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Abstract

Preclinical and clinical studies have demonstrated that berberine (BBR) improves diabetic complications and reduces mortality of patients with congestive heart failure. The therapeutic effects of BBR have been reported to be mediated by its regulation of adenosine monophosphate (AMP)-activated protein kinase (AMPK). We previously reported that BBR protects against ischemia–reperfusion injury via regulating AMPK activity in both ischemic and nonischemic areas of the rat heart. Since diabetic hearts are more sensitive to ischemia–reperfusion injury, we examined whether BBR treatment exhibited cardioprotective effects in the diabetic heart. Type 2 diabetic rats were pretreated plus or minus BBR for 7 days and subjected to 30-minute ischemia followed by 120-minute reperfusion. Pretreatment of type 2 diabetic rats with BBR reduced ischemia–reperfusion injury infarct size and attenuated arrhythmia compared to untreated diabetic controls. Subsequent to ischemia–reperfusion, serum triglyceride, total cholesterol, and malondialdehyde levels were reduced by pretreatment of type 2 diabetic rats with BBR compared to untreated diabetic controls. In contrast, serum glucose and superoxide dismutase levels were unaltered. The mechanism for the BBR-mediated cardioprotective effect was examined. Pretreatment with BBR did not alter AMPK activity in ischemic areas at risk but increased AMPK activity in nonischemic areas compared to untreated diabetic controls. The increased AMPK activity in nonischemic areas was due an elevated ratio of AMP to adenosine triphosphate (ATP) and adenosine diphosphate to ATP. In addition, pretreatment with BBR increased protein kinase B (AKT) phosphorylation and reduced glycogen synthase kinase 3β (GSK3β) activity in nonischemic areas compared to untreated diabetic controls. These findings indicate that BBR protects the diabetic heart from ischemia–reperfusion injury. In addition, BBR may mediate this cardioprotective effect through AMPK activation, AKT phosphorylation, and GSK3β inhibition in the nonischemic areas of the diabetic heart.

Keywords

berberine ischemia–reperfusion AMPK heart type 2 diabetes

Introduction

Ischemic cardiomyopathy remains the leading cause of death in patients with type 2 diabetes. Thus, current pharmacotherapy of patients with type 2 diabetes should be aimed not only at glucose lowering but also at prevention of cardiovascular complications. Berberine (BBR) is an isoquinoline alkaloid extract that has shown promise as a hypoglycemic agent in the management of diabetes in animal and human studies. The beneficial effects of BBR on cardiovascular, liver, and renal disease have been reported in both preclinical and clinical studies.^1

–5 Berberine improves endothelial dysfunction and cardiac dysfunction in diabetic rats and reduces mortality in patients with severe congestive heart failure.^6
–8 These observations suggest that BBR may be a promising drug for the management of myocardial disease related to diabetes.

Recent studies suggest that the beneficial effects of BBR may be mediated by its regulation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a protein kinase that is regulated in response to alterations in intracellular adenine nucleotide concentrations.⁹ Metabolic activators of AMPK include ischemia, oxidative stress, exercise, and glucose deprivation.¹⁰ Adenosine monophosphate-activated protein kinase modulates several cellular processes that are important to cell survival during ischemia. In the ischemic heart, AMPK induces cardiomyocyte autophagy and attenuates endoplasmic reticulum stress, which promotes cell survival.^11,12 Our previous studies using the isolated perfused working heart model in vitro and ischemia–reperfusion rat heart model in vivo demonstrated that BBR provides cardioprotection against ischemia–reperfusion injury through AMPK regulation.¹³ However, whether BBR plays a protective role in ischemia–reperfusion injury in the diabetic heart was unknown. In addition, it was unknown whether such a protective role of BBR involved the protein kinase B (AKT)/glycogen synthase kinase 3β (GSK3β) pathway.

