M E T A B O L I S M C L I N I C A L A N D E X P E R I M E N T A L 6 2 ( 2 0 1 3 ) 1 1 5 9 – 1 1 6 7

Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

Metabolismwww.metabo l i sm jou rna l . com

Berberine improves insulin resistance in cardiomyocytes viaactivation of 5′-adenosine monophosphate-activatedprotein kinase

Wenguang Changa, 1, Ming Zhanga, 1, Jing Li a, Zhaojie Menga, Shengnan Weia,Hongwei Dub,⁎, Li Chena,⁎, Grant M. Hatch c

a Department of Pharmacology, Norman Bethune Medical College, Jilin University, Changchun 130021, Chinab Department of Pediatric Endocrinology, The First Clinical Hospital Affiliated to Jilin University, Changchun 130021, Chinac Department of Pharmacology and Therapeutics, University of Manitoba, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada

A R T I C L E I N F O

Abbreviations: AMP, adenosine monophosdiabetic cardiomyopathy; PBS, phosphate bureceptor substrate-1.⁎ Corresponding authors. Li Chen is to be con

126 XinMin Street, Changchun 130021, ChinaJilin University, 71 Xin Min Street, Changchu

E-mail addresses: dhw_101@126.com (H.1 The first two authors contributed equally

0026-0495/$ – see front matter © 2013 Elsevihttp://dx.doi.org/10.1016/j.metabol.2013.02.00

A B S T R A C T

Article history:Received 12 November 2012Accepted 19 February 2013

Objective. Insulin resistance plays an important role in the pathogenesis of diabeticcardiomyopathy. Berberine (BBR) is a plant alkaloid which promotes hypoglycemia viaincreasing insulin sensitivity in peripheral tissues. Little is known of BBR’s role in regulatingglucose metabolism in heart.

Materials/methods. We examined the effect and mechanism of BBR on glucoseconsumption and glucose uptake in insulin sensitive or insulin resistant rat H9c2cardiomyocyte cells. H9c2 myoblast cells were differentiated into cardiomyocytes andincubated with insulin for 24 h to induce insulin resistance.

Results. BBR-treatment of H9c2 cells increased glucose consumption and glucose uptakecompared to controls. In addition, BBR-treatment attenuated the reduction in glucoseconsumption and glucose uptake in insulin resistant H9c2 cells. Compound C, an inhibitorof AMP-activated protein kinase (AMPK), abolished the enhancement of glucoseconsumption and glucose uptake mediated by BBR in both insulin sensitive and insulinresistant H9c2 cells compared to controls.

Conclusion. BBR significantly increased AMPK activity, but had little effect on the activityof protein kinase B (AKT) in insulin resistant H9c2 cells, suggesting that berberine improvesinsulin resistance in H9c2 cardiomyocytes at least in part via stimulation of AMPK activity.

© 2013 Elsevier Inc. All rights reserved.

Keywords:AMPKGlucose consumptionH9c2BerberineInsulin resistance

1. Introduction

The heart is one of the largest energy consumers in the body,and the balance of the energy metabolism in the heart is

phate; AMPK, 5′-adenosinffered solution; AKT, pro

tacted at: Department of. Hongwei Du, Departmenn 130021, China.Du), zhouweichen@yahooto this and were conside

er Inc. All rights reserved7

important for maintaining its physiological function. Underphysiological conditions, 70% of the energy supply of thenormal heart is provided through oxidation of fatty acid, with30% derived from oxidation of carbohydrate, including 20%

e monophosphate-activated protein kinase; BBR, berberine; DCM,tein kinase B(PKB); GLUT-4, glucose transporter-4; IRS-1, insulin

Pharmacology, Norman Bethune Medical College, Jilin University,t of Pediatric Endocrinology, The First Clinical Hospital Affiliated to

.com (L. Chen).red co-first author.

.

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from glucose oxidation and 10% from glycolysis. However,during the development of cardiovascular diseases, such ascardiomyopathy, myocardial infarction and congestive heartfailure, glucose metabolism becomes the main source ofenergy supply utilized by the heart.

