p75 Neurotrophin Receptor Regulates Energy Balance in Obesity

Abstract

Obesity and metabolic syndrome reflect the dysregulation of molecular pathways that control energy homeostasis. Here, we show that the p75 neurotrophin receptor (p75(NTR)) controls energy expenditure in obese mice on a high-fat diet (HFD). Despite no changes in food intake, p75(NTR)-null mice were protected from HFD-induced obesity and remained lean as a result of increased energy expenditure without developing insulin resistance or liver steatosis. p75(NTR) directly interacts with the catalytic subunit of protein kinase A (PKA) and regulates cAMP signaling in adipocytes, leading to decreased lipolysis and thermogenesis. Adipocyte-specific depletion of p75(NTR) or transplantation of p75(NTR)-null white adipose tissue (WAT) into wild-type mice fed a HFD protected against weight gain and insulin resistance. Our results reveal that signaling from p75(NTR) to cAMP/PKA regulates energy balance and suggest that non-CNS neurotrophin receptor signaling could be a target for treating obesity and the metabolic syndrome.

Figure 1. p75^NTR Deficiency Protects Mice from HFD-Induced Obesity and Metabolic Syndrome

(A) p75^NTR protein (left) and RNA (right) expression in WAT, skeletal muscle (SKM), and liver from WT and p75^NTR−/− mice on ND (normal diet) and, 3 and 8 weeks on HFD. Representative immunoblot is shown from three independent experiments. (B) Body weight of WT and p75^NTR−/− mice on HFD (*P < 0.01, **P < 0.001, two-way ANOVA; n = 7 mice per group). (C) Representative MRI images of WT and p75^NTR−/− mice on HFD (left) and tissue volumes of WT and p75^NTR-/- mice on HFD (right) (**P < 0.01, ns: not significant, unpaired Student's t-test; n = 7 mice per group). (D) Weights of WT and p75^NTR−/− inguinal and intraperitoneal (IP) fat on HFD (*P < 0.05, ***P < 0.001, unpaired Student's t-test; n = 7 mice per group). (E) Basal insulin levels from WT and p75^NTR−/− mice (**P < 0.01, ns, unpaired Student's t-test; n= 4 mice per group). (F) Glucose and (G) insulin tolerance test in WT (n = 13) and p75^NTR−/− (n = 8) mice after 20weeks on HFD (*P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA). (H) Glucose infusion rates from WT (n = 5) and p75^NTR−/− (n = 7) mice on HFD (*P < 0.05, unpaired Student's t-test). (I) Total and insulin-stimulated glucose disposal rate (GDR) of WT (n = 5) and p75^NTR-/- (n = 7) mice on HFD (*P < 0.05, unpaired Student's t-test). (J) Representatives photographs (top) and histological images (bottom) of livers from WT and p75^NTR−/− mice on HFD (n = 3 mice per group). See also Figures S1 and S2.

Figure 2. p75^NTR Deficiency Increases Energy Expenditure, Fat Oxidation, and Lipolysis

(A) Food consumption (left) and energy intake (right) of WT (n = 7) and p75^NTR−/− (n = 8) mice on HFD (ns: not significant, unpaired Student's t-test). (B) Oxygen consumption (left) and energy expenditure (right) normalized to lean body mass in WT (n = 7) and p75^NTR−/− (n = 8) mice on HFD (**P < 0.01, ***P < 0.001, Student's t-test). (C) Fat oxidation normalized to lean body mass in WT and p75^NTR−/− mice on HFD (***P < 0.001, unpaired Student's t-test; n = 6 mice per group). (D) Oxidation of [1-¹⁴C]-palmitate to ¹⁴CO₂ by adipocytes isolated from WT and p75^NTR-/- mice (**P < 0.01, unpaired Student's t-test). Results are from three independent experiments. (E) Ucp1 and Dio2 RNA expression in primary adipocytes from WT and p75^NTR−/− mice (**P <0.01, unpaired Student's t-test; n = 6 mice per group). (F) Serum levels of adiponectin in WT and p75^NTR−/− mice on HFD (*P < 0.05, unpairedStudent's t-test; n = 3 mice per group). See also Figures S3 and S4.

