Abstract
Purpose: Myocardia in diabetic patients exhibit increased vulnerability after ischemia/ reperfusion injury (IRI). It has been demonstrated that glucagon-like peptide-1 (GLP-1) has a protective effect on cardiomyocytes. Protein kinase C (PKC) acts as a key regulator of many signaling pathways including oxidative stress and apoptosis. Our hypothesis is that increased vulnerability of myocardia in diabetic patients is partly due to GLP-1 resistance. The aim of this study was to explore the role of PKC in GLP-1 resistance in diabetic cardiomyocytes. Methods: Cardiac function of diabetic or non-diabetic mice after myocardial IRI was detected with or without administration of GLP-1 analog exendin-4. Impacts of diabetes mellitus on GLP-1R expression in myocardia after IRI were accessed by Western blot. By transfecting PKC isoforms siRNA, in vitro study helped to identify the exact PKC isoforms which contributed to the downregulatio n of GLP-1R or impaired post-receptor signaling pathways in rodent cardiomyocytes (H9C2 cells) cultured by high glucose. 
Introduction
People with diabetes are two to three times more likely to have cardiovascular disease than people without diabetes.
Introduction
1,2 Cardiovascular complications are the major causes of morbidity and mortality in diabetic patients, among which ischemic heart disease is the leading cause of death. 3 It has been proven that myocardial infarction or thrombolytic therapy is the most effective treatment. However, reperfusion introduces a new problem that aggravates myocardial damage: myocardial IRI. 5, 6 Studies also showed that myocardial IRI brings severer damage to diabetic patients. 7, 8 Increased vulnerability of myocardia in diabetic patients has been extensively studied; however, the underlying mechanisms remained ambiguous. GLP-1, an incretin hormone secreted from intestinal L cells, is now clinically used as an anti-diabetic drug. 9, 10 GLP-1 exerts physiological function upon binding to its receptor, which belongs to the class B family of heterotrimeric G-protein-coupled receptors.
Introduction
11 GLP-1R has been proved to be expressed in many tissues, including heart. Research found that GLP-1 improves cardiac function, decreases infarct size after myocardial IRI. 12, 13 However, studies have found that in diabetic mice there exists GLP-1 resistance in several cell types such as pancreatic β cells and endothelial cells. 14, 15 Thus, raises questions about whether increased vulnerability of diabetic myocardia is partly due to GLP-1 resistance. PKC isoforms are a family of serine/threonine kinase which play a crucial role in mediating cell signal pathways as diverse as cell growth, differentiation, apoptosis, oxidation stress and others. 16, 17 PKC isoforms are ubiquitously expressed in all tissues at all times of development. 18 Interestingly, PKCs have demonstrated to have sometimes opposing roles in both normal and diseased states. 19 In diabetic state, it has been proved that activation of PKCs is responsible for GLP-1 resistance in endothelial cells. 20 However, to our knowledge, the possible role of PKC in GLP-1 resistance of diabetic cardiomyocytes remains unclear.
Introduction
In the present study, we provide evidence that in diabetic myocardia there exists GLP-1 resistance. We associate this phenomenon to a decrease in GLP-1R expression induced by PKCβ overexpression and an impairment of GLP-1 postreceptor anti-apoptotic signaling pathways induced by PKCδ overexpression. When PKCβ and PKCδ are specifically knockdown, GLP-1 actions are partially recovered.
Materials And Methods

Animal Models Of Diabetes And Myocardial Ischemia/Reperfusion Injury
All animal studies were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the ethical committee of Air Force Medical University. All commercially available kits and antibodies used in the present study are listed in Table 2 . Spontaneous diabetes model (random blood glucose ≥16.6 mmol/L) and non-diabetes model were achieved using db/db C57BL/6 mice (male, 8 weeks old) and C57BL/6 mice (male, 8 weeks old), respectively. Mice were randomly divided into 6 groups: 1) Diabetes Mellitus (DM); 2) Non-Diabetes Mellitus (Non-DM); 3) Diabetes Mellitus + Ischemia-Reperfusion Injury (DM + IRI); 4) NonDiabetes Mellitus + Ischemia-Reperfusion Injury (Non-DM +IRI); 5) Diabetes Mellitus + Ischemia-Reperfusion Injury + exendin-4 (DM + IRI + exendin-4); and 6) Non-Diabetes Mellitus + Ischemia-Reperfusion Injury + exendin-4 (Non-DM + IRI + exendin-4) (n=6 for each group). Myocardial IRI models were established by ligation of the left anterior descending coronary artery 1-2 mm from the tip of the auricle with a 6-0 polypropylene suture for 30 mins. GLP-1 analog exendin-4 pretreatment (1 nmol/kg) was conducted by intraperitoneal injection 1 hr before the operation and the control group injected saline instead.
