1. Introduction
It is now well known that chronic diabetes is associated with diabetic cardiomyopathy, which is generally characterized by the development of cardiac hypertrophy, metabolic defects, ultrastructural abnormalities and myocardial fibrosis [
1,
2,
3,
4,
5,
6,
7,
8]. Although prolonged hyperglycemia and hyperlipidemia due to insulin deficiency or insulin resistance are the major causes of diabetic cardiomyopathy, mitochondrial (MT) dysfunction and sarcoplasmic reticulum (SR) stress have also been indicated to play crucial roles in the pathogenesis of this devastating health hazard [
9,
10,
11,
12]. In particular, oxidative stress, inflammation, Ca
2+-handling abnormalities and apoptosis are pivotal processes that determine the occurrence of cardiac remodeling and cellular injury in diabetic cardiomyopathy [
13,
14,
15,
16,
17]. In fact, several interventions, such as antihyperglycemic, antioxidant, anti-inflammatory, antifibrotic and antiapoptotic agents, have been shown to exert beneficial effects in preventing diabetic cardiomyopathy [
5,
7,
8,
11,
12,
13,
14,
15,
16,
17]. It should also be mentioned that diabetes is not only associated with insulin deficiency or resistance but also found to affect several endocrine, neuronal and other systems, which result in the elevation of hormones, including norepinephrine, angiotensin II, endothelin, serotonin (5-HT) and thyroid hormones [
18,
19]. Such hormonal imbalances have been reported to promote the development of cardiac remodeling, subcellular alterations, cardiac dysfunction and heart failure in chronic diabetes [
18,
19].
In view of the elevated levels of circulating 5-HT due to the enhancement of platelet aggregation in diabetes [
20,
21,
22,
23], sarpogrelate, a 5-HT
2A receptor antagonist, has been shown to exert beneficial effects in attenuating a wide variety of abnormalities in chronic diabetes [
24,
25,
26,
27,
28,
29,
30,
31]. Previously, we observed that the treatment of diabetic animals with sarpogrelate improved cardiac function by promoting the expression of membrane glucose transporters and releasing insulin from the pancreas [
32]. Since cardiac dysfunction in chronic diabetes is considered to be intimately associated with the remodeling of subcellular organelles such as the sarcolemma (SL), SR, MT and myofibrils (MFs) [
18,
19], the present study was undertaken to examine the effects of sarpogrelate on diabetes-induced changes in subcellular activities. In addition, the hemodynamic parameters and metabolic status of diabetic animals with and without sarpogrelate treatment were evaluated by monitoring the plasma levels of glucose and lipids, as well as myocardial high-energy phosphate content and cardiac function. Furthermore, the mechanism of action of sarpogrelate was investigated by measuring the levels of some biomarkers of oxidative stress in diabetic animals with and without sarpogrelate treatment. Since the beneficial actions of sarpogrelate treatment in diabetic cardiomyopathy were similar to those of insulin treatment [
32], the effects of insulin treatment on subcellular remodeling in diabetic animals were also studied for the purpose of comparison with sarpogrelate.
4. Discussion
In this study, we have shown that body weight, heart weight and plasma insulin lev-els were decreased, whereas several metabolic parameters, such as plasma glucose, cho-lesterol, free fatty acids and triglyceride levels, were increased in diabetic animals. While heart rate and blood pressure were increased, different parameters of cardiac function, such as LVDP, +dP/dT and −dP/dT, were depressed in diabetic animals without any changes in LVEDP. Such hemodynamic and metabolic alterations are in agreement with our previous observations related to chronic diabetes [
32,
34].
Furthermore, the treatment of diabetic animals with insulin or sarpogrelate was observed to attenuate diabetes-induced hemodynamic and metabolic changes either fully or partially, indicating that the beneficial effects of sarpogrelate on diabetic animals may involve mechanisms similar to those of insulin. The observed increase in the plasma level of insulin in diabetic animals upon treatment with sarpogrelate is consistent with our finding that this agent prevented the inhibitory effect of the 5-HT-induced release of insulin from the pancreas [
32]. Furthermore, the treatment of diabetic animals with sarpogrelate, like insulin, was found to lower plasma glucose levels by increasing the membrane glucose transporter GLUT4 protein content, but this agent, unlike insulin, increased GLUT1 content in the heart [
32]. It should be pointed out that sarpogrelate also reduced the elevated levels of glucose in obese mice [
45]. Thus, it appears that sarpogrelate produces antidiabetic insulin-like effects to promote glucose utilization, reduce lipid levels and attenuate diabetes-induced defects in heart function both by acting on 5-HT
2A receptors and through the release of insulin from the pancreas.