The purpose of the present study was to investigate the potential cardioprotective effects of short-term (7 days) BBR treatment in diabetic rat heart against ischemia–reperfusion injury. We observed significant reduction in myocardial infarct size (IS) and incidence of arrhythmia in hearts of type 2 diabetic rats pretreated with BBR, and this was independent of blood glucose (BG) lowering. Furthermore, our results demonstrate that the cardioprotective actions of BBR may be mediated through AMPK and the AKT/GSK3β signaling pathway in the nonischemic areas of the diabetic rat heart.

Materials and Methods

Materials

Antibodies against phos-AMPK, AMPK, phos-AKT, AKT, phos-GSK3β, and GSK3β and antirabbit secondary antibody were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, California). Glyceraldehyde-3-phosphate dehydrogenate was obtained from Epitomics Inc (California), and BBR was purchased from Northeast Pharmaceutical Factory (Beijing, China). Streptozotocin was purchased from Sigma-Aldrich (Beijing, China). Other reagents were purchased from Beijing General Chemical Reagent (Beijing, China).

Animals

Six-week-old male Wistar rats were housed in an environmentally controlled breeding room (temperature: 20 ± 2°C, humidity: 60% ± 5%, 12-hour light/dark cycle). All rats had free access to tap water. All procedures were approved by the Ethics Committee for the Use of Experimental Animals of Jilin University.

Animal Model and Experimental Groups

The diabetic rat model was developed using a high-fat diet plus multiple low doses of streptozotocin, which was similar to that used previously in studies from our laboratory.¹⁴ The rats were fed high-fat diet (consisting of 22% fat, 48% carbohydrate, and 20% protein with total calorific value 44.3 kJ/kg; The Stoyer Center of Experimental Animal Holding, Changchun, China) for 4 weeks. Following 4 weeks of dietary intervention, the diabetic group was injected intraperitoneally (IP) with low doses of streptozotocin (30 mg/kg, dissolved in 0.1 mol/L sodium citrate buffer, pH 4.4). One week later, blood samples were collected by tail cutting for fasting BG measurements by the glucose oxidase–peroxidase method.¹⁴ Rats with a fasting BG <7.8 mmol/L were injected with streptozotocin again (30 mg/kg). Four weeks after the second streptozotocin injection, the fasting BG was measured again, and rats with a fasting BG ≥7.8 mmol/L were considered diabetic. The high-fat diet was maintained for 12 weeks until the ischemia–reperfusion experiment, 1 week before the ischemia–reperfusion experiment. The animals were allocated to 2 groups: (1) DMIR, animals with type 2 diabetes treated with water intragastric gavage (IG) for 7 days prior to the heart perfusion and (2) DMIR + BBR, diabetic rats treated with BBR at a dose of 100 mg/kg (IG) for 7 days prior to the heart perfusion.

Ischemia–Reperfusion

After pretreatment, the diabetic animals were anesthetized with 20% urethane (0.5 mL/100 g, IP), placed face up and fixed on the operating table, and connected to a Lab chart 7.0 biological system (Chengdu Technology & Market Co, Ltd) and functional system electrocardiogram (ECG) wires. After tracheal intubation and connection to a small animal respirator (tidal volume of 8-12 mL, 1:2 breathing, respiratory rate 70-80 times/min), the chest was opened between the left third and fourth rib exposing the heart. A nylon suture was placed around the left anterior descending coronary artery. All groups were made ischemic for 30 minutes by ligating the artery, and this was followed by reperfusion for 120 minutes by loosening the ligature. Arrhythmias were monitored during ischemia–reperfusion by ECG. ST-segment elevation and widening of R wave indicated ischemia. A 50% drop-off in ST-segment elevation was indicative of successful reperfusion.

Measurement of Biological Parameters

Blood glucose, triglycerides (TGs), total cholesterol (TC) levels, malondialdehyde (MDA), and superoxide dismutase (SOD) in rat serum after heart ischemia–reperfusion were determined using assay kits per the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Beijing, China)

Determination of IS

After 120-minute reperfusion, the left coronary artery was ligated again, and then 0.2 mL 1% Evans blue dye was injected through the right common carotid artery in order to distinguish between ischemic and nonischemic areas. The heart was isolated immediately, rinsed with phosphate-buffered solution (PBS; pH 7.4), and the surface dried with filter paper. After Evans blue staining, the left ventricle was cut into 2-mm-thick transverse sections and incubated with 1% triphenyl tetrazoliumchloride phosphate buffer (pH 7.4) at 37°C. Triphenyl tetrazoliumchloride reacts with intracellular dehydrogenases to stain viable tissue red, leaving infarcted areas off-white. Digital photographs were taken of the traverse sections. The IS, area at risk (AAR), and ratio of infarct size to area at risk (IS/AAR) were determined by computer image analysis software (Image plus 6.0 System).