5′-adenosine monophosphate-activated protein kinase(AMPK) is a heterogenous protein trimer composed ofthree subunits; a functional subunit (α) and regulatorysubunits (β γ). AMPK plays an important role in variousintracellular signaling pathways that regulate intracellularenergy balance [1]. Activated AMPK triggers glucose uptake,glycolysis, fatty acid oxidation and mitochondria biogenesis,and decreases protein synthesis, glycogen synthesis andfatty acid synthesis. AMPK has been shown to regulateglucose metabolism through translocation of glucose trans-porter-4 (GLUT4) to the plasma membrane in 3T3-L1adipocytes [2]. Activated AMPK results in enhanced glucosemetabolism via increasing glycolysis in skeletal muscle [3].In addition, activation of hepatic AMPK inhibits hepaticglucose production and lipogenesis, as well as cholesterolsynthesis [4]. Recently, activation of AMPK was found tophosphorylate cAMP response element-binding protein(CREB)-regulated transcription coactivator 2 (TORC2), whichmediates CREB-dependent transcription of peroxisome pro-liferator-activated receptor gamma coactivator 1 (PGC1) andits subsequent gluconeogenic targets phosphoenolpyruvatecarboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase),thus inhibiting the hepatic gluconeogenesis [5]. Further-more, activation of AMPK has been shown to play a criticalrole in glucose metabolism of cardiomyocytes [6]. Thus,AMPK might be an important pharmaceutical target for theregulation of energy metabolism in the heart.

Berberine (BBR), is an isoquinoline alkaloid extractedfrom Coptis chinensis. BBR treatment results in hypoglycemicand insulin-sensitizing activity both in animal models andin patients with type 2 diabetes mellitus [7–11]. Increasedglucose consumption mediated by BBR was shown to be aresult of activated AMPK. BBR increased glucose uptake viaAMP-AMPK-p38 MAPK pathway in L6 rat skeletal muscles[12]. In addition, BBR enhanced glucose metabolism viastimulation of glycolysis due to activation of AMPK. Wepreviously reported that BBR inhibited hepatic gluconeogen-esis via activating AMPK [13]. Despite significant advances inour understanding of the hypoglycemic effect of BBR, therole of BBR in regulating glucose metabolism in cardiac cellsis unknown. In the present study, we show that BBRstimulates glucose consumption and glucose uptake viaactivation of AMPK in normal or insulin resistant H9c2cardiomyocyte cells.

2. Materials and methods

2.1. Materials

DMEM, trypsin-EDTA solution, insulin, 5-amino-1-b-D-ribo-furanosyl-1H-imidazole-4-Carb-oxamide (AICAR), Com-pound C and reagents for western blot analysis wereobtained from Sigma-Aldrich (Beijing, China). FCS wasobtained from Runsheng Biological Materials (Taiyuan,

China). Polyclonal antibodies against phosphorylation ofAKT (Ser473), AKT, phospho-AMPK (Thr172), AMPKα1/2 andMyosin heavy chain (MF20) were obtained from Santa CruzBiotechnology (Santa Cruz, CA., USA). Polyvinylidene fluoride(PVDF) membrane was obtained from Millipore (Beijing,China). 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose, 2-NBDG (Invitrogen).

2.2. Cells

H9c2 rat cardiac myoblastic cells were obtained from CellResource Center (IBMS, Beijing). Cells weremaintained in highglucose Dulbecco's modification of Eagle's medium (DMEM)with 10% fetal calf serum (FCS), 2% L-glutamine, 10% sodiumbicarbonate, 10% sodium pyruvate, 5% Hepes, 1% penicillin/streptomycin and 1% gentamicin in an incubator (37 °C, 5%CO2) in 48 well plates. One day before each experiment, cellswere incubated in cell culturemedium supplemented with 1%FCS to differentiate the cells into cardiomyocytes [14].

2.3. Live fluorescence microscopy of H9c2 Cells labeledwith MF20 and Hoechst 33342

After the differentiation process, H9c2 cells were seeded inglass-bottom dishes (Mat-Tek Corporation) and incubatedwith Myosin heavy chain (MF20, Santa Cruz) antibody (1:200)and Hoechst 33342 (1 μg/ml). The images were obtained usinga confocal microscope.

2.4. Glucose consumption

Glucose consumption was measured as previously described[3,15–17]. H9c2 cardiomyocytes were incubated in serum-free, low-glucose (5.5 mmol/L) DMEM overnight. The cellswere then treated with BBR and/or other reagents (Com-pound C 10 μmol/L, AICAR 1 mmol/L) in fresh serum-freelow-glucose (5.5 mmol/L) DMEM for 24 h, and 10 μl mediumwas removed at 3, 6, 12, and 24 h. The glucose concentrationin medium was measured with glucose assay reagent(Beijing BHKT Clinical Reagent, Beijing, China) and is basedon the glucose oxidase method. The amount of glucoseconsumption (GC) was calculated by the glucose concentra-tions of blank wells subtracting the remaining glucose in cellplated wells.

