Lateral Hypothalamic Neurotensin Neurons Orchestrate Dual Weight Loss Behaviors via Distinct Mechanisms

Abstract

The central mechanism by which neurotensin (Nts) potentiates weight loss has remained elusive. We leveraged chemogenetics to reveal that Nts-expressing neurons of the lateral hypothalamic area (LHA) promote weight loss in mice by increasing volitional activity and restraining food intake. Intriguingly, these dual weight loss behaviors are mediated by distinct signaling pathways: Nts action via NtsR1 is essential for the anorectic effect of the LHA Nts circuit, but not for regulation of locomotor or drinking behavior. Furthermore, although LHA Nts neurons cannot reduce intake of freely available obesogenic foods, they effectively restrain motivated feeding in hungry, weight-restricted animals. LHA Nts neurons are thus vital mediators of central Nts action, particularly in the face of negative energy balance. Enhanced action via LHA Nts neurons may, therefore, be useful to suppress the increased appetitive drive that occurs after lifestyle-mediated weight loss and, hence, to prevent weight regain.

Keywords: drinking; energy balance; feeding; lateral hypothalamic area; locomotor activity; neurotensin; neurotensin receptor; obesity.

Figure 1. Examination of the LHA Nts➔VTA circuit in Nts^Cre;NtsR1⁺⁺ and Nts^Cre;NtsR1^KOKO mice

A) Nts^Cre mice with either intact or developmentally deleted NtsR1 (Nts^Cre;NtsR1⁺⁺ or Nts^Cre;NtsR1^KOKO mice) were injected bilaterally in the LHA with cre-inducible AAV-hm3Dq-mCherry, permitting chemoogenetic activation of LHA Nts neurons by CNO injection. B) i.p CNO treatment induces cFos expression in LHA Nts neurons (white arrows). Scale bar=50μM C) LHA Nts neurons expressing hM3Dq-mCherry in Nts^Cre;NtsR1⁺⁺ and Nts^Cre;NtsR1^KOKO mice (scale bar=200μm). D) hM3Dq-mCherrry-labeled LHA Nts cell bodies in the LHA and terminals in the VTA of Nts^Cre;NtsR1⁺⁺ and Nts^Cre;NtsR1^KOKO mice. E) Both NtsRs are expressed in the VTA, visualized using Ntsr1^Cre;GFP and Ntsr2^Cre;GFP mice. F) Ntsr1 and Ntsr2 mRNA expression in the VTA of Nts^Cre;NtsR1⁺⁺ mice and Nts^Cre;NtsR1^KOKO mice. Data represent mean ± SEM, n=10–11 per group. Data were analyzed by unpaired t-test for each gene, ***p<0.001. For D and E, scale bar=100μM. Abbreviations: f=fornix, ml=medial lemniscus, ip=interpeduncular nucleus.

Figure 2. Acute activation of LHA Nts neurons promotes energy expenditure and suppresses feeding

VEH or CNO-treated Nts^Cre;NtsR1⁺⁺and Nts^Cre;NtsR1^KOKO mice were analyzed in TSE metabolic cages. In A–H, each point represents 108 minutes and gray boxes denote the dark cycle. A, B) water intake, C, D) locomotor activity, E, F) energy expenditure, and G, H) feeding in Nts^Cre;NtsR1⁺⁺ and Nts^Cre;NtsR1^KOKO mice. I) total change in body weight 24 hours post-injection. J) Total weight-adjusted food intake over 24 hours. Data were analyzed by repeated measures two-way ANOVA with Sidak post-tests, and graphed data represent mean ± SEM. **p < 0.01, ***p < 0.001. Nts^Cre;NtsR1⁺⁺ n=10–11; Nts^Cre;NtsR1^KOKO n=9–10.

Figure 3. Acute NtsR1 or D1R blockade abolishes LHA Nts-induced suppression of feeding

Mice were pretreated with PBS, SR48692 (NtsR1 antagonist) or SCH23390 (D1R antagonist) 30 min. prior to VEH or CNO injection. Graphed data represent mean ± SEM over 24 hours post VEH or CNO injection. A–C) water intake, locomotor activity and feeding in Nts^Cre;NtsR1⁺⁺ mice pretreated with the NtsR1 antagonist. D–F) water intake, locomotor activity and feeding in Nts^Cre;NtsR1^KOKO mice pretreated with the NtsR1 antagonist. G–L) same parameters in Nts^Cre;NtsR1⁺⁺ or Nts^Cre;NtsR1^KOKO mice pretreated with a D1R antagonist (SCH23390). Data were analyzed by repeated measures two-way ANOVA with Sidak post-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001. Nts^Cre;NtsR1⁺⁺ n=10–11; Nts^Cre;NtsR1^KOKO n=9–10.