Determination of Ratio of AMP and Adenosine Diphosphate to Adenosine Triphosphate

Adenosine monophosphate, adenosine diphosphate (ADP), and adenosine triphosphate (ATP) levels were determined using enzyme-linked immunosorbent assay (ELISA) kits (Shanghai YanJing Biological Research Technology Co, Ltd, Beijing, China). Briefly, animals were killed and hearts were rapidly removed and frozen for the determination of nucleotide content. Heart tissues were homogenized on ice in homogenization buffer (PBS containing a protease inhibitor, pH 7.4, 4°C), homogenates centrifuged at 3000g for 20 minutes, and AMP, ADP, and ATP amounts in the supernatants were determined by ELISA according to the manufacturer’s instructions.

Western Blot Analysis

After acute left coronary artery ligation, samples were taken from the AAR and nonischemic area (NIA). Quantitative analysis of AMPK, AKT, GSK3β or p-AMPK, Th172 or p-AKT, Ser473 or p-GSK3β, and Ser9 levels was performed as described previously.¹⁵ Briefly, protein samples were prepared from hearts by homogenization with ice-cold radioimmunoprecipitation assay buffer. Protein concentration was measured using the Bradford assay (BioRad protein assay kit). Homogenate proteins (80 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes (BioRad, Beijing, China). Membranes were blocked with 5% (wt/vol) skim milk or 5% bovine serum albumin for 2 hours at room temperature and then incubated with rabbit polyclonal antibodies (p-AKT, p-AMPK, 1:500, GSK3β, p-GSK3β, AMPK, 1:1000; Santa Cruz Biotechnology) with gentle agitation overnight at 4°C. The membranes were washed 3 times for 10 minutes each with 15 mL of TBST (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% [vol/vol] Tween-20) and then incubated with secondary antibody (1:1000 goat antirabbit immunoglobulin G horseradish peroxidase conjugate; Santa Cruz Biotechnology) at room temperature for 2 hours. Protein was then visualized with enhanced chemiluminescence solution and X-ray film. An imaging densitometer was used to scan the protein bands, and these were quantified using image analysis software (Quantity One, Bio-Rad). The results were expressed as phosphorylated protein relative to total protein.

Statistics

All the data are expressed as mean ± standard error (SE). The differences between groups are compared with unpaired Student t test, and P < .05 was considered statistically significant.

Results

Berberine pretreatment decreases serum TG, TC, and MDA levels but does not alter serum glucose and SOD levels after cardiac ischemia–reperfusion injury in diabetic rats.

To examine whether BBR treatment exhibited cardioprotective effects in the diabetic heart, type 2 diabetic rats were pretreated minus (DMIR) or plus BBR (DMIR + BBR) for 7 days, the hearts were subjected to 30-minute ischemia followed by 120-minute reperfusion, blood was collected from the tail vein, and serum levels of TG, TC, and glucose were examined. No difference in body weight was observed between DMIR and DMIR + BBR rats (Table 1). In contrast, serum TG and TC levels were decreased in DMIR + BBR-treated animals compared to DMIR. Serum glucose levels were unaltered between DMIR and DMIR + BBR animals. Serum lipid peroxidation was assessed by analysis of MDA and SOD, biomarkers of oxidative stress. Serum MDA levels were decreased in DMIR + BBR animals compared to DMIR animals. In contrast, serum SOD levels were unaltered between DMIR and DMIR + BBR animals. Thus, BBR pretreatment reduces serum TC, TG, and MDA levels but does not alter serum glucose or SOD levels in type 2 diabetic animals subjected to cardiac ischemia–reperfusion injury.

