Loss of autophagy in hypothalamic POMC neurons impairs lipolysis

Abstract

Autophagy degrades cytoplasmic contents to achieve cellular homeostasis. We show that selective loss of autophagy in hypothalamic proopiomelanocortin (POMC) neurons decreases α-melanocyte-stimulating hormone (MSH) levels, promoting adiposity, impairing lipolysis and altering glucose homeostasis. Ageing reduces hypothalamic autophagy and α-MSH levels, and aged-mice phenocopy, the adiposity and lipolytic defect observed in POMC neuron autophagy-null mice. Intraperitoneal isoproterenol restores lipolysis in both models, demonstrating normal adipocyte catecholamine responsiveness. We propose that an unconventional, autophagosome-mediated form of secretion in POMC neurons controls energy balance by regulating α-MSH production. Modulating hypothalamic autophagy might have implications for preventing obesity and metabolic syndrome of ageing.

Figure 1

POMC neuron-selective loss of autophagy. (A) Immunofluorescence for POMC (green) and Cre (red; n=4), (B) POMC (green) and Atg7 (red; n=4) and (C) POMC (green) and p62 (red; n=6) in MBH sections from Con and Atg7^F/F-POMC-Cre (KO) mice. (D) TUNEL-positivity (green; n=4) and (E) number of POMC-positive neurons (red) in MBH sections from Con and KO mice (n=6). Values are mean±s.e.m. P values are as compared with diet- and age-matched controls. ^*P<0.05, ^***P<0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. White arrows indicate Atg7, Cre, p62 or POMC signal. Yellow arrows indicate merged signal. Con, control; DAPI, 4′-6-diamidino-2-phenylindole; KO, knockout; MBH, mediobasal hypothalamic; NS, not statistically significant; POMC, proopiomelanocortin; TUNEL, TdT-mediated dUTP nick end labelling.

Figure 2

Loss of atg7 in POMC neurons promotes adiposity. (A) Body wt of 4-mo- and 12-mo-old Con and KO mice on RD (n=9–13) or HFD (n=5–7). (B) Total body fat and (C) lean mass of 4-mo-old Con and KO mice on RD (n=7) and 12-mo-old Con and KO mice on HFD (n=6–8). (D) eWAT wt, (E) fat pads and (F) liver wt of 4-mo-old Con and KO mice on RD (n=6–8). (G) Immunoblots for POMC and ACTH, and (H) NPY in MBH from RD-fed Con and KO mice (n=3–4). (I) Immunofluorescence for POMC (green) and α-MSH (red), and (J) α-MSH (green) in MBH from Con and KO mice on RD (n=5). Values are mean±s.e.m. P values are as compared with diet- and age-matched controls. ^*P<0.05, ^**P<0.01, ^***P<0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; DAPI, 4′-6-diamidino-2-phenylindole; eWAT, epididymal fat; HDF, high-fat diet; KO, knockout; MBH, mediobasal hypothalamic; mo, month; MSH, melanocyte-stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; RD, regular chow; wt, weight.

Figure 3

POMC neuronal autophagy is required for lipolysis. (A) Percent loss of body fat and (B) lean mass in 24-h-fasted Con and KO mice on RD or HFD (n=6–8). (C) Histology for eWAT from fed or 6-h-fasted Con and KO mice on RD (n=3–4). (D) Serum fatty acids and (E) glycerol from fed or 6-h-fasted Con and KO mice on RD (n=7–9). (F) Serum fatty acids and (G) glycerol after intraperitoneal isoproterenol (Iso) in 6-h-fasted Con and KO mice on RD (n=5–6). (H) Immunoblot for oxidized proteins in eWAT from RD-fed Con and KO mice (n=3). (I) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD (n=3). Values are mean±s.e.m. P values are as compared with diet- and age-matched controls. ^*P<0.05, ^**P<0.01, ^***P<0.001. Con, control; eWAT, epididymal fat; FACS, fluorescence-activated cell sorting; HDF, high-fat diet; Iso, isoproterenol; KO, knockout; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.

Figure 4

Loss of POMC neuronal atg7 impairs glucose tolerance. (A) Blood glucose levels in 6 mo (n=7–9) and (B) 12 mo Con and KO mice on RD (n=6), and in (C) 12-mo-old Con and KO mice on HFD (n=4). (D) Serum insulin levels in 6 mo, (n=7–9) and (E) 12-mo-old RD-fed Con and KO mice that were fed or 6 h fasted (n=6). (F) HOMA values from 6-h-fasted 6-mo-old (n=7–9) and (G) 12-mo-old Con and KO mice (n=6) on RD. (H) Glucose tolerance tests in 6-h-fasted 6-mo-old mice on RD (n=4) and in (I) 6-h-fasted 12-mo-old Con and KO mice fed HFD for 10 mo (n=4). (J) Insulin tolerance tests in RD-fed 6-mo-old (n=4) and (K) HFD-fed 12-mo-old Con and KO mice (n=4). (L) Insulin tolerance tests and (M) body weights of 1-mo-old Con and KO mice on RD (n=3–5). Values are mean±s.e.m. P values are as compared with diet- and age-matched controls. ^*P<0.05, ^**P<0.01, ^***P<0.001. Con, control; HDF, high-fat diet; HOMA, homeostasis model of insulin resistance; KO, knockout; mo, month; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.

Figure 5

Ageing reduces POMC neuronal autophagy and lipolysis. (A) Immunoblots for Atg7 in MBH from young (3 mo), middle-aged (12 mo) and aged (22 mo) fed and 6-h-fasted (Stv) mice (n=4), and (B) LC3-II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors (Inh) (n=11–13). Mean±s.e.m values for (B) steady-state LC3-II and (C) LC3-II flux are shown (n=11–13). (D) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh (n=11–13). (E) Immunofluorescence for POMC (green) and p62 (red) (n=4), (F) immunoblots for POMC and ACTH (n=4), and (G) immunofluorescence for α-MSH in MBH from 3 mo and 22 mo mice on RD (n=4). (H) Body wt and (I) eWAT wt of 3-mo- and 22-mo-old mice on RD (n=9–13). (J) Serum fatty acids and (K) glycerol from fed or 6-h-fasted (Stv) 3 mo and 22 mo mice (n=7–9), and (L,M) after intraperitoneal Iso in 6-h-fasted 3 mo and 22 mo mice (n=5–6). (N) Immunoblots for adipose triglyceride lipase (ATGL) in eWAT from 3 mo and 22 mo mice. Values are mean±s.e.m. P values are as compared with controls. ^*P<0.05, ^**P<0.01, ^***P<0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; eWAT, epididymal fat; Iso, isoproterenol; MBH, mediobasal hypothalamic; mo, months; MSH, melanocyte-stimulating hormone; POMC, proopiomelanocortin; RD, regular chow; wt, weight.