Incretin attenuates diabetes-induced damage in rat cardiac tissue

Abstract

Glucagon-like peptide-1 (GLP-1), as a member of the incretin family, has a role in glucose homeostasis, its receptors distributed throughout the body, including the heart. The aim was to investigate cardiac lesions following diabetes induction, and the potential effect of GLP-1 on this type of lesions and the molecular mechanism driving this activity. Adult male rats were classified into: normal, diabetic, 4-week high-dose exenatide-treated diabetic rats, 4-week low-dose exenatide-treated diabetic rats, and 1-week exenatide-treated diabetic rats. The following parameters were measured: in blood: glucose, insulin, lactate dehydrogenase (LDH), total creatine kinase (CK), creatine kinase MB isoenzyme (CK-MB), and CK-MB relative index; in cardiac tissue: lipid peroxide (LPO) and some antioxidant enzymes. The untreated diabetic group displayed significant increases in blood level of glucose, LDH, and CK-MB, and cardiac tissue LPO, and a significant decrease in cardiac tissue antioxidant enzymes. GLP-1 supplementation in diabetic rats definitely decreased the hyperglycemia and abolished the detrimental effects of diabetes on the cardiac tissue. The effect of GLP-1 on blood glucose and on the heart also appeared after a short supplementation period (1 week). It can be concluded that GLP-1 has beneficial effects on diabetes-induced oxidative cardiac tissue damage, most probably via its antioxidant effect directly acting on cardiac tissue and independent of its hypoglycemic effect.

Introduction

Diabetes represents a serious risk factor for the development of cardiovascular complications such as coronary heart disease, peripheral arterial disease, hypertension, stroke, cardiomyopathy, and nephropathy, and cardiovascular disease is responsible for 80 % of deaths among diabetic patients. [1].

In diabetes, the circulating free radicals may contribute to the progression of heart disease and possibly mediate the process of apoptosis [2], a state in which increased oxidative stress is documented [3]. Recent reports provide evidence that high ambient glucose can promote apoptosis in vitro, suggesting potential cellular damage as a result of hyperglycemia in diabetes [4].

The incretin system comprises two major gut hormones: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), both of which are released into the circulation in response to food ingestion, and are known to play a crucial role in the normal insulin response after food intake [5].

GLP-1 is released from intestinal L-cells in the distal ileum and colon [6]. The physiological actions most commonly associated with GLP-1 are its ability to enhance glucose-dependent insulin secretion, suppress post-prandial glucagon secretion, slow gastric emptying, and, in animal models, promote β-cell proliferation and decrease β-cell apoptosis [7]. GLP-1 is rapidly inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4) [8].

In addition to its roles in glycemic control and satiety, GLP-1 appears to exert several additional effects on many tissues via the GLP-1 receptor, which is expressed not only in the pancreatic islets but also in the lung, kidney, intestine, and several regions of the central nervous system [9]. This widespread expression of the GLP-1 receptor may help to explain the range of extrapancreatic effects of GLP-1, including the cardiovascular system as GLP-1 receptors are also found in endothelial cells, vascular smooth muscle cells, monocytes–macrophages of the vascular wall [10], and in the heart [11]. Although GLP-1 receptors are widespread in the cardiovascular system, GLP-1’s role in the cardiac tissue is still largely unknown.

Exenatide is a synthetic GLP-1 mimetic, derived from exendin-4, a naturally occurring GLP-1 receptor agonist isolated from the salivary secretions of the reptile Heloderma suspectum (Gila monster). Exenatide activates the GLP-1 receptor similarly to native GLP-1, but is resistant to the proteolytic activity of DPP-4 and consequently has a longer half-life [12].

Accordingly, the purpose of the present study was to investigate cardiac lesions following streptozotocin (STZ)-induced diabetes mellitus. The second goal was to determine the potential effect of exenatide supplementation on this type of lesion. The third goal was to explore the mechanism driving this activity.

