Life Sciences xxx (2014) xxx–xxx

LFS-14021; No of Pages 8

Contents lists available at ScienceDirect

Life Sciences

j ourna l homepage: www.e lsev ie r .com/ locate / l i fesc ie

Compound K protects pancreatic islet cells against apoptosis throughinhibition of the AMPK/JNK pathway in type 2 diabetic mice and inMIN6 β-cells

Feng Ying Guan a,1, Jian Gu a,1, Wei Li b, Ming Zhang a, Yingshi Ji a, Jing Li a, Li Chen a,⁎, Grant M. Hatch c

a Department of Pharmacology, Key Laboratory of Pathobiology, Ministry of Education, Basic Medicine College, Jilin University, Changchun 130021, Chinab College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130018, Chinac Department of Pharmacology & Therapeutics, University of Manitoba, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada

Abbreviations: T2D, type 2 diabetes; AMPK, AMP-activN-terminal kinase; CK, compound K; HFD, high fat diet;insulin-sensitivity index; TG, triglyceride; TC, total cholestlevel.⁎ Corresponding author at: Department of Pharm

Pathobiology, Ministry of Education, Basic Medicine ColleChina. Tel./fax: +86 431 85619799.

E-mail address: chen_lab@163.com (L. Chen).1 Equal contribution.

http://dx.doi.org/10.1016/j.lfs.2014.04.0340024-3205/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Guan FY, et al, Compin type 2 diabetic mice an..., Life Sci (2014),

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 19 February 2014Accepted 24 April 2014Available online xxxx

Keywords:Compound KAMP-activated protein kinaseC-Jun N-terminal kinaseType 2 diabetesβ-cell apoptosis

Aims: Compound K (CK) is known to possess anti-diabetic activities but the mechanism for this action isunknown. The present study observed the protective effect of CK on islet cell apoptosis through the AMP-activated protein kinase (AMPK) mediated C-Jun N-terminal kinase (JNK) pathway.Main methods: Treatment effect of CK on type 2 diabetic (T2D) mice and palmitate-induced MIN6 β-cells injurywas observed. Fasting plasma glucose, triacylglycerol, total cholesterol, insulin levels and glucose tolerance testwere evaluated. The expression of AMPK and JNK was detected in islet and MIN6 cells.Key findings: CK treatment (30 mg/kg) decreased fasting plasma glucose, triacylglycerol, total cholesterol,elevated plasma insulin levels and improved glucose tolerance in T2D mice. CK treatment attenuated islet cellapoptosis and caspase-3 activity accompanied by a decrease in AMPK and JNK activation. Meanwhile, CKtreatment attenuated the palmitate-induced reduction in MIN6 β-cell viability, apoptosis and caspase-3 activity

and activation of AMPK and JNK. The AMPK activator AICAR attenuated the CK-mediated inhibition of palmitate-induced apoptosis.Significance: These data suggest that CK treatment provides a beneficial anti-diabetic effect inmice with T2D andthis protective effect may be mediated through prevention of β-cell apoptosis via inhibition of the AMPK-JNKpathway.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Type 2 diabetes (T2D) is the most common endocrine disease.T2D patients develop insulin resistance and β-cell failure leading toinadequate insulin secretion, finally presenting as elevated bloodglucose level. Experimental evidence indicates that a decrease in pan-creatic β-cell mass and function is a major contributing factor to thereduction in circulating insulin (Chang-Chen et al., 2008). Destructionof pancreatic β-cells is caused by several metabolic stressors includingendoplasmic reticulum stress (Fonseca et al., 2011) and elevated reac-tive oxygen species (Giacca et al., 2011) which occur in hyperlipidemic

ated protein kinase; JUN, C-JunFBG, fasting blood glucose; ISI,erol; FINS, fasting blood insulin

acology, Key Laboratory ofge, Jilin University, Changchun,

oundKprotects pancreatic islhttp://dx.doi.org/10.1016/j.lfs

conditions. Hence, novel strategies to enhance β-cell survival would beof clear therapeutic benefit for patients with T2D.

AMP-activated protein kinase (AMPK) is a regulator of cellular andsystemic energy homeostasis inmammalian cells, activated bymetabol-ic stressors including hypoxia, low glucose and nutrient deprivation(Ryu et al., 2009). Activation of AMPK in skeletal muscle, liver, andadipose tissue was shown to enhance metabolism and improve insulinsensitivity, and may be favorable for the treatment of diabetes(Dziewulska et al., 2010). However, the beneficial effect of AMPK activa-tion in vivo in diabetes is complicated. For example, AMPK activation inβ-cells reduced glucose-stimulated insulin secretion (Okazaki et al.,2010) and increased β-cell death through apoptosis (Fu et al., 2009;Kefas et al., 2004; Riboulet-Chavey et al., 2008; Santos et al., 2011). Inaddition, AMPK activation induced apoptosis of insulin-producingMIN6 cells through stimulation of c-Jun-N-terminal kinase (JNK)(Kefas et al., 2003). Thus AMPKmight serve as an important pharmaco-logical target for enhancement of β-cell function.

Although several drugs are available for the treatment of diabetesthere are many side effects which limit the effectiveness of existingdrugs. Recent research has examined the role of natural products ordietary interventions in the prevention and treatment of diabetes.