Measurement Of Cardiac Function By Echocardiograms
One day after IRI, echocardiography (Sequoia Acuson, Siemens, 15-MHz linear transducer, Erlangen, Germany) were performed. Gaseous anesthesia was conducted and maintained using 3% isoflurane, and the mice were placed on the scanning table. Echocardiographic images were obtained using a dedicated small-animal high-resolution imaging unit and a 30-MHz linear transducer. Left ventricular end-diastolic diameters (LVEDD) and left ventricular end-systolic diameters (LVESD) were measured using a parasternal short-axis view, and LV fractional shortening was calculated as (LVEDD-LVESD)/LVEDD*100. All measurements were averaged over 3 successive cardiac cycles.
Measurement Of Serum GLP-1 Level
The concentrations of GLP-1 were accessed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Abcam, USA). Serum samples were collected at different time points (0 hr, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs) after the surgery in group (1)-(4) and DPP-4 inhibitor was added to the samples immediately to prevent degradation of GLP-1.
Measurement Of Serum GLP-1 Level
Cell Culture And Hypoxia/ReOxygenation Injury 
Assessment Of Apoptosis And Measurement Of ROS
TUNEL staining was performed on H9C2 cells according to the manufacturer's instructions (MEBSTAIN Apoptosis TUNEL kit, Takara). For detection of total nuclei, the slides were covered with the mounting medium containing DAPI (Abcam, UK). Digital photographs were taken at high magnification (×400) using a fluorescent microscopy (Olympus). Cells in which the nucleus was stained were defined as TUNEL positive. Apoptosis index (AI) is defined as cell numbers of apoptotic cells in 100 cells averagely. Each AI was accessed in 20 randomly selected fields. Caspase-3 activity was measured using a caspase-3 assay kit (Clontech, Mountain View, CA) according to the manufacturer's instructions. ROS level was measured with the dye 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA) according to the manufacturer's instructions.
Western Blot Analysis
Both H9C2 cells and myocardial tissues were harvested for Western blot analysis (Western Blot Detection Kit, Elabscience) following standard protocol according to the manufacturer's instructions and Towbin system buffer was used. Primary antibodies used in Western blot were as follows: anti-GLP-1R (1:200, Abcam, UK), anti-Akt , anti-PKCδ (1:500, Abcam, UK) and anti-β-actin (1:500, Santa Cruz, USA). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit lgG (Santa Cruz, USA) at 1:5000 dilution. The results were visualized using an enhanced chemiluminescence system (ECL, Amersham). Densitometric analysis was conducted using ImageJ software (NIH, USA).
Statistical Analysis
Results are represented as mean ± SD. Statistical analyses were performed by one-way analysis of variance (AVONA) and a Student−Newman−Keuls multiple comparison test was performed to identify differences between groups. A P value of less than 0.05 was considered statistically significant.
Results

Impaired Cardioprotective Effects In Response To Increased Endogenous GLP-1 In Diabetic Mice After Myocardial IRI
First, we examined whether endogenous GLP-1 and GLP-1 analog exendin-4 exhibit intact cardioprotective effects in diabetic mice after myocardial IRI. As shown in Figure 1A , diabetic mice showed higher circulating GLP-1 level than non-diabetic mice before (0 hr: 5.10±0.21pM vs 7.42 ±0.32pM) and after (5 hrs: 6.82±0.14pM vs 14.41±0.45Pm) myocardial IRI. However, corresponding left ventricle (LV) fractional shortening of diabetic mice remained lower than non-diabetic mice. Furthermore, addition of exendin-4 brought no significant change in LV fractional shortening of diabetic mice after myocardial IRI, while in contrast, LV fractional shortening of non-diabetic mice significantly increased with administration of exendin-4 after myocardial IRI ( Figure 1B ). Anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) and pro-apoptotic factor Bcl-2 associated X protein (BAX) were accessed by qRT-PCR, the results showed that exendin-4 exerted anti-apoptotic effects in non-diabetic mice after myocardial IRI while in diabetic mice no significant changes in Bcl-2 and BAX were detected ( Figure 1C ). Same conclusion can be made according to caspase-3 activity change ( Figure 1D ).
Impaired Cardioprotective Effects In Response To Increased Endogenous GLP-1 In Diabetic Mice After Myocardial IRI
Essential subunits of NAD(P)H oxidase, NADPH oxidase 4 (NOX4) and p22-phox were analyzed by qRT-PCR. The results indicated that exendin-4 effectively reduced oxidative stress in non-diabetic mice after myocardial IRI while no such effect can be seen in diabetic mice ( Figure 1E) . Results of reactive oxygen species (ROS) measurement showed a similar tendency with NOX4 and p22-phox ( Figure 1F ). Thus indicated that the cardioprotective effects of GLP-1 were impaired in diabetic mice.
