Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 21, 2020; 26(23): 3225-3235
Published online Jun 21, 2020. doi: 10.3748/wjg.v26.i23.3225
Sodium glucose co-transporter 2 inhibition reduces succinate levels in diabetic mice
Lakshini Y Herat, Natalie C Ward, Aaron L Magno, Elizabeth P Rakoczy, Marcio G Kiuchi, Markus P Schlaich, Vance B Matthews
Lakshini Y Herat, Vance B Matthews, School of Biomedical Sciences, Dobney Hypertension Centre, Royal Perth Hospital Unit, University of Western Australia, Perth 6000, Australia
Natalie C Ward, Faculty of Health and Medical Sciences, University of Western Australia, Crawley 6009, Australia
Natalie C Ward, Faculty of Health Sciences, School of Public Health, Curtin University, Bentley 6102, Australia
Aaron L Magno, Research Centre, Royal Perth Hospital, Perth 6000, Australia
Elizabeth P Rakoczy, Department of Molecular Ophthalmology, University of Western Australia, Crawley 6009, Australia
Marcio G Kiuchi, Markus P Schlaich, Dobney Hypertension Centre, School of Medicine, Royal Perth Hospital Unit, University of Western Australia, Perth 6000, Australia
Markus P Schlaich, Department of Cardiology and Department of Nephrology, Royal Perth Hospital, Perth 6000, Australia
Author contributions: Matthews VB and Schlaich MP are equal senior authors and funded the study; Matthews VB designed and coordinated the study; Herat LY, Ward NC and Matthews VB performed the experiments, acquired and analysed data and wrote the manuscript; Magno AL performed the experiments; all authors provided intellectual input; all authors proof read the manuscript and approved the final version of the article.
Supported by the Royal Perth Hospital Medical Research Foundation, No. VMMRF2018.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Harry Perkins Institute for Medical Research (AE141/2019).
Conflict-of-interest statement: Prof. Schlaich is supported by a National Health and Medical Research Council Research Fellowship and has received research support from Medtronic, Abbott, Novartis, Servier, Pfizer, and Boehringer Ingelheim. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. No potential conflicts of interest exist from other authors.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Vance B Matthews, BSc, PhD, Senior Research Fellow, School of Biomedical Sciences, Dobney Hypertension Centre, Royal Perth Hospital Unit, University of Western Australia, Level 3, MRF Building, Rear 50 Murray Street, Perth 6000, Australia. vance.matthews@uwa.edu.au
Received: February 28, 2020
Peer-review started: February 28, 2020
First decision: April 22, 2020
Revised: May 19, 2020
Accepted: June 9, 2020
Article in press: June 9, 2020
Published online: June 21, 2020
Processing time: 114 Days and 9.6 Hours
ARTICLE HIGHLIGHTS
Research background

Type 1 diabetes (T1D) dramatically increases chronic microvascular complications which is a leading cause of diabetes associated morbidity. Both human and murine studies highlight the role of the gut microbiome and gut dysbiosis in the pathogenesis of numerous diseases. It is well-established that diabetes and its complications are of multifactorial aetiology. Recent studies have highlighted the importance of perturbations in the gut microbiota as a contributing factor in the development and progression of diabetes and related complications. Therefore, many studies are now focusing on the gut microbiome as a potential source of biomarkers of diabetes and its complications.

Research motivation

The sodium glucose co-transporter 2 (SGLT2) inhibitors are a novel class of oral antidiabetic medications specifically used in the treatment of type 2 diabetes. It is well established that SGLT2 inhibitors block glucose reabsorption in the renal proximal tubules, thereby resulting in excretion of glucose in the urine and leads to improvements in metabolic and glycaemic parameters. However, preclinical and human studies investigating the beneficial mechanisms of SGLT2 inhibition in T1D and its complications are currently limited. Further pre-clinical investigations are essential to elucidate the underlying mechanisms by which SGLT2 inhibitors may impact the progression of T1D and its related complications. Therefore, we hypothesised that SGLT2 inhibition may exert its protective effects via alterations of the gut microbiome and tested this in a mouse model of T1D and diabetic retinopathy.

Research objectives

To investigate whether the treatment of type 1 diabetic mice with two independent SGLT2 inhibitors (empagliflozin and dapagliflozin) will affect gut health.

Research methods

To address the specific aims of our study, we used two of the most widely investigated SGLT2 inhibitors, empagliflozin or dapagliflozin and administered it to 10 wk old C57BL/6J, Akita, Kimba and Akimba mice for 8 wk via drinking water. At the end of the experiment, all mice were sacrificed and sera was collected. The concentration of succinate and the short-chain fatty acid (SCFA) butyric acid was measured using gas chromatography-mass spectrometry and enzyme immunoassays were conducted to determine insulin, leptin and norepinephrine concentrations. Pancreatic tissue was also wax embedded, sectioned and stained with haematoxylin and eosin and analysed using brightfield microscopy.

Research results

In comparison to C57BL/6J and Kimba mice, both Akita and Akimba mice showed reduced levels of insulin production due to the presence of the Akita allele. In line with this, Akita mice also showed the presence of apoptotic bodies within the pancreatic islets and the acinar cells of both the Akita and Akimba mice displayed swelling which is suggestive of acute injury or pancreatitis. In Akimba mice, SGLT2 inhibition with dapagliflozin for 8 wk significantly reduced succinate levels when compared to vehicle treated mice. Furthermore, succinate levels in Akimba mice treated with the SGLT2 inhibitor empagliflozin showed a similar trend. In diabetic Akita mice, the beneficial SCFA butyric acid was significantly increased after dapagliflozin treatment when compared to vehicle. There was a significant reduction in the kidney norepinephrine content in both dapagliflozin and empagliflozin treated Akita mice. Furthermore, the diabetic Akimba mice also showed a significant reduction in kidney norepinephrine content when treated with empagliflozin. Lastly, both non-diabetic C57BL/6J and Kimba mice showed significantly reduced serum leptin levels after dapagliflozin therapy.

Research conclusions

Our novel study compares and contrasts the effects of SGLT2 inhibition on the main products and intermediate metabolites of gut metabolism particularly in Akita and Akimba mice. We conducted studies using two independent SGLT2 inhibitors and showed that both inhibitors reduced the pathogenic biomarker succinate in our novel T1D Akimba mouse model of retinopathy. However, in relation to succinate levels in Akimba mice, dapagliflozin was more bioactive than empagliflozin, potentially due to factors such as increased half-life, longer duration of action, higher distribution and long retention period in the kidney. Furthermore, we demonstrate for the first time that SGLT2 inhibition is sympathoinhibitory in a T1D mouse model.

Research perspectives

In line with our findings, it would be mechanistically insightful in the future to assess the expression of the succinate specific receptor GPR91 in ocular tissue before and after SGLT2 inhibition as SGLT2 is expressed in the eye. Furthermore, it is important to determine whether our findings can be reproduced in patients with T1D and its complications who are treated with SGLT2 inhibitors.