Table 1.

Body Weight and Blood Parameters After Ischemia–Reperfusion Injury.^a

Group	DMIR	DMIR + BBR
Body weight, g	329 ± 17.1	342 ± 12.4
Blood glucose, mmol/L	27.5 ± 1.6	26.9 ± 0.9
TG, mmol/L	3.89 ± 0.64	2.22 ± 0.49^b
TC, mmol/L	3.04 ± 0.21	2.53 ± 0.13^b
MDA, nmol/mL	9.75 ± 0.72	3.33 ± 0.75^c
SOD, U/mL	272.5 ± 22.15	289.4 ± 43.25

Abbreviations: BBR, berberine; DMIR, diabetic rats subjected to ischemia–reperfusion injury; DMIR + BBR diabetic rats pretreated with BBR subjected to ischemia–reperfusion injury; MDA, malondialdehyde; TC, total cholesterol; TG, triglycerides; SE, standard error; SOD, superoxide dismutase.

^a Data shown are means ± SE (n = 7-9 rats/group).

^b P < .05.

^c P < .01 compared to DMIR.

Berberine Pretreatment Reduces Myocardial IS Following Cardiac Ischemia–Reperfusion Injury in Diabetic Rats

Myocardial IS is a primary determinant of myocardial prognosis with acute myocardial infarction. Type 2 diabetic rats were pretreated minus (DMIR) or plus BBR (DMIR + BBR) for 7 days, the hearts were subjected to 30-minute ischemia followed by 120-minute reperfusion, and IS was determined by Evans blue and 2,3,5-triphenyltetrazolium chloride staining. Ischemia–reperfusion-mediated ratio (IS/AAR) of IS to AAR was reduced 25% (P < .05) in DMIR + BBR animals compared to DMIR animals (Figure 1A). There was no alteration in ratio of AAR to area of left ventricular area (Figure 1B).

Figure 1.

BBR reduces infarct size in diabetic ischemia reperfused rat hearts. A, Percent of infract size to area at risk (IS/AAR). B, Percent area at risk to left ventricular area (AAR/LV). *p < 0.05 compared to DMIR group. AAR indicates area at risk; BBR, berberine; DMIR, animals with type 2 diabetes treated with water (Intragrastic Gavage) for 7 days prior to the heart perfusion; IS, infarct area; LV, total left ventricular area.

Berberine Pretreatment Reduces Arrhythmias During Cardiac Ischemia–Reperfusion Injury in Diabetic Rats

Type 2 diabetic rats were pretreated minus (DMIR) or plus BBR (DMIR + BBR) for 7 days, and the hearts were subjected to 30-minute ischemia followed by 120-minute reperfusion. Heart rate and arrhythmias were recorded during the entire ischemia–reperfusion processes. Heart rate was unaltered between DMIR and DMIR + BBR animals (Table 2). Arrhythmia vulnerability was evaluated as the number of ventricular arrhythmia events (VAEs) and the duration of these VAEs. Premature beats have no significant difference between the 2 groups. The duration of ventricular tachycardia (VT) was decreased 56% (P < .05), ventricular fibrillation reduced 80% (P < .05), and the incidence of VT decreased 60% (P < .05) in DMIR + BBR animals compared to DMIR animals (Table 3). Thus, diabetic animals pretreated with BBR were less prone to the development of cardiac arrhythmias triggered by ischemia–reperfusion.

Table 2.

Heart Rates in DMIR and DMIR + BBR Animals.

Times	DMIR	DMIR + BBR
Preischemic HR, beats/min	342 ± 11.1	345 ± 22.8
Ischemic HR
10 minutes	312 ± 6.0	311 ± 25.2
20 minutes	313 ± 13.9	320 ± 20.6
30 minutes	315 ± 13.7	304 ± 16.4
Reperfusion HR
10 minutes	313 ± 19.2	309 ± 20.3
20 minutes	309 ± 18.8	309 ± 21.5
30 minutes	300 ± 23.4	300 ± 21.6
60 minutes	295 ± 19.5	302 ± 27.6
120 minutes	290 ± 20.6	302 ± 20.5

Abbreviations: BBR, berberine; DMIR, diabetic ischemia–reperfusion; DMIR + BBR, diabetic ischemia–reperfusion plus BBR; HR, heart rate; SE, standard error.

