Melanocortin MC4R receptor is required for energy expenditure but not blood pressure effects of angiotensin II within the mouse brain

Abstract

The brain renin-angiotensin system (RAS) is implicated in control of blood pressure (BP), fluid intake, and energy expenditure (EE). Angiotensin II (ANG II) within the arcuate nucleus of the hypothalamus contributes to control of resting metabolic rate (RMR) and thereby EE through its actions on Agouti-related peptide (AgRP) neurons, which also contribute to EE control by leptin. First, we determined that although leptin stimulates EE in control littermates, mice with transgenic activation of the brain RAS (sRA) exhibit increased EE and leptin has no additive effect to exaggerate EE in these mice. These findings led us to hypothesize that leptin and ANG II in the brain stimulate EE through a shared mechanism. Because AgRP signaling to the melanocortin MC₄R receptor contributes to the metabolic effects of leptin, we performed a series of studies examining RMR, fluid intake, and BP responses to ANG II in mice rendered deficient for expression of MC₄R via a transcriptional block (Mc4r-TB). These mice were resistant to stimulation of RMR in response to activation of the endogenous brain RAS via chronic deoxycorticosterone acetate (DOCA)-salt treatment, whereas fluid and electrolyte effects remained intact. These mice were also resistant to stimulation of RMR via acute intracerebroventricular (ICV) injection of ANG II, whereas BP responses to ICV ANG II remained intact. Collectively, these data demonstrate that the effects of ANG II within the brain to control RMR and EE are dependent on MC₄R signaling, whereas fluid homeostasis and BP responses are independent of MC₄R signaling.

Keywords: angiotensin; blood pressure; energy; melanocortin.

Figure 1.

Schematic representation of experimental design. A: experimental design for study investigating food intake and metabolic rate of mice with transgenic activation of the brain RAS under baseline and leptin-stimulated conditions. B: experimental design for study investigating the consequences of chronic DOCA-salt-mediated activation of the endogenous brain RAS on metabolic rate in mice with genetic deficiency of the MC₄R (Mc4r-TB). C: experimental design for study investigating metabolic rate responses to acute intracerebroventricular (ICV) ANG II administration in Mc4r-TB mice. D: experimental design for study investigating blood pressure responses to acute ICV ANG II administration in Mc4r-TB mice. ANG II, angiotensin II; RAS, renin-angiotensin system.

Figure 2.

Chronic transgenic activation of the brain RAS stimulates energy expenditure. A: body masses of sRA and control littermate mice. Two-way ANOVA indicates genotype P < 0.01, sex P = 0.01, genotype × sex P = 0.53. B: food intake per day. Two-way ANOVA indicates genotype P = 0.26, sex P = 0.32, genotype × sex P = 0.36. C: 24 h average heat production estimated by O₂/CO₂ respirometry. Two-way ANOVA indicates genotype P = 0.34, sex P = 0.01, genotype × sex P = 0.05. D: 24 h average respiratory exchange ratio (RER). Two-way ANOVA indicates genotype P < 0.01, sex P = 0.60, genotype × sex P = 0.83. E: 24 h cumulative photoelectric beam breaks in X and Y planes. Two-way ANOVA indicates genotype P = 0.41, sex P = 0.01, genotype × sex P = 0.59. For all panels, n = 8 control littermates (4 m + 4 f), and n = 8 sRA (4 m + 4 f) mice. Summary data are presented as means ± SE; *P < 0.05 for indicated comparison, †P < 0.05 between sexes within genotype by Holm–Šídák multiple comparison procedure. f, female; m, male; RAS, renin-angiotensin system.

Figure 3.

Leptin stimulates aerobic metabolism, but this effect is not additive to brain RAS activation. A: heat production averaged for 1 h immediately preceding injections of saline vehicle or leptin at 9 am (3 h into the light phase). Males are represented by solid symbols and females by empty symbols. B: heat production time-courses. Empty symbols represent vehicle-treated of each group, whereas filled triangles represent leptin-treated. C: change in heat production in the first 90 min after leptin or saline administration, as shown in B. For C, subject P = ns, genotype P = 0.01, leptin P < 0.01, and leptin × genotype P = 0.02 by two-way repeated-measures ANOVA. Males are represented by solid symbols and females by empty symbols. For all panels, n = 8 control littermates (4 m + 4 f), and n = 8 sRA (4 m + 4 f) mice. For B and C, *P < 0.05 by Holm–Šídák multiple comparison procedure. Summary data are presented as means ± SE; *P < 0.05 by Tukey’s multiple-comparisons procedure (B and C). f, female; m, male; RAS, renin-angiotensin system.

Figure 4.