Figure 4. Chronic activation of LHA Nts neurons induces mild weight loss in chow-fed lean mice

Nts^Cre;NtsR1⁺⁺and Nts^Cre;NtsR1^KOKO mice were treated once daily with VEH or CNO for five consecutive days. A) chow intake, B) body weight, C) 1 hr locomotor activity, and D) water intake in Nts^Cre;NtsR1⁺⁺ mice (n=11). E) chow consumption, F) body weight, G) 1 hr locomotor activity, and H) water intake in Nts^Cre;NtsR1^KOKO mice (n=11). Data were analyzed by repeated-measures two-way ANOVA with Sidak post-tests, except for locomotor activity which was analyzed by paired t-test. Graphed data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.

Figure 5. Repeated activation of LHA Nts neurons does not restrain ad lib palatable food intake in obese or lean mice

LHA Nts neurons of diet-induced obese mice were activated for 5 days. A) daily locomotor activity during the light cycle vs. B) dark cycle, C) plot showing average rate of energy expenditure vs. body weight (p=0.44), D) food intake during light cycle compared to E) dark cycle, F) percent original body weight over five days of chronic VEH or CNO injections. VEH n=6, CNO n=5–6. Data were analyzed by standard two-way ANOVA except for c which was analyzed by ANCOVA. Asterisks indicate significant difference between VEH and CNO at given time point, *p < 0.05, **p < 0.01, ***p < 0.001. In f, (#) represents significant difference in body weight at days 4–5 compared to day 0 in the VEH group, #p <0.05. For g-j, lean Nts^Cre;NtsR1⁺⁺ mice were injected each morning on VEH or CNO for 10 days with ad lib access to HF diet in home cages. G) HF diet intake during the light cycle, H) total food consumed over 10 days, I) 1 hour locomotor activity, and J) body weight in lean Nts^Cre;NtsR1⁺⁺ mice (n=10). *p<0.05, **p<0.01, ***p<0.001 between VEH and CNO from Days 4–10. #p<0.05 for CNO time point compared to Day 1. Daily food intake and body weight were analyzed by repeated-measures two-way ANOVA with Sidak post-tests. Total food intake and locomotor activity was analyzed by paired t-test. Graphed data represent mean ± SEM.

Figure 6. Activation of LHA Nts neurons suppresses fasting-induced ad lib and motivated food intake

Nts^Cre;NtsR1⁺⁺ and Nts^Cre;NtsR1^KOKO mice were food-deprived overnight and received VEH or CNO with food restoration the following morning. A, B) Ad lib chow re-feeding 1 and 24 hours after food restoration. C) Body weight gain during 24 hours of ad lib re-feeding after overnight food-deprivation. D) PR breakpoint for sucrose pellets after VEH or CNO injection in both fed and fasted states. Nts^Cre;NtsR1⁺⁺ n=9, Nts^Cre;NtsR1^KOKO n=12, data were analyzed by repeated measures two-way ANOVA with Sidak post-tests, *p < 0.05, **p < 0.01, ***p < 0.001. Graphed data represent mean ± SEM.

Figure 7. Assessment of reward, anxiety, and repetitive behaviors associated with LHA Nts➔NtsR1 circuit activation

CPP was used assess whether activation of LHA Nts neurons is positively reinforcing or aversive. A) Time-spent in the CNO and VEH-paired sides during the pre- and post-tests reveals that activation of LHA Nts neurons is rewarding in Nts^Cre;NtsR1^KOKO (n=15) but not Nts^Cre;NtsR1⁺⁺ (n=17) mice. B) No differences were detected in Nts^ChR (n=12) and Nts^Cre;NtsR1^{KOKO ChR} (n=10) controls. Anxiety-like behavior was assessed via EPM and no differences were detected in open arm C) entries or D) time spent (Nts^Cre;NtsR1⁺⁺+VEH n=10, Nts^Cre;NtsR1⁺⁺+CNO n=10, Nts^Cre;NtsR1^KOKO +VEH n=6, Nts^Cre;NtsR1^KOKO +CNO n=8). E, F) nestlet weight 90 minutes after VEH or CNO injection in Nts^Cre;NtsR1⁺⁺ and Nts^Cre;NtsR1^KOKO mice (Nts^Cre;NtsR1⁺⁺+VEH n=8, Nts^Cre;NtsR1⁺⁺+CNO n=10, Nts^Cre;NtsR1^KOKO +VEH n=6, Nts^Cre;NtsR1^KOKO +CNO n=8). Nestlet-shredding and EPM data were analyzed by standard two-way ANOVA while CPP was analyzed by repeated-measures two-way ANOVA to compare pretest to post-test, with Sidak post-tests in both analyses. Graphed data represent mean ± SEM. Significant differences between VEH and CNO-treated mice: *p < 0.05, **p < 0.01, ***p < 0.001. Significant differences between genotypes with the same treatment: ###p < 0.001, ####p < 0.0001.