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034

2 F.Y. Guan et al. / Life Sciences xxx (2014) xxx–xxx

Compound K (CK) (also known as M1, IH901) is a terminal metaboliteof protopanaxadiol ginsenosides. CK exhibits various biologicalactivities as an anti-cancer (Kim do et al., 2009a; Lee et al., 2010) andanti-inflammation (Joh et al., 2011) and it has hepatoprotective effects(Lee et al., 2005). In db/db mice CK increased insulin secretion inβ-cells through its action on an ATP-sensitive K+ channel andimproved insulin sensitivity and enhanced plasma adiponectin levels(Han et al., 2007). In addition, CK attenuated hepatic lipid accumulationvia AMPK activation in HepG2 (Kim do et al., 2009b). Previously wedemonstrated that CK down-regulates the key gluconenogenesisenzymes glucose-6-phosphatase and phosphoenolpyruvate carboxy-kinase. Reduction in the activities of these enzymes may serve as oneof the hypoglycemic mechanisms mediated by CK treatment (Li et al.,2012). However, the effect of CK on AMPK activity in β-cell functionand survival remains unclear. In the present study, we examined theprotective effects of CK on pancreatic β-cell function in T2D mice andinMIN6 β-cells following palmitic acid induced cell injury and examinedwhether modulation of AMPK activation via CK was involved in thisprotective effect.

Materials and methods

Materials

Streptozotocin, AMPK activator AICAR and inhibitor Compound Cwere purchased from Sigma; insulin was purchased from Eli Lilly,Changchun, China; glucose, total cholesterol (TC) and triglyceride (TG)test kits were obtained from BHKT Clinical Reagent Co., Ltd, Beijing,China; Iodine [125I] Insulin Radioimmunoassay Kit was purchasedfrom Tianjin Nine Tripods Medical & Bioengineering Co., Ltd, Tianjin,China; and other reagents were purchased from Beijing ChemicalFactory, Beijing, China. Antibodies (AMPK, p-AMPK, JNK, p-JNK, BAX,BCL-2, cytochrome-c, caspase-3, GAPDH) were purchased from SantaCruz, CA, USA. CK used in this study was isolated and purified fromPanax ginseng roots by a series of chromatography procedures in ourlaboratory, and their structures were elucidated by comparison ofspectral data. Its purity was determined to be more than 98.5% byHPLC–UV analysis. The solution of palmitate was prepared by mixingand heating to 90 °C with equal molar amounts of NaOH and palmiticacid to a concentration of 400 mM. It was further diluted with distilledwater and 5% BSA (fatty acid free) to 50 mM, sterilized and stored at4 °C. A suitable amount of palmitate was slowly added to 37 °C culturemedium before use.

Animal models

Male ICR mice (18-22 g) were purchased from the experimentalanimal center of Jilin University (Jilin, China). After one week of regularfeeding, the animals were randomly divided into 2 groups: a controlgroup (CON, N = 12) and a high-fat diet fed group (HFD, N = 50).After 4 weeks of high-fat diet feeding, 100 mg/kg streptozotocindissolved in citrate buffer was injected (i.p.) into HFD animals. Thecontrol animals were injected with citrate buffer alone. 4 weeks afterinjection, fasting blood glucose (FBG) was measured. HFD fed micewith FBG above 7.8 mmol/L were considered T2D and were thenrandomly divided into 2 groups: an untreated group (DM) (n = 12)and a CK-treated group (n = 12). Other mice with FBG less than7.8 mmol/L were sacrificed. CK (30 mg/kg) dissolved in saline wasgiven by gavage. CON and DM were gavaged with saline alone. Micewere treated daily for 4 weeks. All the mice were housed in standardpolypropylene cages (6 mice/cage) and maintained under controlledroom temperature and humidity with 12 h light and dark cycles. Foodintake was recorded daily and body weight was recorded once perweek. At the end of the study, animals were fasted overnight andblood samples obtained from the tailswere collected into EDTA contain-ing tubes and placed on ice. After centrifugation at 3500 ×g for 10 min,

Please cite this article as: Guan FY, et al, CompoundKprotects pancreatic islin type 2 diabetic mice an..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs

plasma was collected and stored at −80 °C. The pancreases wereimmediately separated, collected and stored in liquid nitrogen untilfurther analysis.

Measurement of fasting blood glucose, fasting insulin, triglyceride, totalcholesterol, insulin sensitivity index (ISI) and oral glucose tolerancetest (OGTT)

FBG, TC, and TG in the plasma collected above were measuredaccording to the instructions of the corresponding commercial kits.Fasting insulin was assayed by RIA according to the instructions provid-ed by themanufacturer. The ISI was calculated by the formula [ISI = Ln(FBG × FINS)−1] according to the fasting insulin and glucose concen-tration of each mouse. An OGTT was conducted after an overnight fast(12 h). Mice were gavaged with glucose (2 g/kg BW). Blood sampleswere subsequently obtained at 2 h after gavage and the blood glucosewas measured according to the instructions of the correspondingcommercial kit.

Cell experiments

MIN6 cell line was purchased from XiangYa School of Medicine,Central South University. The cells were cultured in Dulbecco'smodifiedEagle's medium (DMEM 25 mmol/L glucose) equilibrated with 5% CO2

and 95% air at 37 °C. The medium was supplemented with 10% fetalcalf serum, 100 U/ml penicillin sulfate and 50 μg/ml gentamycin. Allexperiments were performed when cells reached 80% confluence. Cellviability was assessed by theMTT assay. Briefly, MIN6 cells were seededin 96 well plates at 1 × 104 cells/well. Cells were incubated with 0–1.0 mM palmitate for 24 h. Cells were then washed twice with coldPBS and then MTT solution was added to the cells at a final concentra-tion of 0.5 mg/ml. After incubating for 4 h at 37 °C, with 5% CO2, thesolution was removed, and 150 μl dimethylsulfoxide was added. Theprecipitate in eachwell was dissolved for 10min and the optical density(OD) was determined at 570 nm using a microplate reader. The cellviability of CK-treated cells was performed as above except cells wereincubated with 2–32 μM CK. The optimum concentration of palmitateand CK used for subsequent experiments was determined after cellviability assays. CK and/or AICAR (1 mM) was added simultaneouslywith palmitate for 24 h. Cells were pre-incubated with 10 μMCompound C for 1 h prior to treatment for 24 h with palmitate. Totalprotein was extracted and probed with antibodies specific for AMPK,pThr172-AMPK, JNK and p-JNK.