Data shown are means ± SE (n = 7-9 rats/group).

Table 3.

Ventricular Arrhythmia Induced by Ischemia–Reperfusion in DMIR and DMIR + BBR Animals.^a

Group	Rate of Death (%)	Permature (times)	Last time of VF (s)	Last time of VT (s)	VF%	VT%
DMIR	28	57.0 ± 12.12	13.1 ± 5.54	44.6 ± 11.87	71	100
DMIR + BBR	25	34.4 ± 7.87	2.7 ± 1.36^b	19.3 ± 7.41^b	28	100

Abbreviations: BBR, berberine; DMIR, diabetic ischemia–reperfusion; DMIR + BBR, diabetic ischemia–reperfusion plus BBR; permature, the number of premature beats; last time of VF, the duration of ventricular fibrillation; last time of VT, the duration of ventricular tachycardia; SE, standard error.

^a Data shown are means ± SE (n = 7-9 rats/group).

^b P < .05 compared to the DMIR group.

Berberine Treatment Increases the Ratio of AMP to ATP and ADP to ATP in Nonischemic Area of the Ischemia–Reperfused Diabetic Heart

To determine whether BBR treatment altered energy status in the ischemia–reperfused diabetic heart, type 2 diabetic rats were pretreated minus (DMIR) or plus BBR (DMIR + BBR) for 7 days, the hearts were subjected to 30-minute ischemia followed by 120-minute reperfusion, and the ratio of AMP to ATP and ADP to ATP were examined in the AAR and NIA in the heart. The ratio of AMP to ATP or ADP to ATP was unaltered in the AAR in DMIR + BBR animals compared to DMIR animals (Figure 2A and B). In contrast, the ratio of AMP to ATP was increased 86% (P < .01) and the ratio of ADP to ATP increased 30% (P < .01) in DMIR + BBR animals compared to DMIR animals (Figure 2C and D). These data suggest that BBR may improve energy status in the NIA of the diabetic heart.

Figure 2.

Berberine increases AMP/ATP and ADP/ATP ratio in NIA of diabetic ischemia–reperfused rat hearts. Adenine nucleotides from homogenate of myocardial tissue extracts of AAR and NIA were determined by ELISA. A, The AMP/ATP ratio in AAR. B, The ADP/ATP ratio in AAR. C, The AMP/ATP ratio in NIA. D, The ADP/ATP ratio in NIA. **P < .01 compared to the DMIR group. AAR indicates area at risk; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; BBR, berberine; DMIR, hearts from diabetic rats subjected to ischemia–reperfusion injury; DMIR + BBR, hearts from BBR pretreatment diabetic rats subjected to ischemia–reperfusion injury; ELISA, enzyme-linked immunosorbent assay; NIA, nonischemic area.

Adenosine Monophosphate-Activated Protein Kinase Activity Is Increased by BBR in Nonischemic Area of the Diabetic Heart

Adenosine monophosphate-activated protein kinase is an energy sensor and activated response to changes in ratio of AMP to ATP or ADP to ATP. To determine whether BBR altered AMPK activity in diabetic hearts, type 2 diabetic rats were pretreated minus (DMIR) or plus BBR (DMIR + BBR) for 7 days, the hearts were subjected to 30-minute ischemia followed by 120-minute reperfusion, and the ratio of phosphorylated AMPK (Thr172) to total AMPK was determined in AAR and NIA of the heart. The ratio of phosphorylated AMPK (Thr172) to total AMPK was unaltered in the AAR of DMIR + BBR animals compared to DMIR animals (Figure 3A). In contrast, the ratio of phosphorylated AMPK (Thr172) to total AMPK was increased 62% (P < .05) in the NIA of DMIR + BBR animals compared to DMIR animals (Figure 3B). Thus, BBR treatment increases AMPK activity in the NIA of the diabetic heart.