Figure 3. p75^NTR Regulates Lipolysis via cAMP/PKA Signaling

(A) Isoproterenol-stimulated FFA and glycerol levels in WAT from WT and p75^NTR−/− mice on HFD (**P < 0.01, ***P < 0.001, two-way ANOVA; n = 8 mice per group). (B) cAMP accumulation in WAT from WT and p75^NTR−/− mice on normal chow (ND) or 10 weeks on HFD (*P < 0.05, ns: not significant, unpaired Student's t-test; n = 4 mice per group). (C) FFA levels in WAT treated with the PKA inhibitor, H-89 from WT, and p75^NTR−/− mice on HFD (WT or p75^NTR−/− vs p75^NTR−/− treated with H-89, ***P < 0.001, two-way ANOVA; n = 5 mice per group). (D) P-HSL, HSL, P-p38, p38, P-CREB, CREB, and p75^NTR protein expression (left) and immunoprecipitation of lysates with anti-perilipin followed by Western blotting with anti-phospho-PKA(p-PKA) to detect all PKA-phosphorylation sites on perilipin and total perilipin expression (n = 2 mice per group) (right) in WAT from WT and p75^NTR−/− mice on HFD. Phospho-perilipin levels normalized to GAPDH were quantified by densitometry (Δ represents fold changes). Representative immunoblots are shown from n = 12 mice per group. (E) Immunoprecipitation of PKA-Cα protein followed by Western blotting to detect PKA RIIβ and RIIα from WT and p75^NTR−/− MEF-derived adipocytes treated or not with isoproterenol (ISO). Representative immunoblot from three independent experiments. (F) PKA-Cα, PKA-RIIβ, and PKA-RIIα protein expression in WT and p75^NTR−/− MEF-derivedadipocytes. Representative immunoblots from three independent experiments. (G) Immunoprecipitation of HA-PKA-Cα protein (top) and myc-PKA-RIIβ (bottom) followed by Western blotting to detect GFP-p75^NTR in 293T cells overexpressing indicated constructs. Representative immunoblots from three independent experiments. (H) Immunoprecipitation of PKA-RIIβ (top) and PKA-Cα (bottom) protein followed by Western blotting to detect p75^NTR in WAT from WT mice. Representative immunoblots from three independent experiments. (I) ELISA binding assays between recombinant His-p75ICD and increasing concentrations of His-PKA-Cα (left) and PKA-RIIβ (right). His-Hsp20 was used as a control. Results are from 3 independent experiments performed with duplicates. K_d values were estimated using a one-site binding model. See also Figure S5.

Figure 4. p75^NTR Interacts with the PKA Catalytic (Cα) Subunit to Regulate Lipolysis

(A) Peptide array mapping of the p75ICD sites required for the interaction with PKA-Cα and PKA-RIIβ. Schematic diagram of p75ICD shows the domain organization and peptide location, length, and sequences. (B) Alanine scanning substitution arrays of the identified interacting p75^NTR peptides 360–384 and 375–399 probed with PKA-Cα. Red amino acids indicate substitutions that block the interaction. (C) Alanine scanning substitution arrays of interacting p75^NTR peptides 368–392, 383–407, and 388–412 probed with PKA-RIIβ. Highlighted in red are the amino acids whose substitution blocks the interaction. (D) p75^NTR mutant constructs generated. (E) Immunoprecipitation of myc-PKA-Cα protein followed by Western blotting to detect HA-p75^NTR wild-type and HA-p75^NTRC379A or HA-p75^NTR 3M in 293T cells overexpressing indicated constructs. Representative immunoblots are shown from three independent experiments. (F) Immunoprecipitation of myc-PKA-RIIβ protein followed by Western blotting to detect HA-p75^NTR wild-type and HA-p75^NTR P380A, HA-p75^NTR R404A, HA-p75^NTR 2M, or HA-p75^NTR 3M in 293T cells overexpressing indicated constructs. Representative immunoblots from three independent experiments. (G) P-HSL, HSL, and p75^NTR protein expression in WT and p75^NTR−/− MEF-derived adipocytes infected with lentiviral vectors overexpressing the indicated constructs and treated or not with isoproterenol (ISO). Representative immunoblots from three independent experiments. See also Figure S6.