Determination of caspase-3 activity

Cell apoptosis was assessed by caspase-3 activity and annexin V/PIstaining. Caspase-3 activitywasmeasured using a Caspase-3 Colorimet-ric Assay kit (Beyotime Institute of Biotechnology, China) accordingto the manufacturer's instructions. Briefly, cells were washed withcold PBS, resuspended in lysis buffer and left on ice for 15 min. Thelysate was centrifuged at 16,000 ×g at 4 °C for 15 min. The productionof p-nitroaniline was measured at 405 nm using a microplate reader.Caspase-3 activity was normalized to the total extracted proteinconcentration.

Apoptosis determination

Quantitative evaluation of apoptosis was performed by flow cytom-etry after double staining with the Annexin V-FITC apoptosis detectionkit (Tianjin Sungene Biotech Co., Ltd. China). Intact (normal) cells(FITC−/PI−), early-stage apoptotic cells (FITC+/PI−), late-stageapoptotic cells (FITC+/PI+) and necrotic cells (FITC−/PI+) werequantified by flow cytometry. In brief, following treatment of MIN6cells under various conditions, cells were harvested and pelleted bycentrifuging at 1000 rpm for 5 min at room temperature. Cells were

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034

3F.Y. Guan et al. / Life Sciences xxx (2014) xxx–xxx

washed twice with cold PBS and resuspended in binding buffer at aconcentration of 1 × 106 cells/ml. Annexin V-FITC (5 μl) and PI (10 μl)were added to 100 μl of cells (1 × 105 cells). The suspension wasmixed gently and incubated in the dark for 15min at room temperature.Binding buffer (400 μl) was added to the suspension and the sampleswere analyzed by fluorescence activated cell sorting (FACS).

Western blot analysis

Lysates of total pancreatic islet or MIN6 cells were prepared withice cold RIPA buffer (50 mM Tris-HCl, PH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with aprotease inhibitor (1 mM PMSF) and phosphatase inhibitors (50 mMNaF/0.1 mM sodium vanadate/10 mM sodium molybdate/20 mM/3-glycerol phosphate/10 mM 4-nitropyrophosphate). Protein wasmeasured using the Bradford assay (Bio-Rad protein assay kit). Totalprotein extracts (80 μg) were separated on 10% SDS-polyacrylamideelectrophoresis gel (SDS-PAGE) and then electroblotted at 4 °C topolyvinylidene difluoride membranes. Membranes were treated with5% (w/v) skim milk or 5% BSA for 2 h at room temperature andthen incubated with primary Bax or Bcl-2 or cytochrome c or cleavedcaspase-3 antibodies (1:800 or 1:1000) with gentle agitation overnightat 4 °C. The membranes were washed 3 times for 10 min with TBST(10 mM Tris–HCl, 150 mM NaCl and 0.1% (v/v) Tween-20) and thenincubated with horse radish peroxidase-conjugated secondary anti-bodies (1:2000) at room temperature for 1 h. The membranes wererewashed 3 times with TBST, as described above. Antigen–antibodycomplexes in all membranes were detected by the chemiluminescenceECL plus kit. An imaging densitometer was used to scan the proteinbands and the densities were quantified using the image analysissoftware. GAPDH was used as loading control.

Statistical analysis

The data are presented as mean ± SE. Statistical significance ofdifferences between groups was analyzed by Student's t test or byone-way analysis of variance (ANOVA) when more than two groupswere compared. All statistics was calculated with software SPSS 13.0(SPSS Inc., USA). The level of significance level was defined as p b 0.05.

Results

CK treatment improves glucose and lipid metabolism in T2D mice

To determine the effect of CK on glucose and lipid metabolicparameters in T2D mice, FBG, fasting blood insulin levels (FINS) andblood lipids were determined and OGTT was performed in control,T2D mice (DM) and T2D mice treated with CK (CK). In addition, theinsulin sensitivity index (ISI) was determined in these animals. Bodyweight was significantly reduced in DM animals compared to control.FBG, TG, TC and 2 h blood glucose were significantly higher in the DManimals compared to controls (Table 1). In contrast, FINS and ISI weresignificantly lower in DM animals compared to controls. These dataindicated that insulin sensitivity was markedly decreased in these T2D

Table 1Body weight, fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), fasting bloodT2D and CK-treated T2D mice.

Groups N Body weight (g) FBG(mmol/L)

FINS(mU/L)

T(

Con 12 38 ± 2.2 4.8 ± 1.0 17.4 ± 1.6 0DM 12 30 ± 2.5* 14.6 ± 1.6** 11.7 ± 2.1** 1CK 12 31 ± 3.2 8.5 ± 1.2# 14.2 ± 2.0# 1

Con, control; DM, diabetic group; and CK, diabetic group treated with CK. Data represent the m

Please cite this article as: Guan FY, et al, CompoundKprotects pancreatic islin type 2 diabetic mice an..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs

mice. Treatment of T2D mice with CK resulted in a significant decreasein FBG, TG, TC and 2 h blood glucose and an increase in FINS comparedto DManimals. The level of FBG, TG, TC, FINS and 2 h blood glucose in CKtreated animals approached the level of those observed in controlanimals. These results indicate that CK may protect the ability of isletβ-cells to secret insulin in T2D mice.

