Glutamatergic Preoptic Area Neurons That Express Leptin Receptors Drive Temperature-Dependent Body Weight Homeostasis

Abstract

The preoptic area (POA) regulates body temperature, but is not considered a site for body weight control. A subpopulation of POA neurons express leptin receptors (LepRb(POA) neurons) and modulate reproductive function. However, LepRb(POA) neurons project to sympathetic premotor neurons that control brown adipose tissue (BAT) thermogenesis, suggesting an additional role in energy homeostasis and body weight regulation. We determined the role of LepRb(POA) neurons in energy homeostasis using cre-dependent viral vectors to selectively activate these neurons and analyzed functional outcomes in mice. We show that LepRb(POA) neurons mediate homeostatic adaptations to ambient temperature changes, and their pharmacogenetic activation drives robust suppression of energy expenditure and food intake, which lowers body temperature and body weight. Surprisingly, our data show that hypothermia-inducing LepRb(POA) neurons are glutamatergic, while GABAergic POA neurons, originally thought to mediate warm-induced inhibition of sympathetic premotor neurons, have no effect on energy expenditure. Our data suggest a new view into the neurochemical and functional properties of BAT-related POA circuits and highlight their additional role in modulating food intake and body weight.

Significance statement: Brown adipose tissue (BAT)-induced thermogenesis is a promising therapeutic target to treat obesity and metabolic diseases. The preoptic area (POA) controls body temperature by modulating BAT activity, but its role in body weight homeostasis has not been addressed. LepRb(POA) neurons are BAT-related neurons and we show that they are sufficient to inhibit energy expenditure. We further show that LepRb(POA) neurons modulate food intake and body weight, which is mediated by temperature-dependent homeostatic responses. We further found that LepRb(POA) neurons are stimulatory glutamatergic neurons, contrary to prevalent models, providing a new view on thermoregulatory neural circuits. In summary, our study significantly expands our current understanding of central circuits and mechanisms that modulate energy homeostasis.

Keywords: DREADD; body temperature; body weight; energy expenditure; food intake; thermoregulation.

Figure 1.

LepRb^POA neurons decrease core temperature and energy expenditure. A, Distribution of LepRb-expressing neurons in the POA of LepRb^EGFP reporter mice. Right panels show corresponding regions in the mouse brain atlas (Paxinos and Franklin, 2004). 3V, Third ventricle; MPA, medial preoptic area; LPO, lateral preoptic area; VOLT, vascular organ of the lamina terminalis; AVPe, anteroventral periventricular nucleus; VMPO, ventromedial preoptic nucleus; VLPO, ventrolateral preoptic nucleus; MnPO, median preoptic nucleus; ac, anterior commissure. B, Representative images showing virus-infected neurons (mCherry, red) and cFos (green) in the POA in LepRb^POA control and LepRb^POA DREADD-Gq mice. CNO (0.5 mg/kg, i.p.) was injected 2–3 h before perfusion. Insets show magnified images of the indicated areas (dotted line boxes). C, Rectal temperature of LepRb^POA control (n = 7) and LepRb^POA DREADD-Gq (n = 9) mice after CNO injection (1.5 mg/kg, i.p.) at RT. D, VO₂ measurement in LepRb^POA DREADD-Gq mice (n = 4) during saline (black) or CNO (0.5 mg/kg, i.p.; red) injections at RT. E, Locomotor activity in the same LepRb^POA DREADD-Gq mice shown in D during the same period. Locomotor activity from 12:00 to 6:00 P.M. is enlarged in the box. F, Change in VO₂ (percentage) in LepRb^POA control (n = 4) and LepRb^POA DREADD-Gq (n = 4) mice at different CNO concentrations at RT. G, Photos capturing postural extension 70 min after CNO injection (1.5 mg/kg, i.p.) in a LepRb^POA DREADD-Gq mouse compared with a control mouse at RT in their home cages. Data are represented as mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.0001 (two-way repeated-measures ANOVA and Bonferroni's pairwise comparisons).

Figure 2.

