Cellular Signalling 25 (2013) 2348–2361

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Cellular Signalling

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In vivo activating transcription factor 3 silencing ameliorates the AMPKcompensatory effects for ER stress-mediated β-cell dysfunction duringthe progression of type-2 diabetes

Ji Yeon Kim a, Keun Jae Park a, Gyu Hee Kim a, Eun Ae Jeong a, Dae Yeon Lee a, Seong Su Lee b, Dae Jin Kim c,Gu Seob Roh d, Jihyun Song a, Sung Hwan Ki e, Won-Ho Kim a,⁎a Division of Metabolic Disease, Center for Biomedical Science, National Institutes of Health, #187 Osong Saengmyeong2-ro, Osong-eup, Cheongwon-gun, Chungbuk, Republic of Koreab Department of Endocrinology, College of Medicine, Catholic University, Republic of Koreac Department of Psychiatry, College of Medicine, Catholic University, Republic of Koread College of Medicine, Kyeongsang National University, Jinju, Republic of Koreae College of Pharmacy, Chosun University, Gwangju, Republic of Korea

⁎ Corresponding author at: Division ofMetabolic DiseasesNational Institutes of Health, #187 Osong Saengmyeong2-rChungbuk, 363–951, Republic of Korea. Tel.: +82 43 719 8

E-mail address: jhkwh@nih.go.kr (W.-H. Kim).

0898-6568/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.cellsig.2013.07.028

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 May 2013Received in revised form 15 July 2013Accepted 29 July 2013Available online 2 August 2013

Keywords:Type 2 diabetesPancreatic β-cellsER stressAMPKATF3In vivo knockdown

In obese Zucker diabetic fatty (ZDF) rats, ER stress is associated with insulin resistance and pancreatic β-celldysfunction; however the exact mechanisms by which ER stress drives type-2 diabetes remain uncertain.Here, we investigated the role of ATF3 on the preventive regulation of AMPK against ER stress-mediatedβ-cell dysfunction during the end-stage progression of hyperglycemia in ZDF rats. The impaired glucosemetabolism and β-cell dysfunction were significantly increased in late-diabetic phase 19-week-old ZDFrats. Although AMPK phosphorylation reduced in 6- and 12-week-old ZDF rats was remarkably increasedat 19 weeks, the increases of lipogenice genes, ATF3, and ER stress or ROS-mediated β-cell dysfunction were stillremained, which were attenuated by in vivo-injection of chemical chaperon tauroursodeoxycholate (TUDCA),chronic AICAR, or antioxidants. ATF3 did not directly affect AMPK phosphorylation, but counteracts the preventiveeffects of AMPK for high glucose-induced β-cell dysfunction. Moreover, knockdown of ATF3 by delivery ofin vivo-jetPEI ATF3 siRNA attenuated ER stress-mediated β-cell dysfunction and enhanced the beneficial effectof AICAR. Our data suggest that ATF3 may play as a counteracting regulator of AMPK and thus promoteβ-cell dysfunction and the development of type-2 diabetes and could be a potential therapeutic targetin treating type-2 diabetes.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Type-2 diabetes (T2D) is one of the most prevalent metabolicdisorders associated with abnormal lipid and glucose metabolismand it is a principal source of morbidity and mortality worldwide[1,2]. Obesity is a major underlying pathology associated with thedevelopment of T2D, which occurs as pancreatic β-cells fail to compen-sate for increased insulin demand when the body becomes insulinresistant and hyperglycemia [3]. Several studies have shown thatprolonged exposure to fatty acids and high glucose levels, as well aslipid accumulation at sites other than adipose tissue, contribute to insulinresistance and β-cell dysfunction [4,5]. Specifically, the aforementionedlipid accumulation is caused by insulin secretory defects, as well as a

, Center for Biomedical Sciences,o, Osong-eup, Cheongwon-gun,691; fax: +82 43 719 9602.

ghts reserved.

loss ofβ-cell mass due to apoptosis [6]. Recently, the inhibition of pancre-atic β-cell function that occurs in obese ZDF rats, a commonly used T2Danimal model [7], was associated with impaired islet lipid homeostasisand glucolipotoxicity, which leads to oxidative stress, ER stress, inflam-mation, and β-cell apoptosis [8]; however, the underlying mechanismsthat lead to β-cell dysfunction are still unclear.

The endoplasmic reticulum (ER) is a specialized cellular organellethat is responsible for the synthesis, packaging, and assembly ofsecretory and membrane proteins [9]. Because the pancreatic β-cell isthe only source of circulating insulin, which is essential for bothstimulating peripheral tissue glucose uptake and inhibiting hepaticglucose production, insulin-producing β-cells are very susceptibleto changes in ER homeostasis and therefore ER stress, which can resultin the accumulation of unfolded and/or aggregated proteins and theactivation of the ER stress sensors PERK, IRE1, and ATF6 [10]. Severeor prolonged ER stress promotes lipid accumulation and ROS production,which subsequently lead to β-cell apoptosis, and consequently, diabetesmellitus [11], which were supported by several studies using geneticintervention or chemical chaperones [12,13]. Although cells elicit anormal insulin secretory response to glucose when the levels are

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within a homeostatic physiological range, chronically elevated glucoseleads to ER stress by the activation of ER stress sensors that alter theexpression of downstream gene and increase ROS production frommultiple sources, which ultimately leads to β-cell dysfunction andapoptosis [14,15]. However, the upstream or downstream mediatorsof the ER stress response that promotes fulminant damage to pancreaticβ-cells in obese diabetic animal models are still unclear.

5′-AMP-activated protein kinase (AMPK), an energy sensor that issensitive to changes in the AMP/ATP ratio, is considered to be animportant regulator of glucose and lipid metabolism in peripheraltissues of humans and rodentswithmetabolic stress, obesity or diabetes[16]. AMPK activity is generally inhibited as glucose concentrations riseabove the physiological range in various cells, which is especially true inislet β-cells [17]. AMPK activation by AICAR inhibits glucose-inducedinsulin release and impaired glucose or lipid homeostasis in pancreaticβ-cells [18,19]. However, these ideas have been recently challenged, assome studies have shown that the sustained activation of AMPK isassociated with the induction of β-cell apoptosis [20,21]. Despitethese discrepancies, an increasing amount of evidence has indicatedthat AMPK activation is inhibitory to the development of obesity andT2D by suppressing hyperglycemia and thus improving β-cell function[22]. In multiple studies using ZDF rats, which are characterized byprogressive β-cell dysfunction, AMPK activation by AICAR preventedthe development of hyperglycemia and preserved β-cell mass [23].Although AMPK has been extensively studied as a potential targetfor the treatment of hyperglycemia, as well as for the pathogenesisof T2D and obesity [24–26], the role and regulatory mechanism ofAMPK hyper-activation observed in hyperglycemia-mediated fulminantdamage to pancreatic β-cells, especially on the post-diabetic phase, arestill unknown.