Figure 3.

Berberine increases expression of AMPK in NIA of diabetic ischemia–reperfused rat hearts. The AMPK activity was determined as ratio of p-AMPK to AMPK. A, Activity of AMPK is not altered by BBR in AAR. B, Berberine increases phosphorylation of AMPK in NIA. *P < .05 compared to the DMIR group. AAR indicates area at risk; AMPK, adenosine monophosphate-activated protein kinase; BBR, berberine; DMIR hearts from diabetic rats subjected to ischemia–reperfusion injury; DMIR + BBR, hearts from BBR pretreatment diabetic rats subjected to ischemia–reperfusion injury; NIA, nonischemic area.

GSK3β Activity Is Inhibited by BBR in Nonischemic Area of the Diabetic Heart

The AKT/GSK-3β pathway plays an important role in regulating cellular survival during ischemia–reperfusion. To determine whether activity of GSK3β and its upstream regulator, AKT, was altered by BBR treatment in the NIA of diabetic hearts, type 2 diabetic rats were pretreated minus (DMIR) or plus BBR (DMIR + BBR) for 7 days, the hearts were subjected to 30-minute ischemia followed by 120-minute reperfusion, and the ratio of phosphorylated GSK3β (Ser9) to GSK3β and phosphorylated AKT (Ser473) to AKT was determined in the NIA of the heart. The ratio of phosphorylated AKT (Ser473) to AKT was increased 120% (P < .05) and the ratio of phosphorylated GSK3β (Ser9) to GSK3β increased 54% (P < .05) in the NIA of DMIR + BBR animals compared to DMIR animals (Figure 4A-C). Total AKT protein was reduced 39% (P < .05) in the NIA of DMIR + BBR animals compared to DMIR animals (Figure 4A and D). In contrast, total GSK3β protein was reduced 20% (P < .01) in the NIA of DMIR + BBR animals compared to DMIR animals (Figure 4A and E). Thus, BBR treatment increases AKT activity and reduces GSK3β activity in the NIA of the diabetic heart.

Figure 4.

Berberine increases phosphorylation of AKT and GSK3β in nonischemic area of diabetic ischemia–reperfused rat hearts. A, Representative Western blot of phosphorylated AKT, AKT, phosphorylated GSK3β, GSK3β, and GAPDH as loading control. B, Phosphorylated AKT to AKT ratio. C, Phosphorylated GSK3β to GSK3β ratio. D, Total AKT to GAPDH ratio. E, Total GSK3β to GAPDH ratio. *P < .05 compared to the DMIR group. AKT indicates protein kinase B; BBR, berberine; DMIR, hearts from diabetic rats subjected to ischemia–reperfusion injury; DMIR + BBR, hearts from BBR pretreatment diabetic rats subjected to ischemia–reperfusion injury; GSK3β, glycogen synthase kinase 3β; GAPDH, glyceraldehyde-3-phosphate dehydrogenate.

Discussion

The beneficial effect of BBR treatment on improvement in diabetic complications, including endothelial dysfunction, nephropathy, and neuropathy, is well documented. However, most studies have utilized long-term BBR treatment (≥4 weeks).^6,16
–18 The protective effect of BBR reported in these studies is likely mediated through reduction in serum glucose levels. We previously demonstrated that BBR treatment reduced IS in nondiabetic rats subjected to ischemia–reperfusion injury and reduced left ventricular myocardium size in rats subjected to experimentally induced cardiac hypertrophy mediated by suprarenal aortic constriction.^19,20 These data suggested that BBR may have additional cardioprotective actions beyond its glucose-lowering effects.