2.5. Glucose uptake in normal and insulin resistantH9c2 cells

In normal cells, H9c2 cardiac myocytes were pre-incubated3 h in the absence or presence of 10 μmol/L BBR and/or10 μmol/L Compound C and/or 1 mmol/L AICAR and then 2-NBD-glucose (200 μmol/L) in PBS were added and the cellswere incubated for an additional 30 min. For measurement ofglucose uptake in insulin resistant cells, H9c2 cells wereexposed to insulin (100 nmol/L) treatments for 24 h and thenwashed 3 times with KRB containing 0.5% BSA at 40-minintervals over 2 h at 37 °C. After 3 h pretreatment of berberine(10 μmol/L) or other reagents, 200 μmol/L 2-NBD-glucose inPBS was added with or without insulin at 100 nmol/L; and thecells were incubated for an additional 30 min. Glucose uptake

Fig. 1 – Myosin heavy chain (MF20) content in differentiatedH9c2 cells. H9c2 cells cultured in a high-serum mediawere treated with 10% FCS or 1% FCS overnight and labeledwith MF20 (green fluorescence), Hoechst 33342 (nuclei,blue fluorescence). Differentiated H9c2 cell cultures inmedium supplemented with 1% of FCS showed increasedfusion and more elongated cells with multiple nucleiand cells expressing myosin heavy chain significantly.Images were collected by confocal microscopy with a400× total magnification.

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was stopped with 3 washes of ice-cold PBS. The fluorescenceintensity of cells was recorded by fluorescent microplate.

2.6. Insulin resistance in H9c2 cells

Insulin resistance in H9c2 cells was induced by incubatingcells in DMEMwith insulin (100 nmol/L) for 24 h [18]. The cellswere then left untreated or treated with BBR and/or otherreagents (Compound C 10 μmol/L, AICAR 1 mmol/L) andcombined with or without insulin (100 nmol/L) for 12 h (forglucose consumption) or 30 min (for glucose uptake) tomeasure the sensitivity to insulin stimulation.

2.7. Cell viability analysis

Cell viability was determined by MTT assay in 96-well tissueculture plates as described previously [19]. After treatment,the culturemediumwas removed from thewells, and 200 μl ofMTT reagent (Sigma) at a concentration of 1 mg/mL in PBSwasadded to each well. After 4 h incubation at 37°C, MTT reagentin PBS was removed and then the blue-colored formazanproduct was solubilized in 0.15 ml of DMSO for 20 min. Theabsorbance of converted dye was measured at a wave lengthof 570 nm.

2.8. Western blot analysis

Quantitative analysis of AKT, P-AKT, AMPK, or p-AMPK(Thr172)expression was performed as previously described [20]. Thecells were lysed in ice-cold lysis buffer (50 mmol/L Tris–HCl,1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/LNaCl, 1 mmol/L sodium orthovanadate, 1 mmol/L NaF,1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L aproti-nin, 1 mmol/L leupeptin, 1 mmol/L pepstatin A) and centri-fuged for 20 min at 14,000×g. Protein concentration wasmeasured using the bicinchoninic acid (BCA) assay (PierceBCA protein assay kit). For western blot detection, proteins(80–100 μg) were separated on 12% SDS polyacrylamide gelelectrophoresis and electro blotted onto PVDF membranes.Membranes were blocked with 5% (w/v) skim milk or 5% BSAfor 2 h at room temperature and then incubated with rabbitpolyclonal antibodies (p-AMPK, 1:500, AMPK, 1:1000, p-AKT,1:1000, AKT, 1:1000, Santa Cruz Biotechnology) with gentleagitation overnight at 4 °C. The membranes were washed 3times for 10 min each with 15 ml of TBST [10 mmol/L Tris–HCl, 150 mmol/L NaCl and 0.1% (v/v) Tween] and thenincubated with secondary antibody (1:1000 goat anti-rabbitIgG horseradish peroxidase conjugate, Santa Cruz Biotech-nology) at room temperature for 2 h. Protein was visualizedwith enhanced chemiluminescence solution and imageswere generated with GENE Imaging system. The imageswere quantified using Image Analysis Software (QuantityOne). The results were expressed as phosphorylated proteinrelative to total protein.