Activating transcription factor 3 (ATF3), a stress-inducible genethat encodes a member of the ATF/cAMP response element-binding(CREB) family of transcription factors, is induced by signals relevantto the pancreaticβ-cell dysfunction such as proinflammatory cytokines,nitric oxide, and high concentrations of glucose and free fatty acid [27].Previously, we demonstrated that ATF3 plays a critical role in β-celldysfunction and apoptosis through glucokinase (GCK) nitration anddownregulation, which could be triggered by enhancing peroxynitrite,iNOS and NO generation [28]. In addition, we reported that ATF3,which consequently induced by ER stress-mediated JNK activation,was involved in the reduction of pancreatic and duodenal homeobox-1 (PDX-1) as well as adiponectin transcription [29,30]. However, thespecific role of ATF3 and associated regulatorymechanism in pancreaticβ-cell dysfunction and apoptosis during the development of T2Dremain elusive. Therefore, it is necessary to identify how AMPKcontributes to impaired glucose metabolism as well as its role inthe ER stress-mediated fulminant damage to β-cells during theprogressive development of obesity and T2D. In our study, wefound that ER stress is a main pathway that promotes the progressionof obese T2D in ZDF rats. Furthermore, AMPK activation is greatlyincreased in end-stage diabetes to serve a protective role againsthyperglycemia-induced ER or oxidative stress. However, these compen-satory roles in restoring and maintaining cellular homeostasis wereoverwhelmed by excessive increases of ER stress or ROS production,thereby resulting in β-cell dysfunction and apoptosis. Finally, we cansuggest that ATF3may play as a counteractionmolecule for the beneficialeffect of AMPK and thus triggers β-cell dysfunction and the developmentof T2D.

2. Materials and methods

2.1. Cell line and animals

Rat INS-1 pancreatic β-cells were cultured in PRMI 1640 containing11.1 mM glucose supplemented with 10% fetal bovine serum, 2 mMglutamine, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol,

10 mM HEPES, and 100 IU/mL penicillin, and 100 μg/mL streptomycin(Life Technologies, Gaithersburg, MD). Male Zucker diabetic fatty(ZDF/Gmi-fa/fa) rats(6, 12, 19 week-old) and their sex- and age-matchedZucker lean control(ZLC/Gmi-+/fa) rats were purchased from GeneticModels(GMI, Indianapolis, IA). Animals were maintained on a commer-cial chow diet ad libitum before being killed. Body weights and bloodglucose levels for the 6-, 12-, and 19-week-old ZDF rats were prediabetic,diabetic, and postdiabetic stage-appropriate, respectively. All animalexperiments were conducted in accordance with guidelines from theKorean National Institutes of Health Animal Facility.

2.2. Glucose and insulin tolerance tests

For GTTs, animals fasted overnight (11 h) were injected intra-peritoneally with 1.5 g glucose/kg, plasma glucose levels weremeasured at 0, 30, 60, 90, and 120 min with glucometer DEX(Bayer). For ITTs, animals fasted for 5 h were injected intraperitoneallywith 2 IU insulin/kg body wt (Novo Nordisk Pharma.) and blood glucoselevels were measured.

2.3. Determination of insulin contents and ATP, and nitrite

Insulin levels in INS-1 cells and islets were determined by radio-immunoassay using mouse insulin as standard. ATP levels were deter-mined using a luminometric assay kit from Promega as describedpreviously [20]. And also, nitrite was determined by the Griess methodas described previously [28].

2.4. AMPK activity assay

Total AMPK was immunoprecipitated from 500 μg of protein usingan antibody against AMPKα, and AMPK activity was assessed by deter-mining the incorporation of 32P into the synthetic SAMS peptide assayas described [20].

2.5. Transient Transfection

The INS-1 cells, 60% to 70% confluence,were transfectedwith pCDNA-empty, pCDNA-AMPK-WT, pCDNA-AMPK-K45R (a generous gift fromDr. M. Birnbaum) as previously described [31], ATF3 (a generousgift from Dr. T Hai), and siRNAs for ATF3 (sc-72029) and AMPK(sc-270142)(Santa CruZ, CA) using a lipofectin reagent (GIBCO,Gaithersburg, MD) following the protocol recommended by the manu-facturer. Briefly, after a 16-h incubation period, the media was replacedwith normal growthmedia and cells were grown for an additional 24 h.

2.6. Immunohistochemistry and Immunocyto-chemistry

After liver and pancreas tissues were fixed, deparaffinized, andwashed, sections were treated with diluted blocking serum for 20 min.Slides were incubated overnight at 4°C in a humidified chamber withspecific antibodies diluted in blocking serum. Following all procedureswere performed as described previously [28].

2.7. Measurement of carnitinepalmitoyltrans-ferase-1 (CPT-1) activity

CPT-1 activity was measured in mitochondria of islet cellsisolated from ZL and ZDF rats by monitoring the formation ofthe palmitoyl-ester of L-[methyl-3H]-carnitine (specific activity 80.0 Ci/mmol), as previously described [32]. Mitochondrial fractionization wasperformed as described previously [33].

2.8. Oxygen consumption

Cellular oxygen consumption in islet cells (50–80 cells/well)and INS-1 cells (2 × 104 cells/well) was measured using a Seahorse

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bioscience XF24 analyzer (Billerica, MA) in 24 well plates at 37°C asdescribed previously [34]. The detailed procedures can be found inSupplementary Material and Methods.

2.9. Administration of drugs

Male ZDF and ZL rats aging at 15 weeks were used since the rats areconsidered as the phase of progressive diabetes with a compensatorymechanism before entering to the late-stage diabetic characteristicsalong with insulin deficiency as well as hyperglycemia [35]. ForTUDCA injection, male ZDF and ZL rats (8 weeks or 15 weeks) wereadministrated intraperitoneally with TUDCA twice daily (0.25 g/kgin PBS for 8 am and 8 pm) for 7 days (Controls, 100 μl PBS) andsacrificed at 12 weeks or 19 weeks, respectively. For insulin injection,15-week rats were administrated with insulin (25 U/kg/day, s.c.) for14 days. For AICAR treatment, the rats were received a single (acute)or daily for 14 days (chronic) subcutaneous injection of AICAR(500 mg/kg; Sigma) dissolved in 0.9% NaCl or saline as describedpreviously [36]. Also, the rats were received 130 mg/kg/twice a day ofNAC (Sigma) and 0.5 mg/kg/twice a day of L-NMMA dissolved in 0.9%saline and distilled water, respectively, subcutaneously for 7 days. Afterfinal injection, at 9 am, blood samples were collected from the tail veinformeasurement of circulating insulin and glucose levels for the indicatedtimes [37].

