Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis

Abstract

Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor agonists on energy and glucose homeostasis are unknown. Here, we show that mice lacking serotonin 2C receptors (Htr2c) specifically in pro-opiomelanocortin (POMC) neurons had normal body weight but developed glucoregulatory defects including hyperinsulinemia, hyperglucagonemia, hyperglycemia, and insulin resistance. Moreover, these mice did not show anorectic responses to serotonergic agents that suppress appetite and developed hyperphagia and obesity when they were fed a high-fat/high-sugar diet. A requirement of serotonin 2C receptors in POMC neurons for the maintenance of normal energy and glucose homeostasis was further demonstrated when Htr2c loss was induced in POMC neurons in adult mice using a tamoxifen-inducible POMC-cre system. These data demonstrate that serotonin 2C receptor-expressing POMC neurons are required to control energy and glucose homeostasis and implicate POMC neurons as the target for the effect of serotonin 2C receptor agonists on weight-loss induction and improved glycemic control.

Figure 1. Generation and validation of 2C^flox/Y mice.

(A) Strategy used to create mice in which serotonin 2C receptors (Htr2c [2C]) could be conditionally ablated using cre-lox technology. 2C^flox/Y mice were validated by crossing with mice expressing cre in zona pellicuda (ZP3-cre) cells to produce whole-body Htr2c-null mice (2C^flox/Y × ZP3-cre). (B–D) Predicted decreases in survival as well as increases in body weight and 4- to 5-hour-fasted blood glucose levels in 2C^flox/Y × ZP3-cre mice versus littermate controls (n = 17–20 mice/genotype). Symbols in B are reduced in size to prevent overlap in the control groups. (E–I) Six- to 8-week-old male mice lacking Htr2c in POMC neurons that coexpress the fluorescent reporter tdTomato were functionally validated using treatment with the serotonin 2C receptor agonist mCPP (4 μM) during electrophysiological recordings. n = 5 POMC-cre (controls) and n = 17 2C^flox/Y × POMC-cre (2C^flox × POMC-cre) littermate mice in E–I. Results are shown as the mean ± SEM. *P < 0.05 versus other genotypes assessed using a Student’s t test.

Figure 2. Mice lacking Htr2c in POMC neurons exhibit altered energy balance and sensitivity to diet-induced obesity.

(A–F) Body weight and metabolic cage data for 8-week-old male mice lacking Htr2c in POMC neurons (2C^flox × POMC-cre) in chow-fed conditions (n = 8–11 mice per genotype). (G–H) Body weight and body composition (Bruker minispec) of 20-week-old mice fed an HFHS diet for 8 weeks. (I–M) Metabolic cage data for 8-week-old mice after 1 week of an HFHS diet (n = 8–11 mice per genotype). Results are shown as the mean ± SEM. *P < 0.05 versus other genotypes assessed using a Student’s t test or ANOVA.

Figure 3. Mice lacking Htr2c in POMC neurons and chronically fed an HFHS diet exhibit obesity and altered energy balance.

(A–E) Body weight, food intake, VO₂, VCO₂, and physical activity over a 1-week period in metabolic cages of 20-week-old male mice fed an HFHS diet for 12 weeks (n = 6–9 mice per genotype). Control genotypes (WT, POMC-cre, and 2C^flox/Y littermates) are combined to improve clarity in C (no differences exist between these groups). Results are shown as the mean ± SEM. *P < 0.05 assessed using Student’s t-tests or ANOVA.

Figure 4. d-Fen and mCPP do not diminish food intake in mice lacking serotonin 2C receptors (Htr2c) in POMC neurons.

Agonists of serotonin 2C receptor signaling (i.p. d-Fen and mCPP, 5 and 3 mg/kg, respectively) were administered in a counterbalanced manner at 1-week intervals to 18-hour overnight-fasted mice constitutively lacking Htr2c specifically in POMC neurons and to control littermates (n = 9–12 mice per genotype). Food was removed at 1800 h, and drug or vehicle was administered at 1130 h the following day. A chow diet was reintroduced 30 minutes later, and food intake was recorded hourly for 6 hours. Data are shown as the means ± SEM. *P < 0.05 versus vehicle treatment within a genotype using Student’s t tests.

