Nuclear receptor 5A2 regulation of Agrp underlies olanzapine-induced hyperphagia

Abstract

Antipsychotic (AP) drugs are efficacious treatments for various psychiatric disorders, but excessive weight gain and subsequent development of metabolic disease remain serious side effects of their use. Increased food intake leads to AP-induced weight gain, but the underlying molecular mechanisms remain unknown. In previous studies, we identified the neuropeptide Agrp and the transcription factor nuclear receptor subfamily 5 group A member 2 (Nr5a2) as significantly upregulated genes in the hypothalamus following AP-induced hyperphagia. While Agrp is expressed specifically in the arcuate nucleus of the hypothalamus and plays a critical role in appetite stimulation, Nr5a2 is expressed in both the CNS and periphery, but its role in food intake behaviors remains unknown. In this study, we investigated the role of hypothalamic Nr5a2 in AP-induced hyperphagia and weight gain. In hypothalamic cell lines, olanzapine treatment resulted in a dose-dependent increase in gene expression of Nr5a2 and Agrp. In mice, the pharmacological inhibition of NR5A2 decreased olanzapine-induced hyperphagia and weight gain, while the knockdown of Nr5a2 in the arcuate nucleus partially reversed olanzapine-induced hyperphagia. Chromatin-immunoprecipitation studies showed for the first time that NR5A2 directly binds to the Agrp promoter region. Lastly, the analysis of single-cell RNA seq data confirms that Nr5a2 and Agrp are co-expressed in a subset of neurons in the arcuate nucleus. In summary, we identify Nr5a2 as a key mechanistic driver of AP-induced food intake. These findings can inform future clinical development of APs that do not activate hyperphagia and weight gain.

Fig. 1. OLZ treatment is associated with elevated Nr5a2 expression.

OLZ treatment of hypothalamic cells results in dose-dependent increase in expression of Nr5a2 (A, B) and Agrp (C, D) as determined by qPCR (A, C) and western blotting (B, D). Mice that are highly Prone to Antipsychotic-Induced Weight Gain (AIWG-P) have significantly elevated hypothalamic levels of Nr5a2 (E, F) and Agrp (G, H) gene expression compared with AIWG-Resistant mice (AIWG-R) at both the gene expression (E, G) and protein level (F, H). E Data passed the Shapiro–Wilk test for normality and is expressed as mean ± SEM and was analyzed using either one-way ANOVA followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli with a false discovery rate of 0.10 (A, C, n = 3–8 replicates per group) or Student’s t test (B, D–H, n = 3–10 replicates per group), * denotes statistical significance at p < 0.05.

Fig. 2. Systemic NR5A2 antagonist treatment reduces food intake and weight gain in mice treated with olanzapine.

A Nr5a2 expression, B average daily food intake, C weight gain, D hypothalamic neuropeptide expression in C57BL/6 WT female mice fed either control diet (CON) or OLZ diet and injected with NR5A2 antagonist (SR1848, “SR”, 30 mg/kg) or vehicle (VEH) for 7 days determined by real time RT-qPCR assays. E NR5A2 expression in nuclear fraction of hypothalamic cells lines treated with SR1848 (5 μM for 24 h). F Quantitative PCR determination of Agrp expression in hypothalamic cells treated with SR1848 (1 or 5 μM) for 6 h. G AGRP protein expression in hypothalamic cells treated with SR1848 (1 μM for 24 h). Data passed the Shapiro–Wilk test for normality and are expressed as mean ± SEM and was analyzed using either one-way ANOVA followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli with a false discovery rate of 0.10 (A–D, H, I, n = 7–10), or Student’s t test (E–G, n = 4–11 replicates per group), * denotes statistical significance at p < 0.05. ** denotes statistical significance at p < 0.01.

Fig. 3. Hypothalamic knockdown of Nr5a2 significantly bunts OLZ-induced food intake and weight gain.

A–C Bilateral injection of AAV2-eSyn-EGFP (120–150 nl) into the ARC (A–P: −1.58 mm from Bregma; M–L ± 0.25 mm from midline; D–V: −5.8 mm into the skull) (Blue: DAPI, Green: GFP, white dotted lines show ARC boundaries). siRNA-mediated knock down of Nr5a2, delivered by stereotaxic injection to the arcuate nucleus, results in D reduced expression of Nr5a2, E blunted OLZ-induced food intake and F, G reduced OLZ-induced weight gain and body fat (H), Data passed the Shapiro–Wilk test for normality and are expressed as mean ± SEM and were analyzed using either one-way ANOVA followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli with a false discovery rate of 0.10 (D–H, n = 4–5), * denotes statistical significance at p < 0.05.

Fig. 4. Agrp KO mice are resistant to OLZ-induced hyperphagia and weight gain.

Generation and characterization of Agrp^−/− mice, A Schematic of the wildtype (WT, +) and knockout (KO, −) alleles. Multiple in-frame (underlined bold) and out-of-frame (underlined) stop codons are inserted into the coding sequences in the exon 5 of Agrp^−/− mice. B PCR genotyping of Agrp^+/+ and Agrp^−/− mice. C Immunostaining of AGRP proteins in the ARC of Agrp^+/+ and D Agrp^−/− mice. 3V the third ventricle, ARC arcuate nucleus of the hypothalamus. Scale bar is 50 µm. E Food intake, F, G weight gain, H hypothalamic gene expression in WT and KO mice treated with CON or OLZ diets. Data passed the Shapiro–Wilk test for normality and are expressed as mean ± SEM and were analyzed using either one-way ANOVA followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli with a false discovery rate of 0.10 (E–H, n = 9–12 per group), * denotes statistical significance at p < 0.05.

Fig. 5. Agrp is a direct transcriptional target of NR5A2.

A Agrp promoter region. Chromatin immunoprecipitation with NR5A2 antibodies followed by PCR (ChIP-PCR) in the hypothalamic cell line (mHypo-59A) result in enrichment of binding to the B. Prox1 promoter (positive control), Prox1 LOCA (negative control), Agrp promoter region compared with beads without antibody. Data are expressed as mean ± SEM and were analyzed using Student’s t test (n = 2–4 replicates per group), ** denotes statistical significance at p < 0.01. C Expression of Agrp and Nr5a2 across ARC-ME neuronal populations from GEO dataset GSE93374. Scaling is relative to each gene’s expression across all cells in a given annotation selection, i.e., cells associated with each column label in the dot plot.