Glutaredoxins concomitant with optimal ROS activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes

Author’s Accepted Manuscript Glutaredoxins concomitant with optimal ROS activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes Kelei Dong, Meiling Wu, Xiaomin Liu, Yanjie Huang, Dongyang Zhang, Yiting Wang, Liang-Jun Yan, Dongyun Shi www.elsevier.com

PII: DOI: Reference:

S0891-5849(16)30449-X http://dx.doi.org/10.1016/j.freeradbiomed.2016.10.007 FRB13022

To appear in: Free Radical Biology and Medicine Received date: 13 September 2016 Revised date: 9 October 2016 Accepted date: 11 October 2016 Cite this article as: Kelei Dong, Meiling Wu, Xiaomin Liu, Yanjie Huang, Dongyang Zhang, Yiting Wang, Liang-Jun Yan and Dongyun Shi, Glutaredoxins concomitant with optimal ROS activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2016.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Glutaredoxins concomitant with optimal ROS activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes

Kelei Donga1, Meiling Wua1, Xiaomin Liua, Yanjie Huanga, Dongyang Zhanga, Yiting Wanga, Liang-Jun Yanb, Dongyun Shia*

a

Department of Biochemistry and Molecular Biology, Shanghai Medical College of

Fudan University, Free Radical Regulation and Application Research Center of Fudan University, Shanghai 200032, People’s Republic of China b

Department of Pharmaceutical Sciences, UNT System College of Pharmacy,

University of North Texas Health Science Center, Fort Worth, Texas, USA *

Correspondence

author:

Dong-Yun

Shi,

tel:

0086-21-54237299,

fax:

0086-21-54237897. email address: [email protected]

Abstract AMPK dysregulation contributes to the onset and development of type 2 diabetes (T2DM). AMPK is known to be activated by reactive oxygen species (ROS) and antioxidant interference. However the mechanism by which redox state mediates such contradictory result remains largely unknown. Here we used streptozotocin-high fat diet （STZ-HFD） induced-type 2 diabetic rats and cells lines (L02 and HEK 293) to 1

These authors contribute equally to this work. 1

explore the mechanism of redox-mediated AMPK activation. We show glutaredoxins (Grxs) concomitant with optimal ROS act as an essential mediator for AMPK activation. ROS level results in different mechanisms for AMPK activation. Under low ROS microenvironment, Grxs-mediated S-glutathionylation on AMPK-α catalytic subunit activates AMPK to improve glucose transportation and degradation while inhibiting glycogen synthesis and keeping redox balance. While, under high ROS microenvironment, AMPK is activated by an AMP-dependent mechanism, however sustained high level ROS also causes loss of AMPK protein. This finding provides evidence for a new approach to diabetes treatment by individual doses of ROS or antioxidant calibrated against the actual redox level in vivo. Moreover, the novel function of Grxs in promoting glucose metabolism may provide new target for T2DM treatment. Abbreviations Allo, allopurinol; AMPK, adenosine monophosphate-activated protein kinase; Apo, apocynin; Cu-Zn-SOD, Cu-Zn-superoxide dismutase; DTT, dithiothreitol; ECAR, extracellular acidification rate; GK, glucokinase; GLUT4, glucose transporter 4; GPxs, glutathione peroxidases; GR, glutathione reductase; Grx-1, glutaredoxin 1, cytosolic; Grx-2, glutaredoxin 2, mitochondrial; Grxs, glutaredoxins; GYS-1, glycogen synthase-1; HPLC, high performance liquid chromatography; LDH, lactate dehydrogenase; Mn-SOD, Mn-superoxide dismutase; NAC, N-acetyl cysteine; NOX, NADPH oxidase; OCR, oxygen consumption rate; OGTT, oral glucose tolerance test;

2

p-AMPK, phospho-AMPK α1/α2; PFK-1, phosphofructokinase-1; p-PFK-2/PFK-2, phospho-PFK-2/PFK-2; p-GYS, phospho-glycogen synthase; PK, pyruvate kinase; Prxs, peroxiredoxins; ROS, reactive oxygen species; R-SOH, sulfenic acid; R-SO2H, sulfinic

acid;

R-SO3H,

sulfonic

acid;

SD,

Sprague-Dawley;

STZ-HFD,

Streptozotocin-high fat diet; T2DM, type 2 diabetes mellitus; TEM, transmission electron microscope; Trxs, thioredoxins; XO, xanthine oxidase.