CK inhibits apoptosis in islet tissue of T2D mice

To determinewhether the CK-mediated increase in FINS in T2Dmicewas due to the protective effects of CK on islet tissue survival, the levelof pro- and anti-apoptotic Bcl-2 family proteins Bax and Bcl-2, cleavedcaspase-3 and cytochrome-c was examined in pancreatic islets isolatedfrom control, DM and CK animals. As shown in Fig. 1, the ratio ofBax/Bcl-2 was increased in islets from DM mice compared to control.Treatment with CK significantly reduced the ratio of Bax/Bcl-2 com-pared to DMmice. The level of cytochrome c was increased in the isletsof DM animals compared to control and reduced by the treatment ofthese mice with CK. Cleaved caspase-3 was increased in the islets ofDM mice compared to control and reduced in T2D animals treatedwith CK. These data suggested that CK treatment may attenuateapoptosis of islet tissue in T2D mice.

CK treatment inhibits AMPK and JNK activity in islet tissue of T2D mice.

AMPK is a potential therapeutic target for T2D as activation of AMPKwas shown to enhance β-cell death through apoptosis (Jung and Chung,2011).We investigated whether the anti-apoptotic effects of CK on isletcells in T2D mice were associated with attenuation of the AMPKpathway. The level of total AMPK protein and the phosphorylated(Thr172) form of AMPK (P-AMPK) protein was examined in pancreaticislets isolated from control, DM and CK animals. As shown in Fig. 2a, thetotal AMPK levels were comparable between control, DM and CK mice.P-AMPK was significantly increased in DM mice compared to control.Treatment with CK significantly attenuated P-AMPK levels comparedtoDMmice. These data indicate that CK-treatment attenuates activationof AMPK in T2D mice.

β-cell apoptosis induced by activation of AMPKmay bemediated viathe activation of JNK (Kefas et al., 2003). We examined the level of totalJNK (T-JNK) andphosphorylated JNK (P-JNK) protein in pancreatic isletsof control, DM and CK mice. As shown in Fig. 2b, the level of T-JNK wasunaltered between control, DM and CK-treated mice. The level of P-JNKwas elevated in DM mice compared to control. Treatment with CKsignificantly attenuated P-JNK levels compared to DM mice. Takentogether, the results from these in vivo studies indicated that CK mayattenuate apoptosis of β-cells in pancreatic islets of T2D mice throughinhibition of the activation of the AMPK/JNK pathway.

CK attenuates palmitate-induced cell death in MIN6 β-cells

Previous studies have shown that chronic free fatty acid exposureinduces pancreatic β-cell injury which is analogous to the condition ofthe pancreas in T2D (Kharroubi et al., 2004). To determine if CK wasprotective against fatty acid induced loss in viability of β-cells, MIN6

insulin level (FINS), 2 h blood glucose levels and insulin sensitivity index (ISI) in control,

Gmmol/L)

TC(mmol/L)

ISI 2 hour blood glucose(mmol/L)

.73 ± 0.08 2.43 ± 0.32 −4.4 ± 0.2 9.3 ± 1.5

.53 ± 0.21** 4.36 ± 1.10** −5.1 ± 0.2** 18.8 ± 1.8**

.14 ± 0.20## 3.18 ± 0.44# −4.79 ± 0.1 11.4 ± 2##

ean ± SE. *p b 0.05 vs Con, **p b 0.01 vs Con, #p b 0.05 vs DM, ##p b 0.01 vs DM.

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034

CON DM CK

Bax

Bcl-2

Cytochrome c

CleavedCaspase-3

GAPDH

23 KDa

26 KDa

15 KDa

17 KDa

35 KDa

Rel

ativ

e co

nte

nt

(%o

f co

ntr

ol)

250

200

150

100

50

0

Bax/Bcl-2

Cytochrome c/GAPDH

Cleaved caspase-3/GAPDH

Fig. 1. Expression of Bax, Bcl-2, cytochrome-c, and cleaved caspase-3 in the pancreaticislets of CK-treated mice. Pancreatic islets were isolated from control, DM and CK animalsand the protein levels of Bax, Bcl-2, cytochrome-c, cleaved caspase-3 and GAPDH weredetermined by western blot analysis as described in Materials and methods. The graphindicates Bax/Bcl-2 ratio, cytochrome c/GAPDH ratio and cleaved caspase-3/GAPDH ratiorelative expression compared to control. CON, control; DM, T2D; CK, CK-treated T2D.Data represent the mean ± SE (n = 6). *p b 0.05, vs. CON; #p b 0.05, vs. DM.

Rel

ativ

e co

nte

nt

(%o

f co

ntr

ol)

P-AMPK

T-AMPK

63KDa

63KDa

CON DM CK

200

150

100

50

0

P-JNK

T-JNK

54 KDa

46 KDa

54 KDa

46 KDa

Rel

ativ

e co

nte

nt

(%o

f co

ntr

ol)

250

200

150

100

50

0

CON DM CK

a

b

Fig. 2. Expression of AMPK and JNK in the pancreatic islets of CK-treated mice. Pancreaticislets were isolated from control, DM and CK animals and the levels of AMPK and P-AMPK(a) or levels of JNK andP-JNK (b)were determined bywesternblot analysis as described inMaterials and methods. The graph indicates ratio of p-AMPK/t-AMPK (a) and p-JNK/t-JNK(b) expressed relative to control. CON, control; DM, T2D; CK, CK-treated T2D. Datarepresent the mean ± SE (n = 6). *p b 0.05, vs. CON; #p b 0.05, vs. DM.