2.9. Statistics

Data are expressed in mean ± SE. Differences between twomeans were analyzed by Student’s t-test. A two-tailed p value<0.05 was considered to be significant.

3. Results

3.1. Differentiation features of H9c2 cells

H9c2 cells are similar to neonatal cardiomyocytes in manyways. For example, themembranestructure, Gprotein signalingsystem, electrophysiological properties and reactivity signalstimulation of cardiac hypertrophy are similar between H9c2cells and neonatal cardiomyocytes. To examine the role of BBRin regulating glucose metabolism in cardiac cells, H9c2 myo-blastswere fixedand labeledwithmyosinheavy chainantibody(MF20) and counterstained with Hoechst as described inMaterials andMethods. H9c2 cells grown inmedium containing10% FCS (growth-promoting medium) exhibited a myoblastphenotype, including a spindle-to-stellate shape and weremononucleated (Fig. 1). Differentiated H9c2 cell cultured inmediumsupplementedwith 1%of FCS showed increased fusionand more elongated cells with multiple nuclei. The differentmorphology was in agreement with previous reports [21,22]. Tofurther characterize differentiation induction, cells were immu-nolabeled with antibodies against myosin heavy chain (MF20)protein, a marker of muscle differentiation. As shown in Fig. 1,differentiated H9c2 cells exhibited significant expression ofmyosin heavy chain.

3.2. Low concentrations of BBR did not affect H9c2cell viability

H9c2 cardiomyoblast cells were incubated in cell culturemedium supplemented with 1% FCS to differentiate the cellsinto cardiomyocytes and the cytotoxicity of BBR was tested inthese cells using the MTT assay. BBR was non-cytotoxicconcentrations at 50 μmol/L or lower (Fig. 2). In contrast,concentrations of 100 μmol/L or higher resulted in significant

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cytotoxicity. No abnormal features were observed in cellstreated with 3.125–50 μmol/L BBR using light microscopeanalysis (data not shown). A 5–20 μM concentration of BBRwas chosen for all subsequent experiments.

3.3. BBR increased glucose consumption and glucoseuptake in H9c2 cells through activated AMPK

To investigate the effect of BBR on glucose consumption inH9c2 cells, cells were treated with various concentrations ofBBR (5–20 μM) for up to 24 h. BBR increased glucose consump-tion with time in a dose-dependent manner (Fig. 3A). Weexamined if expression of p-AMPK was altered in H9c2 cellstreatedwith 5–20 μMBBR for 24 h. Total AMPK expressionwasunaltered between control and BBR treated cells. In contrast,p-AMPK levels and the ratio of p-AMPK to AMPK weresignificantly increased by BBR treatment in a dose dependentmanner (Fig. 3B). We next investigated whether the activatedAMPK contributed to the increased glucose consumption inBBR treated cells. AMPK inhibitor Compound C and activatorAICAR were added to H9c2 cells treated with or without BBR.Glucose consumption in H9c2 cells was 4-fold higher (p < 0.05)in cells treated with BBR for 24 h compared to controls (Fig. 3C). The presence of Compound C diminished the glucoseconsumption induced by BBR. In contrast, AICAR enhancedthe action of BBR on increasing glucose consumption in H9c2cells. Moreover, treatment of H9c2 cells with BBR and AICARfor 24 h activated AMPK and increased the ratio of p-AMPK toAMPK. In contrast, the presence of Compound C blocked theactivation of AMPK even in the presence of BBR (Fig. 3D). Theeffect of BBR on glucose uptake was further tested in H9c2cardiomyocytes. 10 μmol/L BBR increased the glucose uptakecompared to the normal control. The presence of Compound Cdiminished the glucose uptake induced by BBR (Fig. 3E). Theseresults suggest that BBR stimulates glucose consumption andglucose uptake in H9c2 cells by activation of AMPK.

