C-type natriuretic peptide is a pivotal regulator of metabolic homeostasis

Abstract

Thermogenesis and adipogenesis are tightly regulated mechanisms that maintain lipid homeostasis and energy balance; dysfunction of these critical processes underpins obesity and contributes to cardiometabolic disease. C-type natriuretic peptide (CNP) fulfills a multimodal protective role in the cardiovascular system governing local blood flow, angiogenesis, cardiac function, and immune cell reactivity. Herein, we investigated a parallel, preservative function for CNP in coordinating metabolic homeostasis. Global inducible CNP knockout mice exhibited reduced body weight, higher temperature, lower adiposity, and greater energy expenditure in vivo. This thermogenic phenotype was associated with increased expression of uncoupling protein-1 and preferential lipid utilization by mitochondria, a switch corroborated by a corresponding diminution of insulin secretion and glucose clearance. Complementary studies in isolated murine and human adipocytes revealed that CNP exerts these metabolic regulatory actions by inhibiting sympathetic thermogenic programming via Gi-coupled natriuretic peptide receptor (NPR)-C and reducing peroxisome proliferator-activated receptor-γ coactivator-1α expression, while concomitantly driving adipogenesis via NPR-B/protein kinase-G. Finally, we identified an association between CNP/NPR-C expression and obesity in patient samples. These findings establish a pivotal physiological role for CNP as a metabolic switch to balance energy homeostasis. Pharmacological targeting of these receptors may offer therapeutic utility in the metabolic syndrome and related cardiovascular disorders.

Keywords: G protein–coupled receptor; adipogenesis; cardiometabolic disease; natriuretic peptide; thermogenesis.

Fig. 1.

Body weight and WAT accumulation are markedly reduced in gbCNP^−/− mice in vivo. Plasma levels of CNP (A) in WT (^+/+) and gbCNP^−/− (^−/−) mice fed STD chow or HFD. Mean arterial blood pressure (MABP; B) and heart rate (HR; C) measured by radiotelemetry, and endothelium-dependent relaxation to acetylcholine in mesenteric arteries (ACh; D) in WT (^+/+) and gbCNP^−/− (^−/−) mice. Body weight and sum of all WAT (gonadal, perirenal, inguinal, and mesenteric) weight in WT and gbCNP^−/− animals fed STD chow (E and F) or HFD (G and H). Food (I) and water (J) intake. Data are represented as mean ± SEM n = 7 to 18. Statistical analysis by two-way ANOVA with Šídák post hoc test (A, I, and J), two-way repeated-measures ANOVA (B, C, E, and G), two-tailed Student’s t test (F and H). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Fig. 2.

Body temperature and energy expenditure are overtly increased in gbCNP^−/− mice in vivo. Core body temperature (A), activity (B), EE (C), RER (D and E), and resting EE (REE; F) in WT (^+/+) and gbCNP^−/− (^−/−) mice. Expression of the thermogenic marker UCP-1 in the gWAT, reWAT, iWAT, and BAT in STD- (G) and HFD- (H) fed animals. EE (I) and rectal temperature (J) in WT (^+/+) and gbCNP^−/− (^−/−) mice following cold temperature (4 °C) challenge. Expression of the thermogenic markers UCP-1 and PGC-1α in BAT in gbCNP^−/− mice compared to WT (^+/+) littermates (K and L). Data are represented as mean± SEM n = 6 to 8. Statistical analysis by two-way repeated-measures ANOVA (A–D and I), two-way ANOVA with Šídák post hoc test (E), or two-tailed Student’s t test (F–H, J–L). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Fig. 3.

The prothermogenic phenotype in gbCNP^−/− mice is recapitulated in animals with global NPR-C deletion. Body weight and sum of all WAT (gonadal, perirenal, inguinal, and mesenteric) weight in WT and NPR-C^−/− animals fed STD chow (A and B) or HFD (C and D). Food (E) and water (F) intake, and gWAT (G), reWAT (H), iWAT (I), mesWAT (J), and BAT (K) in WT (^+/+) and NPR-C^−/− (^−/−) mice on STD chow or HFD. Body temperature and activity in WT animals following minipump infusion of cANF^4-23 (0.4 mg/kg/d, subcutaneously) (L). Data are represented as mean ± SEM, n = 6 to 10. Statistical analysis by two-way repeated-measures ANOVA (A, C, and L), two-tailed Student’s t test (B and D), or two-way ANOVA with Šídák post hoc test (E–K). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Fig. 4.