Keywords: Glutaredoxins; ROS; AMPK; Type 2 diabetes; Glucose metabolism; Glutathionylation

3

1. Introduction In recent years, diabetes has been characterized as a global epidemic, with numbers expected to reach 300 million by 2025 [1]. Understanding the mechanism underlying pathogenesis of diabetes is critical to developing a cure. Many of the common key driving forces that contribute to the onset and progression of diabetes, including obesity, age and sedentary lifestyle, also contribute to the creation of an oxidizing environment. While oxidative stress has been widely recognized as an important factor in diabetogenesis, contradictory findings, in which many studies have indicated a protective role for ROS in exercise and in the prevention and treatment of diabetes [2, 3], obscure its role in the disease progression. Adenosine monophosphate-activated protein kinase (AMPK) is an important target for the treatment of diabetes [4]. The level of insulin is decreased in mice with AMPK α2-deficiency [5], and AMPK can also improve or ameliorate the symptoms of diabetes through reducing glycogen synthesis, increasing glycolysis, prompting the uptake of glucose in surrounding tissues. Therefore, many of the existing diabetes drugs including rosiglitazone [6] and metformin [7] lower blood glucose by reducing intracellular ROS via the AMPK-FOXO3 pathway [8]. Activation of the AMPK pathway has also been shown to exhibit further protective effects through preventing ROS-mediated mitochondrial division and apoptosis of endothelial cells [9, 10]. These findings contradict with studies that suggest that AMPK is activated by oxidative stress [11, 12]. In a study examining embryos of pregnant mice, AMPK

4

activation was observed in hypoxia-induced oxidative stress. However, these effects were abolished in the presence of antioxidants glutathione ethyl ester and vitamin E. Therefore, it seems that both oxidative stress and antioxidant interventions can lead to AMPK activation. These contradictory mechanisms of AMPK activation highlight a need for further investigation. Hence, the present study was designed to understand the mechanism underlying ROS/antioxidant regulation of AMPK function in diabetes. We hypothesized that different redox states in vivo impact AMPK activation in different mechanisms, thus affecting downstream metabolic pathways. We used streptozotocin-high fat diet (STZ-HFD) induced diabetic rats and liver cells to explore the impact of different redox states on AMPK and its downstream glucose pathways. Our results show that AMPK is activated by either oxidative stress or ROS inhibition. The different amount of cellular ROS could result in different mechanisms for AMPK activation: either by Grx-dependent redox modification or by AMP-dependent allosteric activation. We demonstrated that under low ROS microenvironment Grxs act as an essential mediator to allow S-glutathionylation on AMPK-α catalytic subunit and AMPK activation, thus improving glucose metabolism while keeping redox balance in T2DM.

5

2. Methods

2.1 Diabetic animal models All animal-related procedures were approved by the Fudan University Institutional Laboratory Animal Ethics Committee. Male SD rats (150-160g body weight) were obtained from Fudan University Animal Center (Shanghai, China). Induction of type 2 diabetes by STZ (Sigma Aldrich, USA)-high fat diet (STZ-HFD) was performed as previously described [13-15] and more details were shown in supplementary materials. Normal chow and high-fat diet (HFD) were purchased from Shanghai SLRC laboratory animal Company Ltd (Shanghai, China) and the nutritional composition was shown in supplementary Table S1. Measurement of OGTT was performed according to the procedure previously described [16]. Accu-Chek Performa (Roche Diagnostics, Germany) was used to measure blood glucose levels in rats. Alternatively, after a 12 h fasting period, rats were administered with glucose of 2.5 g/kg body weight. Insulin secretion was detected using an Iodine [125I] Insulin Radioimmunoassay kit (Shanghai Radioimmunoassay Technology Research Co Ltd, Shanghai, China). This study was approved by the Research Ethics Committees of Fudan University and the methods were carried out in accordance with the approved guidelines.

