Melanocortin 3 receptor regulates hepatic autophagy and systemic adiposity

Abstract

Systemic lipid homeostasis requires hepatic autophagy, a major cellular program for intracellular fat recycling. Here, we find melanocortin 3 receptor (MC3R) regulates hepatic autophagy in addition to its previously established CNS role in systemic energy partitioning and puberty. Mice with Mc3r deficiency develop obesity with hepatic triglyceride accumulation and disrupted hepatocellular autophagosome turnover. Mice with partially inactive human MC3R due to obesogenic variants demonstrate similar hepatic autophagic dysfunction. In vitro and in vivo activation of hepatic MC3R upregulates autophagy through LC3II activation, TFEB cytoplasmic-to-nuclear translocation, and subsequent downstream gene activation. MC3R-deficient hepatocytes had blunted autophagosome-lysosome docking and lipid droplet clearance. Finally, the liver-specific rescue of Mc3r was sufficient to restore hepatocellular autophagy, improve hepatocyte mitochondrial function and systemic energy expenditures, reduce adipose tissue lipid accumulation, and partially restore body weight in both male and female mice. We thus report a role for MC3R in regulating hepatic autophagy and systemic adiposity.

Fig. 1. Mc3r insufficiency increases systemic and hepatic adiposity in mice.

a Normalized droplet digital PCR Mc3r transcript levels from Mc3r^+/+ liver (n = 37) at ZT12 (6:00 PM) and from Mc3r^+/+ liver (n = 35) at ZT2 (8:00 AM) and Mc3r^TB/TB liver (n = 18) at ZT12, and from Mc3r^TB/TB liver (n = 19) at ZT2. b Normalized Mc3r transcript levels from Mc3r^+/+ liver (n = 21) and hypothalamus (n = 4, with each RNA extraction sample combining 6 pooled hypothalamus isolations); c Body weight measured from Mc3r^+/+ (n = 8), Mc3r^TB/TB (n = 10), MC3R^hWT/hWT (n = 12) and MC3R^hDM/hDM (n = 11) independent observations. d Total body % fat from Mc3r^+/+ (n = 9), Mc3r^TB/TB (n = 8), MC3R^hWT/hWT (n = 7) and MC3R^hDM/hDM (n = 7) independent observations. e Liver triglycerides (TG) from Mc3r^+/+ (n = 6), Mc3r^TB/TB (n = 5), MC3R^hWT/hWT (n = 7) and MC3R^hDM/hDM (n = 6) independent observations. f Oil Red O images of livers (n = 2/genotype from two independent observations) g Transmission Electron Microscope (TEM) images of livers from mice in the fed state (n = 1–2/genotype from two independent experiments) at 8–12 weeks of age (h) Quantification of lipid droplet (LD) number from TEM images (n = 16–28 images/genotype from two independent observations) shown in (g). Asterisks indicate LD. Data are represented as mean ± SEM. Groups were compared by one-way ANOVA followed by Tukey’s HSD (a), paired two-tailed Student’s t-test (b, h), two-way ANOVA followed by Tukey’s HSD (c–e). * p < 0.05; ** p < 0.01; *** p < 0.001; male mice (8–12 weeks of age) under chow-fed diet were used unless otherwise indicated. Scale bar, 50 µm (f) and 5 µm (g). Source data are provided as a Source data file.

Fig. 2. Altered hepatic autophagy and LD recycling in Mc3r insufficient mice.

a Transmission Electron Microscope images showing liver autophagosomes (AP) from overnight (O/N) fasted Mc3r^+/+ and Mc3r^TB/TB (n = 1–2/genotype from two independent experiments). b Quantification of AP number from TEM images from both Mc3r^+/+ (n = 15–16 images/genotype from two independent observations) shown in (a). c TEM images showing liver AP from overnight (O/N) fasted Mc3r^hWT/hWT and MC3R^hDM/hDM. d Western blot images of liver lysates from Mc3r^+/+, Mc3r^TB/TB, Mc3r^hWT/hWT and MC3R^hDM/hDM. e Quantification of LC3II normalized by actin from both fed-fasted Mc3r^+/+ (n = 4) independent observations, Mc3r^TB/TB (n = 5) independent observations, MC3R^hWT/hWT (n = 10) independent observations, and MC3R^hDM/hDM (n = 9 (fed) and 10 (fasted) independent observations) in (d). f Western blot images of primary hepatocytes from Mc3r^+/+, Mc3r^TB/TB and Mc4r^+/− after 1 h serum starvation or 30 min of 10 mM chloroquine (CQ) treatment. g Quantification of AP flux estimated in (f) and Supplementary Fig. 1c from Mc3r^+/+ (n = 3), Mc3r^TB/TB (n = 3), MC3R^hWT/hWT (n = 4) and MC3R^hDM/hDM (n = 3). h Fluorescent images of primary hepatocytes from Mc3r^+/+, Mc3r^TB/TB and MC3R^hDM/hDM transgenic mice carrying GFP-LC3 after staining with Lysotracker Red. i Quantification of AP volume in (h) (n = ~1500/2 mice/genotype from two independent experiments). Asterisks indicate AP. Arrows and arrowheads indicate mitochondrial outer and inner membranes, respectively. Data are represented as mean ± SEM. Groups were compared by unpaired two-tailed Student’s t-test (b), ordinary one-way ANOVA followed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test or Tukey’s HSD test (e, g, i). * p < 0.05; ** p < 0.01; *** p < 0.001, Scale bar, 1 µm (a, c) and 10 µm (h). Source data are provided as a Source data file.

