Acute and long-term suppression of feeding behavior by POMC neurons in the brainstem and hypothalamus, respectively

Abstract

POMC-derived melanocortins inhibit food intake. In the adult rodent brain, POMC-expressing neurons are located in the arcuate nucleus (ARC) and the nucleus tractus solitarius (NTS), but it remains unclear how POMC neurons in these two brain nuclei regulate feeding behavior and metabolism differentially. Using pharmacogenetic methods to activate or deplete neuron groups in separate brain areas, in the present study, we show that POMC neurons in the ARC and NTS suppress feeding behavior at different time scales. Neurons were activated using the DREADD (designer receptors exclusively activated by designer drugs) method. The evolved human M3-muscarinic receptor was expressed in a selective population of POMC neurons by stereotaxic infusion of Cre-recombinase-dependent, adeno-associated virus vectors into the ARC or NTS of POMC-Cre mice. After injection of the human M3-muscarinic receptor ligand clozapine-N-oxide (1 mg/kg, i.p.), acute activation of NTS POMC neurons produced an immediate inhibition of feeding behavior. In contrast, chronic stimulation was required for ARC POMC neurons to suppress food intake. Using adeno-associated virus delivery of the diphtheria toxin receptor gene, we found that diphtheria toxin-induced ablation of POMC neurons in the ARC but not the NTS, increased food intake, reduced energy expenditure, and ultimately resulted in obesity and metabolic and endocrine disorders. Our results reveal different behavioral functions of POMC neurons in the ARC and NTS, suggesting that POMC neurons regulate feeding and energy homeostasis by integrating long-term adiposity signals from the hypothalamus and short-term satiety signals from the brainstem.

Figure 1.

Selective activation of POMC neurons in the ARC or NTS using DREADD technology. A, B, Designs for AAV DIO hM3Dq-2A-mCherry (A) and AAV DIO mCherry (B) vectors. L-ITR, Left inverted terminal repeat; CAG, CMV enhancer/chicken β-actin promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; and R-ITR, right inverted terminal repeat. C, Stereotaxic infusion of AAV DIO hM3Dq-2A-mCherry vectors into the ARC of POMC-Cre mice labels neurons in the bilateral ARC. 3V, Third ventricle; ME, median eminence; and D, dorsal. D, Vector injection into the medulla labels a population of neurons in the NTS. cc, Central canal. E, Selective expression of hM3Dq/mCherry (red) in POMC-immunopositive neurons (green) in the ARC. F, Whole-cell current-clamp recording reveals that an hM3Dq/mCherry⁺ POMC neuron in the ARC was excited by pressure injection of hM3Dq receptor ligand CNO (horizontal bar). G, Raw recording trace of a single cell and group data show that CNO remains effective in depolarizing hM3Dq/mCherry⁺ POMC neurons in the ARC in the presence of tetrodotoxin. Error bars indicate mean + SEM. H, CNO evokes vigorous firing of action currents from hM3Dq/mCherry⁺ POMC neurons in the NTS. The right panel shows the average peristimulus time histogram of spike firing frequency of five NTS POMC neurons. I, After unilateral expression of hM3Dq (red) in the ARC, CNO injection (1 mg/kg, i.p.) induces c-Fos expression (green) in the ipsilateral but not the contralateral ARC. Right panel shows the overlay of mCherry signals and POMC immunoreactivity within the dashed rectangle area in the two left panels. J, c-Fos immunostaining confirms that CNO activates NTS neurons in vivo. K, In both the ARC and NTS, approximately half of the hM3Dq-expressing POMC neurons express c-Fos in response to a single injection of CNO. L, More than half of c-Fos⁺ neurons lack clear hM3Dq/mCherry expression.

Figure 2.

Chronic but not acute activation of ARC POMC neurons inhibits feeding and body weight. A, Neither a single CNO injection (CNO) nor two injections at 5 h intervals (CNO ×2) affect food intake of ARC^hM3Dq mice. B, Acute CNO administration does not affect the body weight of ARC^hM3Dq mice. C, CNO has no effect on the food intake of ARC^mCherry control mice. D, Multiple CNO injections for 3 d suppress the food intake and reduce the body weight of ARC^hM3Dq mice. Significant effects were observed 1 d after chronic CNO administration (*p < 0.05; **p < 0.01; paired t test between saline control and CNO injections; n = 5 ARC^hM3Dq mice). Asterisks above the lines indicate significant effects on food intake and those below indicate effects on body weight. Error bars indicate mean + SEM.

Figure 3.

Activating POMC neurons in the NTS suppresses feeding behavior immediately. A, Amount of food intake at different time points after first CNO injection into NTS^hM3Dq mice. Food intake is significantly reduced within 2–5 h of the first CNO injection. A second CNO injection 5 h after the initial injection significantly inhibits food intake for an additional 3 h. B, C, Acute CNO administration reduces the meal size (B) and meal number (C) of NTS^hM3Dq mice significantly. D, CNO does not affect the food intake of NTS^mCherry mice. E, Food intake within different time periods after the first CNO injection. F, No effect by acute CNO administration on the body weight of NTS^hM3Dq mice. G, CNO administration does not change the level of locomotor activity within different time periods after the first CNO injection. H, Effects of chronic CNO application on the daily food intake and body weight of NTS^hM3q mice (*p < 0.05; **p < 0.01; paired t test between days of saline control and those of CNO injection; n = 5 mice). Asterisks above the lines indicate significant effects on food intake and those below indicate effects on body weight. Error bars indicate mean + SEM.

Figure 4.

Ablating POMC neurons in the ARC but not the NTS causes obesity and hyperphagia. A, Design for AAV DIO DTR vectors. B, Images illustrating that DT injections eliminate POMC neurons in the ARC of ARC^DTR mice (left) and the NTS of NTS^DTR mice (right). POMC immunostaining (green) was performed to confirm the lesion effect in the ARC. AAV DIO mCherry was injected into NTS^DTR mice to confirm the killing of POMC neurons in the NTS by mCherry labeling. C, After DT injections, ARC^DTR mice but not control ARC^mCherry mice display clear obesity. D, The effect of body weight gain is negatively correlated with the number of remaining POMC neurons in the entire ARC. Each square represents data from a single mouse. E, DT injections increase daily food intake significantly for ARC^DTR mice but not NTS^DTR mice. F, Plots of body weight gain for mice with ablations of POMC neurons in the ARC or the NTS. Eliminating POMC neurons in the ARC but not the NTS results in significant weight gain. Error bars indicate mean + SEM.

Figure 5.

Ablating POMC neurons in the ARC reduces locomotor activity and energy expenditure. A, DT administration reduces the locomotor activity level of ARC^DTR mice significantly during the dark but not the light phases. B, DT lesion of ARC POMC neurons significantly reduces animal oxygen consumption in a metabolic cage. Error bars indicate mean + SEM.

Figure 6.

The effects of ablating POMC neurons on metabolism. A, Ablating POMC neurons in the ARC but not the NTS increases the percentage of fat mass and reduces the percentage of lean mass significantly 1 month after DT treatment. B, Obese ARC^DTR and DT mice exhibit significantly higher levels of serum total cholesterol (CHO), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). The level of triglycerides (TGs) in these mice is comparable to that in control animals. C, Ablating POMC neurons in the ARC but not the NTS produces glucose intolerance. D, Obese ARC^DTR and DT mice exhibit significantly lower serum corticosterone level. Error bars indicate mean + SEM.