2.10. ATF3 gene silencing via siRNA delivery in vivo

Chemically, synthetic siRNA against ATF3 was synthesized byDharmacon RNA Technology. A commercially available cationicpolymer transfection reagent (in vivo-jetPEITM: PolyPlus-Transfection,Illkirch, France) was used to deliver siRNA via intravenous (iv) injection[38,39]. Briefly, 150 μg of siRNA was diluted in 200 μL of 5% glucosesolution was mixed with in vivo-jetPEI solution including transfectionreagent jetPEI. The mixture was incubated for 15 min at room tempera-ture to allow the complexes to form. This mixture was then injectedinto mice once to six times with a three day interval between injections.The scrambled siRNA solution mixed with in vivo-jetPEI solution servedas a control siRNA. Three day interval injection of siRNA was foundto be an optimum to sustain the effects of siRNA via preliminaryexperiments. The in vivo delivery efficiency of siRNA was assessedby performing RT-PCR and western blotting in the removed tissues,lung, liver, or pancreas, and isolated islet cells.

2.11. Statistical analysis

For comparing values obtained in three or more groups, one-factoranalysis of variance was used, followed by Turkey's post hoc test, andp b 0.05 was taken to imply statistical significance.

3. Results

3.1. Pancreatic β-cell dysfunction and apoptosis are correlated with AMPKactivation and lipid accumulation in ZDF rats

We have firstly investigated glucose homeostasis at 6, 12, and19-week-old ZDF rats (Fig. S1). Plasma glucose levels were potentlyincreased with a time in ZDF rats (S1A). Secreted insulin levels peakedat 6 and 12 weeks in the ZDF rats were significantly decreased at19-week (S1B), accompanied by the progressive development ofhypoinsulinemia (not shown) and impaired glucose metabolism leadingto overt diabetes (S1C). Compared with ZL rats, the islets of 6- and 12-week-old ZDF rats were slightly enlarged (S1D, lower right), haddisarrayed islet architecture and had irregular islet boundaries thatbecame worse with age (Fig. 1A), but they were expressed highlevels of insulin similar to ZL rats. In contrast, there was a remarkableloss of insulin expression in the 19-week-old ZDF rats, which exhibited

a reduction in BrdU-positive cells (Fig. 1A, bottom), increase in caspase-3 cleavage and Bax expression, the striking loss of islet cell mass andsize (S1E). In accordance, glucose metabolism-related proteins GCKandGlut2were significantly decreased in 19-week-old ZDF rats, where-as they were slightly increased at 6 weeks (Fig. 1B). AMPK phosphory-lation is significantly reduced in the liver, heart, and muscle of obeseZDF rats and ob/ob mice [40]; therefore, its activation may provide abeneficial effect with regard to the treatment of T2D and obesity. Like-wise, the phosphorylation of AMPK at Thr-172 and its downstreamtarget ACC at Ser-79 were significantly decreased in islet cells from 6-and 12-week-old ZDF rats compared with ZL rats, but their phosphory-lation were specifically increased in 19-week-old ZDF (Fig. 1C). AMPKis known to inhibit key enzymes, such as FAS, ACC, and SREBP1c, thatare involved in lipid and cholesterol synthesis in pancreatic β-cellsof diabetic animals, as well as in the fat, liver, and muscle tissues[17,18]. Expectedly, expression of SREBP1c and its target genes, FASand stearoyl-CoA desaturase-1 (SCD-1), were increased in 6- and 12-week-old ZDF rats (Fig. 1D and S2A). However, despite a reversibleincrease of pAMPK and pACC in 19-week-old ZDF rats, the increase ofSREBP1c, FAS, and SCD1 expression were not conversely decreasedand even further increased. Conversely, the expression and enzymaticactivity of carnitine palmitoyltransferase-1 (CPT-1), which acceleratesfatty acid oxidation, were significantly decreased at 6 weeks andremained low until 19 weeks (Fig. 1D–E).

3.2. ER stress inhibition by TUDCA attenuated β-cell dysfunction andapoptosis in 19-week-old ZDF rats

Obesity induces ER stress through the activation of unfoldedprotein response and in turn plays a central role in the develop-ment of insulin resistance and diabetes via the inhibition of insulinreceptor signaling [39]. To examine the role of ER stress on AMPKactivation and β-cell dysfunction increased in 19-week-old ZDFrats with hyperglycemia and hypoinsulinemia, TUDCA, a chemicalchaperone that reduces ER stress [37], was administrated i.p. for7 days to 8- or 15-week-old ZDF rats. Unexpectedly, AMPK andACC phosphorylation elevated in islet cells of 19-week-old ZDFrats was not inhibited by TUDCA, but they reversely further increased.In contrast, the inhibition of CHOP by TUDCA attenuated the increaseof mature SREBP1c, FAS, and cleaved caspase-3 expression (Fig. 2Aand B) as well as the increase of pancreas and serum triglyceride(Fig. 2C). It was well known that AMPK activation depends on cellularATP contents and the ATP/ADP ratio, which reflects the ability ofmitochondria respiration [41]. So, to examine whether AMPK phosphor-ylation increased in islet cells of 19-week-old ZDF rats was associatedwith the decrease of mitochondria respiration and if so, it may be causedby ER stress, we measured cellular ATP levels and ATP/ADP ratio in theabsence or presence of TUDCA (Fig. 2D). The levels of ATP productionand ATP/ADP ratio were significantly decreased in 19-week-old ZDFrats, which were partially restored by TUDCA treatment. These datasuggest that the reduction in ATP/ADP may not be responsible forAMPK activation since TUDCA also increases AMPK phosphorylationalong with the restoration of ATP/ADP ratio in 19-week ZDF rats. More-over, the hyperinsulinemia and insulin deficiency observed in 12- and19-week-old ZDF, respectively, were reversed by TUDCA (Fig. 2E).Similarly, TUDCA administration also restored glucose metabolism-related proteins decreased in both islet cells and pancreas tissues ofZDF rats (Figs. 2F and S2B). The impaired glucose tolerance (GTT)and increased insulin resistance (ITT) observed in 19-week-oldZDF rats were partially inhibited by TUDCA (Fig. 2G).

