Identification of adipose tissue transcriptomic memory of anorexia nervosa

Abstract

Background: Anorexia nervosa (AN) is a complex debilitating disease characterized by intense fear of weight gain and excessive exercise. It is the deadliest of any psychiatric disorder with a high rate of recidivism, yet its pathophysiology is unclear. The Activity-Based Anorexia (ABA) paradigm is a widely accepted mouse model of AN that recapitulates hypophagia and hyperactivity despite reduced body weight, however, not the chronicity.

Methods: Here, we modified the prototypical ABA paradigm to increase the time to lose 25% of baseline body weight from less than 7 days to more than 2 weeks. We used this paradigm to identify persistently altered genes after weight restoration that represent a transcriptomic memory of under-nutrition and may contribute to AN relapse using RNA sequencing. We focused on adipose tissue as it was identified as a major location of transcriptomic memory of over-nutririon.

Results: We identified 300 dysregulated genes that were refractory to weight restroration after ABA, including Calm2 and Vps13d, which could be potential global regulators of transcriptomic memory in both chronic over- and under-nutrition.

Conclusion: We demonstrated the presence of peristent changes in the adipose tissue transcriptome in the ABA mice after weight restoration. Despite being on the opposite spectrum of weight perturbations, majority of the transcriptomic memory genes of under- and over-nutrition did not overlap, suggestive of the different mechanisms involved in these extreme nutritional statuses.

Keywords: Adipose tissue; Anorexia nervosa; Calm2; Transcriptomic memory; Undernutrition; Vps13d.

Fig. 1

Modified Activity-Based Anorexia model in mice for the study of recidivsm. (A) Diagram of the progressive reduction in food access duration during ABA. (B) Study timeline of ABA with weight restoration period. (C) Body weight changes, as percent of of baseline, (D) Food intake, (E) Time spent running, (F) Distance ran, (G) Average and (H) Maximum running speed of CON (n = 8), ABA (n = 8) and WR (n = 8) mice. Data was expressed as mean SEM and analyzed with two-tailed repeated measures ANOVA followed by two-stage linear step-up procedure of Benjamini, Kreiger and Yekutieli with an FDR of 0.1. Significance was set at p < 0.5

Fig. 2

Weight Restored ABA mice expressed markers that supress food intake. (A) Gonadal adipose tissue weight (CON: n = 8, ABA: n = 7 and WR: n = 8), (B) Leptin plasma concentrations (CON: n = 8, ABA: n = 5 and WR: n = 7), (C) Adipose tissue leptin mRNA expression (CON: n = 6, ABA: n = 6 and WR: n = 6. and the hypothalamic expression of the neuropeptides D.Agrp, E.Npy, F.Pomc, and G.Cart in CON (n = 6–8), ABA (n = 7–8) and WR (n = 8) mice. Data was expressed as mean SEM and analyzed with two-tailed one-way ANOVA followed by two-stage linear step-up procedure of Benjamini, Kreiger and Yekutieli with an FDR of 0.1. Significance was set at p < 0.5

Fig. 3

Metabolic memory of AN. (A) Principal component analysis of all genes CON (n = 6), ABA (n = 6) and WR (n = 6), (B) Venn diagram showing the number of differentially and similarly expressed genes after intersecting the DEGs between ABAvsCON, WRvsCON and WRvsABA. (C) Volcano plot of the DEGs, (D) Heatmap of the top 25 upregulated and downregulated genes filtered using adjusted p value, (E) Top 10 enriched pathways using GSEA in ABAvsCON. (F) Volcano plot of the DEGs, (G) Heatmap of the top 25 upregulated and downregulated genes filtered using adjusted p value, (H) Top 10 enriched pathways using GSEA in WRvsCON, (I) Volcano plot of the DEGs, (J) Heatmap of the top 25 upregulated and downregulated genes filtered using adjusted p value, K. Top 10 enriched pathways using GSEA in WRvsABA. Red points and bars mean upregulated, blue point and bars mean downregulated, black points are not different bewtween the two groups compared