Protein-coding mutation in Adcy3 increases adiposity and alters emotional behaviors sex-dependently in rats

Abstract

Objective: Adenylate cyclase 3 (Adcy3) has been linked to both obesity and major depressive disorder (MDD). Our lab identified a protein-coding variant in the 2^nd transmembrane (TM) helix of Adcy3 in rats, and similar obesity variants have been identified in humans. The current study investigates the role of a TM variant in adiposity and behavior.

Methods: We used CRISPR-SpCas9 to mutate the TM domain of Adcy3 in WKY rats (Adcy3^mut/mut). We also created a heterozygous knockout rat in the same strain (Adcy3^+/-). Wild-type (WT), Adcy3^+/-, and Adcy3^mut/mut rats were fed a high-fat diet for 12 weeks. We measured body weight, fat mass, glucose tolerance, food intake, metabolism, emotion-like behaviors, and memory.

Results: Adcy3^+/- and Adcy3^mut/mut rats weighed more than WT rats due to increased fat mass. There were key sex differences: adiposity was driven by increased food intake in males but by decreased energy expenditure in females. Adcy3^mut/mut males displayed increased passive coping and decreased memory while Adcy3^mut/mut females displayed increased anxiety-like behavior.

Conclusions: These studies show that the ADCY3 TM domain plays a role in protein function, that Adcy3 may contribute to the relationship between obesity and MDD, and that sex influences the relationships between Adcy3, metabolism, and behavior.

Keywords: Obesity; adenylate cyclase; depression; genetics; rat models.

Figure 1.. Development of Adcy3 KO and Adcy3^mut/mut rats and confirmation of Adcy3 expression.

(A) Adcy3 knockout (KO) has a single base pair deletion in the transmembrane domain, causing a frameshift mutation and whole-body KO of the gene. Because Adcy3 KO is lethal, Adcy3^+/− rats were used for experiments. Adcy3^mut/mut has a 3-base pair deletion in the same transmembrane region, causing a F122delV123L protein-coding mutation. (B) Adcy3^+/− males (M) and females (F) express less Adcy3 mRNA than wild type (WT) rats in retroperitoneal white adipose (RetroFat). Adcy3^mut/mut does not have altered Adcy3 mRNA expression in RetroFat. (C) M and F Adcy3^+/− rats express less ADCY3 in RetroFat than WT rats. Adcy3^mut/mut rats do not have altered ADCY3 RetroFat expression. (D) Representative Western Blot image comparing ADCY3 expression across genotypes. GAPDH was used as a loading control. Mean ± SEM. T-test, *p<0.05, **p<0.01, ***p<0.001

Figure 2.. Study design, body weight, and body composition in Adcy3^+/− and Adcy3^mut/mut rats.

(A) Timeline of the study shown in weeks of age. Metabolic phenotyping included weekly measurement of body weight and cage-wide food intake, EchoMRI, intraperitoneal glucose tolerance test (IPGTT), individual housing to measure food intake, TSE PhenoMaster chambers, and euthanasia (sac). Behavioral phenotyping included the open field test (OFT), novel object recognition test (NOR), and forced swim test (FST). (B) Adcy3^+/− males (M) and females (F) gain more weight than wild-type (WT) rats over the course of the study. (C) Adcy3^mut/mut M and F gain more weight than WT rats over the course of the study. (D) There are no differences in fat mass prior to HFD start, except in Adcy3^mut/mut M, which have slightly more fat mass than WT M. (E) After 8 weeks on diet (WOD), all 4 groups have more fat mass than WT rats. Mean ± SEM. T-test or rmANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 3.. Adcy3^+/− and Adcy3^mut/mut fat pads and adipocytes at sac.

(A) Adcy3^+/− males (M) and females (F) have larger retroperitoneal (RetroFat), gonadal, and omental (OmenFat) fat pads at sac than wild-type (WT). (B) Adcy3^mut/mut M and F also have larger RetroFat, gonadal fat, and OmenFat pads at sac than WT. (C) Adcy3^+/− and Adcy3^mut/mut M and F all have significantly larger adipocytes than WT. (D) Representative images of hematoxylin and eosin-stained RetroFat from female rats. Scale bars = 250μm. (E) Relative frequency distributions of adipocyte size for each group. Mean ± SEM. T-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 4.. Sex differences in the cause of adiposity in Adcy3^+/− and Adcy3^mut/mut as measured in TSE PhenoMaster chambers.

Plots of 24 hours of average hourly energy expenditure (EE), cumulative EE, respiratory exchange ratio (RER), and cumulative food intake in (A) Adcy3^+/− males (M), (B) Adcy3^+/− females (F), (C) Adcy3^mut/mut M, and (D) Adcy3^mut/mut F compared to wild-type (WT). Adcy3^+/− and Adcy3^mut/mut M have increased food intake, while Adcy3^+/− and Adcy3^mut/mut F have decreased EE. Only Adcy3^+/− M had increased RER. Gray shading indicates dark cycle. Mean ± SEM. ANCOVA, *p<0.05, **p<0.01

Figure 5.. Cage-wide and individual food intake in Adcy3^+/− and Adcy3^mut/mut.

(A) No differences in cage-wide weekly food intake in Adcy3^+/− males (M) (B) or Adcy3^+/− females (F). (C) No differences in individual food intake over one week in Adcy3^+/− M or F. (D) Adcy3^mut/mut M consume more food than wild-type (WT) M. (E) No differences in cage-wide weekly food intake in Adcy3^mut/mut F. (F) Adcy3^mut/mut M, but not F, consume more food than WT when measured individually over one week. HFD: high-fat diet. Mean ± SEM. T-test or rmANOVA, *p<0.05

Figure 6.. Measures of glucose tolerance and insulin resistance in Adcy3^+/− and Adcy3^mut/mut.

(A) Only Adcy3^+/− males (M) have increased blood glucose compared to wild-type (WT) after an overnight fast. (B) Adcy3^+/− and Adcy3^mut/mut M have increased fasting insulin after an overnight fast, with no differences in females (F). (C) Adcy3^+/− and Adcy3^mut/mut M have increased insulin resistance compared to WT as measured by HOMA-IR. There were no differences in HOMA-IR in F. Mean ± SEM. T-test, *p<0.05, **p<0.01

Figure 7.. Adcy3^mut/mut behaviors in the open field test (OFT), novel object recognition test (NOR), and forced swim test (FST).

(A) Adcy3^mut/mut females (F), but not males (M), spend significantly less time in the center than wild-type (WT). (B) Adcy3^mut/mut M rear significantly more and Adcy3^mut/mut F rear significantly less than WT. (C) Adcy3^mut/mut M tend to groom more than WT M. (D) There were no differences in line crossings in Adcy3^mut/mut M or F. (E) “A”: familiar object, “B”: novel object. Adcy3^mut/mut and WT M both spend significantly more time with the novel object than the familiar object, but Adcy3^mut/mut M tend to spend less time with the novel object compared to WT M, potentially indicating impaired memory. (F) Although Adcy3^mut/mut F also spend more time with the novel object than with the familiar object, there were no differences in NOR behavior compared to WT. (G) Adcy3^mut/mut M spend more time immobile in the FST than WT M. There were no differences in behavior in Adcy3^mut/mut F. Mean ± SEM. T-test, *p<0.05, **p<0.01, ***p<0.001