Figure 5. p75^NTR Deficiency in WAT Protects Mice from HFD-induced Obesity and Insulin Resistance

(A) p75^NTR protein expression in WAT and the central nervous system (CNS) from p75^F/F, Adipocyte-cre, and p75^AKO mice. Representative blot is shown from two independent experiments (n = 5 mice per group). (B) p75^NTR expression in brain, WAT (epididymal fat), SUBC (subcutaneous inguinal fat), BAT (brown adipose tissue), Soleus (skeletal muscle), kidney, and heart from Adipocyte-cre and p75^AKO mice (n = 5 mice per group). (C) Body weight of p75^F/F (n = 18), Adipocyte-cre (n = 7), and p75^AKO (n = 10) mice on HFD (**P < 0.01, *** P < 0.001, two-way ANOVA). (D) Glucose (left) and insulin tolerance (right) tests in Adipocyte-cre (n = 7) and p75^AKO (n = 10) mice after 8 weeks on HFD (*P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA). (E) p75^NTR protein expression in skeletal muscle from p75^F/F (n = 3), SKM-cre (n = 2), andp75^SKMKO mice (left) (n = 3). Quantifications of Western blot analysis for p75^NTR in skeletal muscle from p75^F/F, MCK-cre, and p75 ^SKMKO mice (ns, not significant; *P < 0.05, Tukey'smultiple comparisons test one-way ANOVA analysis) (right). (F) Body weight of p75^F/F (n = 18), SKM-cre (n = 10), and p75 ^SKMKO (n = 15) mice on HFD (notsignificant, two-way ANOVA). See also Figure S7.

Figure 6. p75^NTR Deficiency in WAT Increases Thermogenesis and Lipolysis

(A) Oxygen consumption normalized to lean body mass in Adipocyte-cre and p75^AKO mice on HFD (n = 6 mice per group). (B) Food consumption (left) and energy intake (right) of Adipocyte-cre (n = 10) and p75^AKO (n = 12) mice over 4 days after 10 weeks of HFD (ns: not significant, unpaired Student's t-test). (C) Fat oxidation normalized to lean body mass in Adipocyte-cre and p75^AKO mice on HFD (***P < 0.001, unpaired Student's t test; n = 6 mice per group). (D) Ucp1, Hsl, Pgc-1α, Pparα, and Dio2 RNA expression in WAT from p75^F/F, Adipocyte-cre, and p75^AKO mice on HFD (ns: not significant, *P < 0.05, **P < 0.01, Tukey's multiple comparisons test one-way ANOVA analysis; n = 4 mice per group). (E) P-HSL and HSL protein expression in WAT from p75^F/F (n = 2), Adipocyte-cre (n = 3), andp75^AKO (n = 2) mice on HFD. See also Figure S7.

Figure 7. p75^NTR-deficient WAT Transplantation Reduces Body Weight and Insulin Resistance

(A) Schematic of fat transplantation for WT mice. Epididymal fat from p75^NTR−/−, WT, and no fat (sham) was transplanted in WT mice. All animals after surgery were fed a HFD. (B) Body weight of WT mice transplanted with p75^NTR−/− fat (n = 9), WT fat (n = 9) and sham (n = 5). (WT→WT vs p75^NTR−/−→WT, *P < 0.05, **P < 0.01 by two-way ANOVA). (C) Glucose (left) and insulin tolerance (right) tests in WT-transplanted mice after 8 weeks on HFD. (p75^NTR−/− →WT vs WT→WT, **P < 0.01, ***P < 0.001 by two-way ANOVA; n = 4). (D) Schematic of fat transplantation. Epididymal fat from p75^NTR−/−, WT, and no fat (sham) was transplanted in p75^NTR−/− mice. All animals after surgery were fed a HFD. (E) Body weight of WT and p75^NTR−/− mice transplanted with p75^NTR−/− (n = 9) or WT fat (n = 9)and sham-operated (n = 4). (WT sham vs WT→ p75^NTR−/− **P < 0.01, ***P < 0.001, two-way ANOVA). (F) Glucose (left) and insulin tolerance (right) tests in recipient mice after 10 weeks of HFD (WT sham vs WT→ p75^NTR−/−, *p < 005, **P < 0.01, ***P < 0001, two-way ANOVA; n = 4 mice per group).