Fig 2: Deletion of p62 in ObRb-expressing neurons results in a decrease in energy expenditure and a deterioration of metabolic disorders in both lean and obese mice. The p62flox/flox mice were crossed with ObRb-Cre:tdTomato mice to generate ObRb-p62 KO mice with tdTomato. Eight-week-old male ObRb-p62 KO and ObRb-Cre:tdTomato mice were fed a NCD or HFD for 12 weeks, as indicated in the Methods section. (A) IF staining of ObRb neurons with tdTomato (red) and p62 (green) in the ARC and VMH. (B) Average energy intake (n = 7-8 mice). (C) Body weight (n = 7-9 mice). (D) Body composition (n = 7-9 mice). (E) Rectal temperature (n = 7-9 mice). (F) 24-h oxygen consumption (Vo2) (n = 7-9 mice). (G) Respiratory exchange ratio (RER: VCO2/VO2) (n = 7-9mice). (H) ANCOVA of the total energy expenditure versus body weight (n = 6-8 mice). (I) Blood glucose and AUC during the GTT (n = 7-9 mice). (J) Blood glucose and AUC during the ITT (n = 7-9 mice). (K) H&E staining of WAT (n = 3 mice). (L) UCP1 protein expression in WAT (n = 3 mice). (M) H&E staining of BAT (n = 3 mice). (N) UCP1 immunostaining in BAT (n = 3 mice). (O) IF staining for POMC expression (left) and RFU (right) in the ARC (n = 3 mice). (P) POMC mRNA expression in the hypothalamus (n = 6 mice). (Q) IF staining for phosphorylated STAT3 (left) and RFU (right) in ObRb neurons (n = 3mice). 3V, third cerebral ventricle; ARC, arcuate nucleus. VMH, ventromedial hypothalamus; NCD, normal chow diet; HFD, high-fat diet; GTT, glucose tolerance test; ITT, insulin tolerance test; AUC, the area under the curve; WAT, white adipose tissue; BAT, brown adipose tissue; RFU, relative fluorescent units. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01 vs. ObRb-Cre/NCD; ## p < 0.01 vs. ObRb-Cre/HFD.

Fig 3: Overexpression of p62 in the hypothalamus increases energy expenditure and ameliorates systemic metabolism. (A) UMAP plot showing the clustering of the different cells in the hypothalamus of mice based on snRNA-seq data (n = 3 mice). (B) UMAP plot showing the clustering of the different neurons in the hypothalamus of mice based on snRNA-seq data (n = 3 mice). (C) Violin plot of p62 expression in hypothalamic neurons based on snRNA-seq data (n= 3 mice). (D) Expression of p62 in POMC neurons of db/m and db/db mice (n = 3 mice). (E) IF staining of POMC and p62 in the ARC of db/db and db/m mice (n = 3 mice). (F) Schematic representation of the experimental procedure. Eight-week-old male WT mice were fed an NCD or HFD for 12 weeks, followed by bilateral MBH injection of AAV9-p62/GFP as indicated in the Methods section, and were then maintained on an NCD or HFD for an additional 6 weeks. (G) Daily energy intake (n = 6-8 mice). (H) Body weight (n = 6-8 mice). (I) Body composition (% B.W) (n = 6-8 mice). (J) Rectal temperature (n = 6-8 mice). (K) 24-h oxygen consumption (VO2) (n = 6-8 mice). (L) Respiratory exchange ratio (RER: VCO2/VO2) (n = 6-8 mice). (M) ANCOVA of the total energy expenditure versus body weight (n = 6 mice). (N) Blood glucose and AUC during the GTT (n = 6-8 mice). (O) Blood glucose and AUC during the ITT (n = 6-8 mice). (P) H&E staining of WAT (n = 3 mice). (Q) UCP1 protein expression in WAT (n = 3 mice). (R) H&E staining of BAT (n = 3 mice). (S) UCP1 immunostaining in BAT (n = 3 mice). NCD, normal chow diet; HFD, high-fat diet; MBH, mediobasal hypothalamus; ARC, arcuate nucleus; VMH, ventromedial hypothalamus; 3V, third cerebral ventricle; GTT, glucose tolerance test; ITT, insulin tolerance test; AUC, the area under the curve; WAT, white adipose tissue; BAT, brown adipose tissue. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01 vs. GFP/NCD or db/m; ## p < 0.01 vs. GFP/HFD.

Fig 4: MC4R negatively regulates the expression of Kir2.1. (A) Transcriptome analysis found that the expression of potassium inwardly-rectifying channel subfamily J member 2, KCNJ2, or Kir2.1 was down-regulated. (B) Immunofluorescence for Kir2.1 (green) and merge. Scale bar = 20 μm. (C) Immunofluorescence for Kir2.1 (green), MC4R (purple) and POMC (red) in HFD mice. Scale bar = 200 μm at the top, Scale bar = 100 μm at the bottom. (D-F) Primary hypothalamic neurons were transfected with MC4R shRNA plasmid, and the transfection efficiency and the protein expression of Kir2.1 were analyzed by western blot. (G) Co-IP assay detected the interaction of MC4R and Kir2.1. *P < 0.05, **P < 0.01. One-way ANOVA with Dunnett's post hoc test for (D) relative MC4R protein expression and (E) relative protein expression of Kir2.1. Two-way ANOVA for (B) % Kir2.1 positive of ARC.

Fig 5: Selective overexpression of p62 in POMC neurons increases energy expenditure. (A) Schematic representation of the experimental procedure. 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. (B) IF staining for the distribution of AVV9-DIO-GFP/p62 in the ARC (upper) and p62 expression (lower) in POMC neurons. (C) Energy intake (n = 7-8 mice). (D) Body weight (n = 7-8 mice). (E) Body composition (n = 7-8 mice). (F) Rectal temperature (n = 7-8 mice). (G) 24-h oxygen consumption (Vo2) (n = 7-8 mice). (H) Respiratory exchange ratio (RER: VCO2/VO2) (n = 7-8 mice). (I) ANCOVA of the total energy expenditure versus body weight (n = 6-7 mice). (J and K) Blood glucose and AUC during the GTT (J) and ITT (K) (n = 7-8 mice). (L) H&E staining of WAT (n = 3 mice). (M) UCP1 protein expression in WAT (n = 3 mice). (N) H&E staining of BAT (n = 3 mice). (O) UCP1 immunostaining in BAT (n = 3 mice). NCD, normal chow diet; HFD, high-fat diet; GTT, glucose tolerance test; ITT, insulin tolerance test; AUC, the area under the curve; 3 V, third cerebral ventricle; ARC, arcuate nucleus; WAT, white adipose tissue; BAT, brown adipose tissue. Data are expressed as the mean ± SD. ** p < 0.01 vs. POMC-GFP/NCD; ##p < 0.01 vs. POMC-GFP/HFD.