) Hypothalamic LepR mRNA levels 2 weeks after AAV transduction (normalized to 36B4 levels, n = 6 mice per group). AAV-CAG-DIO-Slug or AAV-CAG-DIO-mCherry vectors were bilaterally microinjected into the MBH of SlugΔLepRb (Slugfl/fl;LepRb-Cre+/–) male mice (9 weeks old, chow diet). (I) LepR promoter luciferase reporter activity (n = 3 per group). (J and K) Hypothalamic Slug occupancy on the LepR promoter (J) and hypothalamic LepR promoter H3K27me2, H3K3me3, and H3K27ac levels (K). Slug+/+ (n = 14) and Slug–/– (n = 14) males were fed a HFD for 14–16 weeks. Data are presented as mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test (A, G, H, J, and K), 1-way (I) and 2-way ANOVA (B, C, and E).