4 F.Y. Guan et al. / Life Sciences xxx (2014) xxx–xxx

β-cells were incubated with increasing concentrations of palmitate orCK or both and cell viability was determined by MTT assay. As shownin Fig. 3a, incubation of MIN6 β-cells with palmitate for 24 h reducedviability of these cells in a dose dependent manner. Viability of MIN6β-cells was decreased to 70% with 0.4 mM palmitate. The possiblecytotoxic effect of CK was examined in MIN6 β-cells by incubatingcells with various concentrations (2–32 μM) of CK for 24 h followedby cell viability assay. As shown in Fig. 3b, 32 μMCKwasmildly cytotox-ic to MIN6 β-cells. Thus, concentrations of CK between 2 and 16 μΜwere used to incubate MIN6 β-cells during to exposure to 0.4 mMpalmitate. As seen in Fig. 3c, incubation of MIN6 β-cells with 8–16 μΜCK significantly attenuated the palmitate-induced decrease in MIN6β-cell viability. Thus, CK attenuates palmitate-induced injury in MIN6β-cells.

CK attenuates palmitate-induced apoptosis in MIN6 cells

To determine whether the protective effect of CK on palmitate-induced cell death in MIN6 β-cells was mediated through a reductionin apoptosis, MIN6 β-cells were incubated plus or minus 0.4 mMpalmitate or incubated with 8 μΜ CK and 0.4 mM palmitate andapoptosis determined by flow cytometry analysis as described inMaterials andmethods. As seen in Fig. 4a and b, the average percentageof early-stage and late-stage apoptotic cells in palmitate-incubatedcells was increased compared to control. CK treatment significantlydecreased the average percent of early-stage and late-stage apoptoticcells in palmitate-incubated cells.

MIN6 β-cells were then incubated plus or minus 0.4 mM palmitateor incubatedwith 8 μΜ CK and 0.4mMpalmitate and caspase-3 activitywas determined. Incubation ofMIN6β-cells with palmitate significantlyincreased the activity of caspase-3 compared to control (Fig. 4c). Treat-ment of palmitate-incubated cells with CK attenuated the increase incaspase-3 activity. Thus, CK treatment of MIN6 β-cells attenuatespalmitate-induced apoptosis.

Please cite this article as: Guan FY, et al, CompoundKprotects pancreatic islin type 2 diabetic mice an..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs

CK treatment inhibits AMPK and JNK activity in MIN6 β-cells

To confirm that the protective effect of CK on palmitate-inducedMIN6 β-cell apoptosis was indeed mediated through inhibition of theAMPK/JNK pathway, apoptosis and caspase-3 activity were determinedin MIN6 β-cells incubated with palmitate or with palmitate plusthe AMPK activator (AICAR, 1 mM), or with palmitate plus the AMPKinhibitor (Compound C, 10 μΜ) or with palmitate, AICAR and CK. Asshown in Fig. 4a–c, treatment of palmitate-incubated MIN6 β-cellswith Compound C significantly attenuated palmitate-induced apoptosisand caspase-3 activity compared to palmitate-treated cells and to levelssimilar to that observed with CK treatment alone. In contrast, AICARinduced apoptosis, increased caspase-3 activity and attenuated theprotective effect of CK in MIN6 β-cells.

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034

Palmitate (mM)

0 0.1 0.2 0.4 0.8 1

140a

b

c

120

100

80

60

40

20

0

Cel

l via

bili

ty(%

of

con

tro

l)

CK (µM)

CK (µM)

0 2 4 8 16 32

140

120

100

80

60

40

20

0

Cel

l via

bili

ty(%

of

con

tro

l)

120

100

80

60

40

20

0

Cel

l via

bili

ty(%

of

con

tro

l)

- - 2 4 8 16

PA - + + + + +

Fig. 3. CK attenuates palmitate-induced cell death in MIN6 β-cells. MIN6 cells wereincubated with various concentrations of palmitate (a) or CK (b) or both (c) and cellviability determined by MTT assay as described in Materials and methods. Data representthe mean ± SE (n = 6). *p b 0.05, **p b 0.01 vs. control; #p b 0.05 vs. palmitate-treatedcells.

5F.Y. Guan et al. / Life Sciences xxx (2014) xxx–xxx

The level of T-AMPK and P-AMPK was then examined in MIN6 β-cells incubated with palmitate or with palmitate plus AICAR, or withpalmitate plus Compound C or with palmitate, AICAR and CK asdescribed above. As shown in Fig. 5a, T-AMPK levels were unalteredby all treatments compared to control. Palmitate-incubation of MIN6β-cells increased P-AMPK levels and the presence of either CK orCompound C attenuated the palmitate-induced increase in P-AMPK.Treatment of MIN6 β-cells with AICAR did not alter the palmitate-induced increase in P-AMPK levels and did not attenuate theCK-mediated reduction in P-AMPK in palmitate-incubated cells.

The level of T-JNK and P-JNK was then examined in MIN6 β-cellsincubated with palmitate or with palmitate plus AICAR, or with

Please cite this article as: Guan FY, et al, CompoundKprotects pancreatic islin type 2 diabetic mice an..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs

palmitate plus Compound C or with palmitate, AICAR and CK asdescribed above. T-JNK levels were unaltered by all treatmentscompared to control (Fig. 5b). Palmitate incubation of MIN6 β-cellsincreased P-JNK levels and the presence of either CK or Compound Cattenuated the palmitate-induced increase in P-JNK level. Treatmentof MIN6 β-cells with AICAR increased the palmitate-induced elevationin P-JNK. CK attenuated the AICAR and palmitate-induced elevation inP-JNK. Together, the above results indicate that the inhibitory action ofCK on P-JNK in MIN6 β-cells is mediated, at least in part by inhibitionof AMPK. Thus, CK inhibits palmitate induced-apoptosis of MIN6β-cells through inactivation of the AMPK mediated JNK pathway.