Fig. 3. Hepatic Mc3r reactivation restores body weight and body composition in Mc3r insufficient mice.

a % Fat mass from Mc3r^+/+ (n = 10), Mc3r^TB/TB (n = 14) and Mc3r^Hep/Hep (n = 10) independent observations. b % Lean mass from Mc3r^+/+ (n = 10), Mc3r^TB/TB (n = 14) and Mc3r^Hep/Hep (n = 10) independent observations. c Fasting serum insulin from Mc3r^+/+, Mc3r^TB/TB and Mc3r^Hep/Hep (n = 7) independent observations/genotype. d Fasting glucose from Mc3r^+/+ (n = 7), Mc3r^TB/TB (n = 6) and Mc3r^Hep/Hep (n = 6) independent observations. e Insulin tolerance test (ITT) measured from Mc3r^+/+ (n = 7), Mc3r^TB/TB (n = 6) and Mc3r^Hep/Hep (n = 7) independent observations. * p < 0.05; ** p < 0.01; *** p < 0.001 group compared to Mc3r^+/+ or ^#p < 0.05 group compared to Mc3r^TB/TB. f ITT AUC measured from (e). g Glucose tolerance test (GTT) measured from Mc3r^+/+ (n = 7), Mc3r^TB/TB (n = 6) and Mc3r^Hep/Hep (n = 6) independent observations. h GTT AUC measured from (g). i Body weight measured from male Mc3r^+/+ (n = 8), Mc3r^TB/TB (n = 9) and Mc3r^Hep/Hep (n = 7) independent observations under chow diets. j Body weight measured from female Mc3r^+/+ (n = 14), Mc3r^TB/TB (n = 8) and Mc3r^Hep/Hep (n = 9) independent observations under chow diets. Data are represented as mean ± SEM. Groups were compared by ordinary one-way ANOVA followed by Tukey’s HSD test (a–d, f, h), two-way ANOVA followed by Tukey’s HSD test (e, g, i, j). * p < 0.05; ** p < 0.01; *** p < 0.001. Source data are provided as a Source Data file.

Fig. 4. Hepatic Mc3r reactivation reversed fat accumulation and fatty acid metabolism in Mc3r insufficient mice.

a H&E staining (b) Oil Red O staining for liver tissue from Mc3r^+/+, Mc3r^TB/TB and Mc3r^Hep/Hep. c Fold change of liver TG levels from Mc3r^+/+ (n = 8), Mc3r^TB/TB (n = 8) and Mc3r^Hep/Hep (n = 7) independent observations. d Liver weight (g) from Mc3r^+/+ (n = 36), Mc3r^TB/TB (n = 11) and Mc3r^Hep/Hep (n = 24) independent observations (e) eWAT weight (g) from Mc3r^+/+ (n = 33), Mc3r^TB/TB (n = 15) and Mc3r^Hep/Hep (n = 24) independent observations (f) Serum TG measured from Mc3r^+/+ (n = 6), Mc3r^TB/TB (n = 7) and Mc3r^Hep/Hep (n = 5) independent observations. g Serum non-esterified fatty acids (NEFA) from Mc3r^+/+ (n = 6), Mc3r^TB/TB (n = 9) and Mc3r^Hep/Hep (n = 9) independent observations. h Serum glycerol from Mc3r^+/+ (n = 6), Mc3r^TB/TB (n = 9) and Mc3r^Hep/Hep (n = 9) independent observations. i Serum TC from Mc3r^+/+ (n = 6), Mc3r^TB/TB (n = 9) and Mc3r^Hep/Hep (n = 6) independent observations. j qPCR mRNA measurements for fatty acid metabolism from Mc3r^+/+ (n = 8 fed) and (n = 7 fasted) independent observations, Mc3r^TB/TB (n = 8), and Mc3r^Hep/Hep (n = 6) independent observations from both fed-fasted conditions. Data are represented as mean ± SEM. Groups were compared by one-way ANOVA followed by Tukey’s HSD test (c–f), one-way ANOVA followed by Fisher’s LSD test (g–i), and two-way ANOVA followed by Tukey’s HSD test (j). * p < 0.05; ** p < 0.01; *** p < 0.001, Scale bar, 50 µm (a, b). Source data are provided as a Source Data file.

