Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis

Abstract

Melanocortin-4 receptor (MC4R) mutations cause dysregulation of energy balance and hyperinsulinemia. We have used mouse models to study the physiological roles of extrahypothalamic MC4Rs. Re-expression of MC4Rs in cholinergic neurons (ChAT-Cre, loxTB MC4R mice) modestly reduced body weight gain without altering food intake and was sufficient to normalize energy expenditure and attenuate hyperglycemia and hyperinsulinemia. In contrast, restoration of MC4R expression in brainstem neurons including those in the dorsal motor nucleus of the vagus (Phox2b-Cre, loxTB MC4R mice) was sufficient to attenuate hyperinsulinemia, while the hyperglycemia and energy balance were not normalized. Additionally, hepatic insulin action and insulin-mediated suppression of hepatic glucose production were improved in ChAT-Cre, loxTB MC4R mice. These findings suggest that MC4Rs expressed by cholinergic neurons regulate energy expenditure and hepatic glucose production. Our results also provide further evidence of the dissociation in pathways mediating the effects of melanocortins on energy balance and glucose homeostasis.

Figure 1

Generation of ChAT-IRES-Cre and Phox2b-Cre BAC Transgenic Mice (A) To generate mice expressing Cre-recombinase specifically in preganglionic sympathetic and parasympathetic neurons, an optimized internal ribosome entry sequence (IRES) fused to Cre recombinase was inserted after the stop codon of the ChAT gene using homologous recombination techniques. (B) To target Cre-expression in preganglionic parasympathetic neurons, a Cre-recombinase cassette was introduced into the ATG translation start codon of the Phox2b BAC, using bacteria dependent recombination. (C, left column) GFP immunohistochemistry in ChAT-Cre, Rosa GFP mice. Prominent GFP activity was observed in dorsal motor nucleus of vagus (DMV) and in the intermediolateral cell column (IML). (C, right column) Beta-galactosidase immunohistochemistry in Phox2b-Cre, RosalacZ mice. Strong Cre mediated lacZ expression was observed in DMV. No immunoreactivity is observed in IML. Scale bar=100μm.

Figure 2

Expression of Mc4r mRNA in ChAT-Cre, loxTB MC4R and Phox2b-Cre, loxTB MC4R Mice (A) Mc4r mRNA in situ hybridization in hindbrain of wild-type mouse. Note Mc4r mRNA expression in both DMV and NTS. (B, C) In ChAT-Cre, loxTB MC4R and Phox2b-Cre, loxTB MC4R mice Mc4r mRNA is re-expressed specifically in the DMV. (D) Mc4r mRNA in situ hybridization in wild-type mice IML of the thoracic spinal cord. (E) In ChAT-Cre, loxTB MC4R mice Mc4r mRNA is re-expressed in rostral-caudal axis of longitudinal section of thoracic IML (arrowheads), comparably to wild-type mice (D). (C) In Phox2b-Cre mice, Mc4r mRNA is not re-expressed in IML. Scale bar=100μm.

Figure 3

Body weight curve and body composition in ChAT-Cre, loxTB MC4R and Phox2b-Cre, loxTB MC4R mice. (A) Body weight curve in ChAT-Cre, loxTB MC4R male mice (n=7–11/group, mean±SEM, *p<0.05, **p<0.01). (B) Body weight curve in Phox2b-Cre, loxTB MC4R mice (n=6–12/group, mean±SEM) (C–E) Body composition and body length in 7–8-week old ChAT-Cre, loxTB MC4R male mice (8–14/group, mean±SEM, *p<0.05, ***p<0.001). (F–H) Body composition and body length in 7–8-week old Phox2b-Cre, loxTB MC4R mice (n=7–10/group, mean±SEM, ***p<0.001).

Figure 4

Weekly food intake and energy expenditure in ChAT-Cre, loxTB MC4R and Phox2bCre, loxTB MC4R mice. (A) Weekly food intake in 8-week-old male ChAT-Cre, loxTB MC4R mice (n=8–11/group, mean±SEM, **p<0.01). (B and C) Average oxygen consumption was calculated for both light (B) and dark (C) periods in 6–7-week old ChAT-Cre, loxTB MC4R mice (n=8–9/group, mean±SEM, *p< 0.05). (D) Weekly food intake in 8-week-old male Phox2bCre, loxTB MC4R mice (n=6–12/group, mean±SEM, ***p<0.001). Energy expenditure in 6–7-week-old Phox2b-Cre, loxTB MC4R during light (E) or dark (F) cycle (n=6/group, mean±SEM).

Figure 5

Glucose and insulin metabolism in ChAT-Cre, MC4RloxTB and Phox2b-Cre, loxTB MC4R mice. (A and B) Plasma glucose and insulin in fed 5–6 week-old male ChAT-Cre, loxTB MC4R mice (n=8–12/group, mean±SEM, *p<0.05, **p<0.01, ***p<0.001). (C and D) Plasma glucose and insulin in fed 5–6 week-old male Phox2b-Cre, loxTB MC4R mice (n=11–18/group, mean±SEM, *p<0.05, **p<0.01). (E) Glucose infusion rate (GIR) during hyperinsulinemic-euglycemic clamp in chow-fed ChAT-Cre, loxTB MC4R mice (n= 6–7/group, mean±SEM, *p<0.05 between WT vs. loxTB MC4R and ChAT-Cre, loxTB MC4R mice, #p<0.05 between loxTB MC4R vs. ChAT-Cre mice). (F) Glucose infusion rate (GIR) during hyperinsulinemic-euglycemic clamp in chow-fed Phox2b-Cre, loxTB MC4R mice (n= 6–7/group, mean±SEM, *p<0.05 between WT vs. loxTB MC4R and Phox2b-Cre, loxTB MC4R mice). (G and H) Hepatic glucose production (EndoRa) at basal or hyperinsulinemic conditions (n= 6–7/group, mean±SEM, *p<0.05, **p<0.01, ***p<0.001). (I and J) Glucose disposal rate (Rd) at basal and hyperinsulinemic conditions (n= 6–7/group, mean±SEM, *p<0.05, **p<0.01, ***p<0.001).