Materials and methods

Ethical approval

The local ethics committee in our university approved this animal experiment protocol, and it was conducted in compliance with the NIH Guide for Care and Use of Laboratory Animals [13].

Experimental groups and animals

Adults Sprague–Dawley male albino rats (8–10 weeks) weighing between 200 and 250 g were used throughout the present study. Rats were housed at room temperature with a 12 h light/dark cycle, and with a supply of a standard diet of commercial rat chow and water ad libitum. Animals were left to acclimatize to the environment for 2 weeks prior to inclusion in the experiment. The rats were divided into three different groups (n = 20): Group 1: control rats (rats with injection of vehicle buffer for 5 weeks); Group 2: diabetic rats (rats with diabetic induction by injection of 50 mg/kg STZ single intraperitoneal injection) [14]; Group 3: high-dose exenatide-treated diabetic rats (rats treated with 5.0 µg/kg single subcutaneouse daily dose of exenatide on the 4th day of STZ treatment for 4 weeks) [15]; Group 4: low-dose exenatide-treated diabetic rats (rats treated with 1.0 µg/kg single subcutaneouse daily dose of exenatide on the 4th day of STZ treatment for 4 weeks); Group 5: 1-week exenatide-treated diabetic rats (rats treated with 5.0 µg/kg single subcutaneouse daily dose of exenatide on the 4th day of STZ treatment for 1 week). The animals received the appropriate treatment at the same time every day. We began the experiment with 20 rats in each group. However, some animals died during the experiment, so the number of analyzed samples in each group was 15.

Chemicals

Phosphate-buffered saline (Oxford, Mumbai, India), Triton X-100 (t-octylphenoxy-polyethox-yethanol) (Sigma-Aldrich, Steinheim, Germany), sodium citrate buffer (pH 4.5) (Oxford), STZ (Sigma, St. Louis, USA), reagents for determination of serum glucose (Spectrum, Egyptian Company for Biotechnology, Egypt), reagents for determination of serum insulin (Bio-Diagnostic, Cairo, Egypt), reagents for determination of serum lactate dehydrogenase (Diamond Diagnostics, Cairo, Egypt), reagents for determination of; serum creatine kinase, serum creatine kinase MB isoenzyme, cardiac tissue lipid peroxide, cardiac tissue glutathione reductase, cardiac tissue superoxide dismutase, and cardiac tissue catalase (Bio-Diagnostic).

Animal sacrifice and sample collection

Overnight-fasted rats, deprived of food and given only water for 16 h overnight, were sacrificed by decapitation at the end of the experimental period. The blood samples were immediately collected in 10-ml Eppendorf tubes, allowed to clot, and then delivered into centrifuge tubes (3,000 rpm for 20 min); serum samples were separated in 2-ml Eppendorf tubes to be used immediately as fresh samples (preferred) or to be stored at –20 °C until used. The hearts of the rats were dissected and stored at −80 °C until used. Serum samples were used to determine glucose, insulin, lactate dehydrogenase (LDH), creatine kinase (CK), and creatine kinase MB isoenzyme (CK-MB) levels, while cardiac samples were used to determine lipid peroxide (LPO), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) levels.

Parameters measured

At the end of the experimental protocol, the following parameters were measured: serum glucose by enzymatic colorimetry [16], serum insulin by ACTIVE Insulin Enzyme-Linked Immunosorbent assay (ELISA) [17], serum LDH by a kinetic method [18], serum CK-MB by ELISA, serum CK by a kinetic method [19], cardiac tissue LPO—the malondialdehyde (MDA)—by colorimetry [20], cardiac tissue antioxidant enzymes: GR by a UV method [21], SOD by colorimetry [22], and CAT by colorimetry [23].

Induction of diabetes

Diabetes mellitus was induced by STZ (50 mg/kg). STZ was freshly dissolved in sodium citrate buffer (pH 4.5) and injected intraperitoneally in a single dose [14]. After 4 days of STZ injection, blood samples were taken from the tail vein for serum glucose levels assay. Rats with a fasting serum glucose level higher than 180 mg/DL (10 mmol/l) were considered diabetic in this experiment.