3.3. Role of ROS and ATF3 on the differential regulation of AMPK activationin the progression of diabetes

The iNOS/NO pathway were closely associated with AMPK activa-tion although the reciprocal regulation of these pathways is still

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Fig. 1. Pancreatic β-cell dysfunction is correlated with the upregulation of AMPK activation and lipogenic gene expression in ZDF rats. Male Zucker lean (ZL) control and Zucker diabeticfatty (ZDF) rats were sacrificed at 6, 12, and 19 weeks. (A) Immunohistochemistry analysis for insulin (left) and BrdU staining (right) in pancreas tissues of ZL or ZDF rats (6, 12, 19 weeks,n = 8). The size of islet cells were calculated and quantified (*p b 0.01, ** b 0.05). (B) Expression (upper) and IHC (lower, 19 weeks) of GCK and Glut2 in pancreas tissues. Scale bar: 100μm. (C) AMPK activation was determined by pAMPK and pACC in islet cells isolated from the pancreas of each-aged ZL and ZDF rats. Quantitation of AMPK phosphorylation levelsnormalized to total AMPK expression (*p b 0.01, **p b 0.05). (D) Upregulation of mSREBP1c, FAS, and SCD1, and downregulation of CPT-1 in islet cells. The quantified data are shownin supplementary Fig. 2A. (E) CPT-1 activity wasmeasured inmitochondria of islet cells isolated from each rats (*p b 0.05). The results represent the average ± S.E.M. from three independentexperiments.

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controversial [42]. Thus, we examined whether the differential regula-tion of AMPK activation in 19-week-old ZDF rats are due to ER stress-mediated increases in iNOS/NO and/or ROS levels. The iNOS expressionand NO production were increased in 12-week-old ZDF rats, whichwere potently enhanced at 19 weeks (Fig. 3A). Similarly, stress-inducible transcription factor ATF3, which plays a critical role in pan-creatic β-cell dysfunction and apoptosis [28], was concomitantlyincreased in 12- and 19-week-old ZDF rats (Figs. 3B and S2B). The in-duction of oxidative stress, as indicated by iNOS and ATF3 in ZDF rats,

was predominantly mediated by an ER stress-dependent pathway, astheir inductions were markedly inhibited by TUDCA (Fig. 3C), whichwere in line with the decreases in NO and ROS contents (Fig. 3D).Furthermore, to confirm the role of oxidative stress on AMPK activationand β-cell dysfunction and apoptosis, the 19-week-old ZDF rats weresubcutaneously injected with iNOS inhibitor L-NMMA and antioxidantNAC for 7-days. L-NMMA or NAC strongly attenuated the induction ofiNOS and ATF3, ER stress (p-eIF2α and CHOP), and lipogenic genes(SREBP1c and FAS) expression in the 19-week-old ZDF rats, whereas

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Fig. 2. ER stress inhibition by TUDCA attenuated β-cell dysfunction and apoptosis in 19-week-old ZDF rats. The 8- or 15-week-old ZL or ZDF rats were administrated intraperitoneally withTUDCA for 7 days and maintained by 12 weeks or 19 weeks, thereafter the islet cells were isolated. (A) Effects of TUDCA on AMPK activation and lipogenic gene expression. (B) Data inFig. 2Awere quantified by densitometry analysis (*p b 0.01, **p b 0.05). (C) Triglyceride levels in pancreas tissues (left) and serum (right) (*p b 0.01, **p b 0.05). (D) Cellular ATP production(left) and ATP/ADP ratio (right) (*p b 0.01). (E) Plasma insulin levels (*p b 0.01, **p b 0.05). (F) Glucosemetabolism-related protein expression in islet cells and pancreas tissues. (G) Glucose(1.5 g/kg, top) and insulin (2 IU/kg, bottom) tolerance tests (n = 6, each group). Compared to saline-treated ZDF rats, *p b 0.05. The results represent the average ± S.E.M. from three inde-pendent experiments.

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AMPK phosphorylation was potently increased by antioxidants (Figs. 3Eand S2C). Consistently, pancreatic β-cell dysfunction and apoptosis inthe 19-week-old-ZDF rats were also strongly inhibited by the L-NMMA-mediated reduction in iNOS and ROS (Fig. 3F). These results suggestthat AMPK activation may be inhibited by ROS produced during theprogression obese type 2 diabetes.

3.4. Chronic AICAR treatment inhibits pancreatic β-cell fulminant damagein ZDF rats

To further define the role of AMPK activation in ER stress-mediatedpancreatic β-cell dysfunction and apoptosis in 19-week-old ZDF rats,15-week-old ZDF rats were administrated with AICAR, an AMPK

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Fig. 3. iNOS/NO/ATF3-mediated oxidative stress plays an important role in β-cell dysfunction and apoptosis of 19-week ZDF rats. (A) iNOS expression (top, western blot; bottomleft, immunohistochemistry; bottom right, quantified iNOS(*p b 0.01, **p b 0.05) and NO production in isolated islet cells (*p b 0.05, **p b 0.01). (B) ATF3 protein expression in pancreastissues. (C) TUDCA inhibits the induction of iNOS and ATF3 in 19-week-old ZDF rats. (D) TUDCA inhibits the production of NO (left) and ROS (right) (*p b 0.05, **p b 0.01). (E) Afterinjection subcutaneously with L-NMMA and NAC, the isolated islet cells or pancreas tissues were subjected to western blot analysis. The quantified data are shown in supplementaryFig. 2B. (F) Immunohistochemistry for insulin and iNOS (pancreas tissues, 100 μm) (left), TUNEL and ROS production assay (islet cells, 100x) (right). The results representthe average ± S.E.M. from three independent experiments.

2353J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

activator, subcutaneously either with a single injection (acute) oronce a day for 14-days (chronic). In ZDF rats thatwere chronically treat-ed with AICAR, AMPK phosphorylation was increased compared withthe saline-treated ZDF rats, correlated with a significant reduction inapoptosis-related proteins, such as activated caspase-3 and Bax, aswell as ER stress-related proteins (Fig. 4A and S2C). In contrast, thelevels of GCK and Glut2 reduced in 19-week-old ZDF rats were stronglyrestored by chronic administration of AICAR, but not by a single injection.Furthermore, the positive effects of chronic AICAR treatment werealso confirmed by performing TUNEL assay and immunohistochemistryanalysis for CHOP and insulin in the islet cells of ZDF rats (Fig. 4B). Theseresults suggest that a moderate increase in AMPK activation is notsufficient to prevent ER stress-mediated β-cell dysfunction and apopto-sis in the 19-week-old ZDF rats; however, chronic AMPK activation by