6

2.2 Cell culture Human liver cell line L02 and human emborynic kidney cell line 293T were obtained from Chinese Academy of Sciences (China). Culture of cells was performed as previously described [17]. Cells were maintained in Dulbecco’s modified Eagle’s medium (GIBCO, USA), low glucose culture medium and were incubated in 5% CO2 cell incubator at 37°C. The medium was supplemented with 10% fetal bovine serum (Solarbio, Beijing Solarbio Science & Technology Company Ltd, Beijing, China), 105 unit/L Penicillin and Streptomycin (North China Pharmaceutical Company Ltd, China).

2.3 Transmission electron microscope analysis After rats were anesthetized, part of their liver tissues were cut and fixed by glutaraldehyde. Ultra-structures of liver cells were then further analyzed by transmission electron microscope at Fudan University (Shanghai, China).

2.4 Assay for GK, PFK-1, PK, LDH and aconitase activity Liver glucokinase (GK) and PFK-1 activities were measured as described previously [18]. PK activity was measured as previously described [18]. LDH activity was

measured as previously described [20]. Aconitase activity was determined spectrophotometrically[21].

Enzyme activity was standardized using protein

quantification.

7

2.5 GSH/GSSG ratio measurement Mouse liver tissues were homogenized into 50μl 1M HPO3 for GSH detection. The suspension was centrifuged with 12,000rpm for 10min at 4°C and then assayed as previously described[22, 23].

2.6 ATP and AMP content analysis ATP and AMP were measured using high performance liquid chromatography (HPLC) as previously described [24]. About 20-30 mg liver tissues were homogenized on ice, the homogenate was treated with perchloric acid. Homogenized samples were centrifuged for 12000 rpm at 4°C for 30min. Supernatant was then collected and mixed with potassium carbonate, followed by further centrifugation for 12000 rpm at 4°C for 20min. Supernatant was obtained for the determination of ATP, and AMP content by HPLC. The detection wavelength was 254nm.

2.7 S-Glutathionylation of AMPK and detection of GSS-AMPK adduct formation. GSS-AMPK adduct were measured as described previously [25]. L02 cells (2×106/ml) were incubated with ethyl ester GSH-biotin (6mM) for 1.5 h. Cells were then washed twice with culture buffer to remove the excess of GSH and treated with H2O2 for 15 or 30 min. Cell lysates were prepared in the presence of N-ethylmaleimide (5mM) and then passed through Bio-Gel P10 to remove free GSH-biotin and N-ethylmaleimide. 8

The level of GSS-protein conjugates was determined using non-reducing Western blot analysis with streptavidin-HRP, whereas GSS-AMPK subunit levels were measured after pull-down with streptavidin-agarose (60 min at 4°C), followed by reducing SDS-PAGE and Western blot analysis with antibodies against AMPK α, AMPK β, or AMPK γ subunits, respectively.

2.8 Metabolic Assays.

Respirometry (oxygen consumption rate, OCR, indicative of mitochondrial OXPHOS) and the extracellular acidification rate (ECAR, indicative of glycolysis) of cells were measured using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience) as described previously [26]. For OCR analysis, 104cells per well were seeded in complete growth medium in 96-well plates designed for the XF24. Grx-1/2 siRNA (500nM) were transfected to L02 cells for 48h in a CO2-free incubator for measurement. A program with a typical 8-min cycle of mix (3min), dwell (2min), and measurement (3min) was used. During measurement, oligomycin (OM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and antimycin A (AA) were added to a final concentration of 10μM, 1μM and 10μM, respectively. For ECAR analysis, Grx-1/2 siRNA-treated cells (104 cells per well) were used for the measurement. The default standard glycolysis stress-test program was selected. Measurements were conducted using final concentrations of 10mM glucose, 1μM

9

oligomycin and 50mM 2-deoxyglucose, respectively.