Figure 5. Impaired glucoregulation in mice lacking serotonin 2C receptors (Htr2c) specifically in POMC neurons.

(A–C) Blood glucose, plasma insulin, and plasma glucagon in chow- and HFHS-fed conditions respectively; n = 9–12 mice per genotype per diet. Ad libitum (Ad lib) blood glucose values in A represent measurements taken at 0700 h. Postabsorptive values represent a 4- to 5-hour morning fast, and 12- and 24-hour fasts denote removal of food at 2100 h or 1800 h, respectively, the night before in A–C. HFHS values in A–C are from 4- to 5-hour-fasted mice. (D and E) Basal and clamp plasma insulin and glucagon levels in 4- to 5-hour morning-fasted (0800–0900 h to 1300 h; n = 7–8 mice per genotype), chronically catheterized, conscious mice during a 120-minute hyperinsulinemic-euglycemic (4 mU/kg/minute, 150 mg/dl, respectively) clamp. (F) Exogenous GIR needed to clamp blood glucose. (G and H) Endogenous rates of endo R_a and R_d were determined using a constant infusion of [3-³H]glucose and steady-state calculations. (I–M) Similar clamp parameters for 12-hour overnight-fasted (2100 h–0900 h; n = 7–8 mice per genotype). Blood was taken from the cut tail, and results are shown as the mean ± SEM. *P < 0.05 versus other genotypes, ^#P < 0.05 versus ad libitum and/or 5-hour-fasted chow-fed conditions within a genotype, and ^†P < 0.05 versus basal values during hyperinsulinemic-euglycemic clamp using Student’s t tests.

Figure 6. Inducible deletion of serotonin 2C receptors (Htr2c) in POMC neurons of adult mice dysregulates energy homeostasis.

Tamoxifen-inducible POMC-cre (POMC-cre:ER^T2) mice that coexpress the cre-stimulated fluorescent reporter tdTomato were treated with 0.15 mg/kg tamoxifen (T) i.p. daily for 5 days, and immunohistochemistry was used to assess the expression of Tomato (A and C) and β-endorphin (B and C). Male POMC-cre:ER^T2 (C) and 2C^flox/Y x POMC-cre:ER^T2 (KO) littermate mice were then treated with tamoxifen or vehicle (V) at 11 weeks of age to assess their chow-fed body weight (D). This was repeated to assess metabolic cages studies in chow-fed mice (E–G; n = 7–9 mice per genotype). Male POMC-cre:ER^T2 (WT) and 2C^flox/Y x POMC-cre:ER^T2 (2C^flox/Y) littermate mice were also fed an HFHS diet to assess body weight (H). (I–K) Metabolic cage data following 1-week exposure to an HFHS diet (n = 7–9 per genotype). Results are shown as the means ± SEM. *P < 0.05 versus other genotypes using Student’s t tests.

Figure 7. Inducible deletion of serotonin 2C receptors (Htr2c) in POMC neurons of adult mice dysregulates glucose homeostasis.

Tamoxifen-inducible POMC-cre (POMC-cre:ER^T2) mice and their respective controls (n = 8 mice per genotype) were treated with tamoxifen (0.15 mg/kg i.p. daily for 5 days) or vehicle. Blood samples from the cut tail were taken immediately before the first dose (week 0) as well as 2 and 4 weeks after treatment to measure plasma glucagon and insulin levels. Results are shown as the means ± SEM. *P < 0.05 versus other genotypes and P < 0.05 versus other genotypes; ^†P < 0.05 versus pre-tamoxifen treatment values within the genotype; ^#P < 0.05 versus tamoxifen-treated control mice.