Inhibition of sympathetic thermogenic activity by G_i-coupled NPR-C is responsible for the metabolic regulatory role of CNP. Forskolin (Fsk; 10 μM)-induced cAMP production in the absence and presence of CNP (100 nM) and Pertussis toxin (Ptx; 100 ng/mL) in primary adipocytes isolated from WT or NPR-C^−/− mice (A). PGC-1α mRNA expression in the presence and absence of NA (1 µM) and/or the selective NPR-C agonist, cANF^4-23 (100 nM; B). Representative images (40x objective; C) and quantification (D) of Tom20 staining for mitochondria density in isolated adipocytes from WT and NPR-C^−/− mice in the absence and presence of CNP (100 nM) or cANF^4-23 (100 nM). Results are shown as percentage of untreated control and expressed as the mean of >100 cells analyzed from three different fields per sample. Expression of the thermogenic markers PGC-1α and UCP-1 mRNA in the absence and presence of CNP (100 nM) in isolated adipocytes from WT mice (E). Effect of β₃-adrenoreceptor blockage with L-748,337 (0.144 mg/kg/d) on body weight (F). PDE2 activity in brain homogenates (G). Plasma NT-proCNP concentrations (H). Correlation between the expression of plasma NT-proCNP and body fat mass in human patients (I). CNP (J) and NPR-C (K) mRNA expression from VAT from human patients. Correlation between CNP and NPR-C mRNA in VAT from human patients (L). Data are represented as mean± SEM. Statistical analysis by one-way ANOVA with Šídák post hoc test (A–D), two-tailed Student’s t test (E, G, H, J, and K), two-way ANOVA (F), or Spearman’s rank correlation (I and L). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Fig. 5.

The prothermogenic actions of CNP deletion are associated with reduced adipogenesis. Mean adipocyte diameter in gWAT (A), reWAT (B), and iWAT (C) fat pads from WT (^+/+) and gbCNP^−/− (^−/−) mice fed STD chow or HFD. Representative images of H&E staining of WAT (D). Representative images of oil red-O staining from WT (^+/+) and gbCNP^−/− (^−/−) primary isolated adipocytes (E) and cellular triglyceride content (F). CNP in the culture media of murine primary adipocytes prior to the addition of the differentiation mixture (preadipocyte) and on day 1 (d1), day 4 (d4), and day 7 (d7) thereafter (G). Cellular triglyceride content in isolated murine adipocytes in the absence and presence of CNP (100 nM; H). Expression of the adipogenic markers adiponectin and PPAR-γ mRNA in isolated murine adipocytes in the absence and presence of CNP (100 nM; I). Cellular triglyceride content in human adipocytes following NPR-B (NPR-B KD) or NPR-C (NPR-C KD) knockdown in the absence (−) and presence of CNP (100 nM; J). Expression of the adipogenic markers adiponectin and PPAR-γ mRNA in isolated human adipocytes in the absence (−) and presence of CNP (100 nM; K). Data are represented as mean ± SEM, n = 5 to 13. Statistical analysis by two-way ANOVA with Šídák post hoc test (A–C) or two-tailed Student’s t test (F–I), or one-way ANOVA with Šídák post hoc test (J and K). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Fig. 6.

CNP/NPR-B/PKG signaling results in the healthy expansion of WAT in vivo. Body weight (A), body weight change (B), gWAT, reWAT, and iWAT (C), and mRNA expression of adipogenic (PPAR-γ and adiponectin), thermogenic (PGC-1α and UCP-1), and NPR-B (D) in iWAT of NPR-C^−/− mice in the absence and presence of CNP (0.2 mg/kg/d, subcutaneously). Effect of CNP (100 nM) on the triglyceride content of isolated murine adipocytes after NPR-B knockdown (KD; E) or in the absence and presence of the protein kinase G inhibitor KT-5823 (2 µM) (F and G). CREB phosphorylation in isolated murine adipocytes in the absence and presence of CNP, cANF^4-23 (both 100 nM) and/or KT5823 (2 µM) (H and I). Data are represented as mean ± SEM, n = 3 to 6. Statistical analysis by two-way repeated-measures ANOVA (A) or one-way ANOVA with Šídák post hoc test (E–H), or two-tailed Student’s t test (B–D). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Fig. 7.

Schematic representation of the pathways involved in CNP control of energy homeostasis. Adenylyl cyclase, AC; adenosine triphosphate, ATP; cyclic adenosine monophosphate, cAMP; cyclic guanosine monophosphate, cGMP; cAMP-response element binding protein, CREB; extracellular signal-regulated kinase 1/2, ERK1/2; G_i protein α-subunit, G_αi guanosine triphosphate, GTP; mitogen-activated protein kinase kinase, MEK; p38 mitogen-activated protein kinases, P38 MAPK.