Corresponding author: Neng Fisheri Kurniati (
Cardiovascular benefit of new anti-hyperglycemic agent such as glucagon like peptide-1 receptor agonist (
Diabetes mellitus (
According to guidelines by American Diabetes Association (
Although the cardiovascular and kidney benefits of both classes are well-established, the molecular mechanism of this benefits are still being elucidated, and whether the combination will provide added risk reduction remains questioned.
Not only on cardiovascular risk factor, some studies may also suggest a direct positive effect of the combination on cardiovascular. One study suggested the benefit of dual therapy on vascular and cardiac function in T2DM patient with high
Therefore, in this study we investigated the cardioprotective effect of both agents against several cardiac biomarkers that usually elevated in damaged heart, to suggest whether the combination is able to protect the heart better than
Thirty male Wistar rats, aged 10–12 weeks, weighing 200–250 g, were purchased from ITB School of Life Sciences and Technology and housed in the animal laboratory at ITB School of Pharmacy in separate cages under controlled environmental condition (12 h light/dark cycle and temperature 24–26 °C) with free access of standard chow and water. The research protocols were approved by the ethics committee for animal research of Bandung Institute of Technology (No.01/KEPHP-ITB/2-2022, 18 February 2022).
The rats were acclimated for 7 days before the study, then randomly divided into 6 groups as follows:
Normal control group: nondiabetic rats, given CMC-Na 0.5% via intragastric (i.g.) and NaCl 0.9% subcutaneously (s.c.) for 30 days;
DM group: diabetic rats, given CMC-Na 0.5% (i.g.) and NaCl 0.9% (s.c.) for 30 days;
DM+ISO group: diabetic rats, given CMC-Na 0.5% (i.g.) and NaCl 0.9% (s.c.) for 30 days, and received isoproterenol (ISO) intraperitoneally (i.p.) at dose 85 mg/kg BW on day 29 and 30 (at 24 hours interval) to induce AMI;
GLP-1RA group: diabetic rats, given liraglutide (Victoza, Novo Nordisk, Denmark) at a dose of 0.062 mg/kg BW (dissolved in NaCl 0,9%) via s.c. for 30 days, and received ISO (i.p.) at dose 85 mg/kg BW on day 29 and 30 (at 24 hours interval) to induce AMI (
SGLT2i group: diabetic rats, given empagliflozin (Jardiance, Bohringer Ingelheim Pharmaceuticals, USA) at a dose of 1 mg/kg BW (dissolved in CMC-Na 0,5%) via i.g. for 30 days, and received ISO (i.p.) at dose 85 mg/kg BW on day 29 and 30 (at 24 hours interval) to induce AMI (
Combination group: diabetic rats, given liraglutide (Victoza, Novo Nordisk, Denmark) at a dose of 0.062 mg/kg BW (dissolved in NaCl 0,9%) via s.c. and empagloflozin (Jardiance, Bohringer Ingelheim Pharmaceuticals, USA) at a dose of 1 mg/kg BW (dissolved in CMC-Na 0,5%) via i.g. for 30 days, and received ISO (i.p.) at dose 85 mg/kg BW on day 29 and 30 (at 24 hours interval) to induce AMI.
T2DM are characterized by the reduction of insulin sensitivity with impaired insulin secretion. Therefore, this study used the combination model to mimic those pathology (
The blood glucose control profile was obtained from
The blood samples were collected from tail (before MI induction) and from heart (after MI induction, as a terminal procedure), then serum samples were separated for the estimation of cardiac biomarkers. The serum levels of CK, CK-MB, LDH, AST and ALT were measured using commercially kits according to the manufacturer’s instructions (DiaSys Diagnostic Systems GmbH, Holzheim, Germany for CK, CK-MB, LDH, and ALT; Glory Diagnostic, Spain for AST). All the biomarkers were assessed using Microlab 300, ELITech Group, France.
The rat’s heart was dissected, washed with 0.9% cold saline, then fixed in 10% neutral buffered formalin. The left ventricle then embedded in paraffin and sectioned at 3 µm thickness using microtome. The tissue specimens were stained using hematoxylin and eosin (H&E). Characteristic of myocytes abnormality such as coagulative necrosis with absent nuclei, hyper eosinophilia of cytoplasm, and an interstitial neutrophil infiltration were observed using light microscope (Olympus BX51) at 400× magnifications (
The data obtained in this study were processed using Minitab version 20 and analyzed using one-way ANOVA with Tukey’s post hoc test. Considered significantly different at p<0.05.
The animal study protocol was approved by the ethics committee for animal research of Bandung Institute of Technology (No.01/KEPHP-ITB/2-2022, 18 February 2022).
