Fearful dogs have increased plasma glutamine and γ-glutamyl glutamine

Abstract

Anxiety-related disorders, including fearfulness are common and leading welfare problems among the worldwide dog population. The etiology of anxieties is complex and affected by genetic and environmental factors. Thus, there is a need for more comprehensive approaches, such as metabolomics, to understand the causes of anxiety and to identify anxiety-related biomarkers for more efficient diagnostic and treatment options. To study metabolic alterations related to canine fearfulness, a non-targeted plasma metabolite profiling was performed in a cohort of 20 fearful and 21 non-fearful dogs. The results showed that nine metabolic features were significantly associated with fearfulness. The most prominent change included increased plasma glutamine and γ-glutamyl glutamine (γ-Glu Gln) in fearful dogs across breeds. Alterations in glutamine metabolism have previously been associated with several psychiatric disorders, indicating the relevance of this finding also in dogs. In addition, we describe a novel breed-specific association between renal biomarker symmetric dimethylarginine (SDMA) and canine fearfulness. These observed metabolic alterations may result from high levels of prolonged psychological stress in fearful dogs.

Figure 1

Summary of data processing and statistical analysis steps prior to metabolite identification. N_m, number of metabolites; PLS-DA, partial least-squares discriminant analysis; VIP, variable importance on projection; FC, fold change.

Figure 2

Distributions of VIP scores and fold changes among statistically significant metabolites. (a) Distribution of VIP scores among statistically significant metabolites. (b) Distribution of fold changes among statistically significant metabolites. Dots indicate positive fold changes whereas squares indicate negative fold changes. SDMA, symmetric dimethylarginine; γ-Glu Gln, γ-glutamylglutamine; VIP, variable importance on projection.

Figure 3

Breed-specific heat maps of metabolic differences between fearful and non-fearful dogs. (a) Metabolic differences between fearful and non-fearful German Shepherds. (b) Metabolic differences between fearful and non-fearful Great Danes. Heat maps were generated with hierarchical clustering. Included are statistically significant metabolic features having VIP >1, FC ≥ ±1.2 and Mann-Whitney U p_FDR < 0.05. The metabolites are listed at the left side of each row, and the subjects are shown at the bottom of each column. The colour scale indicates high (red) or low (blue) metabolite abundance. SDMA, symmetric dimethylarginine; γ-Glu Gln, γ-glutamylglutamine.

Figure 4

Statistically significant metabolites and their association with fearfulness. Included are metabolites that show statistical significance (p < 0.05) in standard logistic regression models. Error bars indicate 95% confidence limits. γ-Glu Gln, γ-glutamylglutamine; SDMA, symmetric dimethylarginine.

Figure 5

Effects of glutamine and SDMA peak areas on fearfulness in Great Danes and German Shepherds. (a) Association between glutamine peak area and fearfulness in Great Danes. The higher the glutamine amount is, the higher is the dog’s probability of being fearful when the dog is Great Dane. (b) Association between glutamine peak area and fearfulness in German Shepherds. The higher the glutamine amount is, the higher is the dog’s probability of being fearful when the dog is German Shepherd. (c) Association between SDMA peak area and fearfulness in Great Danes. The higher the SDMA amount is, the higher is the dog’s probability of being fearful when the dog is Great Dane. (d) Association between SDMA peak area and fearfulness in German Shepherds. SDMA has a slight, non-significant negative association with fearfulness when the dog is German Shepherd. Dashed lines indicate 95% confidence limits. SDMA, symmetric dimethylarginine.