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

<div><p>Objective</p><p>In 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.</p><p>Methods</p><p>Type 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).</p><p>Results</p><p>Treatment 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.</p><p>Conclusions</p><p>Empagliflozin 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.</p></div

Effects of SGLT2i treatment on aortic protein expression of the NO/cGMP signaling cascade as well as oxidative stress and inflammatory pathways in diabetic rats.

<p>Expression of endothelial nitric oxide synthase (eNOS, <b>A</b>), serine1177 phosphorylated eNOS (<b>B</b>), dihydrofolate reductase (DHFR, <b>C</b>), ratio of cGK-I and serine239 phosphorylated VASP (<b>D</b>) were assessed by Western blotting analysis and specific antibodies. Expression of NADPH oxidases Nox1 (<b>E</b>) and Nox2 (<b>F</b>), heme oxygenase-1 (HO-1) (<b>G</b>) and monocyte-chemoattractant-protein-1 (MCP-1 or CCL-2, <b>H</b>) were assessed by Western blotting analysis and specific antibodies. Representative blots for all proteins are shown in supplemental Figure S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112394#pone.0112394.s001" target="_blank">File S1</a>. The data are expressed as % of control and are the means ± SEM from 8–9 (<b>A</b>), 5–6 (<b>B</b>), 7 (<b>C</b>), 4 (<b>D</b>), 6–7 (<b>E</b>), 7–9 (<b>F</b>), 7–9 (<b>G</b>) and 4–6 (<b>H</b>) animals/group. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected and ^$, p<0.05 vs. low dose SGLT2i treated.</p

Weight gain, and blood and serum parameters in controls and diabetic rats.

a<p>Weight gain was calculated from the difference of values prior and 8 weeks post STZ injection. Blood glucose was determined three days after STZ injection without SGLT2i treatment; fasting and non-fasting blood glucose as well as HbA1c levels were measured 8 weeks post STZ injection. The data are the means ± SEM of the indicated number of animals/group; n.d. means not detectable.</p><p>*, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected and ^§, p<0.05 vs. low dose SGLT2i treated.</p>b<p>Separation of HDL and LDL using HF5 (Hollow Fiber Flow Field Flow Fractionation).</p><p>Weight gain, and blood and serum parameters in controls and diabetic rats.</p

Summary of beneficial effects of SGLT2i treatment on diabetes induced vascular dysfunction and other adverse effects of hyperglycemia in rats.

<p>Summary of beneficial effects of SGLT2i treatment on diabetes induced vascular dysfunction and other adverse effects of hyperglycemia in rats.</p

Effects of SGLT2i treatment on vascular parameters in diabetic rats.

<p>Microscopic determination of wall thickness (grey) and collagen content (black) by sirius red staining of paraffinated aortic sections (<b>A</b>). Representative microscope images are shown along with the densitometric quantification. Effects of SGLT2i therapy on endothelium-dependent and independent vascular relaxation by the vasodilators acetylcholine (ACh, <b>B</b>) and nitroglycerin (GTN, <b>C</b>), respectively. Data are the means±SEM from 6–7 (<b>A</b>) and 9–12 (<b>B,C</b>) animals/group. Each single value for an animal corresponds to the means of 4 individual aortic rings from this animal. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected group. For the vascular function data the significance levels were determined by two-way-ANOVA and significances for the entire curves are indicated when at least one compared concentration condition showed significant differences. Vasodilator potency (EC₅₀, pD₂) and efficacy (max. relaxation) were also calculated and subjected to statistical analysis using one-way-ANOVA. The data and results are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112394#pone-0112394-t001" target="_blank">Table 1</a>.</p

Effects of SGLT2i treatment on AGE/RAGE signaling in diabetic rats.

<p>Quantification of AGE-positive proteins by dot blot analysis (<b>A</b>) and RAGE expression was assessed by Western blotting analysis with specific antibodies (<b>B</b>) and quantitative RT-PCR analysis (<b>C</b>). Representative blots are shown at the bottom of the densitometric quantifications. Serum methylglyoxal levels were assessed by HPLC-based quantification (<b>D</b>). Representative chromatograms are shown in supplemental Figure S7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112394#pone.0112394.s001" target="_blank">File S1</a>. The data are expressed as % of control and are the means ±SEM from 7 (<b>A</b>), 6–7 (<b>B</b>) and 8–11 (<b>C,D</b>) animals/group. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ–injected.</p

Effects of SGLT2i treatment on oxidative stress parameters in diabetic rats.

<p>Leukocyte-derived ROS (oxidative burst) in whole blood at 30 min upon zymosan A (<b>A</b>) stimulation along with the effects of the NADPH oxidase isoform 2 (Nox2) inhibitor VAS2870 and the intracellular calcium chelator BAPTA-AM (<b>B</b>) and upon PDBu stimulation (<b>C</b>). <b>(D)</b> Quantification of cardiac NADPH oxidase activity in membrane preparations by lucigenin (5 µM)-derived chemiluminescence. Dihydroethidium (DHE, 1 µM)-fluorescence microtopography was used to assess the effects of SGLT2i treatment on vascular (<b>E</b>) and endothelial (<b>F</b>) ROS production with and without incubation with the eNOS inhibitor L-NAME. Representative microscope images are shown along with the densitometric quantification. Red fluorescence indicates ROS formation whereas green fluorescence represents basal laminae autofluorescence. Data are the means±SEM from 6–7 (<b>A,B</b>), 5 (<b>C</b>), 7–8 (<b>D</b>), 9 (<b>E</b>) or 5–6 (<b>F</b>) animals/group. *, p<0.05 vs. control and ^#, p<0.05 vs. STZ-injected and ^§, p<0.05 vs. low dose SGLT2i treated and ^$, p<0.05 vs. w/o L-NAME.</p