Adiponectin overexpression improves metabolic abnormalities caused by acid ceramidase deficiency but does not prolong lifespan in a mouse model of Farber Disease

Abstract

Farber Disease is a debilitating and lethal childhood disease of ceramide accumulation caused by acid ceramidase deficiency. The potent induction of a ligand-gated neutral ceramidase activity promoted by adiponectin may provide sufficient lowering of ceramides to allow for the treatment of Farber Disease. In vitro, adiponectin or adiponectin receptor agonist treatments lowered total ceramide concentrations in human fibroblasts from a patient with Farber Disease. However, adiponectin overexpression in a Farber Disease mouse model did not improve lifespan or immune infiltration. Intriguingly, mice heterozygous for the Farber Disease mutation were more prone to glucose intolerance and insulin resistance when fed a high-fat diet, and adiponectin overexpression protected from these metabolic perturbations. These studies suggest that adiponectin evokes a ceramidase activity that is not reliant on the functional expression of acid ceramidase, but indicates that additional strategies are required to ameliorate outcomes of Farber Disease.

Keywords: Adiponectin; Ceramidase; Ceramide; Farber Disease; Lipotoxicity.

Fig. 1

Adiponectin and receptor agonists (AdiopoRon) deplete ceramides in fibroblasts from a patient with Farber Disease. Fibroblasts were treated with 0.3 μg/ml (10.4 nM) of adiponectin, 25 μM of Adiporon, or a PBS control. Ceramide measurements were completed 24 h after stimulation. Ceramide content was normalized relative to PBS-control-treated fibroblasts. N = 5 technical replicates per group. Data are representative of 3 independent experiments. Individual values reported with mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

Adiponectin overexpression does not increase lifespan in Farber Disease mice. (A) Adiponectin protein immunoblot from plasma of male ASAH1^+/+ and ASAH1^P361R/P361R mice with and without adiponectin overexpression. (B) Growth curves measured in weights versus age, *ASAH1^+/+ versus ASAH1^P361R/P361R, #ASAH1^+/+ΔGly^Tg versus ASAH1^P361R/P361RΔGly^Tg,n = 4–10 for each genotype, males; (C) Fed blood glucose curve, *ASAH1^+/+ versus ASAH1^P361R/P361R, #ASAH1^+/+ΔGly^Tg versus ASAH1^P361R/P361RΔGly^Tg,n = 4–10 for each genotype, males; (D) Kaplan-Meier survival analysis in Farber Disease mice with and without adiponectin overexpression, males; (E) Kaplan-Meier survival in either vehicle-treated or Myriocin-treated Farber Disease mice, males. Individual values reported with mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Adiponectin overexpression lowers ceramides in plasma, but not in tissues of ASAH1^P361R/P361RΔGly^Tg male mice. Plasma and tissues from 10-week-old males on a normal chow diet were collected for lipid analysis. (A) Ceramide concentrations in plasma; (B) heart; (C) liver; (D) spleen; (E) brain all analyzed with two-way ANOVAs with multiple comparisons; (F) sum of ceramide species in liver, heart, spleen, and brain analyzed with individual one-way ANOVAs with multiple comparisons. (G) Ceramidase activity assay showing the labeled sphingosine to labeled ceramide ratio in livers of male mice. n = 4 per genotype, male mice. Individual values reported with mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

Adiponectin overexpression in ASAH1^P361R/P361RΔGly^Tg mice decreases pro-inflammatory cytokines. Tissues and plasma were harvested 10-week-old males on normal chow diet. Relative mRNA expression of pro-inflammatory cytokines (TNFα, IL-1β, IL-6, and MCP-1) was measured in (A) heart; (B) liver; (C) spleen; and (D) brain (n = 3 per genotype), analyzed with two-way ANOVAs with multiple comparisons; (E) Plasma cytokine concentrations (n = 4 per genotype); (F) H&E staining of heart, liver, spleen, and brain tissues at 200× zoom (arrow indicates infiltrating macrophages) from male mice; Quantification of infiltrating macrophages in (G) heart; (H) liver; (I) spleen; (J) brain. Individual values reported with mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001 using male mice.

Fig. 5

Metabolic abnormalities observed in heterozygous ASAH1^P361R/+ males on a high fat diet are improved with adiponectin overexpression. 8-week-old males were placed on a HFD for 12 weeks. (A) blood glucose concentrations during fed state (n = 8–10 per genotype); (B) body mass during fed state, *ASAH1^+/+ versus ASAH1^P361R/+; (C) Fold change of tissue mass normalized to body weight, n = 6 per genotype; (D) plasma ceramide concentrations after 12 weeks on HFD (n = 4 per genotype); (E) glucose tolerance after 4 weeks on HFD, n = 3–8 per genotype, *ASAH1^+/+ versus ASAH1^P361R/+; (F) area under the curve (AUC) for glucose tolerance test; (G) insulin concentrations during glucose tolerance test (n = 4 per genotype); (H) insulin tolerance displayed by percent of initial glucose concentration after 5 weeks on HFD (n = 3–8 per genotype; (I) AUC insulin tolerance test; (J) pyruvate tolerance after 6 weeks on HFD, #ASAH1^P361R/+ versus ASAH1^P361R/+ΔGly^Tg (n = 3–8 per genotype; (K) AUC pyruvate tolerance test; (L) glucose infusion rate (GIR) and (M) hepatic glucose production during hyperinsulinemic-euglycemic clamps after 9 weeks on HFD (n = 3–5 per genotype). Individual values reported with mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001.