Development and initial characterization of a novel ghrelin receptor CRISPR/Cas9 knockout wistar rat model

Abstract

Background/objectives: Ghrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts by binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing.

Methods: Using CRISPR/Cas9, we developed a GHSR KO in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood.

Results: The RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT; p = 0.0322).

Conclusions: We have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.

Figure 1. Sequences for gRNAs and Genomic structure of GHSR knockout allele

(A) gRNA sequences used to target GHSR gene, with the RNAscope GHSR mRNA probe specified. (B) The coding region for GHSR consists of two exons and spans 3.1 kilobases. Four gRNAs, one “upstream” pair and one “downstream” pair were chosen to flank the first exon of GHSR in rats. The GHSR(KO) allele (T-1M) removes 846 nucleotides and encompasses exon 1. Arrows indicate relative locations of Forward and Reverse genotyping primers (sequence provided in text). GHSR: growth hormone secretagogue receptor; KO: knockout; WT: wild type.

Figure 2. GHSR mRNA expression

WT rats exhibit GHSR mRNA expression in midbrain, hypothalamus, hippocampus but no detectable expression in dorsal striatum and prefrontal cortex. In GHSR KO rats, no detectable mRNA expression was found in any brain region examined. Low magnification scale bar = 1 mm, High magnification scale bar = 20 μm. GHSR: growth hormone secretagogue receptor; KO: knockout; WT: wild type.

Figure 3. Blood GH concentrations after IP ghrelin challenge

GH concentrations (mean + SEM) in GHSR KO (purple; n = 8) and WT (blue; n = 8) rats 15 min after IP injections of saline or acyl-ghrelin (4.5 nmol/kg). In the WT group, IP ghrelin administration increased blood GH concentrations compared to vehicle (p = 0.001). There was no effect of IP ghrelin on blood GH concentrations in the GHSR KO group. The GHSR KO group had lower concentrations of GH compared to the WT group regardless of vehicle or ghrelin treatment (p < 0.01). GH: growth hormone; GHSR: growth hormone secretagogue receptor; IP: intraperitoneal; KO: knockout; WT: wild type.

Figure 4. Food and water intake after IP ghrelin challenge

Food intake (mean + SEM) in GHSR KO (purple; n = 8) and WT (blue; n = 8) rats after IP injections of saline or acyl-ghrelin (3 nmol/kg or 6 nmol/kg) measured at 1 (A), 2 (B) and 6 h (C) post injection. In the WT group, both doses of ghrelin increased food consumption at the 1 h time point compared to vehicle (p < 0.05). There was no effect of IP ghrelin administration on food consumption in the GHSR KO group. Across time points, the GHSR KO group consumed less food than the WT regardless of vehicle or ghrelin treatment (p < 0.05, overall genotype effect; p < 0.05, different chow consumption from 0 nmol ghrelin (saline)). There was no effect of IP ghrelin administration on water intake at 1 (D), 2 (E) or 6 h (F) post injection, and there was no difference in water intake between the GHSR KO and WT groups. GHSR: growth hormone secretagogue receptor; IP: intraperitoneal; KO: knockout; SEM: standard error of the mean; WT: wild type.

Figure 5. Locomotion and anxiety-like behavior

(A) There was no difference between locomotor activity as measured by crossover beam breaks over 12 h in activity chambers (mean ± SEM) between GHSR KO (purple; n = 8) and WT rats (blue; n = 8). (B) There was no difference in distance moved in either the center or the periphery of the open field (mean ± SEM) between GHSR KO (purple; n = 8) and WT rats (blue; n = 8). GHSR: growth hormone secretagogue receptor; KO: knockout; WT: wild type.

Figure 6. Body weight, BAT, gonadal fat, and inguinal fat

(A) Body weight (mean ± SEM) of GHSR KO (purple; n = 8) and WT (blue; n = 8) rats in the juvenile/early adulthood stage of development and during the experimental phase. GHSR KO rats weighed significantly less than WT controls in both stages (p < 0.05, overall genotype effect; see main text for details). (B) GHSR KO rats (purple; n = 8) had significantly more BAT as a percentage of body weight than GHSR WT (blue; n = 8). (C) There was no difference between concentrations of gonadal fat as a percentage of body weight between GHSR KO (purple; n = 8) and WT rats (blue; n = 8). (D) There was no difference between concentrations of inguinal fat as a percentage of body weight between GHSR KO (purple; n = 8) and WT rats (blue; n = 8). GHSR: growth hormone secretagogue receptor; KO: knockout; WT: wild type; BAT: brown adipose tissue.