Fig 2: Neonatal induction of intestinal G6pc1 influences the development of AgRP and POMC neural projections. (A) Schematic representation of hypothalamic nuclei that were quantified (with their corresponding bregma). Representative confocal images and quantification of (B) AgRP-immunoreactive and (C) POMC-immunoreactive fibers in P20 I.G6pcoverexp−P1 (white squares) and control littermates (black squares) (mean ± SEM; n = 5–6). Scale bar = 100 μm. 3V: 3rd ventricle; PVHant: anterior part of the paraventricular nucleus; PVHpost: posterior part of the paraventricular nucleus; LH: lateral hypothalamic area; DMH: dorsomedial nucleus. Two-way ANOVA, followed by multiple comparisons and Sidak's post-hoc test were performed as statistical analyses. ∗p < 0.05; ∗∗p < 0.01.

Fig 3: Ablation of MC4R in the hypothalamus leads to obesity. (A) Immunoflurescent staining for POMC and MC4R of the hypothalamus of POMC-MC4Rflox/flox mice. Scale bar = 500 μm above the picture, Scale bar = 200 μm below the picture. (B) Body-weight in MC4R WT and KO mice fed a chow or HFD from 8 wk of age. (C) Food intake assessed during the first 13 wk of diet treatment. (D) Fat mass at 21 wk old. Data are presented as means ± SD. *P < 0.05, **P < 0.01, two-tailed Student’s t test was used for comparison for (A) MC4R-positive POMC cells; two-way ANOVA for (D) fat volume. Repeated measures ANOVA for multiple comparisons for (B) body weight and (C) food intake

Fig 4: Effect of p62 overexpression in the hypothalamus on the expression of POMC and AgRP and on leptin signaling. Eight-week-old male WT mice were fed an NCD or HFD for 12 weeks, followed by bilateral MBH injection of AAV9-p62/GFP, and were then maintained on an NCD or HFD for an additional 6 weeks. Brain sections were cut and collected as indicated in the Methods section. (A) IF staining for POMC (left) and RFU (right) in the ARC (n = 3 mice). (B) POMC mRNA expression in the hypothalamus (n = 6 mice). (C) IF staining for AgRP (left) and RFU (right) in the ARC (n = 3 mice). (D) IF staining for α-MSH (left) and RFU (right) in the PVN (n = 3 mice). (E) IF staining for STAT3 phosphorylation (left) and RFU (right) in the ARC (n = 3 mice). (F) IF staining for FOXO1 (left) and RFU (right) in the ARC (n = 3 mice). NCD, normal chow diet; HFD, high-fat diet; MBH, mediobasal hypothalamus; ARC, arcuate nucleus; PVN, paraventricular nucleus; 3V, third cerebral ventricle; RFU, relative fluorescent units. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01 vs. GFP/NCD group; ##p < 0.01 vs. GFP/HFD group.

Fig 5: Effects of p62 overexpression in POMC neurons on JAK2 and STAT3 phosphorylation. Eight-week-old male POMC-Cre mice received a bilateral ARC injection of AVV9-DIO-p62/GFP and were fed a NCD or HFD for 12 weeks. Two subgroups of mice were intraperitoneally injected with leptin/saline twice a day for 3 days, as indicated in the Methods section. (A) IF staining of POMC (left) and RFU (right) in the ARC (n = 3 mice). (B) POMC mRNA expression in the hypothalamus (n = 6 mice). (C) IF staining for α-MSH (left) and RFU (right) in the PVN (n = 3 mice). (D) IF staining for c-Fos (left) and RFU (right) in the ARC (n = 3 mice). (E) IF staining of phosphorylated JAK2 (left) and RFU (right) in the ARC (n = 3 mice). (F) IF staining for phosphorylated STAT3 (left) and RFU (right) in the ARC (n = 3 mice). NCD, normal chow diet; HFD, high-fat diet; 3V, third cerebral ventricle; ARC, arcuate nucleus; PVN, paraventricular nucleus; RFU, relative fluorescent units. Data are expressed as the mean ± SD. **p < 0.01 vs. POMC-GFP/NCD or POMC-GFP/saline; ##p < 0.01 vs. POMC-GFP/HFD or POMC-GFP/leptin.