Overexpression Of PKCβ Induced By High Glucose Downregulated GLP-1R Expression In H9C2 Cells
Then, we examined whether impaired cardioprotective effects of GLP-1 in diabetic mice were due to decreased GLP-1R expression in cardiomyocyte. As shown in Figure 2A , compare to non-diabetic mice, GLP-1R expression was lower in diabetic mice before and after myocardial IRI. Since PKC is activated by high glucose in various cell types, 21, 22 we hypothesized that PKC activation is involved in the reduction of GLP-1R expression by high glucose. H9C2 cell line was originated from rat heart myoblast. In the present study, H9C2 cells were cultured for in vitro experiment. As shown in Figure 3B , partially restored the downregulation of GLP-1R ( Figure 3C ). It has been proven that PKCα, -β, -γ, and -δ were expressed in cardiomyocytes, [23] [24] [25] [26] so we examined the effects of each PKC isoforms on GLP-1R expression in H9C2 cells cultured by high glucose. Western blot analysis showed that PKCα, -β, -γ, and -δ expression significantly increased in high glucosecultured H9C2 cells ( Figure 2D ). To figure out the exact isoform of PKC that was responsible for the downregulation of GLP-1R, we knockdown PKCα, -β, -γ, and -δ, respectively, in H9C2 cells. As shown in Figure 2E , PKCα, -β, -γ, and -δ were successfully knockdown. In H9C2 cells cultured by high glucose, knockdown of PKCβ resulted in significantly increased GLP-1R expression, while in contrast, knockdown of PKCα, -γ, and -δ did not result in significant change of GLP-1R expression ( Figure 2F ). Addition of PKCβ-specific inhibitor Ruboxistaurin (RBX) in H9C2 cells increased GLP-1R expression in a similar way as PKCβ siRNA did, and the upregulation effect of PKCβ siRNA on GLP-1R expression can be blocked by PMA ( Figure 2G ).
Overexpression Of PKCβ Induced By High Glucose Downregulated GLP-1R Expression In H9C2 Cells
Thus indicated that downregulation of GLP-1R expression in H9C2 cells was mediated by PKCβ overexpression induced by high glucose. Results of reactive oxygen species (ROS) measurement consistent with the findings above ( Figure 3B ). Anti-apoptotic effects of GLP-1R were also evaluated. As seen in representative TUNEL images and corresponding apoptosis index (AI) ( Figure 4C ), a significantly higher AI was observed in H/R group compared with control group (5.2±0.3% vs 43.4±2.5%). In H9C2 cells cultured by high glucose, addition of exendin-4 alone partially reversed AI (32.0±1.3%) that elevated by H/R. However, knockdown of PKCβ brought no significant change in AI (28.6±0.7%) compared with exendin-4. Similar conclusion can be deduced by results of qRT-PCR analyzed antiapoptotic markers ( Figure 4A and B) . Taken together, increased GLP-1R expression hardly intensifies anti-apoptotic effects of exendin-4 in H9C2 cells cultured by high glucose. Thus arises questions about whether GLP-1 resistance in the diabetic condition is in part due to impaired GLP-1 post-receptor anti-apoptotic signaling pathways.
Overexpression Of PKCδ Induced By
High Glucose Impaired Post-Receptor Anti-Apoptotic Signaling Pathways Of GLP-1R In H9C2 Cells By Inhibition Of Akt Phosphorylation Next, we examined whether and why post-receptor apoptosis-related signal pathways of GLP-1R were impaired.
Overexpression Of PKCδ Induced By
As shown in Figure 5A and B, caspase-3 activity and BAX decreased significantly in response to knockdown of PKCδ in H9C2 cells while Bcl-2 increased in the meantime. Thus implicated that high glucose-induced overexpression of PKCδ is involved in impaired anti-apoptotic effects of GLP-1 in H9C2 cells. It has been reported that GLP-1 exerts its anti-apoptotic effects through activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and phosphorylation of Akt results in downstream activation of anti-apoptotic factors such as Bcl-2. 27 Thus, we examined whether high glucose-induced overexpression of PKCδ impairs post-receptor anti-apoptotic signaling pathways of GLP-1 by blocking PI3K/Akt pathway. Figure 5C shows that knockdown of PKCδ in H9C2 cells cultured by high glucose increased phosphorylation level of Akt, addition of exendin-4 on the basis of PKCδ knockdown further increased phosphorylation level of Akt. Thus indicating that high glucose-induced overexpression of PKCδ impairs anti-apoptotic effects of GLP-1 by inhibition of Akt phosphorylation. Further studies showed that neither inhibition nor activation of PI3K by wortmannin and insulin-like growth factor 1 (IGF-1) respectively influenced PKCδ level, and change in PKCδ level did not influence PI3K level either ( Figure 5D ). Thus indicating that PKCδ and PI3K are mutually independent and both of them affect Akt phosphorylation.