Chronic stimulation of resting metabolic rate (RMR) by DOCA-salt requires melanocortin MC₄R receptors. A: regression analysis of RMR determined by O₂/CO₂ respirometry in animals at thermoneutrality (30°C). Circles represent data from pretest, and triangles represent data from animals treated with DOCA-salt. Solid color symbols indicate males, symbols that are bicolor represent females. B: RMR corrected for body mass by generalized linear modeling (GLM), using body mass covariate at 37.72 g. Body mass P < 0.01, genotype P < 0.05, DOCA-salt P < 0.01, genotype × DOCA-salt interaction P < 0.05. C: spike frequency of direct multifiber efferent brown adipose tissue (BAT) sympathetic nerve activity (SNA) recording. D: total daily fluid intake. Dotted line represents typical fluid intake in C57BL/6J mice under baseline conditions. Males are represented by solid symbols and females by empty symbols. For A and B, n = 6 (5 m + 1 f) pretest control, 5 (4 m + 1 f) DOCA-salt control, 10 (5 m + 5 f) pretest Mc4r-TB, and 8 (3 m + 5 f) DOCA-salt Mc4r-TB mice. Summary data presented as means ± SE; *P < 0.05 by Tukey’s multiple comparisons procedure (B) or two-tailed Student’s t test (C). DOCA, deoxycorticosterone acetate; f, female; m, male.

Figure 5.

Acute stimulation of resting metabolic rate (RMR) by intracerebroventricular (ICV) ANG II requires melanocortin MC₄R receptors. A: regression analysis of baseline RMR, determined by gradient-layer direct calorimetry in animals at thermoneutrality (30°C) versus body mass. Solid color symbols indicate males and symbols that are bicolor represent females. B: RMR corrected for body mass by GLM, using body mass covariate at 28.94 g. Body mass P < 0.01, genotype P = ns. C: RMR response to acute ICV injection of 5 µg ANG II. Genotype P = 0.07. Males are represented by solid symbols and females by empty symbols. D: change in RMR corrected for body mass by GLM, using body mass covariate at 28.94 g. Body mass P < 0.01, genotype P < 0.01. For all panels, n = 7 (5 m + 2 f) littermate control, and 6 (2 m + 4 f) Mc4r-TB mice. Summary data presented as means ± SE; *P < 0.05 by GLM (D), and †P < 0.05 vs. zero by one-sample t test. ANG II, angiotensin II; f, female; GLM, generalized linear model; m, male.

Figure 6.

Acute stimulation of BP by intracerebroventricular (ICV) ANG II does not require melanocortin MC₄R receptors. A: average mean arterial pressure over 30 min following successive escalating doses of ICV ANG II. Repeated-measures two-way ANOVA indicates genotype P = 0.74, ANG II dose P < 0.01, and genotype × ANG II interaction P = 0.27. B: average heart rate responses over 30 min following successive escalating doses of ICV ANG II. Repeated-measures two-way ANOVA indicates genotype P = 0.81, ANG II dose P < 0.01, and genotype × ANG II interaction P = 0.33. For both panels, *P < 0.05 vs. baseline within control, and †P < 0.05 vs. baseline within Mc4r-TB by Holm–Šídák multiple comparison procedure. Four-parameter nonlinear regression analyses indicate that curves are indistinguishable (P = 0.28 for MAP, P = 0.86 for HR), and therefore only the global curve fits are shown as a thick, solid black line. Because calculated Hillslope values do not significantly differ from 1, other parameters were estimated from a four-parameter curve fit with Hillslope constrained to 1. For MAP: min = 123.2 ± 3.2 and max = 161.1 ± 8.8 mmHg, and EC₅₀=12.9 ± 9.0 µg. For HR: min = 584.4 ± 15.1 and max = 496.8 ± 19.7, and EC₅₀=3.2 ± 3.1 µg. For both panels, circles indicate males, and triangles represent females. For both panels, n = 5 (3 m + 2 f) littermate control, and 7 (2 m + 5 f) Mc4r-TB mice. ANG II, angiotensin II; f, female; HR, heart rate; m, male; MAP, mean arterial pressure.

Figure 7.

Working model. Schematic highlighting hypothesized neural networks, utilizing shared molecular players such as ANG II, AT_1A, and MC₄R to provide dissociable control of RMR, BP, and ingestive behaviors. In specific, the current study supports a role for AT_1A on AgRP neurons in the control of RMR but not BP or intake behaviors. Further, the current study demonstrates a required role for MC₄R in this mechanism, thereby positively implicating AgRP neurotransmission in this mechanism. ANG II, angiotensin II; BP, blood pressure; RMR, resting metabolic rate.