Both creatine phosphate and ATP contents were observed to be decreased, whereas the contents of ADP and AMP were increased in diabetic animals. Such changes in high-energy phosphate stores may be due to the depression of MT state 3 respiration and oxidative phosphorylation activities in the diabetic heart. The observed changes in cardiac energy stores and MT function in chronic diabetes are in agreement with previous reports [
34,
38]. The depressed activity of oxidative metabolism has also been reported in MT from genetically diabetic mice [
46]. On the other hand, the treatment of diabetic rats with sarpogrelate or insulin was found to attenuate diabetes-induced alterations in high-energy phosphate stores as well as changes in MT state 3 respiration and oxidative phosphorylation. It should be pointed out that sarpogrelate has been reported to attenuate ischemia–reperfusion-induced alterations in cardiac high-energy phosphate stores [
34]. It is also noted that MT Ca
2+-uptake and Mg
2+-ATPase activities in the hearts of animals with chronic diabetes were found to be decreased. Although the treatment of diabetic animals with sarpogrelate or insulin attenuated alterations in MT Ca
2+-uptake, changes in MT Ca
2+-ATPase activity were not affected by these treatments. Such depressed changes in MT Mg
2+-ATPase in chronic diabetes may represent an irreversible state of the MT membrane. In fact, dramatic changes in MT ultrastructure and permeability have been reported to occur in diabetic cardiomyopathy as well as due to ischemia–reperfusion [
34,
46]. Thus, in view of the well-established role of MT function and energy stores in maintaining cardiac function and structure, it is evident that sarpogrelate, like insulin, may improve cardiac performance in diabetic cardiomyopathy.
Previously, we have reported varying degrees of depression of SL Na
+-K
+ ATPase, Na
+-Ca
2+ exchange, ATP-dependent Ca
2+-uptake, Ca
2+-channel density and Ca
2+-stimulated ATPase activities, whereas the sensitivity of Ca
2+ channels was increased and Mg
2+-ATPase activity was unchanged in the diabetic heart [
35,
36]. The observed alterations in these SL activities in the hearts of animals with chronic diabetes in this study are similar to these reports. In addition, we have found that the treatment of diabetic animals with sarpogrelate or insulin attenuated diabetes-induced changes in SL activities. In view of the direct or indirect roles of SL Na
+-K
+ ATPase, Na
+-Ca
2+ exchange, Ca
2+-uptake, Ca
2+-stimulated ATPase and Ca
2+-channel activities in Ca
2+ entry and Ca
2+ removal from cardiomyocytes, alterations in these SL activities in the diabetic heart are considered to account for the occurrence of intracellular Ca
2+ overload, metabolic defects and the development of diabetic cardiomyopathy [
18,
19]. Thus, the attenuation of diabetes-induced changes in SL activities by both sarpogrelate and insulin treatments may be associated with the beneficial effects of these interventions on myocardial metabolism, cardiac function and cardiac ultrastructure as a consequence of preventing the occurrence of intracellular Ca
2+ overload. Such antidiabetic actions of sarpogrelate may partly be mediated by the blockade of 5-HT
2A receptors [
32], which are activated by the elevated levels of 5-HT in diabetes [
20,
21,
22,
23].
