Acute Metabolic Influences on the Natriuretic Peptide System in Humans

Abstract

Background: The cardiac natriuretic peptides (NPs), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), have central roles in sodium and blood pressure regulation. Extracardiac factors (e.g., obesity and diabetes) influence NP production, potentially altering cardiovascular responses to volume and pressure stress.

Objectives: This study examined the effects of acute carbohydrate intake on the NP system in humans, and investigated underlying mechanisms.

Methods: Normotensive subjects (N = 33) were given a high-carbohydrate shake. Venous blood was sampled to measure N-terminal (NT)-proANP and NT-proBNP levels. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and HepG2 cells were treated with glucose, and expression levels of NPs and micro ribonucleic acid 425 (miR-425), a negative regulator of ANP, were examined. The role of nuclear factor kappa B (NF-κB) in the glucose-mediated effects was investigated using a NF-κB inhibitor and expression plasmids encoding NF-κB subunits.

Results: We observed a 27% reduction in the levels of circulating NT-proANP (p < 0.001, maximal at 6 h) after carbohydrate challenge, with no effect on NT-proBNP levels in our human subjects. Glucose treatment of hESC-CMs for 6 h and 24 h increased levels of the primary transcript of miR-425 (pri-miR-425) and mature miR-425. A corresponding decrease in NPPA messenger RNA levels was also observed at both time points. Overexpression of NF-κB subunits in H9c2 cardiomyocytes increased miR-425 levels, whereas inhibition of NF-κB abrogated the glucose-mediated increase in pri-miR-425 levels in HepG2 cells.

Conclusions: Acute carbohydrate challenge is associated with a reduction in ANP production. The mechanism appears to involve a glucose-induced increase in the expression of miR-425, mediated by NF-κB signaling.

Keywords: cardiomyocyte; glucose; micro ribonucleic acid; obesity.

Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Figures

FIGURE 1. Carbohydrate Challenge: Plasma Glucose and Insulin Levels

Plasma glucose (A) and insulin (B) levels are shown at baseline and in response to the carbohydrate challenge in lean (n = 19) (solid lines with squares) and overweight or obese (n = 14) (dotted lines with circles) individuals. Data are expressed as mean ± SEM. BMI = body mass index.

FIGURE 2. Carbohydrate Challenge: Plasma NT-proANP and NT-proBNP Levels

Concentrations of plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) levels (A) and plasma N-terminal pro–B-type natriuretic peptide (NT-proBNP) levels (B) at baseline and at 15, 30, 60, 90, 120, 240, and 360 min after the carbohydrate challenge. The solid line with squares represents lean subjects, and the dotted line with circles represents overweight or obese subjects. Data are expressed as mean ± SEM. BMI = body mass index.

FIGURE 3. Effect of Glucose Treatment on Cardiomyocytes

Human embryonic stem cell–derived cardiomyocytes (hESC-CMs) were cultured overnight in medium containing 1 mmol/l glucose and then treated with 5 mmol/ glucose for 6 h and 24 h. Primary transcript of micro ribonucleic acid 425 (pri-miR-425) levels (A), mature miR-425 levels (B), and NPPA messenger ribonucleic acid (mRNA) levels (C) are relative to those in untreated cells. Data are expressed as mean ± SEM (n = 6).

FIGURE 4. Luciferase Activity

Glucose treatment increased the luciferase activity encoded by the micro ribonucleic acid 425 (miR-425) promoter-luciferase construct (miR-425 promoter-Luc). The ratio of firefly luciferase activity to renilla luciferase activity was normalized to that in HepG2 cells transfected with a plasmid directing expression of luciferase without the miR-425 promoter sequence (relative luciferase activity). Data are expressed as mean ± SEM (n = 6).

FIGURE 5. Effect of NF-κB on miR-425

Cardiomyocytes were transfected with constructs expressing either nuclear factor kappa B (NF-κB) p50 or p65. After 24 h, cells were harvested. Overexpression of the NF-κB p50 subunit alone or together with the p65 subunit increased mature micro ribonucleic acid 425 (miR-425) levels in H9c2 cardiomyocytes. Mature miR-425 levels are relative to those in cells transfected with an empty construct (Control). Data are expressed as mean ± SEM (n = 3).

FIGURE 6. Effect of SN50 on pri-miR-425

HepG2 cells were cultured overnight in medium containing 1 mmol/l glucose and 50 μg/ml SN50, an NF-κB inhibitor, followed by treatment with 30 mmol/l glucose for 6 h. Glucose-mediated increase in pri-miR-425 levels is abrogated in the presence of SN50. pri-miR-425 levels are relative to those in untreated cells (Control). Data are expressed as mean ± SEM (n = 6). Abbreviations as in Figure 5.

FIGURE 7. Effect of Glucose on Transgenic Mice

After a 16-h fast, C57BL/6J mice expressing a human NPPA transgene (NPPAtg/+ mice) were administered glucose at 2 g/kg body weight or phosphate-buffered saline (PBS) as a control. Oral administration of glucose increased cardiac miR-425 levels in NPPAtg/+ mice compared with controls. Data are expressed as mean ± SEM (n = 13). miR-425, micro ribonucleic acid 425.

CENTRAL ILLUSTRATION. Metabolic Influences on NPs: Model of a High-Carbohydrate Challenge in Humans

A high glucose load stimulates nuclear factor kappa B (NF-κB) signaling, which induces the expression of micro ribonucleic acid 425 (miR-425), a negative regulator of atrial natriuretic peptide (ANP) production, in cardiomyocytes. Suppression of ANP production could have adverse short- and long-term cardiometabolic consequences, because ANP plays a role in blood pressure reduction, salt excretion, activation of brown fat, and increased energy expenditure. mRNA = messenger ribonucleic acid; NP = natriuretic peptide; pri-miR-425 = primary transcript of miR-425; UTR = untranslated region.