LepRb^POA neurons inhibit β3 adrenergic receptor-dependent BAT thermogenesis. A, 24 h VO₂ measurement in LepRb^POA DREADD-Gq mice (n = 4) after injections of saline (black), CNO (0.5 mg/kg, i.p.; red), CL316,243 (1.0 mg/kg, i.p.; blue), or CNO + CL316,243 (green). B, The average VO₂ during 6 h of postinjection was compared between injections. Values inside columns represent percentages compared with the saline injection. C, The average locomotor activity during 6 h after injection was compared between injections. Values inside columns represent percentages compared with the saline injection. Data are represented as mean ± SEM. Bars with different letters denote significant differences at p < 0.05 (one-way repeated-measures ANOVA and Fisher's least significant difference pairwise comparisons).

Figure 3.

Chronic activation of LepRb^POA neurons. A, The experimental scheme showing timings of body weight measurement and injections for chronic CNO treatment. CNO was injected intraperitoneally twice daily at 0.3 mg/kg for 6 consecutive days following 3 d of saline injections, and no injection was made during 3 d of recovery. B, Body weight of LepRb^POA control (n = 5) and LepRb^POA DREADD-Gq (n = 5) mice during chronic CNO treatment. C, CNO reduced energy expenditure during 6 d of CNO treatment in LepRb^POA DREADD-Gq (n = 5) but not in control (n = 5) mice. D, Average daily energy expenditure (kcal/g/d) was compared between groups and treatments. E, Chronic CNO reduced daily food intake in LepRb^POA DREADD-Gq (n = 5) but not in control (n = 5) mice. F, Average daily food intake (kcal/g/d) was compared between groups and treatments. G, Change in food intake (percentage) in LepRb^POA control (n = 4) and LepRb^POA DREADD-Gq (n = 4) mice at different CNO concentrations at RT. Data are represented as mean ± SEM *p < 0.05, **p < 0.01, and bars with different letters denote significant differences at p < 0.05 (two-way repeated-measures ANOVA and Bonferroni's pairwise comparisons).

Figure 4.

LepRb^POA neurons modulate food intake. A, Average daily locomotor activity was compared between groups and treatments during chronic CNO treatment. B, Total travel distance during 5 min was compared between groups (control, n = 5; DREADD-Gq, n = 4) and treatments 30 min after injections. C, The experimental scheme for restricted feeding. D, The first 2 h food intake during restricted feeding was measured in LepRb^POA control (n = 5) and LepRb^POA DREADD-Gq (n = 4) mice. Only LepRb^POA DREADD-Gq mice reduced food intake by CNO injection. E, Average 2 h food intake during the restricted feeding (control, n = 5; DREADD-Gq, n = 4). Saline data are from days 3 and 5, and CNO data are from days 4 and 6. Data are represented as mean ± SEM. *p < 0.01 (paired t test). Data with different letters denote significant differences at p < 0.05 (two-way repeated-measures ANOVA and Bonferroni's pairwise comparisons).

Figure 5.

LepRb^POA neurons are activated by warm exposure, but not by cold exposure. A, Comparisons of energy expenditure and food intake between mice housed at 22°C (n = 6) and 30°C (n = 6). B, Representative immunohistochemical images showing the distribution of cFos (red, top panels) and cFos/LepRb^POA-EGFP (bottom panels) after 3 h of cold (4°C; n = 6), RT (22°C; n = 9), and warm (30°C; n = 6) exposure in LepRb^EGFP mice. Insets show magnified images of the indicated areas (dotted line boxes). C, The percentage of LepRb^POA-EGFP cells that express cFos was calculated for each temperature condition. D, E, Total number of EGFP⁺ (D) and EGFP⁺/cFos⁺ (E) cells in the POA counted in each temperature condition. *p < 0.001 (independent t test). Data with different letters denote significant differences at p < 0.01 for C and p < 0.05 for E (one-way ANOVA and Fisher's least significant difference pairwise comparisons). n.s., Not significant.

Figure 6.

Pharmacogenetic activation of LepRb^POA neurons at warm and cold ambient temperature. A, The experimental scheme showing timings of temperature changes and injections for indirect calorimetry experiment. B, At 30°C, LepRb^POA DREADD-Gq mice further decreased VO₂ by CNO injection compared with saline injection. C, At 10°C, CNO injection greatly attenuated cold-induced increase in VO₂ in LepRb^POA DREADD-Gq mice. D, Average VO₂ during 4 h postinjection was calculated and the change from baseline (ΔVO₂) was compared between saline and CNO in each temperature condition. RT value is calculated from Figure 1D. E, ΔVO₂ Saline − ΔVO₂ CNO was calculated for each temperature condition and normalized to 30°C. F, G, Locomotor activity in the same LepRb^POA DREADD-Gq mice shown in B and C during the same period. Locomotor activity from 12:00 to 6:00 P.M. is enlarged in the box. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (two-way repeated-measures ANOVA and Bonferroni's pairwise comparisons for B–D, F, and G, and one-way repeated-measures ANOVA and Fisher's least significant difference pairwise comparisons for E).