2.8 Other methods.

Human siRNA sequences were designed by Shanghai GenePharma Pharmaceutical Technology Company Ltd (Shanghai, China) and shown in supplementary Table S2 and transfected by Lipofectamine 2000 Reagent (Invitrogen, Life Technologies). Western blot was performed according to standard procedures and protein extracts from liver and cells were prepared as previously described [27]. Protein expression was analyzed with antibodies against GK(19666-1-AP, Proteintech, USA), PFK-1(55028-1-AP, Proteintech, USA), PFK-2(17838-1-AP, Proteintech, USA), LDH(19987-1-AP,

Proteintech,

USA),

GR(18257-1-AP,

Proteintech,

USA),

Grx-1(15804-1-AP, Proteintech, USA), Grx-2(13381-1-AP ， Proteintech, USA), Trx-1(14999-1-AP, Proteintech, USA), Trx-2(13089-1-AP, Proteintech, USA), Prx-1(15816-1-AP, Proteintech, USA), Mn-SOD(24127-1-AP, Proteintech, USA), AMPK α1(10929-2-AP, Proteintech, USA), AMPK α2 (Proteintech, USA), acetyl-Mn-SOD (Abcam, UK), phospho-AMPK α1/α2(#11183, Signalway Antibody, USA), phospho-GYS (#11945, Signalway Antibody, USA), phospho-PFK-2(#13064, Cell Signaling Technology, USA) and β-actin (#3700, Cell Signaling Technology, USA).

10

2.9 Statistical analysis

Linear regression statistical analysis was performed using GraphPad Prism 5.0. Unpaired Student t-tests were used to calculate the significant difference between two sample groups. For datasets containing more than two groups that required analysis of changes overtime, one-way ANOVA and Tukey post hoc test calculations were performed instead. All data are expressed as mean ±SD. A value of P < 0.05 was considered statistically significant.

3. Results 3.1 Enhanced antioxidant system contributes to dramatic AMPK upregulation and activation in AMP-independent manner in diabetic rats STZ has been widely used to induce experimental diabetes in animals [13, 14, 28]. By using STZ combined with high fat diets (HFD), we established HDF-STZ induced type 2 diabetic rats model as we recently reported [13]. Rats given HFD alone maintained normal level of blood glucose (Fig.S1A). However, rats given a combined regimen of HFD and STZ injection exhibited elevated blood glucose levels over the course of 3 weeks. Specifically, significant elevation was observed at 35 and 50 mg/kg STZ, respectively (Fig.S1B). Type 2 diabetic conditions were also confirmed by plasma insulin levels and oral glucose tolerance test (OGTT) (Fig.S1C and S1D). In the high dosage (STZ dosage 50mg/kg BW) group, the fasting and blood glucose levels elevated significantly as compared to that of the control. With increasing STZ dosage, 11

hyperglycemia in diabetic rats also aggravated (Fig.S1E). We investigated the effect of inhibiting ROS on the diabetic rats. We utilized an antioxidant apocynin (Apo) which also acts as NADPH oxidase (NOX) selective inhibitor [29], and xanthine oxidase (XO) specific inhibitors allopurinol (Allo) [30], to inhibit ROS in rats. Apo and Allo intervention significantly alleviated diabetic conditions by reducing the blood sugar levels in diabetic rats (Fig.S1F). Studies have shown that inactivation of AMPK plays an important role in the occurrence of diabetes [31]. Consistent with findings in the literatures [5], our results showed that the expression of AMPK α2, but not AMPK α1, in diabetic rats was reduced significantly. However, contrary to the declined protein level, the ratio of phosphorylated AMPK between α1 and α2 was elevated (Fig.1A), indicating that oxidative stress does activate AMPK phosphorylation. It is known that AMPK is activated when ATP level in the body decreases. Our HPLC analysis showed that AMP output elevated significantly while ATP output and ATP/AMP ratio decreased in STZ-induced diabetic rats (Fig.1C). This result suggests that AMPK activation in STZ-induced diabetic rats was caused by lack of ATP. When ROS were inhibited by Apo or Allo, both AMPK protein and phosphorylation level increased significantly (Fig.1B). However, Apo or Allo intervention did not reduce ATP output, on the contrary it raised ATP output and ATP/AMP ratio in diabetic rats (Fig.1D), indicating that AMPK activation induced by Apo or Allo intervention is not due to lack of ATP, suggesting that antioxidant treatment could activate AMPK in an AMP-independent

12

mechanism.

