Empagliflozin improves primary haemodynamic parameters and attenuates the development of atherosclerosis in high fat diet fed APOE knockout mice
Introduction
Type 2 diabetes (T2DM) prevalence rates have been increasing during the past decades, in parallel to the documented obesity epidemic [NCD-RisC, 2016; Zimmet and Alberti, 2016]. T2DM comprises up to 90% of all diabetic cases in adults, with the most recent International Diabetes Federation (IDF) estimates indicating that 415 million adults (1 in 11 adults) have diabetes, a number predicted to reach 642 million (1 in 10 adults) by 2040 [IDF, 2015]. Furthermore, close pathogenic links exist between diabetes and cardiovascular disease (CVD), with CVD currently representing the main cause of morbidity/mortality in diabetic patients (up to 80% of all diabetic patients die from CVD-related events) [ESC 2013; Matheus et al. 2013]. It becomes evident that T2DM-related cardio-metabolic disease poses a significant challenge in clinical practice, requiring new effective treatment options which will lower the disease burden and particularly the associated CVD risk.
Despite advances in our understanding of T2DM pathophysiology, so far the applied glucose-lowering strategies have had little or no impact on CVD progression/outcomes in T2DM patients, whilst only a limited number of new medications have been added to the arsenal against the current diabesity epidemic [e.g. incretin mimetics and sodium/glucose cotransporter 2 (SGLT2) inhibitors] [Bolen et al., 2016; Thompson and Davis, 2016]. Moreover, the complete spectrum of effects of these new anti-diabetic agents has not been fully clarified yet. In this context, current research is focused on exploring the exact effects/outcomes of the new anti-diabetic medications [e.g. of glucagon-like peptide-1 (GLP-1) agonists and SGLT2 inhibitors] not only in controlling hyperglycaemia, but also on T2DM-related CVD. Indeed, following the lack of success in significantly controlling CVD progression in T2DM with previous anti-diabetic medications and tight glycaemic control, there is now increasing evidence indicating that certain newer anti-diabetic agents, particularly GLP-1 agonists (e.g. liraglutide) and SGLT2 inhibitors (e.g. empagliflozin and dapagliflozin) will offer significant CVD benefits independent of glycaemic control [Thompson and Davis, 2016; Flory et al. 2016].
SGLT2 inhibitors constitute the newest class of anti-diabetic medications which act by increasing urinary glucose excretion, and, hence, improve glycaemic control independently of insulin secretion [Heerspink et al. 2016; Marx and McGuire, 2016]. Recently, the EMPA-REG OUTCOME trial, a key CVD outcome trial, showed that empagliflozin significantly lowered the combined CVD endpoint of CVD death, non-fatal stroke and non-fatal myocardial infarction in T2DM patients with prevalent CVD [Zinman et al. 2015]. Moreover, empagliflozin unexpectedly induced a significant reduction in the individual endpoints of CVD death, heart failure hospitalization and overall mortality in this high CVD risk population of T2DM patients. Of note, a recent meta-analysis by Zelniker et al. showed that the benefits of SGLT-2 inhibitors are more pronounced in patients with established atherosclerotic CVD [Zelniker et al. 2019]. Thus, much research focus has been placed on elucidating the mechanisms responsible for these beneficial CVD effects of SGLT2 inhibition, which appear to extend beyond glucose control, potentially including mechanisms relating to weight loss, blood pressure lowering and sodium depletion, neuro-hormonal and renal haemodynamic effects, and effects on myocardial energetics/signalling [Heerspink et al. 2016; Marx and McGuire, 2016].
In the present study, we aimed to investigate the long-term effect of empagliflozin using a dose of 10 mg/kg/day on atherosclerosis development in the aorta of the APOE (−/−) atherosclerosis mouse model focusing particularly on its role in local factors related to atheroma plaque stability. Furthermore, classic CVD risk factors such as hyperlipidemia, hypertension, weight gain and inflammation were evaluated.
Section snippets
Animals
APOE (−/−) mice (C57BL/6J-ApoEtm1Unc) were originally purchased from “The Jackson Laboratory” and bred in the animal facility of National and Kapodistrian University of Athens. Mice were kept at specific pathogen free (SPF) controlled environment (22–26 °C temperature, 40–60% humidity and 12 h light/dark cycle). Animal experiments were approved by the local Animal Care and Use Committee.