CK-MB relative index measurement

CK-MB relative index (%) = [CK-MB (ng/mL) ÷ total CK (Units/L)] × 100 [24].

Statistical analysis

All values are presented as mean ± SEM. Data were evaluated by use of the SPSS statistical software ( v.11.0, SPSS, Chicago, IL, USA), and independent samples t test; p < 0.05 being considered statistically significant.

Results

Effects of diabetes induction

STZ administration, in a single intra-peritoneal dose level of 50 mg/kg, induced diabetes in all male rats and produced a significant (p < 0.05) decrease in fasting serum insulin level, with a significant (p < 0.05) increase in the fasting serum glucose to the diabetic level (Table 1).

Table 1 Effect of induction of diabetes on fasting serum glucose and fasting serum insulin in male albino rats

Diabetes induction also leads to obvious detrimental effects on the cardiac tissues, this being proved by measuring some of the cardiac injury markers in the serum and cardiac tissue. STZ administration in all male rats produced significant (p < 0.05) rises in serum LDH, total CK, CK-MB and CK-MB relative index compared to the control group. Moreover, it showed a cardiac oxidative stress injury indicated by a significant (p < 0.05) increase in the cardiac tissue LPO level, as well as significant (p < 0.05) decreases in cardiac antioxidant enzymes GR, SOD, and CAT levels compared to the control group (Table 2).

Table 2 Effect of induction of diabetes on some cardiac injury markers in male albino rats

Effect of exenatide supplementation in diabetic male albino rats

Rats treated with 5.0 µg/kg single subcutaneouse daily dose of exenatide on the 4th day of STZ treatment for 4 weeks produced a significant (p < 0.05) increase in fasting serum insulin level, with a significant (p < 0.05) decrease in the fasting serum glucose level (Fig. 1).

Fig. 1

Effect of low- and high-dose of long-term exenatide supplementation (for 4 weeks) on fasting serum glucose, fasting serum insulin, and some cardiac injury markers in diabetic male albino rats. Values are mean ± SEM. n = 15. *Significant (p < 0.05) difference from diabetic group. LD exenatide low-dose exenatide, HD exenatide high-dose exenatide, LDH lactate dehydrogenase, CK creatine kinase, CK-MB creatine kinase MB isoenzyme, LPO lipid peroxide, GR glutathione reductase, SOD superoxide dismutase, CAT catalase

Exenatide supplementation in diabetic rats in the above dose also abolished several detrimental effects of diabetes on the cardiac tissue since it produced significant (p < 0.05) decreases in serum LDH, total CK, CK-MB, CK-MB relative index, and cardiac tissue LPO, and produced significant (p < 0.05) increases in cardiac tissue antioxidant enzymes GR, SOD, and CAT levels (Fig. 1).

Rats treated with 1.0 µg/kg single subcutaneous daily dose of exenatide for 4 weeks produced a significant (p < 0.05) change in the measured parameters. However, this effect was weak and exenatide in this low dose failed to return these parameters to the normal, non-diabetic level (Fig. 1).

Rats treated with 5.0 µg/kg single subcutaneous daily dose of exenatide for 1 week produced a significant (p < 0.05) increase in fasting serum insulin level, with a significant (p < 0.05) decrease in the fasting serum glucose level (Table 3). Also, it produced significant (p < 0.05) decreases in serum LDH, total CK, CK-MB, CK-MB relative index, and cardiac tissue LPO, and produced significant (p < 0.05) increases in cardiac tissue antioxidant enzymes GR, SOD, and CAT levels (Table 4).