Fig. 5. Assessment of energy intake and energy expenditure at ambient and thermoneutral temperatures by mice indirect calorimetry.

a Cumulative energy intake (kcal) monitored for light and dark phase at ambient 22 °C and thermoneutral temperature 30 °C. b Daily energy intake (kcal) for light, dark, and total (24 h) at 22 °C from Mc3r^+/+, Mc3r^TB/TB and Mc3r^Hep/Hep (n = 20) independent observations. c Daily energy intake (kcal) for light, dark, and total (24 h) at 30 °C from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep (n = 20) independent observations. d Total energy expenditure (kcal/mouse/h) for light and dark phase e TEE (kcal/mouse/h) for light, dark, and total (24 h) at 22 °C from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep (n = 20) independent observations. f TEE (kcal/mouse/h) for light, dark, and total (24 h) at 30 °C from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep (n = 20) independent observations. g Oxygen consumption (VO₂) (ml/mouse/h) for light and dark phase h VO₂ (ml/mouse/h) for light, dark, and total (24 h) at 22 °C from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep (n = 20) independent observations. i VO₂ (ml/mouse/h) for light, dark, and total (24 h) at 30 °C from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep (n = 20) independent observations. j Respiratory exchange ratio (RER) (VCO_2/VO₂) for light and dark phase (k) RER (VCO_2/VO₂) for light, dark, and total (24 h) at 22 °C from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep (n = 20) independent observations. l RER (VCO_2/VO₂) for light, dark, and total (24 h) at 30 °C from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep (n = 20) independent observations. In (a, d, g, and j), data from the first day of adaptation are not shown. Chow-fed, n = 20 mice/group (10 males and 10 females) mice 3–4 months of age. Data are represented as mean ± SEM. Groups were compared using Friedman’s test by Dunn’s paired analysis (b, c, e, f, h, i, k, l) and post hoc test for adjusted estimated marginal means in Supplementary Table 1. * p < 0.05; ** p < 0.01; *** p < 0.001. Source data are provided as a Source Data file.

Fig. 6. Hepatic Mc3r reactivation restores adaptative energy expenditure, cellular respiration, and lipid droplet autophagy.