Our results showed that Apo and Allo intervention activated the majority of redox proteins including glutathione reductase (GR), Grx, thioredoxins (Trxs), glutathione peroxidases (GPxs), peroxiredoxins (Prxs) and Cu-Zn-superoxide dismutase (Cu-Zn-SOD) (Fig.2A), of which Grx increased most dramatically, whereas GSH/GSSG ratio was either unchanged or slightly decreased (Fig.2B). Mitochondrial aconitase is known to be highly sensitive to ROS [21, 32]. Our results show that STZ-induced diabetic rats exhibited impairment in aconitase activity (Fig.2C), suggesting that these rats were undergoing oxidative stress attack. Oxidative stress could impair mitochondria and cause less ATP generation as well as more protein degradation. We proposed that both reduced AMPK protein level and increased phospho-AMPK in diabetic rats were caused by oxidative stress. Apo and Allo intervention rescued aconitase activity (Fig.2C), suggesting that an enhanced antioxidant system protects cells from ROS attack and prevents protein inactivation or degradation. Based on the above results, we conclude that the significant increase in the antioxidant system of diabetic rats contributes enormously to AMPK protein upregulation (Fig.2D).

3.2 Grxs were involved in promoting AMPK activation We further explored the mechanism by which antioxidant system contributes to AMPK activation. Grxs upregulation and AMPK activation demonstrated a significant 13

positive correlation when cytosolic ROS was inhibited in diabetic rats. We speculated that Grxs upregulation may be related to AMPK activation. By creating an oxidative stress condition in L02 liver cells with diamide, we found that oxidation could activate AMPK phosphorylation and Grx expression (Fig.3A). This result seems to be contradictory to the observation that suppression of cytoplasmic ROS led to AMPK activation and Grxs upregulation. Further studies indicated that oxidative stress could regulate Grxs in a bidirectional manner (Fig.3B-3D). Grx protein levels showed slight increase under low concentrations of H2O2 (10μM), but being inhibited by high concentrations of H2O2 (1mM) (Fig.3B). We found that intermediate concentration of H2O2 at 0.5mM was a threshold level, at which the H2O2 mediated oxidative stress switched from protecting to damaging. Initially N-acetyl cysteine (NAC) could reverse the upregulated-Grx induced by low concentrations of H2O2 (10μM). However, with increased NAC concentrations, Grx expression elevated accordingly (Fig.3C, upper). On the other hand, NAC resumed the Grx expression which was downregulated by high concentrations of H2O2 (1mM). Similar results were also obtained by diamide/DTT. DTT resumed diamide-induced Grxs downregulation (Fig.3D). These results suggest that at a certain threshold, ROS and antioxidant treatment can both achieve optimal ROS level and upregulate Grxs. We found that changes in AMPK activity were very similar to those of Grxs expression. Oxidative stress activated AMPK, whereas excessive oxidative stress impaired AMPK activity (Fig.3E). Furthermore, declined AMPK activity could be rescued by DTT (Fig.3F).

14

We investigated whether there was a correlation between Grxs upregulation and AMPK activation. We found that phosphorylation of AMPK and AMPK’s downstream target protein acetyl-CoA carboxylase (ACC) was significantly inhibited when Grx-1/2 expression was subjected to siRNA interference (Fig.3G). On the other hand, AMPK silence had little effect on Grxs expression (Fig.3H), suggesting that AMPK is a downstream target of Grxs, which is probably why Grxs have a regulatory effect on AMPK phosphorylation. We also found that, with AMPK α2 being partly inhibited, addition of diamide restored AMPK phosphorylation (Fig.S2A), indicating that oxidative stress indeed plays a role in activating AMPK. Similarly, addition of diamide could also lead to rescue partly inhibited Grx-1/2 (Fig.S2B). With restoration of Grxs expression, AMPK phosphorylation was also restored accordingly; nonetheless, there was no change in AMPK content (Fig.S2B). This result further confirmed that Grxs could influence AMPK phosphorylation.