Table 3 Effect of short-term exenatide supplementation (for 1 week) on fasting serum glucose and fasting serum insulin in diabetic male albino rats

Table 4 Effect of short-term exenatide supplementation (for 1 week) on some cardiac injury markers in diabetic male albino rats

Discussion

In this study, STZ produced a significant decrease in fasting serum insulin levels associated with a significant increase in fasting serum glucose to the diabetic levels. Streptozotocin (STZ, 2-deoxy-2-(3-(methyl-3-nitrosoureido)-D-glucopyranose) is synthesized by Streptomycetes achromogenes and is used to induce diabetes mellitus by destruction of β-cells of the islets, which leads to a reduction in insulin release, which in turn leads to hyperglycaemia [25].

Cytosolic enzymes, such as CK-MB and LDH, which leak out from damaged tissues to the blood stream when the cell membrane becomes permeable or ruptures, serve as diagnostic markers of myocardial cell injury [26]. In the present study, the levels of LDH, total CK, absolute CK-MB, and CK-MB relative index were significantly increased in the diabetic rats indicating the obvious detrimental effects of diabetes on the cardiac tissue. These results are in line with previous reports which found that serum LDH and CK-MB levels increased in STZ diabetic rats, possibly due to myocardial dysfunction [27]. Huang et al. [28] also reported that the serum CK-MB and LDH levels were increased in diabetic patients, and may serve as a marker for cardiovascular risk and cardiac muscle damage. The significant elevation in CK-MB relative index with diabetic induction in this study indicating cardiac source of elevated CK-MB as reported by previous studies, such as Panteghini [29], who reported that CK-MB relative index is more specific than absolute CK-MB as a marker of myocardial damage.

The results from the present study have also demonstrated that diabetes induction caused a significant elevation in the levels of cardiac tissue LPO and a significant depression in the levels of the cardiac antioxidant enzymes SOD, CAT, and GR, indicating the onset of oxidative stress in the heart of the diabetic animals.

Our results are in line with previous reports which found that hyperglycemia results in the production of reactive oxygen species (ROS), and coexists with a reduction in antioxidant enzymes, and an increase in lipid peroxidation in the heart [30–32].

A significant reduction in myocardial glucose supply and utilization has been observed in isolated diabetic cardiomyocytes, which could be the primary injury in the pathogenesis of this specific heart muscle disease [33, 34]. Also, carbohydrate and lipid metabolic abnormalities, such as hyperglycemia and hyperlipidemia, may contribute to the development of cardiac dysfunction in diabetes mellitus [35]. This metabolic stress in tissue causes the generation of oxidative stress due to overproduction of ROS [36, 37]. The polyunsaturated fatty acids undergo lipid peroxidation by ROS under stress condition, and the end product is MDA [38]. So, the increase in the level of MDA indicates the increased level of oxidative stress induced by the toxicant in the key tissues of the body, such as brain, liver, heart, and other organs, and the plasma membrane of RBCs [39–43]. On the other hand, the antioxidant enzymes are best characterized by their first line of defense against oxidative stress as they are believed to maintain balance between the production of ROS and antioxidant defense system as a consequence of oxidative stress [44], and the reduction in these antioxidant enzymes demonstrated the failure of the antioxidant system, which may result in cardiac toxicity [45].

In the present study, treatment of diabetic animals with exenatide produced significant decreases in elevated serum glucose levels and significant increases in the depressed serum insulin level. These results agree with previous results which found that Harlan Sprague–Dawley rats administered exendin-4 exhibit a glucose-stimulated, dose-dependent increase in insulin levels [46], In diabetic mice, fasting glucose is normalized after 12 weeks of exendin-4 therapy, and hemoglobin A_1c was 4.1 % lower in treated mice than in control mice [47]. Exendin-4 also significantly and dose-dependently lowered fasting glucose in four diabetic rhesus monkeys by up to 37 % [48].