Experimental protocols
20 male APOE (−/−) mice were kept on a standard rodent chow. At the age of 5 weeks, mice were switched to
Quantification of atherosclerotic lesion area
Aortic tissues were fixed and embedded in paraffin. The 4-μm-thick sections were stained with hematoxylin–eosin (H&E) and used for histopathological analysis whereas Masson's trichrome stained sections were used to quantify tissue section's collagen content. The degree of pathological changes was evaluated microscopically by measuring the area of atheromatous plaque. Results are reported as the percentage of the neointima area containing the lesion. Threshold was set and the positively stained
Oral administration of empagliflozin for 10 weeks improved diastolic blood pressure, heart rate and reduced fasting blood glucose levels
No significant difference in daily food intake was observed between the two groups. Body weight was significantly increased in both groups after feeding HFD and 10 weeks of oral Empagliflozin/vehicle administration compared to the value measured at experiment baseline. No significant difference in weight gain was observed between Empagliflozin and control group (data not shown but available in supplementary material-Supplementary Fig. 1).
Fasting blood glucose (8 h of fasting) and serum lipid
Discussion
Although clinical trials have showed the anti-atherogenic effects of SGLT2-i in patients with T2DM, providing data for primary and secondary prevention for CVD, the exact mode of their direct and/or indirect actions mediating this effect is not fully explored yet [Heerspink et al. 2016; Marx and McGuire, 2016]. Interestingly, according to the EMPA-REG OUTCOME Trial, reductions in key CV outcomes and mortality with empagliflozin vs placebo were consistent across the wide spectrum of CV risk [
Conclusions
Although more mechanistic studies need to be performed, our study suggests that empagliflozin has anti-atherogenic properties affecting traditional CVD factors such as HDL-cholesterol, hypertension and heart rate, though not inducing weight loss. Undoubtedly, glucose lowering effects contribute to this result; however, there is sufficient evidence in the literature, suggesting that empagliflozin may have pleiotropic actions. In this study, we commenced empagliflozin administration after 5 weeks
Conflicts of interest
The authors declare that they have no competing interests.
Ethics approval and consent to participate
This study was approved by the Athens University Medical School Ethics Committee and the Veterinary Directorate of Attica Region in agreement with Directive 2010/63/EU and all animal experiments were performed in compliance with the European Guideline for experimental animal research.
Funding
This work was supported by Novo-Nordisk Hellas.
Acknowledgements
The authors are grateful to Vasiliki Kalotychou for her technical assistance.
References (50)
- et al.
Novel anti-glycemic Drugs and reduction of cardiovascular risk in diabetes: Expectations realized, Promises unmet
Curr. Atheroscler. Rep.
(2016 Dec) - et al.
The SGLT2 inhibitor empagliflozin improves insulin sensitivity in db/db mice both as monotherapy and in combination with linagliptin
Metabolism
(2016 Feb) - et al.
The SGLT2 inhibitor empagliflozin improves the primary diabetic complica‐ tions in ZDF rats
Redox Biol.
(2017) - et al.
SGLT1 in pancreatic α cells regulates glucagon secretion in mice, possibly explaining the distinct effects of SGLT2 inhibitors on plasma glucagon levels
Mol. Metab.
(2019 Jan) - et al.
Prevention of progression of diabetic nephropathy by the SGLT2 inhibitor ipragliflozin in uninephrectomized type 2 diabetic mice
Eur. J. Pharmacol.
(2018 Jul 5) - et al.
SGLT2 inhibitors for primary and secondary prevention of cardiovascular and renal outcomes in type 2 diabetes: a systematic review and meta-analysis of cardiovascular outcome trials
Lancet
(2019 Jan 5) - et al.
Empagliflozin, calcium, and SGLT1/2 receptor affinity: another piece of the puzzle
ESC Heart Fail
(2018 Aug) - et al.
Glycemic control by the SGLT2 inhibitor empagliflozin decreases aortic stiffness, renal resistivity index and kidney injury
Cardiovasc. Diabetol.
(2018 Jul 30) - et al.
Effects of sodium-glucose co-transporter 2 inhibitors on blood pressure: a systematic review and meta-analysis
J. Am. Soc. Hypertens.
(2014 Apr) - et al.
Mechanism of increased LDL (Low-Density lipoprotein) and decreased triglycerides with SGLT2 (Sodium-Glucose cotransporter 2) inhibition
Arterioscler. Thromb. Vasc. Biol.
(2018 Sep)
Diabetes Medications for Adults with Type 2 Diabetes: an Update [Internet]
ESC Guidelines on diabetes, pre-diabetes, and cardiovascular diseases developed in collaboration with the EASD: the Task Force on diabetes, pre-diabetes, and cardiovascular diseases of the European Society of Cardiology (ESC) and developed in collaboration with the European Association for the Study of Diabetes (EASD)
Eur. Heart J.
SGLT-2 inhibitors: pharmacokinetics characteristics and effects on lipids
Expert Opin. Drug Metabol. Toxicol.