Chronic diabetes has been reported to depress cardiac SR Ca
2+-pump and Ca
2+-release activities, which are considered to explain the impaired relaxation of the heart [
18,
37]. On the other hand, defects in MF and myosin ATPases have been shown to be associated with depressed cardiac contraction in chronic diabetes [
18,
39,
40,
41]. The results of this study regarding depressed SR Ca
2+-uptake, Ca
2+-stimulated ATPase and Ca
2+-release activities without any changes in Mg
2+-ATPase in the chronic diabetic heart are in agreement with our previous observations [
37]. Likewise, the data on changes in cardiac MF Ca
2+-stimulated ATPase, MF Mg
2+-ATPase, myosin Ca
2+-ATPase and myosin Mg
2+-ATPase in chronic diabetes are also consistent with earlier observations [
39]. Furthermore, sarpogrelate and insulin treatments were observed to partially or fully attenuate diabetes-induced alterations in both SR and MF activities. Thus, it is evident that the improvement of cardiac function upon the treatment of diabetic animals with sarpogrelate or insulin may be attributed to a reduction in the SR and MF defects. In view of the marked increase in 5-HT levels in diabetes [
20,
21,
22,
23], sarpogrelate might also produce beneficial effects on SR and MF activities by blocking 5-HT
2A receptors. It should be pointed out that the activities of both MF and myosin Mg
2+-ATPase, like that of MT Mg
2+-ATPase, were depressed in the diabetic heart; however, MF and myosin Mg
2+-ATPase activities, unlike that of MT Mg
2+-ATPase, in the diabetic heart were increased significantly by the treatment of diabetic animals with sarpogrelate or insulin. On the other hand, the activities of cardiac SR Mg
2+-ATPase and SL Mg
2+-ATPase were unaltered in diabetic animals with or without sarpogrelate or insulin treatment. Such differences in the responsiveness of MF, MT, SL and SR Mg
2+-ATPases may be due to differences in the structure and function of the enzyme molecules in various subcellular organelles. It should also be mentioned that the beneficial effects of insulin treatment on most of the parameters measured in this study were somewhat greater, but not significantly, than those observed with sarpogrelate treatment. Such differences in the beneficial effects of these treatments may be due to differences in the doses of these interventions.
Earlier, we showed that a well-known 5-HT
2A receptor antagonist, sarpogrelate, releases insulin from the pancreas, and its effect in promoting glucose uptake was additive with that insulin [
32]. In this study, we observed that this agent produces beneficial effects on hemodynamic, metabolic and subcellular alterations in diabetic animals, which are similar to those seen upon treatment with insulin. In addition to the plasma levels of 5-HT being elevated in diabetes, this hormone has also been reported to induce diabetes-like metabolic alterations [
20,
21,
22,
23]. Thus, it is likely that sarpogrelate may exert its effects on diabetes through both the blockade of 5-HT
2A receptors and the release of insulin from the pancreas. It should be noted that diabetes is a highly complex disease that is known to affect several systems in the body. Particular effects include the aggregation of platelets for releasing 5-HT, the activation of the sympathetic nervous system for releasing catecholamines and the activation of the renin–angiotensin system for the formation of Ang II in diabetes; all of these vasoactive hormones are known to promote oxidative stress in the diabetic heart [
18,
19]. In addition, both high levels of plasma glucose and excessive utilization of free fatty acids by MT due to insulin deficiency or insulin ineffectiveness in diabetes have been reported to generate oxidative stress [
18,
19]. Thus, it appears that the beneficial insulin-like effects of sarpogrelate treatment on hemodynamic, metabolic and subcellular activities in the present study may have occurred as a consequence of a reduction in the development of oxidative stress in diabetic animals. This view is supported by our observations that the treatment of diabetic animals with sarpogrelate or insulin markedly attenuated the altered levels of some biomarkers of oxidative stress, such as malondialdehyde and oxidized glutathione, glutathione peroxidase and superoxide peroxidase, in the diabetic heart. Since the levels of catalase were not changed in the diabetic heart with or without treatment, it is likely that the modification of oxidative stress biomarkers by diabetes, as well as due to insulin or sarpogrelate treatment, is site-specific in the oxidative stress pathway. Nonetheless, on the basis of the attenuation of diabetes-induced defects in SL, SR and MF activities by treatments with vitamin E, propranolol and losartan, it has been indicated that oxidative stress plays a critical role in inducing metabolic and subcellular abnormalities during the development of diabetic cardiomyopathy [
18,
19]. Accordingly, in view of the results of the present study, as well as our previous observations, it is evident that the improvement of cardiac performance, metabolic defects and alterations in subcellular activities by sarpogrelate or insulin treatment may be a consequence of a reduction in the level of oxidative stress.