These results can be referred to the finding of the previous report that GLP-1 potentiates glucose-induced insulin secretion and improves the function of pancreatic β-cells by promoting the genesis and proliferation, and by inhibiting apoptotic signals and glucagon secretion from pancreatic α-cells, thus resulting in the regulation of glucose homeostasis [49]. GLP-1 synergistically acts with glucose to promote insulin gene transcription, MRNA stability, and biosynthesis, increasing the expression of the transcription factor Pancreas duodenum homeobox 1 (Pdx-1) and the binding of Pdx-1 to the insulin promoter. Furthermore, GLP-1 has been shown to improve glucose sensitivity to glucose-resistant β cells [50–52]. GLP-1 also inhibits hepatic glucose production and stimulates glucose uptake in fat and muscle and increases glycogen synthase activity and glucose metabolism in skeletal muscle [53–55].

Studying the effects of exenatide (5.0 µg/kg for 4 weeks) on the cardiac tissue, we found that the increased levels of serum LDH, total CK, CK-MB and CK-MB relative index observed with diabetic induction were significantly suppressed by exenatide supplementation, indicating good cardioprotective effects of exanitide in diabetic animals that can be refered to its antioxidant action, because we found that there was a significant decrease in cardiac tissue LPO associated with a significant increase in cardiac tissue antioxidant enzyme levels in the exenatide-treated diabetic rats.

Low doses of exenatide (1.0 µg/kg for 4 weeks) to the diabetic rats produced significant, but weak changes in blood glucose and serum insulin levels but failed to return them to the normal non-diabetic level. The same results were found in the measured serum/cardiac parameters related to cardiac tissue injury. So, as shown in the present study, long-term treatment with exenatide dose definitely down-regulates diabetic-elevated blood glucose levels and the detrimental effect on the heart.

Exenatide administration to diabetic rats for a short period (5.0 µg/kg for 1 week) can correct their blood glucose level. Moreover, it produced significant beneficial effects on serum/cardiac parameters related to cardiac tissue injury. This supports the idea that exenatide exerts its protective effects on the heart via directly acting on cardiac tissue and independent of its hypoglycemic effect, since exenatide produced its effect on the heart in the early period of treatment.

Importantly, beneficial actions of GLP-1 receptor agonists on CVD have also been reported in both experimental models and in human patients, either in the presence or absence of diabetes [56–59].

Recently, the crucial role of GLP-1 in cardiovascular disease has been suggested by both preclinical and clinical studies [60]. It has been reported that GLP-1 may have cardioprotective effects, being capable of reducing the ischemia/reperfusion injury and also cardiac dysfunction in various animal models and humans [60, 61]. Several mechanisms may explain the effect of GLP-1 in reducing cardiac dysfunction, one of them being the possibility that the GLP-1 can reduce oxidative stress and can increase antioxidants, leading to decreased apoptosis [62, 63]. Therefore, it is reasonable that, in our study, GLP-1 should, by reducing oxidative stress generation, improve cardiac tissue damage generated by diabetes induction. A recent study also reported that the GLP-1 receptor agonist, exendin-4, increased SOD levels and decreased MDA levels in neonatal rats with hyperglycemia-induced cardiomyocytes injury [64].

Another study confirmed that GLP-1 enhances phosphatydil inositol 3 kinase, which plays a key role in activating the antiapoptotic pathway and promotes cardio-protection. Therefore, a direct effect of GLP-1 against apoptosis in cardiac cells is possible [65]. At present, the precise mechanisms underlying these potentially beneficial actions of GLP-1 on the heart remain largely unknown and appear to be complex.

In conclusion, the prominent finding of this study was that GLP-1, besides its usefulness in the management of diabetes, exerts significant cardioprotective effects against oxidative stress induced by diabetes induction. The mechanisms involved may be related to its ability to increase the concentration of antioxidant defense enzymes. The preliminary results of this study suggest that GLP-1 can exert its protective effects on the heart via a direct effect on the heart independently of correcting the hyperglycemia and consequent removal of glucotoxicity from cardiac tissue. These insights afford the opportunity to design therapeutic approaches targeted at specific pathogenic mechanisms, including improving diabetic control, antioxidant, and insulin-stimulatory drugs that might be effective for preventing or delaying the development of diabetic cardiomyopathy. However, it is clear that further basic mechanistic research is required before any potential therapeutic benefits may be realized.