Empagliflozin reduced mortality and hospitalization for heart failure across the spectrum of cardiovascular risk in the EMPA-REG OUTCOME trial. Circulation
Inhibition of kidney proximal tubular glucose reabsorption does not prevent against diabetic nephropathy in type 1 diabetic eNOS knockout mice
PLoS One
Sodium glucose transporter 2 (SGLT2) inhibition with empagliflozin improves cardiac diastolic function in a female rodent model of diabetes
Cardiovasc. Diabetol.
Empagliflozin improves left ventricular diastolic dysfunction in a genetic model of type 2 diabetes
Cardiovasc. Drugs Ther.
The beneficial effects of empagliflozin, an SGLT2 inhibitor, on atherosclerosis in ApoE (-/-) mice fed a western diet
Diabetologia
The Role of MCP-1 in atherosclerosis
Stem Cell.
Sodium glucose cotransporter 2 inhibitors in the treatment of diabetes mellitus: cardiovascular and kidney effects, potential mechanisms, and clinical applications
Circulation
IDF Diabetes Atlas
Tofogliflozin, a selective inhibitor of sodium-glucose cotransporter 2, suppresses renal damage in KKAy/Ta mice, obese and type 2 diabetic animals
Diabetes Vasc. Dis. Res.
Effects of canagliflozin on weight loss in high fat diet‐induced obese mice
PLoS One
24-Hour blood pressure-lowering effect of an SGLT-2 inhibitor in patients with diabetes and uncontrolled nocturnal hypertension: results from the randomized, placebo-controlled SACRA study
Circulation
Empagli‐ flozin lessened cardiac injury and reduced visceral adipocyte hypertro‐ phy in prediabetic rats with metabolic syndrome
Cardiovasc. Diabetol.
Cited by (34)
Targeting inflammatory signaling pathways with SGLT2 inhibitors: Insights into cardiovascular health and cardiac cell improvement
2024, Current Problems in CardiologySodium-Glucose Cotransporter-2 Inhibitors: Impact on Atherosclerosis and Atherosclerotic Cardiovascular Disease Events
2022, Heart Failure ClinicsCitation Excerpt :Although results from animal studies suggest some potential improvement in microcirculatory function,38,39 human studies evaluating changes in flow-mediated dilatation and markers of arterial stiffness such as pulse wave velocity have been conflicting40–44 and may instead relate to associated changes in blood pressure or body weight.45–49 Animal models have shown SGLT-2i can reduce atherosclerotic plaque number and size,50 with conflicting data as to the role of diabetes in these observations.21,25,51,52 Whether SGLT-2i modifies the propensity for plaque formation to a greater degree in the presence of glycemia/diabetes status, or whether this relates to the length of SGLT-2i treatment remains unclear.
Antidiabetic drugs and oxidized low-density lipoprotein: A review of anti-atherosclerotic mechanisms
2021, Pharmacological ResearchCitation Excerpt :In rodents, SGLT2 inhibitors have also been found to significantly suppress various inflammatory mediators that are possibly induced by oxLDL during the development of atherosclerosis. Oral administration of empagliflozin to ApoE-/- mice with, or without diabetes, resulted in lower levels of TNF-α, IL-6, MCP-1, ICAM-1, and VCAM-1 [183–185]. One study specifically related this finding to lower oxLDL receptor CD36 in heart tissue [182].
Endothelial function and dysfunction: Impact of sodium-glucose cotransporter 2 inhibitors
2021, Pharmacology and TherapeuticsCitation Excerpt :This reduction was associated with lower levels of high-sensitivity C-reactive protein (hsCRP), TNF-α, IL-6, and CCL-2 (Han et al., 2017). Another study involving treatment of ApoE−/− mice with empagliflozin (10 mg/kg/day orally for 10 weeks) showed a reduction in the atherosclerotic lesion area and decreased mRNA expression of VCAM-1 and monocyte chemoattractant protein-1 (MCP-1) in the aortic root lesion (Dimitriadis et al., 2019). Empagliflozin (20 mg/kg/day orally for eight and 12 weeks) also suppressed atherosclerotic lesion development, macrophage accumulation and expression of inflammatory molecules (MCP-1, VCAM-1, CD68) and NOX subunits (NOX-2 and p22phox) in the aorta of diabetic ApoE−/− mice (Ganbaatar et al., 2020).
Anti-inflammatory effects of sodium-glucose co-transporter 2 inhibitors on atherosclerosis
2020, Vascular PharmacologyCitation Excerpt :Empagliflozin (10 mg/kg/day) has also been confirmed to attenuate the progression of atherosclerosis in the aortae of ApoE−/− mice. Histomorphometry revealed that empagliflozin treatment widened the lumen area in aortic roots by nearly 50%, and reduced atherosclerotic lesion formation at near-statistically significant levels (P = 0.06) [48]. Canagliflozin (10 mg/kg/day) administration for 5 weeks significantly reduced the progression of atherosclerosis by 25%.