AICAR is enough to prevent oxidative stress by inhibiting the expressionof stress-inducible ATF3 and iNOS, as well as the production of NO andROS (Fig. 4C and D). Hyperglycemia and hypoinsulinemia caused byinsulin deficiency in the 19-week-old ZDF rats were also corrected bythe chronic AICAR treatment but not by the acute treatment (Fig. 4E–F).Similarly, chronic AICAR administration remarkably improved glucosetolerance and insulin sensitivity in 19-week-old ZDF rats, correlatedwith an inhibition of TG accumulation in islet cells (Fig. 4G–H), indicatingthat AMPK activation by AICAR is associated with the enhancing ofoxygen consumption via mitochondrial respiration and thus may triggerenergy expenditure. In addition, AMPK activation in 19-week-old ZDFrats may serve as one of the compensatory mechanisms to prevent theER stress-mediated pancreatic β-cell dysfunction and apoptosis duringthe development of T2D, but its beneficial effects were prevented by

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Fig. 4. Chronic treatment of AICAR inhibits ER stress-mediated pancreatic β-cell dysfunction and apoptosis. Male 15-week-old ZDF rats (n = 8, each group) were subcutaneously admin-istrated with AICAR single injection (acute) or once a day for 14 days (chronic treatment), thereafter they were maintained until they were 19 weeks of age and were then sacrificed orisolated islet cells. (A) Representative western blot was shown. The quantified data are shown in supplementary Fig. 2C. (B) TUNEL assay (in isolated islet cells, 100×) and immunohis-tochemistry for insulin and CHOP (pancreas tissues, 100 μm) were performed. (C) Effects of chronic AICAR treatment on ATF3 and iNOS expression in islet cells. (D) Effects of chronicAICAR treatment on NO (left, *p b 0.01) and ROS production (right, *p b 0.05). (E and F) Blood glucose and plasma insulin (*p b 0.01, **p b 0.05). (G) Glucose (left) and insulin (right)tolerance tests (n = 6, each group). Compared to chronic-saline injected ZDF rats, *p b 0.01. (H) Effects of chronic AICAR treatment on triglyceride (TG) levels in islet cells (*p b 0.05).The results represent the average ± S.E.M. from three independent experiments.

2354 J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

excessive ER stress or ROS production. The protective effects ofAMPK on ER stress- or ROS-mediated β-cell dysfunction and apo-ptosis were confirmed based on INS-1 cells that had increased ER stressas induced by chronic high glucose exposure or the addition oftunicamycin in the presence or absence of AICAR (S3). We havealso obtained similar results by using wild-type-AMPK or dominantnegative-AMPK cDNAs, as well as AMPK siRNA (S4). Concomitantly,AICAR administration for 14-days into 8-week-old ZDF rats can also

strongly reduce ER stress-mediated pancreatic β-cell dysfunction andapoptosis associated with obese T2DM (data not shown).

3.5. ATF3 counteracts the beneficial effects of AMPK against high glucose-induced pancreatic β-cell dysfunction and apoptosis

Based on the above data, we hypothesized that ATF3 could be acounteraction molecule against the beneficial effects of AMPK and

2355J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

thus promote high glucose-mediated β-cell dysfunction and apoptosis.So, INS-1 cells were transfected with ATF3 siRNA and exposedto 28 mM glucose. Interestingly, ATF3 depletion did not affectphosphorylation of AMPK and ACC decreased by high glucose(Figs. 5A and S5A), but there was a marked attenuation of pancreaticβ-cell dysfunction and apoptosis induced by high glucose (Figs. 5B andS5B). Also, the counteraction of ATF3 and iNOS on insulin and GCK ex-pression in high glucose-treated cells was remarkably attenuated byATF3 siRNA (Figs. 5C). Next, to confirm the role of ATF3 in blockingthe beneficial effects of AMPK for high glucose-induced β-cell dysfunc-tion and apoptosis, the cells were treated with 28 mM glucose or AICARin the absence or presence of ATF3 or ATF3 siRNA (Figs. 5D). AMPKactivation by AICAR inhibited high glucose-induced β-cell dysfunction(downregulation of GCK, Glut2, and insulin) and apoptosis (Bax and

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Fig. 5. ATF3 counteracts the preventive effects of AMPK for chronic high glucose-induced pancsiRNA and the cells starvedwith 2% serum and 2 mMglucose-containingmediumwere treateddepletion inhibitsβ-cell dysfunction and apoptosis. (C) Immunocytochemistry analysis for insulcounteracts the compensatory effects of AICAR for high glucose-induced β-cell dysfunction orglucose in the presence or absence of AICAR. The quantified data for western blots are shown inFinal merged images (100×). (F) After treatmentwith 28 mMglucose or AICAR in the presenceflow cytometer (M1, percentage of ROS production). The results represent the average ± S.E.M

caspase-3 cleavage) (lane 3), but the inhibitory effects by AICAR wereabolished by ATF3 overexpression (lane 5) and conversely, were potentlyincreased byATF3 siRNA (lane 7). ATF3 depletion also inhibited highglucose-induced β-cell apoptosis (Figs. 5E and S5C). Correlatively, highglucose-induced ROS production (M1, 91.87) was significantly de-creased by AICAR (M1, 56.25), but these inhibitory effects of AMPKwere prevented by ATF3 overexpression (M1, 81.88), not by ATF3knockdown (M1, 63.65) (Fig. 5F).

3.6. The counteraction of ATF3 and AMPK on high glucose-induced β-cellfulminant damage

To further confirm the counteraction of ATF3 against the preventiveeffects of AMPK on high glucose-induced β-cell dysfunction and

C

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reatic β-cell dysfunction and apoptosis. INS-1 cells were transiently transfected with ATF3with 28 mMglucose for 2 days. (A) Effects of ATF3 depletion on AMPK activation. (B) ATF3in (red), ATF3 (green), GCK (red), and iNOS (green). Finalmerged images (100x). (D)ATF3apoptosis. After transfection with ATF3 or ATF3 siRNA, the cells were treated with 28 mMsupplementary Fig. 5. (E) Immunocytochemistry for ATF3 (Green) and TUNEL assay (red).or absence of ATF3 or ATF3 siRNA, ROS productionwasmeasured by using the FACSCalibar. from three independent experiments.