Discussion

CK is a final metabolite of protopanaxadiol ginsenosides and hasvarious biological activities including anti-cancer (Kim do et al.,2009a; Lee et al., 2010), anti-inflammatory (Joh et al., 2011) andhepatoprotective effects (Lee et al., 2005). CK was shown to improveinsulin sensitivity by enhancing insulin secretion by acting on a ATP-sensitive K+ channel (Han et al., 2007). We previously demonstratedthat a HFD combined with streptozotocin injection could induce T2Din mice. This experimental model exhibited several metabolic disordersincluding impaired insulin secretary capacity of pancreatic β-cells,glucose intolerance and hyperglycemia and resembled the charac-teristics of human T2D. We have utilized this in vivo model toexamine the hypoglycemic effects of CK. We previously observedthat CK down-regulated the key gluconenogenesis enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, and that thiscould serve, in part, as one of the hypoglycemic mechanisms of CK(Li et al., 2012). Since accumulating evidence indicated that a decreasein pancreatic β-cell mass and function is a major contributing factor tothe pathogenesis of diabetes, in the present study we examined whatrole, if any, CK plays in protection of pancreatic β-cell mass and functionin vivo and in vitro.

Our results clearly indicate that treatment of T2D mice with CKresulted in significant reduction in FBG, plasma TG and TC levels andimproved insulin sensitivity as evidenced by an improved OGTT. Thisresult is consistent with our previous study and that observed in db/dbmice (Li et al., 2012; Yoon et al., 2007). Our T2D mice exhibited anenhanced mitochondrial death signaling pathway which includedelevated cytochrome C, pro-apoptotic protein Bax, executor caspase-3and reduced anti-apoptotic protein Bcl-2 in pancreatic islets. Theplasma insulin levels were significantly increased in our T2D miceafter CK treatment. In addition, CK treatment decreased the ratio ofBax to Bcl-2 and attenuated the increase in caspase-3 and cytochromeC and this attenuated pancreatic islet cell apoptosis. The anti-apoptosis effect of CK was confirmed in MIN6 β-cells incubated withpalmitate. Treatment of these cells with CK inhibited palmitate-induced apoptosis and the activity of caspase-3. These in vivo andin vitro studies clearly indicate that CK treatment has beneficial effectsin the protection of pancreatic β-cells from apoptosis, a key process inthe pathogenesis of T2D.

AMPK acts as an integrator of regulatory signalsmonitoring systemicand cellular energy status and is considered a novel therapeutic targetfor the treatment of T2D. Activation of AMPK in skeletal muscle, liver,and adipose tissue enhances metabolism, improves insulin sensitivity,and is favorable for the treatment of diabetes (Dziewulska et al.,2010). However, accumulating evidence indicates that AMPK activationin β-cells exerts detrimental effects in vitro and in vivo. For example,AMPK activation was responsible for an inhibition of insulin release incell lines derived from pancreatic β-cells (Salt et al., 1998). Activationof AMPK resulted in apoptosis in primary β-cells (Kefas et al., 2003).In addition, overexpression of AMPK impaired β-cell function in vivo(Kim et al., 2007). In contrast, inhibition of AMPK protected β-cellsfrom cytokine-induced apoptosis in MIN6 β-cells (Riboulet-Chaveyet al., 2008). In the present study CK treatment significantly inhibited

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034

PA - + + + + +

CK - - + + - -

AICAR - - - + + -

Compound c - - - - - +

Ap

op

toti

c ce

lls (

%)

100

80

60

40

20

0

a

b

PA - + + + + +

CK - - + + - -

AICAR - - - + + -

Compound c - - - - - +

Cas

pas

e-3

acti

vity

(%o

f co

ntr

ol)

3

2

1

0

c

0.48 34.22

43.53

0.1

64.04 14.26

1.09 36.41

52.93

1.74 25.49

24.53

21.77 9.57

48.24

21.600.85 52.16

16.79 30.20

89.93 2.15

4.510.27

CON PA PA+CK

PA+AICAR+CK PA+AICAR PA+Compound C

Fig. 4. CK protectsMIN6 cells from palmitate-induced apoptosis. MIN6 cells were incubated plus orminus 0.4mMpalmitate for 24 h or with 8 μΜ CK and palmitate for 24 h orwith AICAR(1mM)and palmitate for 24 h orwith 10 μMCompound C for 1 h followed by palmitate for 24 orwith CK, AICAR andpalmitate for 24 h and the percent apoptotic cellswere determinedbyFACS analysis (a and b) and caspase-3 activities (c)were determined as described inMaterials andmethods. Caspase-3 activity is expressed relative to control. Data represent themean±SE (n = 6). *p b 0.05, vs. control; #p b 0.05, vs. palmitate-treated; $p b 0.05 vs. co-treatment with palmitate and CK.

6 F.Y. Guan et al. / Life Sciences xxx (2014) xxx–xxx

AMPK activity in T2D mice and in MIN6 β-cells treated with palmitate.The AMPK activator AICAR attenuated the protective effect of CK againstpalmitate induced-apoptosis in MIN6 β-cells. These results indicatedthat CK may prevent islet β-cell dysfunction through inhibition ofAMPK activation.

