Impact of Serotonin Transporter Absence on Brain Insulin Receptor Expression, Plasma Metabolome Changes, and ADHD-like Behavior in Mice fed a Western Diet

Abstract

The impaired function of the serotonin transporter (SERT) in humans has been linked to a higher risk of obesity and type 2 diabetes, especially as people age. Consuming a "Western diet" (WD), which is high in saturated fats, cholesterol, and sugars, can induce metabolic syndrome. Previous research indicated that mice carrying a targeted inactivation of the Sert gene (knockout, KO) and fed a WD display significant metabolic disturbances and behaviors reminiscent of ADHD. These abnormalities might be mediated via a dysfunction in insulin receptor (IR) signaling, which is also associated with adult ADHD. However, the impact of Sert deficiency on IR signaling and systemic metabolic changes has not been thoroughly explored. In this study, we conducted a detailed analysis of locomotor behavior in wild-type (WT) and KO mice fed a WD or control diet. We investigated changes in the blood metabolome and examined, via PCR, the expression of insulin receptor A and B isoforms and key regulators of their function in the brain. Twelve-month-old KO mice and their WT littermates were fed a WD for three weeks. Nuclear magnetic resonance spectroscopy analysis of plasma samples showed that KO mice on a WD had higher levels of lipids and lipoproteins and lower levels of glucose, lactate, alanine, valine, and isoleucine compared to other groups. SERT-KO mice on the control diet exhibited increased brain levels of both IR A and B isoforms, accompanied by a modest increase in the negative regulator ENPP. The KO mice also displayed anxiety-like behavior and reduced exploratory activity in an open field test. However, when the KO animals were fed a WD, the aberrant expression levels of IR isoforms in the KO mice and locomotor behavior were ameliorated indicating a complex interaction between genetic and dietary factors that might contribute to ADHD-like symptoms. Overall, our findings suggest that the lack of Sert leads to a unique metabolic phenotype in aged mice, characterized by dysregulated IR-related pathways. These changes are exacerbated by WD in the blood metabolome and are associated with behavioral abnormalities.

Keywords: ADHD; Sert-deficient mice; aging; glucose tolerance; insulin receptors; metabolomics; serotonin transporter; western diet.

Figure 1

Experiment design: Flow diagram of experimental paradigm paradigm assessing the impact of the WD on WT and SERT-KO mice. (A) In the first study, a glucose tolerance test was carried out on Day 22, blood was harvested for biochemical assay on Day 24. (B) In the second study, behavioral evaluation and the open field test was performed on day 21. After the last behavioral assessment, mice were sacrificed on Day 22, and the brains were dissected, blood was collected. Brain was harvested to be used for a subsequent RNA isolation and RT-qPCR assay blood was used in the metabolome assay.

Figure 2

Physiological and metabolic changes in SERT-KO and WT mice on a WD. We found an effect of genotype on body weight at week 1 (A) and week 2 (B). At week 3 (C), significant diet and genotype effects were observed, with body weight increased in the normalized KO/WD group compared to 100%. There was an effect of diet on glucose tolerance (D), with the normalized WT/WD group showing an increased AUC compared to 100%. Significant diet and genotype effects on leptin levels in the blood were also found (E); both WT and KO mice normalized to their respective CD groups had increased leptin levels compared to 100%. Statistical analyses were performed using two-way ANOVA with Tukey’s post-hoc test, t-test (& p < 0.05), and one-sample t-test (# p < 0.05). (WT-CD group, n = 6, WT-WD group, n = 7, KO-SERT-CD group, n = 8, KO-SERT-WD group, n = 8). Black and diagonal bars represent WD-fed groups whose measurements were normalized to the respective genotype control diet group. All data are presented as mean ± SEM.

Figure 3

Distinct plasma metabolic profile changes in SERT-KO and WT mice housed on the WD. (A) PCA plot depicting how PC1 accounts for 40.8% of the variance in the data and PC2 for 25.8% which separated the data into four clusters. (B) OPLS-DA plots of the pairwise comparison revealed that the average accuracy for the models was high (*** p < 0.05, t-test) The true models performed significantly better than random chance in categorizing the samples in the test sets (* p < 0.001, Kolmogorov–Smirnov test) (C) A heatmap of the principal metabolites responsible for the group showing the relative relationship of each metabolite in each group. We found significant differences in the lipids, energy metabolites and amino acids in each group (n = 7 mice for each group).

Figure 4

Behavioral changes in the open field between SERT-KO and WT mice fed a WD. (A) Velocity normalized to respective CD groups was decreased in WT/WD and increased in KO/WD mice. (B) There was a significant genotype effect in the number of transitions between central zone and periphery. (C) Time spent in stretched posture normalized to CD and duration of stretched posture was decreased in WT/WD compared to 100%. (D) Interaction between diet and genotype in duration of grooming was revealed, this parameter when normalized to CD was increased in WT/CD compared to 100%. Two-way ANOVA and Tukey’s post-hoc test (* p < 0.05), t-test (& p < 0.05) and one sample t-test (# p < 0.05). Black and diagonal bars represent WD-fed groups whose measurements were normalized to the respective genotype control diet group. (WT-CD group, n = 6, WT-WD group, n = 7, KO-SERT-CD group, n = 8, KO-SERT-WD group, n = 8). All data are mean ± SEM.

Figure 5

Distinct expression of IR subtypes in the brain of SERT-KO and WT mice housed on WD. IRA expression levels were increased in KO/CD compared to WT/CD mice and decreased in KO/WD compared to KO/CD in (A) hippocampus and (B) dorsal raphe. (C) Significant diet and genotype effects were revealed in IRA expression level in prefrontal cortex. Both WT and KO groups normalized to respective CD had this measure decreased compared to 100%. (D) In hypothalamus, IRA expression levels were increased in KO/CD compared to WT/CD mice and decreased in KO/WD compared to KO/CD. (E) IRB expression was significantly decreased in hippocampus of KO/WD compared to KO/CD. Significant genotype effect was found for IRB expression in (F) dorsal raphe and (G) prefrontal cortex. In both structures it was decreased in normalized KO mice compared to 100%. (H) In hypothalamus, IRB expression was significantly higher in KO/CD mice compared to WT/CD and KO/WD animals. Two-way ANOVA and Tukey’s post-hoc test (* p < 0.05), t-test (& p < 0.05) and one sample t-test (# p < 0.05; n = 7 mice for each group). Black and diagonal bars represent WD-fed groups whose measurements were normalized to the respective genotype control diet group. All data are mean ± SEM.

Figure 6

WD-induced changes in brain expression of transcription factors in SERT-KO and WT mice. ACSL1 expression in the (A) hippocampus, (B) dorsal raphe, (C) prefrontal cortex, and (D) hypothalamus. ENPP expression in the (E) hippocampus, (F) dorsal raphe, (G) prefrontal cortex and (H) hypothalamus. PTPN1 expression in the (I) hippocampus, (J) dorsal raphe, (K) prefrontal cortex, (L) hypothalamus. PTEN expression in the (M) hippocampus, (N) dorsal raphe, (O) prefrontal cortex, and (P) hypothalamus. CD36 expression in the (Q) hippocampus, (R) dorsal raphe, (S) prefrontal cortex, and (T) hypothalamus. Two-way ANOVA and Tukey’s post-hoc test (* p < 0.05), t-test (& p < 0.05) and one sample t-test (# p < 0.05; n = 7 mice for each group). Black and diagonal bars represent WD-fed groups whose measurements were normalized to the respective genotype control diet group. All data are mean ± SEM.