2356 J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

apoptosis, the INS-1 cells were transfected with the constructedfull-length GFP-ATF3(FL), N-terminal (1–100) domain-deletedGFP-ATF3(ΔN) or GFP-ATF3(ΔC), a construct with a deletion in theC-terminal (101–181), which is necessary for interactions with otherproteins and then treated with 28 mM glucose (Fig. 6A). AMPK phos-phorylation (Fig. 6B) and its kinase activity (Fig. 6C) decreased in highglucose-treated cells did not changed by the overexpression of full-length or deleted-mutant ATF3 constructs. However, the downregulationof GCK and insulin and the induction of Bax and CHOP by 28 mMglucose depends on the C-terminal domain of ATF3 since these arepotentiated by ATF3(FL) or ATF3(ΔN), whereas they were remarkablyattenuated by ATF3(ΔC) (S5). Correlatively, the reduction of insulincontent and ATP production (Fig. 6D), and TUNEL-positive apoptosis(Fig. 6E) induced by 28 mM glucose were dependently regulated bythe C-terminal domain of ATF3. To further confirm the counteractionbetween ATF3 and AMPK for high glucose-mediated β-cell dysfunctionand apoptosis, the cells were treated with AICAR in the presence ofATF3 constructs (Fig. 6D–E). The attenuating effects by AICAR weredetected in GFP-empty vector-transfected cells, but not in full lengthor N-terminal deleted ATF3-transfected cells (Fig. 6D and E). Onthe other hand, the functional counteraction between ATF3 andAMPK may not due to their direct interactions since the phosphorylat-ed AMPK or total AMPK was not present in the complexes with ATF3,despite endogenous PDX-1 interacts with GFP-ATF3 (S6A) as like inour previous studies [29]. Concomitantly, glutathione S-transferase(GST)-pull down assay showed that purified GST-ATF3 does not interact

ATF3(ΔC)

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with AMPK despite endogenous AMPKwas highly expressed or activatedby AICAR (S6B).

3.7. In vivo ATF3 knockout ameliorates the impaired glucose metabolismand β-cell damages during the progression of T2D

To further confirm the role of ATF3 as an antagonizing regula-tor for the compensatory effects of AMPK during the development ofT2D, a loss-of-function study in vivo was performed throughin vivo-injection with ATF3-specific siRNA. Among the constructedfour-different siRNA candidates for rat-specific ATF3 (Supplemen-tal Table 1), 275-ATF3 siRNAhas the highest efficiency for ATF3 silencingin various cell lines (S7A). Next, to determine the functional conse-quences of ATF3 as a counteraction regulator of AMPK for the develop-ment of T2D, 15-week ZDF rats were intravenously injected with 5%glucose or 150 μg of 275-ATF3 siRNA using in vivo-jetPEI PolyPlusreagents. Similar to lung and liver tissues (S7B), the levels of ATF3mRNA and protein in both pancreas tissues and islet cells isolatedfrom ATF3 siRNA-injected ZDF rats were significantly decreased(Fig. 7A). Hyperglycemia (Fig. 7B) and triglyceride accumulation(Fig. 7C) increased in ZDF rats were strongly inhibited by chronicadministration of ATF3 siRNA but not by single. The remarkableimprovement of impaired glucose tolerance and insulin resistance(Fig. 7D–E) was also observed. Correlatively, insulin levels decreased in19-week ZDF rats were also recovered by chronic administration ofATF3 siRNA (Fig. 7F). As well, the increased TUNEL-apoptosis was

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icβ-cell dysfunction and apoptosis. (A) Constructs of full-length (FL), C-terminal (ΔC) orN-M glucose for 2 days and then subjected to western blot. (C) AMPK kinase activity. Aftertates were subjected to the AMPK kinase assay using SAMS peptide (*p b 0.05). (D) Effects, INS-1 cells were treated with 28 mM glucose along with AICAR for 2 days (*p b 0.01,ts represent the average ± S.E.M. from three independent experiments.

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Fig. 7. In vivo ATF3 knockdown ameliorates the impaired glucose metabolism, insulin insensitivity, and β-cell apoptosis in 19-week ZDF rats. Male 15-week-old ZDF rats(n = 8, each group) were intravenously administrated with in vivo-jetPEI ATF3 siRNA single injection (acute) or six times 3-day a three day interval between injections(chronic treatment), thereafter maintained to the 19 weeks and isolated islet cells. (A) RT-PCR (top) and western blot (bottom) analysis of ATF3 in pancreas tissues andislet cells from control siRNA- and ATF3 siRNA-injected ZDF rats. (B–C) ATF3 silencing inhibits hyperglycemia (B) and triglyceride accumulation (C) in 19 weeks ZDFrats. (*p b 0.05). (D–E) Effects of ATF3 silencing on glucose (GTT, D) and insulin (ITT, E) tolerance. (*p b 0.05). (F) Plasma insulin levels (*p b 0.01, **p b 0.05). (G) TUNEL assay wereperformed in isolated islet cells (Left, 100×). TUNEL-positive apoptotic cells were quantified (Right, *p b 0.05). The results represent the average ± S.E.M. from three independentexperiments.

2357J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

significantly decreased in ATF3 siRNA-injected ZDF rats, which wasemphasized by chronic administration of ATF3 siRNA (Fig. 7G).

3.8. ATF3 silencing protects the production of RNS and ER stress-mediatedβ-cell dysfunction and apoptosis

We next investigated the mechanisms by which in vivo ATF3 genesilencing inhibited ER stress-mediated β-cell dysfunction and apoptosisduring T2D development. As shown in Fig. 8A and B, in vivo transfectionof ATF3 siRNA, but not control siRNA, significantly lowered the levels

of ER stress-, apoptosis-, or lipid accumulation-related proteins. Incontrast, the levels of insulin and GCK reduced in ZDF rats wereremarkably restored in the islet cells of ATF3 siRNA-injected mice.Moreover, the reversible regulation by ATF3 knockdown for theexpression of insulin and CHOP in islet cells were further confirmedby immunohistochemical analysis (Fig. 8C–F). ATF3 knockdownalso significantly reduced iNOS expression and NO or ROS productionincreased during the progression of T2D (Fig. 8G–H) and the effects ofATF3 siRNA ameliorating pancreatic β-cell dysfunction and apoptosiswere more effective in chronically injected rats.

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Fig. 8. In vivo ATF3 knockdown attenuates the increases of ER stress and ROS in 19-week ZDF rats. Male 15-week-old ZDF rats (n = 8, each group)were intravenously administratedwithin vivo-jetPEI ATF3 siRNA. (A)Western blottingwas performed in pancreas tissues of ATF3 siRNA-injected rats. (B) Densitometric quantification of 8Awere normalized by β-actin, respec-tively. (*p b 0.01, **p b 0.05). (C–F) Immunohistochemistry analysis (Scale bars, 100 μm). The decreased insulin and increased CHOP protein expression were attenuated in pancreastissues of ATF3 siRNA-injected ZDF rats. (G) iNOSexpressionwasdecreased inATF3 siRNA-injected rats.Western blotting (top) and immunohistochemistry analysis (bottom). (H) Chronicinjection of ATF3 siRNA inhibits the production of NO and ROS (*p b 0.01). The results represent the average ± S.E.M. from three independent experiments.