3.4. BBR increased glucose consumption and glucoseuptake in H9c2 cells with insulin resistance

Insulin resistance in H9c2 cells was established by addinginsulin (100 nmol/L) to the culturemedium for 24 h. As shown

Fig. 2 – Cytotoxicity of BBR in H9c2 cells. H9c2 cardiacmyocyteswere incubatedwith various concentrations of BBRfor up to 24 h and cell viability was determined byMTT assayas described in Materials and Methods. Data represent themean ± SE of six experiments. *p < 0.05, **p < 0.01 comparedto control.

in Fig. 4A and Fig. 4B, insulin stimulated glucose consumptionand glucose uptake in insulin-sensitive H9c2 cells, but cellscultured with insulin (insulin-resistant H9c2 cells) wereunresponsive to insulin stimulation. Since Ser473 phosphory-lation of AKT is a molecular indicator of insulin resistance, weexamined if expression of AKT, a key protein in the insulinsignaling pathway, was altered in insulin resistant H9c2 cells.Total AKT expression was unaltered between insulin-sensi-tive and insulin-resistant H9c2 cells. Treatment of insulin-sensitive H9c2 cells with insulin increased p-AKT levels. Incontrast, treatment of insulin-resistant H9c2 cells with insulindid not alter p-AKT levels (Fig. 4C). Thus, adding insulin(100 nmol/L) to the culture medium for 24 h resulted in theestablishment of insulin-resistant H9c2 cells. We examined ifBBR-treatment altered glucose consumption in H9c2 cells withinsulin resistance. BBR-treatment of insulin-resistant H9c2cells significantly increased basal and insulin simulated-glucose consumption in insulin-resistant H9c2 cells (Fig. 4D).To examine whether BBR affected the glucose uptake ininsulin-resistant cells, BBR at 10 μmol/Lwas incubatedwith orwithout the addition of insulin at 100 nmol/L for 30 min. Asshown in Fig. 4E, BBR treatment at 10 μmol/L also increasedthe glucose uptake in the insulin-resistant cells.

3.5. BBR improved insulin-induced insulin resistancethrough activation of AMPK

Since the BBR-mediated stimulation in glucose consumptionand glucose uptake in insulin-sensitive H9c2 cells wasmediated through AMPK, we examined the possibility thatBBR improved insulin sensitivity through AMPK activation ininsulin resistant H9c2 cells. The effects of BBR on glucoseconsumption and glucose uptake were examined in thepresence of the specific AMPK inhibitor Compound C. Asshown in Fig. 5A and 5B, Treatment of insulin resistant H9c2cells with BBR significantly increased the glucose consump-tion and glucose uptake in either the absence or presence ofinsulin. The presence of Compound C did not affect glucoseconsumption and glucose uptake in insulin-resistant H9c2cells in either the absence or presence of insulin. In contrast,CompoundC attenuated but did not completely block the BBR-mediated stimulation of glucose consumption and glucoseuptake in either the absence or presence of insulin. Moreover,BBR significantly stimulated phosphorylation of AMPK in theabsence or presence of insulin, this stimulation was sup-pressed by Compound C (Fig. 5C).We next examined the effectof BBR on p-AKT in insulin-resistant H9c2 cells. Expression ofp-AKT in insulin resistant H9c2 cells was slightly, but notsignificantly increased by BBR treatment in either the absenceor presence of insulin (Fig. 5D). Taken together, BBR improvesinsulin resistance in H9c2 cardiomyocytes at least in part viastimulation of AMPK activity.

4. Discussion

In the present study, the effect and mechanism of BBR onglucose metabolism were examined in both insulin-sensitiveand insulin-resistant rat H9c2 cardiomyocytes. The resultsindicate that BBR stimulates glucose consumption and

Fig. 3 – Effect of BBR, Compound C, AICAR on glucose consumption and AMPK activation in insulin sensitive H9c2cardiomyocytes. (A) H9c2 cardiac myocytes were incubated in the absence or presence of various concentrations of BBR andglucose consumption determined at various times as described in Materials and Methods. (B) H9c2 cardiac myocytes wereincubated with various concentrations of BBR for 24 h and activation of AMPK (MW: 62 kDa) was determined by western blotanalysis as described in Materials andMethods. A representative blot is depicted. (C) H9c2 cardiac myocytes were incubated inthe absence or presence of 10 μmol/L BBR and/or 10 μmol/L Compound C and/or 1 mmol/L AICAR and glucose consumptionwas determined at various times as described in Materials and Methods. (D) H9c2 cardiac myocytes were incubated in theabsence or presence of 10 μmol/L BBR and/or AICAR and/or CompoundC and activation of AMPK (MW: 62 kDa)was determinedbywestern blot analysis as described inMaterials andMethods. (E) H9c2 cardiacmyocyteswere preincubated in the absence orpresence of 10 μmol/L BBR and/or 10 μmol/L Compound C and/or 1 mmol/L AICAR and then 2-NBD-glucose in PBS was addedand the cells were incubated for an additional 30 min. Glucose uptake was stopped with 3 times of ice-cold PBS washing. Thefluorescence intensity of cells was recorded by fluorescent microplate. A representative blot is depicted. Data are expressed asmean ± SE of six experiments. *p < 0.05, **p < 0.01 compared with control, #p < 0.05 compared with BBR treatment group.

