Elevated body fat increases amphetamine accumulation in brain: evidence from genetic and diet-induced forms of adiposity

Abstract

Despite the high prevalence of obesity, little is known about its potential impact on the pharmacokinetics of psychotropic drugs. In the course of investigating the role of the microRNA system on neuronal signaling, we found that mice lacking the translin/trax microRNA-degrading enzyme display an exaggerated locomotor response to amphetamine. As these mice display robust adiposity in the context of normal body weight, we checked whether this phenotype might reflect elevated brain levels of amphetamine. To assess this hypothesis, we compared plasma and brain amphetamine levels of wild type and Tsn KO mice. Furthermore, we checked the effect of diet-induced increases in adiposity on plasma and brain amphetamine levels in wild type mice. Brain amphetamine levels were higher in Tsn KO mice than in wild type littermates and correlated with adiposity. Analysis of the effect of diet-induced increases in adiposity in wild type mice on brain amphetamine levels also demonstrated that brain amphetamine levels correlate with adiposity. Increased adiposity displayed by Tsn KO mice or by wild type mice fed a high-fat diet correlates with elevated brain amphetamine levels. As amphetamine and its analogues are widely used to treat attention deficit disorder, which is associated with obesity, further studies are warranted to assess the impact of adiposity on amphetamine levels in these patients.

Fig. 1. Localization of TN and TX to DA neurons and striatal neurons.

A Double immunostaining of midbrain sections for TH (red) and TN or TX (green) shows TN or TX expression located in the VTA or substantia nigra. B Immunostaining in striatal medium spiny neurons shows prominent nuclear TX staining (green) in DARPP-32 positive neurons (red) (top row). TX staining of sections from mice expressing tdTomato in D1R + neurons demonstrates that TX is expressed in both D1R-positive neurons as well as in D1R-negative neurons (bottom row). Scale bar, 30 μm.

Fig. 2. Tsn KO mice display increased locomotor response to amphetamine (AMPH) but normal levels of tissue DA, TH, DAT and D2R.

A Amphetamine-induced (2.5 mg/kg, i.p.) locomotor activity is increased in male KO mice. Locomotor activity was monitored every 5 min and arrowheads indicate the time of injections. n = 8/group. Two-way ANOVA with RM revealed a significant effect of time (p < 0.0001), a significant effect of genotype (p = 0.0095), as well as a significant interaction between time X genotype (p < 0.0001). Bonferroni post-hoc testing was done to identify individual time points that were significantly different. **p < 0.01, and ***p < 0.001. B Immunostaining shows comparable TH staining of DA neurons (left panel) and striatal terminals (right panel) in WT and translin KO mice. Scale bar, 500 μm. C Tissue DA levels in the nucleus accumbens (NAc) and striatum (STR) are similar in WT and translin KO mice. n = 5–6/group. D, E Immunoblotting shows normal TH, DAT, D2R expression levels in NAc and STR in translin KO mice. Image J was used for quantification. n = 6/group. Data are expressed as mean ± SEM. Statistical significance was assessed by Student’s t-test.

Fig. 3. Correlation of fat mass with AMPH-induced locomotor activity and AMPH levels in plasma and brain.

A Global, conditional deletion of Tsn during adulthood does not affect the locomotor response to AMPH (2.5 mg/kg, i.p.). B The total locomotor activity induced by AMPH (2.5 mg/kg i.p.) in WT and Tsn KO mice was correlated with % fat mass (r = 0.5571, P = 0.025). n = 8/group. C Plasma AMPH levels (in nmol/mL) (left), and correlation with % fat mass (right; r = 0.744, P = 0.0055). 2.5 mg/kg AMPH was injected i.p. based on body weight. n = 6/group. D Brain AMPH levels (in nmol/g) (left), and correlation with % fat mass (right; r = 0.919, p < 0.0001). 2.5 mg/kg AMPH was injected i.p. based on body weight. n = 6/group. E Plasma and brain AMPH levels when 3.5 mg/kg AMPH was injected i.p. based on lean mass. n = 6/group. F Prior to i.p. injection of 3.5 mg/kg based on lean mass, baseline locomotor activity monitored following saline injection was lower in Tsn KO mice than in WT littermates. G The normalized locomotor response to AMPH shown as a percentage of the baseline level (100%) is comparable between groups. n = 7-8/group. Data are expressed as Mean ± SEM. Statistical significance indicated by asterisks was assessed by Student’s t-test. **p < 0.01.

Fig. 4. Effect of HF diet and adiposity on AMPH-induced locomotor activity and AMPH levels in plasma and brain.

A The baseline locomotor activity was lower in WT mice fed the HF diet (HF), compared to those that were given limited access to HF diet (LMHF). n = 8/group. B The normalized locomotor response to AMPH is higher in HF mice compared to Chow or LMHF mice. Two-way ANOVA with RM revealed a significant effect of time (p < 0.0001), a significant effect of group (p = 0.0015), as well as a significant interaction between the time X group (p = 0.0001). Different letters indicate statistically significant differences (p < 0.05) at individual time points based on Bonferroni post-hoc testing. C Adiposity (% Fat Mass) values for each mouse used for locomotor testing. D Correlation between % fat mass and AMPH locomotor activity for mice in all three groups falls just outside the significance threshold (p = 0.0568). E Limiting correlation to HF mice yields a p value (0.0466) just within the threshold for significance. F Adiposity (% Fat Mass) values for each mouse used for determination of amphetamine levels are shown. Adiposity of the HF group is significantly greater than that of the LMHF group. n = 6/group. G Panel at left shows that plasma amphetamine levels are higher in Chow or HF groups compared to LMHF group. The right panel shows a strong correlation between % fat mass and plasma amphetamine levels (p < 0.0001). H Panel at left shows that brain amphetamine levels in the HF group are higher than those in the LMHF group. The panel at right shows a strong correlation between % fat mass and brain amphetamine levels (p = 0.0005).