2358 J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

4. Discussion

In these studies, we demonstrate that ATF3 plays as a counteractionregulator against a preventive response of AMPK for ER stress-mediatedpancreatic β-cell fulminant damage during the progression to T2D. ZDFrats serve as amimicmodel of human T2D that is characterized by insulinresistance, β-cell defects, and hyperglycemia [43]. Although the animalshave early compensatory responses to hyperglycemia-induced fulminantdamage to pancreaticβ-cells, the exactmolecularmechanisms associated

with the development of T2D, especiallywith regard toβ-cell dysfunctionand apoptosis, are still not clear. Here, we have shown that AMPKphosphorylation decreased during the progression of prediabetes(6 weeks) to diabetes (12 weeks) in ZDF rats and was specificallyincreased in late diabetes (19 weeks). However, despite remarkableincrease of AMPK activity in 19-week-old ZDF rats, there is no a com-pensatory response of AMPK to prevent pancreatic β-cell fulminantdamage by hyperglycemia-induced ER or oxidative stress. We demon-strate that the oxidative stress and ER stress excessively increased in

2359J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

late-diabetic phase of T2D progression may contribute to overridethe protective effects of AMPK activation on hyperglycemia-mediatedpancreatic β-cell dysfunction and apoptosis.

Several studies suggest that ER stress contributes to obesity andpromotes lipid accumulation and insulin resistance in insulin-responsive tissues, subsequently leading to diabetes with pancreaticβ-cell dysfunction and apoptosis [40], but the exact regulatory mecha-nisms involved in this progression remain elusive. Our data reveal thatan ER stress-associated pathway is a major pathway that contributes tothe development of diabetes in ZDF rats. First, the impaired glucosetolerance or insulin resistance observed in the processing of obeseT2D was strongly inhibited by TUDCA injection (Fig. 2H). Likewise,the increased apoptosis-related proteins and lipid accumulation and thedecreased glucose metabolism-related proteins were also attenuated byTUDCA treatment. Similar to previous studies using obese and diabeticmodels [43], different to the pattern of 6- and 12-week-old ZDF rats,AMPK activity was specifically increased in 19-week-old ZDF rats withsevere diabetes (Fig. 1C). However, the expression of the lipogenicgenes SREBP1c, FAS, and SCD1, was not compensated by activatedAMPK pathway, and even more increased in the 19-week-old ZDFrats (Fig. 1C and D). Additionally, activated AMPK did not inhibitand conversely, potentiated ER stress-mediated pancreatic β-cell dys-function and apoptosis (Figs. 1 and S1). These are contradictory to theprevious studies demonstrating that AMPK has an established role inrestoring and maintaining cellular homeostasis when it is responsiveto a variety of metabolic stresses that change the cellular AMP/ATPratio, either by increasing ATP consumption or reducing ATP produc-tion, as is seen following hypoxia, glucose deprivation, or the inhibitionof mitochondrial oxidative phosphorylation [44]. Until now, most ofstudies are focused in roles of AMPK as an early defense mechanismused to compensate for impaired glucose and insulin tolerance. Howev-er, the function or role of AMPK reversely increased in 19-week-oldZDF rats, a late-diabetes phase, was not clearly defined. So, we can

Insulin sensitivity Gluconeogenesis

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ZDF rats: Progression of obese T2DM

Fig. 9. The proposedmodel by which in vivo therapeutic treatment inhibits excessive ER stress/of obese T2DM. Black lines, the reduced pAMPK and the increased ER stress, ATF3 or iNOS/NOptosis. Red lines, excessive increase of ER stress and oxidative stress (ATF3 and iNOS/NO) in 19-wZDF rats may play as a final compensatory preventive effects for ER stress or oxidative stress-meER stress and oxidative stress overwhelmed the compensatory preventive effects of AMPK. Riweek-old ZDF rats were injected with TUDCA (i.p.), AICAR (s.c.), antioxidants (NAC and L-NMline, drug treatment and ATF3 knockdown inhibits ER stress and oxidative stress (ROS), subseβ-cell dysfunction and apoptosis.

hypothesize that the increase of AMPK activation in 19-week-old ZDFrats with the status of hyperglycemia and hypoinsulinemia could playas a final compensatory protecting or death-triggering mechanism forthe attenuation of metabolic dysfunction and apoptosis. However,our several data support the former hypothesis. First, AMPK phosphory-lation increased in 19-week-old ZDF rats was potently increased byTUDCA-induced ER stress inhibition, accompanied with the reduction oflipogenic genes or cleaved caspase-3 expression and triglyceride accumu-lation (Fig. 2). Second, ZDF rats chronically injected with AICAR, but notby single, strongly attenuated the increase of glucose or insulin toleranceand β-cell dysfunction, as well as apoptosis-related and ER stress-associated proteins (Fig. 4), indicating that only mild activation ofendogenous AMPK may not be sufficient to prevent ER stress-mediatedβ-cell dysfunction and apoptosis in 19-week-old ZDF rats. Furthermore,the functional inhibition of the compensatory effects of AMPK in the 19-week-old ZDF rats may be due to excessive ER stress or ROS. In supportof this, iNOS and ATF3 expression was significantly increased in the 19-week-old ZDF rats, correlated with the increase of NO and ROS produc-tion, which were strongly attenuated by a potent increase of AMPKactivation in cells treated with TUDCA, AICAR, L-NMMA, or NAC (Figs. 3and 4). The compensatory preventive responses of AMPK were alsosupported by INS-1 cells using constitutively active-AMPK or dominantnegative-AMPK cDNAs, as well as AMPK siRNA (S4). From these results,we demonstrated that the higher endogenous AMPK levels than AMPKactivation observed in 19-week-old ZDF rats could be required foroverwhelming the excessive ER stress or oxidative stress-mediatedpancreatic β-cell dysfunction. On the other hand, the increase ofAMPK activity in 19-week-old ZDF rats may be due to the decreaseof cellular ATP levels and ATP/ADP ratio through the reduction ofoxygen consumption, which reflects the state of mitochondrial res-piration ATP production [41,45]. In our data, AMPK phosphorylationincreased in 19-week-old ZDF rats was correlated with the reductionof cellular ATP and ATP/ADP ratio, which were strongly attenuated

