SIRT1 accelerates the progression of activity-based anorexia

Abstract

Food consumption is fundamental for life, and eating disorders often result in devastating or life-threatening conditions. Anorexia nervosa (AN) is characterized by a persistent restriction of energy intake, leading to lowered body weight, constant fear of gaining weight, and psychological disturbances of body perception. Herein, we demonstrate that SIRT1 inhibition, both genetically and pharmacologically, delays the onset and progression of AN behaviors in activity-based anorexia (ABA) models, while SIRT1 activation accelerates ABA phenotypes. Mechanistically, we suggest that SIRT1 promotes progression of ABA, in part through its interaction with NRF1, leading to suppression of a NMDA receptor subunit Grin2A. Our results suggest that AN may arise from pathological positive feedback loops: voluntary food restriction activates SIRT1, promoting anxiety, hyperactivity, and addiction to starvation, exacerbating the dieting and exercising, thus further activating SIRT1. We propose SIRT1 inhibition can break this cycle and provide a potential therapy for individuals suffering from AN.

Fig. 1. SIRT1 BSKO and BSOX baseline phenotypes.

a Brain-specific knockout (BSKO) mice lack functional SIRT1 in the brain, while the brain-specific overexpressing (BSOX) mice overexpress SIRT1 in the brain. This is representative of three independent experiments. b BSKO mice (red) are significantly smaller than their littermates, while there are no differences between WT (black) and BSOX (green) mice. c, d No differences were observed in baseline food intake or activity of WT, BSKO, or BSOX mice. P values were calculated using unpaired two-tailed t tests. The box-plots represent the median, 25th, and 75th percentiles of the data and the whiskers represent 5–95% of the data. Six mice were used in each cohort. NS, not significant. Source data are provided as a Source Data file.

Fig. 2. SIRT1 activity amplifies AN-like phenotypes in animals.

a BSKO mice (red) subjected to ABA are resistant to the model and lose weight slower compare to their WT littermates (black). Weight loss in fraction of initial body weight over time is presented. b BSOX mice (green) are more susceptible to ABA and showed faster weight loss. c The BSKO mice show protection from the decline in food intake typically observed during the ABA model. d BSOX mice show no difference in food intake compared to their WT littermates. e The BSKO mice also do not upregulate their activity as much as their WT littermates. f The BSOX mice have an elevated physical activity throughout the ABA model. g Selisistat-treated mice (blue), similarly to the BSKO mice, show protection from body weight loss throughout the ABA experiment. h Selisistat-treated mice consume roughly similar amounts of food during ABA. i Mice treated with selisistat do not upregulate physical activity during ABA. Statistical analysis was completed using two-way ANOVA with SEM error bars. n = 6 for the BSKO and BSOX mice and their WT littermates. n = 6 for the vehicle control and n = 7 for the selisistat-treated mice. Source data are provided as a Source Data file.

Fig. 3. SIRT1 activity, mediated by NRF1, suppresses the expression of Grin2A.

a RT-qPCR expression analysis of Grin2A in the cortex of BSKO- (red), BSOX- (green), and selisistat- (selis.) (blue) treated mice, n ≥ 12. b Expression analysis of Grin1A gene in the same mice as in a, n ≥ 7. c, d Modulation of SIRT1 abundance in vitro in Neuro-2a cells directly influences the activity of the Grin2A promoter. c SIRT1 protein levels in Neuro-2a cells are shown, together with an example of a typical SDS-PAGE, n ≥ 5. d The activity of the Grin2A-luciferase reporter in the same cells as in c is presented, n ≥ 12. e Using siRNAs for eight transcription factors (TFs), we transfected Neuro-2a cells along with the 2 kb Grin2A promoter, and T1OX or T1KO constructs. NRF1 was the only TF that eliminated the impact of SIRT1 on Grin2A transcription, n ≥ 6. f Transfecting Neuro-2a cells with a Grin2A promoter with its NRF1-binding site mutated and the T1KO and T1OX constructs resulted in a complete loss of responsiveness to SIRT1, n ≥ 9. P values were calculated using unpaired two-tailed t tests, *P < 0.05, **P < 0.005. Error bars are SEM. The box-plots represent the median, 25th, and 75th percentiles of the data and the whiskers represent 5–95% of the data. NS, not significant. Source data are provided as a Source Data file.

Fig. 4. Grin2A partially mediates multipronged action of SIRT1 in progression of ABA.

a Schematic representation of Grin2A knockout mouse generation using CRISPR/Cas9 technology. b RT-PCR quantification of Grin2A (orange) mRNA in brains of transgenic mice is presented. Truncated Grin2A mRNA is expressed at nearly WT levels. P values were calculated using unpaired two-tailed t tests, error bars are SEM, n = 6. c SDS-PAGE analysis of Grin2A expression in brains of Grin2A knockout mice. No Grin2A protein is expressed in transgenic line of mice. This is representative of three independent experiments. d Grin2A knockout mice (orange) do not benefit from pharmaceutical SIRT1 inhibition during ABA progression. Both selisistat- and saline-treated animals lose weight at identical rate (contrast to WT mice (blue) treated with selisistat, shown in Fig. 2g, and e have similar food consumption. f Selisistat injections have modest impact on ABA-induced hyperactivity in Grin2A-null background. Statistical analysis is done using two-way ANOVA with SEM error bars, n = 8. g Proposed multipronged mechanism of SIRT1 action in progression of AN. Source data are provided as a Source Data file.