In the current study, we utilized a 100 mg/kg/d dose of BBR. This dose exhibited a much greater BG-lowering effect and reduction in diabetic complications in a rat model of type 2 diabetes when compared to a lower 50 mg/kg/d dose.²¹ In addition, a 100 mg/kg/d dose of BBR was shown to reduce cardiac IS and improve cardiac function in a nondiabetic cardiac ischemia–reperfusion rat model.¹³ The dose of BBR utilized in our study was similar to the dose utilized for controlling BG in a previous clinical study, which recommended 1000 mg/60 kg/d for optimum BG control in diabetic patients.²²

In the current study, short-term (7 days) BBR treatment profoundly reduced myocardial IS in the NIA of the heart in rats subjected to cardiac ischemia–reperfusion injury. We measured glucose concentration after ischemia–reperfusion injury in this experiment. Interestingly, the glucose level was much higher than normal BG levels (>25 mmol/L), and BBR had no effect on glucose lowering after ischemia–reperfusion. The elevated BG was likely due to the acute stress response to ischemia–reperfusion injury. Thus, BBR treatment does not reduce glucose levels immediately after acute cardiac stress induced by ischemia–reperfusion injury in the heart.

In the current study, BBR treatment of diabetic rats decreased serum TG and TC levels, suggesting that a reduction in serum lipid levels may also participate in the cardioprotective action of BBR. The hypolipidemic effect of BBR has attracted much attention. Oral administration of BBR (100 mg/d) for 2 weeks significantly reduced plasma cholesterol in the hyperlipidemic hamster.²³ In fact, one clinical study showed that the lipid-lowering effect of BBR was even greater than of metformin and comparable with statins.^24
–26 The outstanding action of BBR as a lipid-lowing drug in clinic application to patients with hypercholesterolemia could be applied as alterative treatment for patients with statin-resistant dyslipidemia. The mechanism of the lipid-lowering effect of BBR may be due to its stabilization of the hepatic low-density lipoprotein receptor (LDLR) mediated through increased extracellular signal-regulated kinase-dependent pathway and increased transcriptional activity of the LDLR promoter.⁴ In addition, BBR treatment was shown to modulate turnover of bile acids and the metabolism of cholesterol and reduced liver phosphatidate phosphohydrolase,²⁷ an important enzyme in the synthesis of TG.

Oxidative stress induced by lipoperoxidation is involved in ischemia–reperfusion-mediated injury.²⁸ Fatty acids such as palmitate may damage the myocardium, thus enhancing enzyme release, severely impairing myocardial mechanical function and provoking arrhythmias.^29,30 These adverse effects of fatty acids are especially marked in the postischemic reperfusion period. In the current study, BBR treatment of diabetic rats reduced MDA levels, a marker of lipoperoxidation. In addition, BBR treatment reduced cardiac arrhythmia in diabetic rats subjected to ischemia–reperfusion injury. These data suggest that an antioxidative effect of BBR may participate in its cardioprotective action. Thus, BBR treatment appears to have multiple beneficial effects for functional recovery of the diabetic heart after ischemia–reperfusion injury, and this may decrease the risk of cardiac hypertrophy and subsequent development of heart failure.

One possible mechanism by which BBR may protect the myocardium is via regulation of AMPK activity. Activation of AMPK is regulated by changes in the AMP to ATP or ADP to ATP ratio.^31,32 As a cellular fuel gauge, AMPK is an important regulator of myocardial energy metabolism during ischemia and reperfusion.^33,34 During cardiac ischemia, AMPK is activated, and this results in an increase in myocardial glucose uptake, glycolysis, and fatty acid oxidation. This activation of AMPK has the potential to increase energy production, thereby protecting the heart during ischemic stress. However, in the diabetic state, expression of AMPK is inhibited by high levels of fatty acids.^35,36 In addition, ischemia-induced blockade of AMPK activation was shown to increase myocardial cell apoptosis in the diabetic rat heart.⁷ Under ischemia–reperfusion stress, changes in both oxygen and nutrient supply result in temporal variation in AMPK activity as well as changes in high-energy nucleotide levels. In the current study, BBR treatment of diabetic animals increased the AMP to ATP ratio and the ADP to ATP ratio in the NIA of hearts subjected to ischemia–reperfusion injury. Thus, BBR treatment may help maintain energy supply for the diabetic heart during ischemia–reperfusion injury.