AMPK is a kinase that transmits extracellular signals leading toJNK activation. JNK activation contributes to induction of apoptosisin pancreatic β-cells under adverse environmental conditions. Forexample, cytokines (IL-1β, tumor necrosis factor-α, and interferon-γ)produced β-cell apoptosis through activation of JNK (Kim et al., 2005;

Please cite this article as: Guan FY, et al, CompoundKprotects pancreatic islin type 2 diabetic mice an..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs

Maedler et al., 2008). Palmitate or oleate incubation of pancreatic NIT-1 cells resulted in JNK activation, reduction in insulin and ultimatelydysfunction and apoptosis of these cells (Yuan et al., 2010). Selectiveinhibition of the JNK pathway reduced β-cell apoptosis elicited by IL-1β (Ferdaoussi et al., 2008). Inhibition of JNK protected β-cells fromthe deleterious effects of high glucose in diabetes (Maedler et al.,2008). In the present study, JNK activation was increased in the pancre-atic islets of T2D mice and in palmitate-treated MIN6 β-cells andthis activation was markedly inhibited by CK treatment. To furtherdetermine whether the reduction in JNK activity mediated by CK was

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034

PA - + + + + +CK - - + + - -

AICAR - - - + + -

Compound c - - - - - +

P-AMPK

T-AMPK

Rel

ativ

e co

nte

nt

(%o

f co

ntr

ol)

250

200

150

100

50

0

63KDa

63KDa

P-JNK

T-JNK

54 KDa

46 KDa

54 KDa

46 KDa

PA - + + + + +CK - - + + - -

AICAR - - - + + -

Compound c - - - - - +

Rel

ativ

e co

nte

nt

(%o

f co

ntr

ol)

800

600

400

200

0

a

b

Fig. 5. CK inhibits palmitate induced-apoptosis in MIN6 cells through inactivation ofthe AMPK mediated JNK pathway. a. MIN6 cells were incubated as indicated in Fig. 4and T-AMPK and P-AMPK were determined as described in Materials and methods.The ratio of the P-AMPK/T-AMPK was expressed relative to control. Data represent themean ± SE (n = 6). *p b 0.05, vs. control; #p b 0.05, vs. palmitate-treated. b. MIN6 cellswere incubated as indicated in Fig. 4 and T-JNK and P-JNK were determined as describedinMaterials andmethods. The ratio of P-JNK/T-JNKwas expressed relative to control. Datarepresent the mean ± SE (n= 6). *p b 0.05, vs. control; #p b 0.05, vs. palmitate-treated;$p b 0.05 vs. co-treatment with palmitate and CK.

7F.Y. Guan et al. / Life Sciences xxx (2014) xxx–xxx

due to inhibition of AMPK activation, the AMPK activator (AICAR) orinhibitor (Compound C) was added to palmitate treated-MIN6 β-cellsin the presence of CK. Compound C or CK inhibited JNK activation,while AICAR attenuated the inhibitory effect of CK on JNK activation.These studies indicated that CK induced inhibition of AMPK activityand this resulted in reduced JNK activation.

Conclusion

Our results provide new insight into the mechanism of the protec-tive effect of CK against apoptosis in T2D mice in vivo and palmitate-treated MIN6 β-cells in vitro through the demonstration that CK treat-ment inhibits apoptosis and themitochondrial death signaling pathwayand this is accompanied by a decrease in activation of AMPK and JNK.Thus CK may prevent apoptosis of β-cells and prevent β-cell dysfunc-tion through inhibition of the AMPK/JNK pathway. These data and ourprevious conclusion that CK treatment may be useful in the treatmentof T2D (Li et al., 2012) certificated CK has both insulin sensitizationand β-cell conservation.

Please cite this article as: Guan FY, et al, CompoundKprotects pancreatic islin type 2 diabetic mice an..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs

Conflict of interest statement

There are no any competing interests.

Acknowledgments

The research in this study was supported by a grant from theJilin Provincial Science & Technology Department (2011Z060,20140414052GH), the Opening Project of State Key Laboratory ofSupramolecular Structure and Materials of Jilin University under GrantNo. SKLSSM201317, and in part by funding from the Heart and StrokeFoundation 000510 (GM Hatch), the Canadian Institutes of HealthResearch MOP-106428 (GM Hatch), and the Canada Research Chair inMolecular Cardiolipin Metabolism grant (GM Hatch).

References

Chang-Chen KJ, Mullur R, Bernal-Mizrachi E. β-cell failure as a complication of diabetes.Rev Endocr Metab Disord 2008;9:329–43.

Dziewulska A, Dobrzyn P, Dobrzyn A. The role of AMP-activated protein kinase in regula-tion of skeletal muscle metabolism. Postepy Hig Med Dosw (Online) 2010;64:513–21.

Ferdaoussi M, Abdelli S, Yang JY, Cornu M, Niederhauser G, Favre D, et al. Exendin-4 pro-tects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway. Diabetes 2008;57:1205–15.

Fonseca SG, Gromada J, Urano F. Endoplasmic reticulum stress and pancreatic beta-celldeath. Trends Endocrinol Metab 2011;22:266–74.

Fu A, Ng AC, Depatie C, Wijesekara N, He Y, Wang GS, et al. Loss of Lkb1 in adult beta cellsincreases beta cell mass and enhances glucose tolerance inmice. Cell Metab 2009;10:285–95.

Giacca A, Xiao C, Oprescu AI, Carpentier AC, Lewis GF. Lipid-induced pancreatic beta-celldysfunction: focus on in vivo studies. Am J Physiol Endocrinol Metab 2011;300:E255–62.

Han GC, Ko SK, Sung JH, Chung SH. Compound K enhances insulin secretion withbeneficial metabolic effects in db/db mice. J Agric Food Chem 2007;55:10641–8.