Figure 7.

LepRb^POA neurons are glutamatergic. A, B, Representative immunohistochemical images showing leptin-induced pSTAT3 (black, DAB staining) in the POA of Vglut2^EYFP (A, n = 3) and Vgat^EYFP (B, n = 3) mice. Areas within dotted line indicate areas used for pSTAT3 cell counting. C, D, Representative immunohistochemical images showing leptin-induced pSTAT3 (red) and EYFP (green) in the POA of Vglut2^EYFP (C) and Vgat^EYFP (D) mice. Insets show magnified images of the indicated areas (dotted-line boxes) to show colocalization of pSTAT3 and EYFP. E, F, Schematic drawings for the distribution of pSTAT3 and EYFP double-positive cells in the POA in Vglut2^EYFP and Vgat^EYFP mice. G, H, At RT, CNO injection (1.5 mg/kg, i.p.) decreased rectal temperature in Vglut2^POA DREADD-Gq but not in Vgat^POA DREADD-Gq mice. Data are represented as mean ± SEM. *p < 0.01 and **p < 0.001 (two-way repeated-measures ANOVA and Bonferroni's pairwise comparisons).

Figure 8.

Glutamatergic, not GABAergic, POA neurons mediate adaptations to ambient temperature changes. A–F, VO₂ was measured in Vglut2^POA and Vgat^POA DREADD-Gq mice (n = 4 each) at RT, 30°C, and 10°C. Saline (black) or CNO (0.5 mg/kg, i.p.; red) was injected at 11:00 A.M. (CT5, arrows; Fig. 6A). Orange and blue boxes indicate temperature change to 30 and 10°C, respectively. Only Vglut2^POA DREADD-Gq mice, but not Vgat^POA DREADD-Gq mice, further decreased VO₂ by CNO injection compared with saline injection in all temperature conditions. G, H, Average VO₂ during 4 h after injection was calculated and the change from baseline (ΔVO₂) was compared between saline and CNO in each temperature condition. I, ΔVO₂ Saline − ΔVO₂ CNO was calculated for each temperature condition and normalized to 30°C. Data for LepRb^POA DREADD-Gq mice are from Figure 6E. J, K, Locomotor activity in the same mice shown in A and D during the same period at RT. Locomotor activity was not affected by CNO (0.5 mg/kg, i.p.) in either Vglut2^POA (n = 4) or Vgat^POA (n = 4) DREADD-Gq mice. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (two-way repeated-measures ANOVA and Bonferroni's pairwise comparisons for A–H, J, and K, and one-way repeated-measures ANOVA and Fisher's least significant difference pairwise comparisons for I).

Figure 9.

A new view for BAT-related POA circuits. A, The original view suggests direct GABAergic innervation from POA neurons to the DMH and RPa. Here, warm-sensitive GABAergic POA neurons directly inhibit BAT-related neurons in the DMH/DHA and RPa. Upon cold exposure, POA GABAergic neurons are inhibited and DMH and RPa neurons are disinhibited, which causes activation of SNA to BAT to counteract heat loss from cold exposure. B, The new view proposes glutamatergic inputs to the DMH and RPa, which must be indirect to BAT-activating DMH and RPa neurons. Alternatively, LepRb^POA neurons might directly innervate warm-sensitive DMH neurons that inhibit BAT-activating RPa neurons via acetylcholine (Ach; Jeong et al., 2015). Here, warm-sensitive glutamatergic LepRb^POA neurons modulate both energy expenditure and food intake in a temperature-dependent manner to regulate body temperature and body weight. Solid lines indicate direct neuronal connections and dashed lines indicate indirect connections. Connections with question marks require further verification. cs, Cold-sensitive neurons; EE, energy expenditure; FI; food intake; Glut; glutamate; NE, norepinephrine; ws, warm-sensitive neurons.