The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis

Abstract

Objective: Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown.

Methods: We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies.

Results: We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions.

Conclusions: GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

Keywords: AAC, area above the curve; AMPARs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; ANCOVA, analysis of covariance; ANOVA, analysis of variance; AUC, area under the curve; AgRP; AgRP, agouti-related peptide; CNS, central nervous system; DAB, 3,3′-diaminobenzidine; DIO, diet-induced obesity; DREADD, Designer Receptor Exclusively Activated by Dedigner Drugs; EPSCs, excitatory post-synaptic synaptic currents; GABA, gamma-aminobutyric acid; GTT, glucose tolerance test; GluN2B; Glycemia; HFD, high-fat diet; HSD, honestly significant difference; ITT, insulin tolerance test; KO, knockout; LTD, long-term depression; LTP, long-term potentiation; Lepob/ob mice, obese leptin-deficient mice; Leptin; Metabolism; NMDAR; NMDARs, N-methyl-d-aspartate receptors; PBS, phosphate-buffered saline; POMC, pro-opiomelanocortin; PVN, paraventricular nucleus; RT, room temperature; hrGFP, humanized renilla GFP.

Figure 1

GluN2B in AgRP neurons is required for normal body weight and food intake. (A–D) Body weight (males (A) and females (B)) and cumulative food intake (weeks 3–15) (males (C) and females (D)) of control mice (GluN2B^flox/flox) and AgRP-GluN2B KO mice. (E and F) Body composition (16 weeks of age) (E) and blood leptin levels (F) of control (GluN2B^flox/flox) and AgRP-GluN2B KO female mice. Data are shown as means ± SEM (n = 4–11/group). P values in the line graphs are expressed as differences between groups (Student's t test) at the end of the study (week 16). Repeated measures two-way ANOVA and post hoc Tukey HSD tests were conducted to evaluate effect of GluN2B deletion from AgRP neurons on body weight. Post hoc multiple comparisons were performed using Tukey HSD test. Cumulative food intake and body composition at week 15 and leptin levels at week 17 were evaluated with Student's t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 2

GluN2B in POMC neurons is not required for normal regulation of body weight and food intake. (A and B) Body weight (A) and cumulative food intake (weeks 3–15) (B) of control (GluN2B^flox/flox) and POMC-GluN2B KO female mice. (C and D) Body composition (15 weeks of age) (C) and blood leptin levels (D) of control (GluN2B^flox/flox) and POMC-GluN2B KO female mice. Data are shown as means ± SEM (n = 6–9/group). NS: Not significant.

Figure 3

GluN2A in AgRP and POMC neurons is not required for normal body weight and energy intake. (A and C) Body weight (A) and cumulative food intake (C) (weeks 3–12) of control (GluN2A^flox/flox) and AgRP-GluN2A KO female mice. (B and D) Body weight (B) and cumulative food intake (D) at week 12 of control (GluN2A^flox/flox) and POMC-GluN2A KO female mice. Data are shown as means ± SEM (n = 7/group). NS: Not significant.

Figure 4

Deletion of GluN2B in AgRP and POMC neurons: Pair-feeding and high-fat diet studies. (A) Body weight and cumulative food intake (weeks 3–9) of control (GluN2B^flox/flox) and AgRP-GluN2B KO female mice (n = 4–6/group). (B) Body weight and cumulative food intake (weeks 4–10) of control (GluN2B^flox/flox) and AgRP-GluN2B KO male mice and control (GluN2B^flox/flox) and POMC-GluN2B KO female mice during high-fat diet feeding. Data are shown as means ± SEM (n = 4–11/group). Repeated measures two-way ANOVA or Student's t test were conducted to evaluate differences between groups. NS: Not significant.

Figure 5

Deletion of GluN2B in AgRP and POMC neurons: Blood glucose and insulin, and glucose- and insulin-tolerance. (A–D) Fed blood glucose and insulin levels (15–17 weeks of age) in animals from AgRP (A and C) and POMC (B and D) studies, respectively. (E–H) Glucose tolerance test (GTT) following 15 h food removal (2 mg/g d-glucose, intraperitoneal) and insulin tolerance test (ITT) following 5 h food removal (1.2 U/kg insulin, intraperitoneal) (15–17 weeks of age) of AgRP (E and G) and POMC (F and H) studies, respectively. AgRP and POMC data are from female and male mice, respectively, and are shown as means ± SEM (n = 3–9/group). P > 0.05 for all experiments applying ANCOVA or repeated measures two-way ANCOVA. Logarithmic transformations were done for GTT values of AgRP study and ITT values of POMC study. Controls represent GluN2B^flox/flox mice. NS: Not significant.

Figure 6

Elimination of GluN2B in AgRP neurons of Lep^ob/obmice normalizes hyperglycemia independently body weight. (A–D) Body weight (A), body composition (8 weeks of age) (B), average daily food intake (week 7–8) (C), and blood glucose (D) in ob/ob controls (ob/ob;AgRP-ires-cre and ob/ob;GluN2B^flox/flox) and ob/ob;AgRP-GluN2B KO female mice. (E and F) Body weight (E) and food intake (F) of female mice (9 weeks of age) after implantation of osmotic pumps loaded with leptin (500 ng/h) (9 days). Values in E and F are shown as percent of initial body weight and food intake, respectively. Data are shown as means ± SEM (n = 3–12 mice/group). Repeated measures two-way ANOVA or Student's t test were conducted to evaluate differences between groups. *P ≤ 0.05, **P ≤ 0.01. NS: Not significant.

Figure 7

Deletion of GluN2B from AgRP and POMC neurons: Neuron numbers (A–F) Representative microphotographs (hrGFP immunohistochemistry) and cell counts of AgRP (A and C) and POMC (B and D) neurons in brain sections from body weight-matched (E and F) male control and KO animals (8 weeks of age). Data in Figure 8C and D are presented as the mean neuron number ± SEM (n = 4–5 mice/group). Differences between groups were evaluated with Student's t test. NS: Not significant. ARC: Arcuate nucleus. 3V: Third ventricle. Images in (A) and (B) were captured at 20× magnification.

Figure 8

Deletion of GluN2B from AgRP Neurons: Dendritic length. (A) Representative fluorescence microphotograph (presented in grey tone) of dendrites of AgRP neurons in AgRP-GluN2B KO and control (GluN2B^flox/flox) male mice at 8 weeks of age. (B) Dendritic length of AgRP-GluN2B KO and control (GluN2B^flox/flox) male in 8 week old mice. (C) Body weight-matched groups of control and KO mice were pre-selected for these studies. Bar graph in Figure B shows the mean dendritic length ± SEM (n = 4–9 dendrites/group and n = 2–4 mice/group). *P ≤ 0.05, Student's t test between neuron-specific GluN2B-deleted mice versus control mice (GluN2B^flox/flox) at week 8. Scale bar in (A) represents 50 μm.