a Energy expenditure (kcal/mouse/h) at 6 °C from Mc3r^+/+(n = 15), Mc3r^TB/TB (n = 16), and Mc3r^Hep/Hep (n = 12) independent observations. b EE (kcal/mouse/h) at 6 °C from Mc3r^+/+ (n = 15), Mc3r^TB/TB (n = 16), and Mc3r^Hep/Hep (n = 12) independent observations. c Oxygen consumption (VO₂) (ml/mouse/h) at 6 °C from Mc3r^+/+ (n = 15), Mc3r^TB/TB (n = 16), and Mc3r^Hep/Hep (n = 12) independent observations. d VO₂ (ml/mouse/h) at 6 °C from Mc3r^+/+ (n = 15), Mc3r^TB/TB (n = 16), and Mc3r^Hep/Hep (n = 12) independent observations. e Oxygen consumption rate (OCR) from cultured primary hepatocytes from Mc3r^+/+, Mc3r^TB/TB and Mc3r^Hep/Hep from 3 independent experiments. f Basal respiration OCR (pmol/min/5000 cells) from 3 independent experiments. g Maximal respiration OCR (pmol/min/5000 cells) from 3 independent experiments. h Bulk RNA-Sequencing analysis of mouse liver samples from Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep genotypes, all on a normal chow diet (n = 4) independent observations. Pairwise scatter plot illustrates the Log₂ fold change (Log₂FC) values for all significantly differentially expressed genes across three genotype comparisons. The Log₂FC for Mc3r^TB/TB vs Mc3r^+/+ is plotted against Mc3r^Hep/Hep vs Mc3r^+/+ (blue-filled circles), and linear regression fit lines are shown for the comparisons between Mc3r^TB/TB vs. Mc3r^+/+ and Mc3r^Hep/Hep vs Mc3r^+/+ (blue line). i The Log₂FC for Mc3r^TB/TB vs Mc3r^+/+ is plotted against Mc3r^Hep/Hep vs Mc3r^TB/TB (orange-filled circles). Linear regression fit lines are shown for the comparisons between Mc3r^TB/TB vs. Mc3r^+/+ and Mc3r^Hep/Hep vs Mc3r^TB/TB (orange line), with the black line shows best fit perfect correlation lines included for visualization. j Heatmap depicting significant gene ontology (GO) biological pathways related to lipid droplet autophagy, lipid metabolism, and energy homeostasis. The negative log₁₀ P values for these pathways were compared across the Mc3r^TB/TB vs. Mc3r^+/+, Mc3r^Hep/Hep vs Mc3r^+/+ and Mc3r^Hep/Hep vs Mc3r^TB/TB comparisons (adjusted p-value ≤ 0.05). k Western blot images of liver lysates from Mc3r^+/+, Mc3r^TB/TB and Mc3r^Hep/Hep. l Quantification of LC3II normalized by β-actin in (g) from Mc3r^+/+ (n = 12), Mc3r^TB/TB (n = 10), and Mc3r^Hep/Hep (n = 11) independent observations. m Quantification of LC3II normalized by LC3I in (g) from Mc3r^+/+(n = 12), Mc3r^TB/TB (n = 10), and Mc3r^Hep/Hep (n = 11) independent observations. Data are represented as mean ± SEM. Groups were compared by one-way ANOVA followed by Fisher’s LSD test (b, d), one-way ANOVA followed by Tukey’s HSD test (f, g), two-tailed simple linear regression (h, i). p < 0.05; ** p < 0.01; *** p < 0.001. Source data are provided as a Source Data file.

Fig. 7. γ-MSH induces hepatic autophagy and nuclear TFEB activity.

a Representative fluorescent images of primary hepatocytes from Mc3r^+/+ transgenic mice carrying GFP-LC3 under 2 nM D-Trp⁸-γ-MSH (γ-MSH) or 10 nM rapamycin (Rap) after staining with Lysotracker Red and DAPI. b Time-lapse images of primary hepatocytes from Mc3r^+/+ transgenic mice carrying GFP-LC3 under 2 nM γ-MSH after staining with Lysotracker Red. c Representative western blot images of primary hepatocytes from Mc3r^+/+ and Mc3r^TB/TB after 2 nM γ-MSH treatment. d Representative super resolution microscope images of primary hepatocytes from Mc3r^+/+ transgenic mice carrying GFP-LC3 under 100 nM MC3R-specific (versus MC4R) agonist (NDP42) for 60 min after staining with Lysotracker Red compared to control. e Measurements of total area of GFP-LC3 after 60 min of stimulation (d). f Representative western blot images of primary hepatocytes treated with serum starvation (HBSS) for 60 min, 1 µM Torin for 60 min, or 100 nM NDP42 (MC3R-specific agonist versus MC4R) for 30 min. g Quantification of LC3II normalized by β-actin in (f). Groups were analyzed between Control (n = 5), Starvation (n = 3), Torin (n = 5), and NDP42 (n = 5) independent experiments. h Representative western blot images of liver lysates from Mc3r^+/+ with intraperitoneal γ-MSH injection. i Quantification of LC3II normalized by β-actin in (h) (n = 10) independent observations. j Representative western blot images of nuclear fractions (top panels) and cytosolic fractions (bottom panels) from primary Mc3r^+/+ hepatocytes after 1 h starvation (HBSS) or 2 nM γ-MSH treatment. k qPCR measurement from Mc3r^+/+ and Mc3r^TB/TB hepatocytes after 2 nM γ-MSH (n ≥ 5 independent observations). l Heatmap of Log10 normalized counts for TFEB target genes and lysosomal genes in mouse liver RNAseq analysis across Mc3r^+/+, Mc3r^TB/TB, and Mc3r^Hep/Hep genotypes on a normal chow diet (n = 3) independent observations. Data are represented as mean ± SEM. Groups were compared by paired two-tailed Student’s t-test (e), unpaired two-tailed Student’s t-test (i, k), one-way ANOVA followed by Fisher’s LSD test (g). p < 0.05; ** p < 0.01; *** p < 0.001, Scale bar, 10 µm (a, b, d). Source data are provided as a Source Data file.