In vivo Therapeutic treatment(Drug and Gene silencing)

see 2tes

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Apoptosis

n

ROS-mediated β-cell fulminant damage in 19-week-old ZDF rats. Left scheme: Progressionin 6 and 12-week-old ZDF rats are associated with pancreatic β-cell dysfunction and apo-eek-old ZDF rats. Pink lines, AMPK phosphorylation specifically increased in 19-week-olddiated pancreatic β-cell dysfunction and apoptosis. Red-dotted lines, excessive increase ofght Scheme: In vivo therapeutic treatment using drugs and gene silencing. Blue lines, 15-MA, s.c.), or in vivo-jetPEI ATF3 siRNA (i.v.). The rats were sacrificed at 19 week. Greenquently the development of type 2 diabetes were prevented by attenuation of pancreatic

2360 J.Y. Kim et al. / Cellular Signalling 25 (2013) 2348–2361

by TUDCA treatment (Fig. 2A and D). These are suggest that AMPKactivation increased in 19-week ZDF rats may not due to the reduc-tion of ATP/ADP ratio since TUDCA restored the ATP levels but didnot suppress the activity of AMPK. AMPK activation increased in19-week ZDF rats may be a last effort for cytoprotection against tothe ecxcessive oxidative stress. In fact, these data seem contradictoryto our previous report [20] showing that AMPK activation by AICAR orAMPK overexpression potentiated chronic high glucose-inducedpancreatic β-cell dysfunction and apoptosis, although the experimentalparadigm (cell type and condition) was different. In fact, the pattern ofAMPK activation is biphasic and the roles of AMPK activated in acutephase and chronic late phase are different although their roles are stillcontroversial. Our data show that ATF3 counteracts the effects of AMPKand thus promote chronic high glucose-induced β-cell dysfunction andapoptosis via iNOS induction and NO or ROS production (Fig. 5).Conversely, ATF3 andβ-cell dysfunction increased during the progressionof obese T2Dwere significantly inhibited by AICAR injection (Figs. 4and 5, S2 and S3). Although ATF3 did not directly affect AMPK phos-phorylation, its depletion attenuates ER stress-mediated the impairedglucose or insulin tolerance and β-cell dysfunction and apoptosis, resultsin the prevention of type 2 diabetes (Fig. 5) and conversely, its over-expression promotes these events (Fig. 6). Conversely, our data alsoshow that ATF3, especially via C-terminal domain, counteracts thebeneficial effects of AMPK for glucotoxicity (Fig. 6) and their counterac-tion was not due to the direct interaction (S4). Although not shown inhere, it is possible that ATF3 may counteract the inhibitory effects ofAMPK through the defect of mitochondrial respiration chain and thustriggers ER stress or oxidative stress-mediated β-cell dysfunctionand apoptosis. Furthermore, we couldn't exclude the possibility thatATF3 may be in a parallel pathway with AMPK and they can act the an-tagonistic machinery in an opposite direction. So, to confirm the criticalrole of ATF3 antagonizing the compensatory preventive effects of AMPKduring the development of T2D, we have performed a loss-of-functionstudy in vivo through in vivo delivery with ATF3-specific siRNA (Figs. 7and 8). The in vivo inhibition of ATF3 using in vivo-jetPEI PolyPlus reagent[38,39] ameliorates the impaired glucose metabolism and pancreaticβ-cell dysfunction and apoptosis, and thus prevents the development ofT2D at least in part by inhibiting ER stress and reducing the productionof RNS. However, in our using models, the injected ATF3 siRNA includ-ing TUDCA (Figs. 2 and 3) and AICAR (Fig. 4) may act on several organssuch as liver and muscle, not specific to pancreatic β-cells. So, it is pos-sible that the therapeutic effects of ATF3 siRNA on pancreatic β-celldysfunction and apoptosis could be indirectly regulated through liveror muscle metabolism. As well, it is possible that the counteraction ofATF3 and AICAR may be associated with AMPK-independent pathwaysince AICAR has multiple AMPK-independent effects. Therefore, to de-fine the precise mechanisms for the regulation of ATF3 on ER stress-mediated β-cell fulminant damage during the development of T2D,we need to do further studying by using pancreas-specific knockoutor silencing models. Taken together, these results show that ER stressis the main pathological pathway for the progression of T2D in obeseZDF rats (Fig. 9). Here, we found that increased AMPK activation duringend-stage diabetes may serve as a final compensative mechanismto prevent hyperglycemia-induced ER stress and oxidative stress.However, it is likely that its compensatory role may be overwhelmedby excessive increases in ER stress and oxidative stress via ATF3 induc-tion, whereby resulting in cell dysfunction and apoptosis. These aresupported by that in vivo silencing of ATF3 using in vivo-jetPEIdelivery system ameliorates the preventive effects of AMPK forthe ER stress- or ROS-mediated β-cell dysfunction. Therefore, we showforwhatwe believe is thefirst time that ATF3may play as a counteractionmolecule against the compensatory preventive effects of AMPK via thereduction of mitochondrial oxygen consumption and excessiveinduction of iNOS or ROS during the development of T2D. Also, ourfindings suggest that strategies based on the inhibition of ATF3 mightbe of benefit in the treatment of the development of obese T2D.

Acknowledgment

We thank Dr. M. Birnbaum and Dr. T. Hai for providing plasmid cDNA(M.B., pcDNA-empty, pcDNA-AMPK-WT, and pcDNA-AMPK-K45R; T.H.,ATF3 cDNA).

This work was supported by research grants from the KoreanNational Institutes of Health (4845-302-210-13).

No potential conflicts of interest relevant to this article were reported.J.Y.K. and W.H.K. researched data, contributed to discussion, wrote,

reviewed, and edited the manuscript. K.J.P., G.H.K, E.A.J., and D.Y.L.researched data. S.S.L., D.J.K., G.S.R., J.S., and S.H.K. contributed discus-sion and reviewed the manuscript. W.H.K. is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the dataanalysis.

This manuscript was reviewed and edited by American Journal ofExperts.

The abbreviations used are: ROS, reactive oxygen species; TUDCA,tauroursodeoxycholate; NAC, N-acetyl-L-cysteine; L-NMMA, L-NG-nitro-arginine methyl acetate; FACS, fluorescent-activated cell sorter; AMPK,5′-AMP-activated protein kinase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; DCFH-DA, dichloro-dihydrofluorescein diacetate; FAS, fatty acid synthase, SREBP, sterolregulatory element binding protein; ACC, acetyl-CoA carboxylase;PEPCK, phosphoenolpyruvate carboxykinase; UPR, unfolded proteinresponse; IRE1α, inositol-requiring protein-1α, JNK, jun nuclear kinase;iNOS, inducible nitric oxide synthase; NO, nitric oxide; IRS-1, insulinreceptor signaling-1.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cellsig.2013.07.028.

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