Joh EH, Lee IA, Jung IH, Kim DH. Ginsenoside Rb1 and its metabolite compound K inhibitIRAK-1 activation—the key step of inflammation. Biochem Pharmacol 2011;82:278–86.

Jung MS, Chung SH. AMP-activated protein kinase: a potential target for ginsenosides?Arch Pharm Res 2011;34:1037–40.

Kefas BA, Cai Y, Ling Z, Heimberg H, Hue L, Pipeleers D, et al. AMP-activated protein kinasecan induce apoptosis of insulin-producingMIN6 cells through stimulation of c-Jun-N-terminal kinase. J Mol Endocrinol 2003;30:151–61.

Kefas BA, Cai Y, Kerckhofs K, Ling Z, Martens G, Heimberg H, et al. Metformin-inducedstimulation of AMP-activated protein kinase in β-cells impairs their glucoseresponsiveness and can lead to apoptosis. Biochem Pharmacol 2004;68:409–16.

Kharroubi I, Ladriere L, Cardozo AK, Dogusan Z, Cnop M, Eizirik DL. Free fatty acids andcytokines induce pancreatic beta-cell apoptosis by different mechanisms: role ofnuclear factor-kappaB and endoplasmic reticulum stress. Endocrinology 2004;145:5087–96.

Kim DY, Park MW, Yuan HD, Lee HJ, Kim SH, Chung SH. Compound K induces apoptosisvia CAMK-IV/AMPK pathways in HT-29 colon cancer cells. J Agric Food Chem2009a;57:10573–8.

KimDY, Yuan HD, Chung IK, Chung SH. Compound K, intestinal metabolite of ginsenoside,attenuates hepatic lipid accumulation via AMPK activation in human hepatoma cells.J Agric Food Chem 2009b;57:1532–7.

Kim WH, Lee JW, Gao B, Jung MH. Synergistic activation of JNK/SAPK induced by TNF-αand IFN-γ: apoptosis of pancreatic β-cells via the p53 and ROS pathway. Cell Signal2005;17:1516–32.

Kim WH, Lee JW, Suh YH, Lee HJ, Lee SH, Oh YK, et al. AICAR potentiates ROS productioninduced by chronic high glucose: roles of AMPK in pancreatic beta-cell apoptosis. CellSignal 2007;19:791–805.

Lee HU, Bae EA, Han MJ, Kim NJ, Kim DH. Hepatoprotective effect of ginsenoside Rb1 andcompound K on tert-butyl hydroperoxide-induced liver injury. Liver Int. 2005;25:1069–73.

Lee IK, Kang KA, Lim CM, Kim KC, KimHS, KimDH, et al. Compound K, a metabolite of gin-seng saponin, induces mitochondria-dependent and caspase-dependent apoptosisvia the generation of reactive oxygen species in human colon cancer cells. Int J MolSci 2010;11:4916–31.

Li W, Zhang M, Gu J, Meng ZJ, Zhao LC, Zheng YN, et al. Hypoglycemic effect ofprotopanaxadiol-type ginsenosides and compound K on Type 2 diabetes mice in-duced by high-fat diet combining with streptozotocin via suppression of hepaticgluconeogenesis. Fitoterapia 2012;83:192–8.

Maedler K, Schulthess FT, Bielman C, Berney T, Bonny C, Prentki M, et al. Glucose andleptin induce apoptosis in human beta-cells and impair glucose-stimulated insulinsecretion through activation of c-Jun N-terminal kinases. FASEB J. 2008;22:1905–13.

Okazaki Y, Eto K, Yamashita T, Okamoto M, Ohsugi M, Noda M, et al. Decreased insulinsecretion and accumulation of triglyceride in beta cells overexpressing a dominant-negative form of AMP-activated protein kinase. Endocr J 2010;57:141–52.

Riboulet-Chavey A, Diraison F, Siew LK, Wong FS, Rutter GA. Inhibition of AMP-activatedprotein kinase protects pancreatic beta-cells from cytokine-mediated apoptosis andCD8+ T-cell-induced cytotoxicity. Diabetes 2008;57:415–23.

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034

8 F.Y. Guan et al. / Life Sciences xxx (2014) xxx–xxx

Ryu GR, Lee MK, Lee E, Ko SH, Ahn YB, Kim JW, et al. Activation of AMP-activated proteinkinase mediates acute and severe hypoxic injury to pancreatic beta cells. BiochemBiophys Res Commun 2009;386:356–62.

Salt IP, Johnson G, Ashcroft SJ, Hardie DG. AMP-activated protein kinase is activated bylow glucose in cell lines derived from pancreatic beta cells, and may regulate insulinrelease. Biochem J 1998;335(Pt 3):533–9.

Santos GJ, Oliveira CA, Boschero AC, Rezende LF. CNTF protects MIN6 cells against apopto-sis induced by Alloxan and IL-1beta through downregulation of the AMPK pathway.Cell Signal 2011;23:1669–76.

Please cite this article as: Guan FY, et al, CompoundKprotects pancreatic islin type 2 diabetic mice an..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs

Yoon SH, Han EJ, Sung JH, Chung SH. Anti-diabetic effects of compound K versus metfor-min versus compound K-metformin combination therapy in diabetic db/dbmice. BiolPharm Bull 2007;30:2196–200.

Yuan H, Zhang X, Huang X, Lu Y, TangW, Man Y, et al. NADPH oxidase 2-derived reactiveoxygen species mediate FFAs-induced dysfunction and apoptosis of beta-cells viaJNK, p38 MAPK and p53 pathways. PLoS One 2010;5:e15726.

et cells against apoptosis through inhibition of the AMPK/JNKpathway.2014.04.034