In addition to AMPK, the AKT signaling pathway is crucial in energy sensing, growth factor signaling, and the establishment of cardiomyocyte number and size.³⁷ Glycogen synthase kinase 3β is a downstream protein regulated by AKT. Inhibition of GSK3β has been shown to be protective in classic cardiac preconditioning, an important mechanism of cardioprotection against ischemia–reperfusion injury.^38,39 Glycogen synthase kinase 3β is active in resting cells and is inactivated by phosphorylation at Ser9.⁴⁰ Inhibition of GSK3β has been shown to reduce apoptosis and enhance cell survival.^41,42 Thus, phosphorylation and inhibition of GSK3β may mediate cardioprotection. Crosstalk between AKT and AMPK signaling is still not clear. However, it is interesting to note that in cardiac myocytes and in vascular endothelial cells, AKT activation appears to be regulated by the AMPK pathway, as inhibition of AMPK activity either by compound C or by small interfering RNA-mediated knockdown of AMPK α1 decreased agonist-induced AKT phosphorylation.^43,44 In the current study, BBR treatment resulted in activation of AMPK in the NIA of diabetic hearts subjected to ischemia–reperfusion injury, and this was accompanied by phosphorylation of both AKT and GSK3β. These data suggest that phosphorylation of AKT/GSK3β in the diabetic heart subjected to ischemia–reperfusion injury may be regulated, in part, through AMPK.

In summary, our findings demonstrate that short-term (7 days) BBR treatment exhibits cardioprotective effects against ischemia–reperfusion injury in type 2 diabetic animals. Furthermore, a BBR-mediated increase in AMP to ATP ratio, ADP to ATP ratio, AMPK activation, phosphorylation of AKT, and inhibition of GSK3β in the NIA may contribute to its cardioprotection against ischemia–reperfusion injury in the diabetic heart.

Footnotes

Authors’ Note

This work was conducted in Preclinical Pharmacology R&D Center of Jilin Province and Key Lab of Traditional Medicine for Diabetes of Jilin Province.

Author Contributions

W. G. Chang contributed to conception and design, contributed to interpretation, drafted the manuscript, and agrees to be accountable for all aspects of work ensuring integrity and accuracy. K. Li contributed to conception and design, contributed to acquisition, analysis, and interpretation, drafted the manuscript, and agrees to be accountable for all aspects of work ensuring integrity and accuracy. F. Y. Guan contributed to acquisition, analysis, and interpretation and drafted the manuscript. F. Yao contributed to acquisition and analysis. Y. Yu contributed to acquisition and analysis. M. Zhang contributed to conception and design, critically revised the manuscript, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy. G. M. Hatch critically revised the manuscript, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy. L. Chen critically revised the manuscript, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: G.M.H. is a Canada Research Chair in Molecular Cardiolipin Metabolism.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Natural Science Foundation of China (81170745, 81200598), Jilin Science & Technology Development Plan (20140203011YY), and the Heart and Stroke Foundation of Canada.

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Berberine Pretreatment Confers Cardioprotection Against Ischemia–Reperfusion Injury in a Rat Model of Type 2 Diabetes

Abstract

Keywords

Introduction

Materials and Methods

Materials

Animals

Animal Model and Experimental Groups

Ischemia–Reperfusion

Measurement of Biological Parameters

Determination of IS

Determination of Ratio of AMP and Adenosine Diphosphate to Adenosine Triphosphate

Western Blot Analysis

Statistics

Results

Berberine Pretreatment Reduces Myocardial IS Following Cardiac Ischemia–Reperfusion Injury in Diabetic Rats

Berberine Pretreatment Reduces Arrhythmias During Cardiac Ischemia–Reperfusion Injury in Diabetic Rats

Berberine Treatment Increases the Ratio of AMP to ATP and ADP to ATP in Nonischemic Area of the Ischemia–Reperfused Diabetic Heart

Adenosine Monophosphate-Activated Protein Kinase Activity Is Increased by BBR in Nonischemic Area of the Diabetic Heart

GSK3β Activity Is Inhibited by BBR in Nonischemic Area of the Diabetic Heart

Discussion

Footnotes

Authors’ Note

Author Contributions

Declaration of Conflicting Interests

Funding

References