Endothelin-1 as a novel target for the prevention of metabolic dysfunction with intermittent hypoxia in male participants

Abstract

We examined the effect of intermittent hypoxia (IH, a hallmark feature of sleep apnea) on adipose tissue lipolysis and the role of endothelin-1 (ET-1) in this response. We hypothesized that IH can increase ET-1 secretion and plasma free fatty acid (FFA) concentrations. We further hypothesized that inhibition of ET-1 receptor activation with bosentan could prevent any IH-mediated increase in FFA. To test this hypothesis, 16 healthy male participants (32 ± 5 yr, 26 ± 2 kg/m²) were exposed to 30 min of IH in the absence (control) and presence of bosentan (62.5 mg oral twice daily for 3 days prior). Arterial blood samples for ET-1, epinephrine, and FFA concentrations, as well as abdominal subcutaneous adipose tissue biopsies (to assess transcription of cellular receptors/proteins involved in lipolysis), were collected. Additional proof-of-concept studies were conducted in vitro using primary differentiated human white preadipocytes (HWPs). We show that IH increased circulating ET-1, epinephrine, and FFA (P < 0.05). Bosentan treatment reduced plasma epinephrine concentrations and blunted IH-mediated increases in FFA (P < 0.01). In adipose tissue, bosentan had no effect on cellular receptors and proteins involved in lipolysis (P > 0.05). ET-1 treatment did not directly induce lipolysis in differentiated HWP. In conclusion, IH increases plasma ET-1 and FFA concentrations. Inhibition of ET-1 receptors with bosentan attenuates the FFA increase in response to IH. Based on a lack of a direct effect of ET-1 in HWP, we speculate the effect of bosentan on circulating FFA in vivo may be secondary to its ability to reduce sympathoadrenal tone.

Keywords: adipose tissue; bosentan; epinephrine; fatty acids; intermittent hypoxia.

Figure 1.

Study timeline and effect of intermittent hypoxia and endothelin-1 receptor inhibition on main outcome variables in healthy male participants. A: plasma endothelin-1 (ET-1) increased with acute intermittent hypoxia (IH). Treatment with oral bosentan increased ET-1. B: total free fatty acids (FFAs) increased with IH and were lower after bosentan treatment. C: epinephrine increased with IH. Lower epinephrine was observed after bosentan treatment. Data are from n = 16 male participants, presented as means ± SD and analyzed using a linear mixed model with pairwise comparisons via Student’s t test. *P < 0.05 pairwise comparison vs. Control; †P < 0.05 pairwise comparison vs. Pre-IH (Control, Bosentan); ^P < 0.05 pairwise comparison vs. Pre-IH (Control only). HVR, hypoxic ventilatory response. Arrows in study schematic depict blood sampling time points.

Figure 2.

Glycerol measured in conditioned media from differentiated human white preadipocytes. Insulin (1 nM)-mediated suppression of basal lipolysis and Bt₂cAMP (dibutyryl cAMP, 1 mM)-mediated activation of lipolysis were observed. ET-1 (10 nM for 24 h) does not induce lipolysis in differentiated HWPs as indicated by free glycerol release in conditioned media. Data from at least eight independent experiments are presented as means ± SD. Overall P value from Wilcoxon/Kruskal–Wallis test. *P < 0.05 vs. control as determined by paired Wilcoxon test. ET-1, endothelin-1; HWPs, human white preadipocytes.

Figure 3.

Endothelin-1 induces phosphorylation of cellular signaling pathways in a time-dependent manner. Representative Western blot images showing ET-1 (10 nM)-mediated phosphorylation of ERK (A) and AKT (B) in differentiated HWPs over time (min). Data are presented as means ± SD from four independent experiments. Values normalized to 0 min. Overall P value from Wilcoxon/Kruskal–Wallis test. *P < 0.05 vs. time 0 as determined by paired Wilcoxon test. ERK, extracellular signal-regulated kinase; ET-1, endothelin-1; HWPs, human white preadipocytes.

Figure 4.

Chronic exposure to ET-1 and bosentan does not alter Bt₂cAMP-mediated activation of lipolysis. Differentiated HWPs were exposed to ET-1 (1 µM) and bosentan (10 µM) for 72 h before induction of lipolysis by Bt₂cAMP as determined at glycerol release. Data from at least six independent experiments are presented as means ± SD. Overall P value from Wilcoxon/Kruskal–Wallis test. *P < 0.05 vs. untreated cells stimulated by Bt₂cAMP as determined by Wilcoxon test. Bt₂cAMP, dibutyryl cAMP; ET-1, endothelin-1; HWPs, human white preadipocytes.

Figure 5.

Chronic exposure to ET-1 does not alter transcription of cellular adrenergic receptors. Graphs showing mRNA expression of α₁-adrenergic receptor (ADRA1A, A), α₂-adrenergic receptor (ADRA2A, B), β₁-adrenergic receptor (ADRB1, C), and β₂-adrenergic receptor (ADRB2, D) after 72-h exposure to increasing concentration of ET-1. mRNA expression was calculated as ratio to endogenous control (GAPDH) and is presented as percentage change from control (0 nM, ET-1). Data from at least five independent experiments are presented as means ± SD. Overall P value from Wilcoxon/Kruskal–Wallis test. *P < 0.05 vs. Control (0) as determined by paired Wilcoxon test. ET-1, endothelin-1.

Figure 6.

Chronic exposure to bosentan increases transcription of some cellular adrenergic receptors. Graphs showing mRNA expression of α₁-adrenergic receptor (ADRA1A, A), α₂-adrenergic receptor (ADRA2A, B), β₁-adrenergic receptor (ADRB1, C), and β₂-adrenergic receptor (ADRB2, D) after 72-h exposure to increasing concentration of bosentan. Effects of bosentan (1 µM) in presence of ET-1 (1,000 nM) were also evaluated. mRNA expression was calculated as ratio to endogenous control (GAPDH) and is presented as percentage change from control (0 µM, bosentan). Data from at least three independent experiments are presented as means ± SD. Overall P value from Wilcoxon/Kruskal–Wallis test *P < 0.05 vs. Control (0) as determined by paired Wilcoxon test. ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7.

Chronic exposure to bosentan increases transcription of ET-1 receptors. Graphs showing mRNA expression of ET_A-receptor (A) and ET_B-receptor (B) after 72-h exposure to increasing concentration of bosentan. mRNA expression was calculated as ratio to endogenous control (GAPDH) and is presented as percentage change from control (0 µM, bosentan). Data from at least three independent experiments are presented as means ± SD. Overall P value from Wilcoxon/Kruskal–Wallis test. *P < 0.05 vs. Control (0) as determined by paired Wilcoxon test. ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.