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glucose uptake in both insulin-sensitive and insulin-resistantH9c2 cardiomyocytes and the mechanism underlying im-provement of BBR on insulin resistant in H9c2 cells may beassociated with activation of AMPK.

Myocardial insulin resistance and hypoglycemia have beenidentified as major determinants in the development ofdiabetic cardiomyopathy. In type 2 diabetes there is a failureto increase glucose disposal into peripheral tissues in

Fig. 4 – Effect of BBR on glucose consumption and AKT activation in insulin-sensitive and insulin-resistant H9c2cardiomyocytes treated plus orminus insulin. (A) Insulin-sensitive or insulin-resistant H9c2 cardiomyocyteswere incubated inthe absence or presence insulin and glucose consumption determined as described inMaterials andMethods. (B) 200 μmol/L 2-NBD-glucose was added to H9c2 cells with or without insulin (100 nmol/L) for 30 min to test the ability of glucose uptake; thefluorescence intensity of cells was recorded by fluorescent microplate. (C) Insulin-sensitive or insulin-resistant H9c2cardiomyocytes were incubated in the absence or presence insulin and AKT (MW: 60 kDa) activation determined by westernblot analysis as described in Materials and Methods. A representative blot is depicted. (D) Insulin-resistant H9c2cardiomyocytes were incubated in the absence or presence of BBR and the absence or presence of insulin and glucoseconsumption was determined as described in Materials and Methods. (E) H9c2 cells were exposed to insulin (100 nmol/L) for24 h to induce insulin resistance and then washed 3 times with KRB containing 0.5% BSA at 40-min intervals over 2 h at 37 °C.After 3 h pretreatment of berberine (10 μmol/L), 200 μmol/L 2-NBD-glucose was added to insulin-resistant H9c2 cells with orwithout insulin (100 nmol/L) for 30 min to test the ability of glucose uptake; the fluorescence intensity of cells was recorded byfluorescentmicroplate. Data represent themean ± SE of six experiments. *p < 0.05 compared with control, #p < 0.05 comparedwith insulin resistant group in presence of insulin.

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response to insulin leading to chronically elevated levels ofglucose in the circulation followed by a compensatory rise ininsulin [23,24]. The elevated glucose and insulin levels in turnexacerbate insulin resistance, which results in dysfunction ofmyocardium and contributes to the pathogenesis of diabeticcardiomyopathy [25].

BBR has been studied as an anti-diabetic drugwidely due toits remarkable hypoglycemic effects [9,26]. Recently, thecardiovascular protective effects of BBR have attracted much

attention [27–30]. It was initially reported that BBR couldsignificantly improve cardiac function in patients with severecongestive heart failure [31]. The anti-arrhythmic actions ofBBR were attributed to its inhibitory effects on IK1, IK, andHERG channel [28]. Recently, it was reported that the BBRattenuation of ischemic arrhythmias maybe related to recov-ery of depressed I(to) and I(Ca) currents in diabetic rats. Inaddition, BBR was shown to modulate the sympatheticnervousactivity of ratswith experimental cardiachypertrophy