Insulin sensitivity was determined at baseline (before the induction), after the induction, and after receiving one or both agents for 4 weeks. The KITT values are shown in Table
KITT profile at baseline, after induction, and after treated with
Groups | KITT (%/minute) | ||
---|---|---|---|
Baseline | After induction | After treated | |
Normal control | 1.01±0.13 | 1.09±0.21 | 0.98±0.08 |
|
1.14±0.10 | 0.69±0.08a,c | 0.61±0.14a,c |
Liraglutide | 0.94±0.04 | 0.69±0.17a,c | 0.85±0.06c |
Empagliflozin | 1.00±0.16 | 0.57±0.15a,c | 0.93±0.14b,d |
Combination | 1.05±0.27 | 0.67±0.16a,c | 0.97±0.10b |
The data are expressed as mean ± SD. One-way ANOVA with Tukey Post hoc test. The letter shows signifcantly different (p<0.05) compared to anormal control, b
Before the induction, there were no significant difference of KITT value between groups that indicates similar insulin sensitivity at baseline. After given lipid emulsion for 14 days, all the tested groups showed significantly lower KITT value compared to normal control and baseline, indicate the reduction of blood glucose disappearance rate from blood (%/min) after given insulin (reduction of insulin sensitivity). The group treated with empagliflozin and/or liraglutide for 4 weeks showed the increase of KITT value. The KITT value of all treated group were not significantly different to normal control group. However, only empagliflozin and combination group that significantly higher than
Blood glucose control was obtained from
Group | Fasting blood glucose (mg/dL) | |||||
---|---|---|---|---|---|---|
Baseline | After induction | After treated (Week-) | ||||
1 | 2 | 3 | 4 | |||
Normal control | 93.80±7.53 | 101.00±12.53 | 91.60±12.10 | 91.20±13.88 | 98.00±12.19 | 89.00±8.28 |
|
93.25±3.50 | 259.00±109.77a,c | 269.00±93.68a,c | 312.50±135.93a,c | 350.25±121.93a,c | 407.25±89.11a,c |
Liraglutide | 93.00±6.98 | 266.00±85.81a,c | 115.25±16.58b,d | 123.00±24.18b,d | 122.00±12.94b,c,d | 106.50±9.18b,d |
Empagliflozin | 97.25±8.77 | 267.25±75.59a,c | 112.00±18.67b,d | 127.75±19.31b,c,d | 130.50±8.43b,c,d | 106.5±11.27b,d |
Combination | 94.20±13.52 | 253.40±26.84a,c | 96.00±5.66b,d | 108.60±22.32b,d | 126.60±22.03b,d | 95.20±7.09b,d |
The data are expressed as mean ± SD. One-way ANOVA with Tukey Post hoc test. The letter shows significantly different (p<0.05) compared to anormal control, b
Cardiac biomarkers were obtained from serum collected before and after
Effect of empagliflozin and/liraglutide on cardiac biomarkers in diabetic rats.
Groups | Cardiac biomarkers (U/L) | ||||
---|---|---|---|---|---|
CK | CK-MB | LDH | AST | ALT | |
Normal Control | 123.81±16.16b | 121.19±10.56b | 307.93±63.04b | 99.32±20.97b | 52.80±20.32 |
|
361.64±84.44a | 244.53±24.60a | 914.53±196.28a | 168.98±51.12a | 101.56±46.82 |
Liraglutide | 192.48±44.06b | 184.75±24.01a | 428.22±44.84b | 148.40±10.34 | 55.01±18.34 |
Empagliflozin | 149.19±70.24b | 147.00±18.68b | 508.97±129.63b | 142.40±36.57 | 70.93±7.67 |
Combination | 103.59±30.74b | 99.07±18.38b,c | 325.10±103.73b | 125.32±11.86 | 53.29±10.76 |
The data are expressed as mean ± SD. One-way ANOVA with Tukey Post hoc test. The letter shows significantly different (p<0.05) compared to anormal control, b
The CK, CK-MB, LDH, and AST levels were significantly elevated in
Percentage inhibition of serum cardiac biomarkers elevation in treated vs
Groups | Inhibition of cardiac biomarker serum elevation (%) | ||||
---|---|---|---|---|---|
CK | CK-MB | LDH | AST | ALT | |
Liraglutide | 46.77 | 23.48 | 53.18 | 12.18 | 45.83 |
Empagliflozin | 58.74 | 39.11 | 44.37 | 15.73 | 30.16 |
Combination | 71.36 | 58.97 | 64.45 | 25.83 | 47.53 |
The biomarkers level after
Effect of empagliflozin and/liraglutide on cardiac biomarkers in diabetic rats induced with AMI.