Knockdown Of Both PKCβ And PKCδ Significantly Restored Anti-Apoptotic Effects Of GLP-1 Analog On H9C2 Cells
To further confirm these results, H9C2 cells were cotransfected with PKCβ and PKCδ siRNA. QRT-PCR analysis of HMOX-1, NQO-1, NOX4, p22-phox and TXNIP showed that knockdown of PKCβ restored anti-oxidative stress effects of GLP-1 analog in H9C2 cultured by high glucose, and knockdown of PKCδ exhibited no significant contribution in restoration of anti-oxidative stress effects of GLP-1 analog. Results of ROS measurement arrived at the same conclusion ( Figure 6 ).
Knockdown Of Both PKCβ And PKCδ Significantly Restored Anti-Apoptotic Effects Of GLP-1 Analog On H9C2 Cells
Representative TUNEL images and corresponding apoptosis index showed that, on the basis of HG + H/R + Ex-4 group (40.4±2.1%), addition of PKCβ knockdown hardly changed apoptosis index (38.3±1.9%) while addition of PKCδ knockdown decreased apoptosis index (33.5±1.3%). Notably, knockdown of both PKCβ and PKCδ caused a significant reduction in AI (14.5±1.2%) ( Figure 7C ). Analysis of apoptosis-related markers such as Bcl-2, BAX and Caspase-3 activity pointed to the same conclusion ( Figure 7A, B) . Thus indicating an ideal method to restore the anti-apoptotic effects of GLP-1 in diabetic condition.
Discussion
The current study provides a new perspective that increased vulnerability in diabetic myocardium is in part due to GLP-1 resistance. These data demonstrated that hyperglycemia via overexpression of PKCβ and PKCδ can increase cardiomyocyte vulnerability through a dual pathway including downregulation of GLP-1R expression and impairment of GLP-1 post-receptor anti-apoptosis signaling pathway.
Discussion
Previously, downregulation of GLP-1R has been proven to exist in several cell types in the diabetic state. Gang Xu et al, reported that downregulation of GLP-1R in pancreatic β cells is due to overexpression of PKCα in diabetes. 15 The relationship between reduced responsiveness to GLP-1 and changes in GLP-1 post-receptor signaling pathways is needed to be examined. GLP-1 exerts antiapoptotic effects by inhibition of apoptosis through the PI3K-Akt pathway. 12 PKCs are known to be major mediators of signal transduction pathways and have been shown to regulate sets of biological functions including apoptosis. 16 Significant effort has been made in understanding the role of specific PKCs in myocardium ischemia/reperfusion injury over the past two decades, but it is still a subject of debates. 29, 30 One hypothesis suggests that post G-protein-coupled receptor signaling pathways lead to the activation of PKC via diacylglycerol, after which, activated PKC then phosphorylates a secondary effector protein, which is thought to be induce protection. 31 However, numerous research have shown that PKC inhibition at reperfusion is protective. 32 Thus indicate a variability of actions that PKC isoform exhibited in myocardial ischemia/reperfusion condition. However, the above-mentioned researches did not take diabetes into consideration. In the present study, our results demonstrated that in diabetic cardiomyocyte, overexpression of PKCδ impaired GLP-1 post-receptor anti-apoptosis signaling pathways by inhibition of Akt phosphorylation. Knockdown of PKCδ partly restored the anti-apoptotic effects of GLP-1R. Overexpression of PKCδ inhibited downstream signaling pathways of GLP-1R, and that explains why knockdown of PKCβ alone hardly restored the antiapoptotic effects of GLP-1R. By knockdown of both PKCβ and PKCδ, we restored GLP-1 actions significantly in diabetic condition, thus implicated that PKCβ and PKCδ contributed to GLP-1 resistance through different mechanisms. Further studies explored the relationship between PI3K and PKCδ, the results showed that they exert no influences to each other ( Figure 8 ). To further explore the anti-apoptotic effects of GLP-1 in diabetic cardiomyocytes, signaling pathways involve PKCδ and Akt are need to be elucidated.
Conclusion
In the present study, we found that increased myocardial vulnerability is in part due to GLP-1 resistance in diabetic condition. We suggest that downregulation of the GLP-1R induced by PKCβ overexpression and impaired GLP-1 postreceptor anti-apoptotic signaling pathways induced by PKCδ overexpression are responsible for GLP-1 resistance in diabetic condition. This finding provides a new perspective in developing novel therapeutic strategy in treating myocardial IRI in diabetic condition based on activation of the GLP-1R. 

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