Fig. 5 – Effect of BBR, Compound C and insulin on glucose consumption and AKT activation in insulin-resistant H9c2cardiomyocytes. (A) Insulin-resistant H9c2 cardiomyocytes were incubated in the absence or presence of BBR and/orCompound C and/or insulin and glucose consumption was determined as described in Materials and Methods. (B) After 3 hpretreatment of berberine(10 μmol/L) or other reagents in insulin resistant H9c2 cells, 200 μmol/L 2-NBD-glucose in PBS wasadded with or without insulin at 100 nmol/L; and the cells were incubated for an additional 30 min. Glucose uptake wasstopped with 3 washes of ice-cold PBS. The fluorescence intensity of cells was recorded by fluorescent microplate. (C) Insulin-resistant H9c2 cardiomyocytes were incubated in the absence or presence of BBR in the absence or presence of insulin andAMPK (MW: 62 kDa) activation was determined by western blot analysis as described in Materials and Methods. Arepresentative blot is depicted. (D) Insulin-resistant H9c2 cardiomyocytes were incubated in the absence or presence of BBR inthe absence or presence of insulin and AKT (MW: 60 kDa) activation was determined by western blot analysis as described inMaterials andMethods. A representative blot is depicted. Data represent themean ± SE of six experiments. *p < 0.05 comparedwith insulin resistant group absence of insulin, #p < 0.05 compared with insulin resistant group in presence of insulin, $,p < 0.05 compared with insulin resistant group treatment with BBR in absence of insulin, ★, p < 0.05 compared with insulinresistant group treatment with BBR in presence of insulin.

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and improve pressure-load induced left ventricular hyper-trophy [27,32]. We previously showed that BBR improvedcardiac vascular endothelial function [7] and attenuated lossof cardiac function in acute ischemia reperfusion injury [33].The present study demonstrates that BBR not only increasesglucose consumption and glucose uptake in normal insulin-sensitive H9c2 cells but also enhances both in H9c2 cells withinsulin resistance.

Both insulin signaling and AMPK pathways are involvedin glucose uptake and consumption in peripheral tissues.Insulin activates insulin receptor substrate-1 (IRS-1) andincreases the Glucose transporter type 4 (GLUT-4) transloca-tion to membranes by phosphorylation of AKT [34], whileAMPK stimulates GLUT-4 translocation to membranes aswell through activation of AS160 [35]. The effects of BBR onthe insulin-stimulated glucose uptake pathway are varied

and sometimes conflicting which may, in part, be attributedto the variety of cell types and treatment times utilized inthese studies. It was reported that BBR exerted its hypogly-cemic effect in alloxan-induced diabetic C57BL/6 mice viaactivated AKT pathway [9]. In contrast, a number of studiesindicated that BBR improved glucose metabolism in insulinsensitive cells, including HepG2, C2C12, L6, 3T3-L1 cells, in aninsulin independent manner [12,15,36]. BBR stimulatedglucose uptake in L6 myotubes through AMPK activation[12]. BBR treatment resulted in increased AMPK activity in3T3-L1 adipocytes and L6 myotubes, and increased GLUT4translocation in L6 cells in a PI3K-independent manner [37].These findings suggest that BBR displays beneficial effects inthe treatment of diabetes via stimulation of AMPK activity. Inthe present study, BBR only weakly, but not significantlystimulated the phosphorylation of AKT, a key molecule in

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the insulin signaling pathway, but strongly promoted thephosphorylation of AMPK. Moreover, BBR-stimulated glucoseconsumption and glucose uptake were diminished by theAMPK inhibitor Compound C in insulin-resistant H9c2 cells.These data suggest that BBR improves insulin resistance inH9c2 cardiomyocytes at least in part via enhancement ofAMPK activity.

In conclusion, BBR stimulates glucose consumption andglucose uptake in insulin-sensitive and insulin-resistantH9c2 cardiomyocytes. The mechanism underlying improve-ment of BBR on insulin resistance in H9c2 cells is mediated,at least in part, by an increase in AMPK activity. We suggestthat BBR might provide cardioprotection in insulin resis-tance and diabetes.

Authors’ contributions

Conceived and designed the experiments: Wenguang Chang,Ming Zhang, Li Chen, Hongwei DU. Performed the experi-ments: Wenguang Chang, Ming Zhang, Jing Li, Zhaojie Meng,Shengnan Wei. Analyzed the data: Wenguang Chang, MingZhang. Contributed reagents/materials/analysis tools: LiChen, Hongwei Du. Wrote the paper: Grant M. Hatch.

Acknowledgment

This work was supported by the National Natural ScienceFoundation of China (81170745 and 81200598), Jilin Science &Technology Development Plan (2012747), Jilin Province Ad-ministration of Traditional Chinese medicine science andtechnology projects (2010-093), and the Heart and StrokeFoundation of Manitoba. G.M.H. is a Canada Research Chair inMolecular Cardiolipin Metabolism.

Conflict of interest

The authors have declared that no competing interests exist.

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