Liraglutide treatment improves the coronary microcirculation in insulin resistant Zucker obese rats on a high salt diet

Abstract

Background: Obesity, hypertension and prediabetes contribute greatly to coronary artery disease, heart failure and vascular events, and are the leading cause of mortality and morbidity in developed societies. Salt sensitivity exacerbates endothelial dysfunction. Herein, we investigated the effect of chronic glucagon like peptide-1 (GLP-1) receptor activation on the coronary microcirculation and cardiac remodeling in Zucker rats on a high-salt diet (6% NaCl).

Methods: Eight-week old Zucker lean (+/+) and obese (fa/fa) rats were treated with vehicle or liraglutide (LIRA) (0.1 mg/kg/day, s.c.) for 8 weeks. Systolic blood pressure (SBP) was measured using tail-cuff method in conscious rats. Myocardial function was assessed by echocardiography. Synchrotron contrast microangiography was then used to investigate coronary arterial vessel function (vessels 50-350 µm internal diameter) in vivo in anesthetized rats. Myocardial gene and protein expression levels of vasoactive factors, inflammatory, oxidative stress and remodeling markers were determined by real-time PCR and Western blotting.

Results: We found that in comparison to the vehicle-treated fa/fa rats, rats treated with LIRA showed significant improvement in acetylcholine-mediated vasodilation in the small arteries and arterioles (< 150 µm diameter). Neither soluble guanylyl cyclase or endothelial NO synthase (eNOS) mRNA levels or total eNOS protein expression in the myocardium were significantly altered by LIRA. However, LIRA downregulated Nox-1 mRNA (p = 0.030) and reduced ET-1 protein (p = 0.044) expression. LIRA significantly attenuated the expressions of proinflammatory and profibrotic associated biomarkers (NF-κB, CD68, IL-1β, TGF-β1, osteopontin) and nitrotyrosine in comparison to fa/fa-Veh rats, but did not attenuate perivascular fibrosis appreciably.

Conclusions: In a rat model of metabolic syndrome, chronic LIRA treatment improved the capacity for NO-mediated dilation throughout the coronary macro and microcirculations and partially normalized myocardial remodeling independent of changes in body mass or blood glucose.

Keywords: Endothelial dysfunction; Inflammation; Liraglutide; Nitric oxide; Oxidative stress.

Fig. 1

Representative synchrotron angiograms of the coronary vasculature in Zucker rats after 8 weeks on a high-salt diet. a Images during baseline, infusions of acetylcholine (ACh). b Acute infusion protocol during coronary vasculature imaging. c Vessel internal diameter categorized by branching order during baseline ringer’s lactate infusion. d Visualized vessel number during baseline. Calibration wire (yellow arrow) = 50 μm. The data are shown as the mean ± SEM; N = 5–6 rats per group. The significance of group differences was determined by ANOVA for factors of genotype (G), drug treatment (T) and their interaction (I)

Fig. 2

LIRA treatment improved the capacity to increase perfused segments by NO-mediated dilation in Zucker fa/fa and +/+ rats after 8 weeks on a high-salt diet. a–d Percentage change in 1st to 4th order vessel caliber and visible vessel number during infusions of ACh and ACh stimulation during blockade of prostaglandin production and uncoupling of gap junctions from baseline. Mean ± SEM. The significance of group differences was determined by ANOVA for factors of genotype (G), drug treatment (T) and their interaction (I)

Fig. 3

Mean arterial pressure (MAP) and mean heart rate (MHR) and their change during the acute infusion. a Change in MAP and change in MHR during baseline and b, c infusions of ACh, indomethacin + carbenoxolone (blockade) and post-blockade + ACh relative to vehicle Ringer’s lactate solution (baseline). Mean ± SEM. N = 5–6 rats per group. The significance of group differences was determined by 2-way ANOVA for factors of genotype (G) and drug treatment (T) and their interaction (I)