Groups | Cardiac biomarkers (U/L) | ||||
---|---|---|---|---|---|
CK | CK-MB | LDH | AST | ALT | |
Normal control | 136.85±28.27b | 134.61±12.87b | 325.37±90.41b | 115.79±17.25b | 51.86±16.89b |
|
361.13±103.63a,b | 263.51±33.41a,b | 1012.08±191.39a,b | 189.15±50.86a | 96.63±41.72 |
596.76±28.18a,c | 438.29±50,79a,c | 1679.11±255.76a,c | 247.89±56.45a | 123.98±59.32a | |
Liraglutide | 338.93±26.61a,b | 238.39±26.33a,b | 763.63±83.46a,b | 179.15±21.89 | 64.48±16.28 |
Empagliflozin | 275.17±145.08b | 214.28±18.81a,b | 671.68±102.99a,b | 180.40±12.19 | 69.54±19.12 |
Combination | 175.99±35.94b,* | 125.06±19.32b,# | 452.60±151.38b | 145.72±20.84b | 64.60±7.43 |
The data are expressed as mean ± SD. One-way ANOVA with Tukey Post hoc test. The letter shows significantly different (p<0.05) compared to anormal control, b
As for CK-MB and LDH parameter, the levels in all three treated group were significantly lower than
All these findings may indicate better inhibition on the elevation of these biomarkers in combination group. The data then transformed into percentage of inhibition, shown in Table
Percentage inhibition of serum cardiac biomarkers elevation in treated vs
Groups | Inhibition of cardiac biomarker serum elevation (%) | ||||
---|---|---|---|---|---|
CK | CK-MB | LDH | AST | ALT | |
Liraglutide | 43.21 | 45.61 | 54.52 | 27.73 | 47.99 |
Empagliflozin | 53.89 | 51.11 | 59.99 | 27.23 | 43.91 |
Combination | 70.51 | 71.48 | 73.05 | 41.22 | 47.90 |
In histopathological examination using H&E staining, regular arrangement of myocardial fibers was shown in normal control group (Fig.
Histopathological examination of heart tissue stained with H&E staining and 400× magnification.
Several in vivo studies have been conducted to confirmed cardiovascular benefit of both classes. Liraglutide, dapagliflozin, and empagliflozin showed cardioprotective effect in diabetic cardiomyopathy model (
Streptozotocin (
Lipid emulsion given for two weeks was significantly lowering the insulin sensitivity based on KITT value calculated from IPITT. This lipid emulsion contained triglycerides (
After the induction was confirmed and have been treated with either or both classes for 4 weeks, the
There are few differences in human and animal heart structure, that is challenging to design a good model to mimic pathophysiology of MI in human. In this study, we used isoproterenol (
Cardiomyocytes death causing the leak of several proteins into the circulation, thus the level of cardiac enzymes tends to be elevated in the serum after MI (
The biomarker measurement before
Glucose-lowering therapies alone were already proven to reduce microvascular event, while it is more challenging to prevent macrovascular complication in T2DM (
Several studies suggested other cardioprotective effects of GLP-1RAs through the attenuation of atherosclerosis, reduced inflammation, and some beneficial action on myocardial function which improved cardiovascular outcomes (
Even though the SGLT2 is not expressed in heart, its direct cardioprotective effect might be due to the modulation of autophagy. Autophagy is a complex process in response of several stimuli and is important to eliminate defect organelle associated with oxidative stress. Insufficient or excessive activation of autophagy may be harmful in MI.
This study has several limitations, which only focuses on the effect of both agent against several biomarkers that could leaked to circulation when myocardial damage or death occur. The biomarkers used in this study were relatively non- to almost specific to cardiomyocytes. In the future, the measurement of Troponin-I and the isomers of LDH (to calculated LDH-1/LDH-2 ratio), which more specific to cardiomyocyte, might be used. Several oxidative stress markers also haven’t been measured yet to support the mechanism or pathway related to cardiomyocyte survival for each agent and combination therapy. This study also may be limited to liraglutide and empagliflozin, not other drugs in the same class.
This study suggested better cardioprotective effect of liraglutide and empagliflozin combined in diabetic subjects induced with AMI, based on its inhibition against the elevation of several cardiac biomarkers. These results may be used as a consideration for clinical study to provide definitive evidence regarding the additional reduction of cardiovascular outcomes for the combination therapy. GLP-1RAs and SGLT2is are categorized as an expensive antihyperglycemic agents, thus cost-effectiveness study to support the combination to be used earlier in a newly diagnostic T2DM patient with high risk or established ASCVD is needed.
This research was funded by P2MI ITB.