The sodium-glucose co-transporter 2 inhibitor empagliflozin improves diabetes-induced vascular dysfunction in the streptozotocin diabetes rat model by interfering with oxidative stress and glucotoxicity.

Matthias Oelze,Eric Mayoux,Michael Hausding,Elena Zinßius,Saule Agdauletova,Andreas Daiber,Anna Gottschlich,Michael Mader,Yuliya Mikhed,Thomas Jansen,Paul Stamm,Eberhard Schulz,Thomas Münzel,Swenja Kröller-Schön,Philipp Welschof,Serge P Bottari,Sebastian Steven

doi:10.1371/journal.pone.0112394

Abstract

ObjectiveIn diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress.MethodsType I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE).ResultsTreatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy.ConclusionsEmpagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.

Highlights

Diabetes mellitus is one of the major risk factors for the development of cardiovascular disease [1]
Diabetic animals had higher triglyceride and total cholesterol levels compared to controls and to diabetic animals treated with SGLTi
Protein expression We have shown previously that eNOS and dihydrofolate reductase (DHFR) expression was increased in aorta from diabetic animals suggesting compensatory up-regulation of these enzymes due to the dysfunction/uncoupling of eNOS and oxidative depletion of its co-factor tetrahydrobiopterin (BH4) with subsequent induction of dihydrofolate reductase, the BH2 recycling enzyme

Summary

Introduction

Diabetes mellitus is one of the major risk factors for the development of cardiovascular disease [1]. The vascular NADPH oxidase and an uncoupled endothelial nitric oxide synthase (eNOS, type 3) have been identified as enzymatic sources of increased vascular production of reactive oxygen species (ROS) [3]. Adverse phosphorylation of eNOS at Thr495 and Tyr657 may potentially play a significant role as well [7,8]. Another major concept of diabetic pathology is based on direct glucotoxicity, including increased formation of advanced glycation end products (AGE) and their signaling via specific receptors (RAGE) leading to vascular dysfunction and end organ damage [9,10]. Increased vascular oxidative stress can lead to immune cell activation [14,15] or is even mediated by inflammatory cells, as was recently demonstrated in the angiotensin II infusion model [16]

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Conclusion