Fig. 4

The effects of LIRA treatment on expression of CD-68, NF-κB, TGF-β1 and IL-1β in the left ventricle. a Protein expression levels of CD-68, NF-κB, TGF-β1 and IL-1β detected by Western blotting. β-actin was used as an internal control. Bands are representative of four separate experiments. b–e Relative density values of CD-68, NF-κB, TGF-β1 and IL-1β. f mRNA expression of Vcam-1 in Zucker rat myocardium. GAPDH gene was used as an internal control. The ΔΔcT values are shown as the mean ± SEM. N = 5–6 rats per group. The significance of group differences was determined by ANOVA for factors of genotype (G), drug treatment (T) and their interaction term (I)

Fig. 5

The effects of LIRA treatment on nitrosative, oxidative stress marker and anti-oxidant levels. a Protein expression levels of NT in left ventricle detected by Western blotting. β-actin was used as an internal control. Bands are representative of two separate experiments. b Relative density values of NT. c, d mRNA expressions of NOX-1 and Gpx-1 in Zucker rat myocardium. GAPDH gene was used as an internal control. The ΔΔcT values are shown as the mean ± SEM. Effects of LIRA on Reduced GSH (e), oxidized GSSG (f), and GSH/GSSG ratio (g). N = 5–6 rats per group. The significance of group differences was determined by ANOVA for factors of genotype (G), drug treatment (T) and their interaction term (I)

Fig. 6

Changes in mRNA abundance of genes related to NO-signaling pathway. a–c mRNA expressions of VEGF, eNOS and ET-1 in Zucker rat myocardium. GAPDH gene was used as an internal control. The ΔΔcT values are shown as the mean ± SEM. d–h The effects of LIRA treatment on expression of VEGF, total-eNOS, ET-1 and GLP-1R in the left ventricle. d Protein expression levels of VEGF, total-eNOS, ET-1 and GLP-1R detected by Western blotting. β-actin was used as an internal control. Bands are representative of five separate experiments. e–h Relative density values of VEGF, total-eNOS, ET-1 and GLP-1R. N = 5–6 rats per group. The significance of group differences was determined by ANOVA for factors of genotype (G), drug treatment (T) and their interaction term (I)

Fig. 7

Azan-Mallory staining for perivascular collagen expression. a Azan-Mallory stained myocardial sections from a vehicle-treated +/+ and LIRA-treated +/+ rats showing low levels of collagen deposition (blue fibrils); broad adventitial ring of strong blue fibrotic tissue reactivity in a vehicle-treated fa/fa rats; LIRA-treated fa/fa rats showing moderate blue fibrous tissue positivity around a coronary artery. Scale bar are 20 µm. b Quantification of perivascular fibrosis (%). c Protein expression levels of OPN detected by Western blotting. β-actin was used as an internal control. Bands are representative of two separate experiments. Relative density values of OPN, data are shown as the mean ± SEM, N = 5–6 rats per group. The significance of group differences was determined by ANOVA for factors of genotype (G), drug treatment (T) and their interaction term (I)

Fig. 8

a Hematoxylin and eosin stained sections of myocardium illustrating cardiomyocytes in cross-section (×400). Scale bars are 20 µm. b Bar graph showing quantitative analysis of cross -sectional area of cardiomyocytes. c The effects of LIRA treatment on expression of ANP in left ventricle. c Protein expression levels of ANP detected by Western blotting. β-actin was used as an internal control. Bands are representative of two separate experiments. Relative density values of ANP, data are shown as the mean ± SEM, N = 5–6 rats per group. The significance of group differences was determined by ANOVA for factors of genotype (G), drug treatment (T) and their interaction term (I)

Fig. 9

Proposed schematic representation for the beneficial effects of LIRA in coronary dysfunction associated with the additive effects of obesity and a high-salt diet. High-salt diet induced coronary dysfunction is characterized by increased RNS-production, inflammation and reduced NO bioavailability. LIRA treatment improved endothelial function, oxidative stress and inflammation. These effects are mediated through the downregulation of ET-1, TGF-β1 and upregulation of eNOS and to a variable extent VEGF