3.3 Glutaredoxins activate AMPK through S-glutathionylation

Several studies have already demonstrated that increased intracellular concentrations of H2O2 result AMPK activation [33, 34]. H2O2 is also reported to cause oxidative-modification and S-glutathionylation of cysteine residues in AMPK, leading to AMPK activation [25]. This gives us the insights that GRX may contribute to

15

AMPK activation through S-glutathionylation. Given that Grxs have a unique function of catalyzing glutathionylation, we hypothesized that a potential mechanism by which optimal intracellular concentrations of H2O2 could activate AMPK through forming the S-glutathionylation cysteine residues in AMPK α subunit with the help of Grxs. As shown in Fig.4A, exposure to 0.01mM H2O2 led to an increase of GSS-protein adduct concomitant with Grxs upregulation, while the GSS-protein adduct decreased under increased concentrations of H2O2 (0.1mM) treatment in parallel with Grxs downregulation, suggesting that glutathionylation requires an optimal ROS level. Previous

studies

showed that H2O2

exposure produced GSS-phospho-Thr

172-AMPKα subunit S-glutathionylation and activation [25]. Consistence with these findings,

we

(GSS-AMPKα)

showed and

that

GSH-biotin-bound GSH-biotin-bound

(S-glutathionylated)

AMPKα

phospho-Thr172-AMPKα

(GSS-phospho-Thr172-AMPKα) increased in response to a lower concentration of H2O2 (0.01mM) (Fig.4B). This indicates that optimal ROS induced AMPK activation is associated with increased S-glutathionylation of phospho-AMPKα. We have shown that ATP did not change in those cells exposed to the lower concentrations of H2O2 (below 0.1mM) (Fig.4E). This further confirmed that AMPK activation by low ROS is due to redox modification rather than gamma subunit-mediated activation stimulated by ATP deficiency, as outlined in Fig.S3. Grxs mediated-glutathionylation is known to be regulated by GSH/GSSG ratio [35].

16

With GSH/GSSG ratio being decreased, Grxs could enhance glutathionylation; otherwise it could promote deglutathionylation. We found both 0.01mM H2O2 group and control group displayed a lower GSH/GSSG ratio (Fig.4C). With increase of H2O2, GSH/GSSG ratio compensatorily elevated. However, when H2O2 was increased to 1mM, GSH/GSSG ratio was significantly decreased (Fig.S4A), and the GSS-AMPK was also decreased(Fig.S4B). Our results show that the optimal GSH/GSSH ratio for glutathionylation occurred at 1.2 which is close to the optimal GSH/GSSG ratio for p53 glutathionylation in the literature [35]. In the case of 1mM H2O2 treatment group, although GSH/GSSH ratio was in the optimal range, we speculated that the lower Grx level in response to an excessive oxidative stress, could impair the glutathionylation process. This might be the reason why the GSS-AMPK was lower in those groups (Fig.S4B). These results confirmed that cellular redox states could affect the AMPK glutathionylation process.

To determine whether Grxs-catalyzed glutathionylation is involved in the AMPK activation, HEK 293 cells loaded with ethyl ester GSH-biotin were incubated with H2O2. By using siRNA interference to knockdown Grx, we found that the Grx downregulation prevented S-glutathionylation of phospho-AMPK (Fig4E). Moreover, phosphorylated AMPKα in the pull down (GSH-biotin-bound AMPKα) was decreased by Grxs siRNA interference when normalized to the amount of AMPKα in the

pull

down

(GSH-biotin-bound

AMPKα),

suggesting

Grxs

increased

S-glutathionylation of p-AMPKα. This indicates that the critical role of Grx in 17

promoting AMPK glutathionylation and activation.

3.4 AMPK was instantly activated in AMP-dependent manner under high level ROS microenvironment Previous study showed that ROS-induced AMPK activation is AMP-dependent [36]. We further investigated why ROS displayed such different mechanisms. Interestingly, we found that higher doses of H2O2 caused a rapid depletion of cellular ATP (Fig.5B and Fig.S5B), which increased AMP/ATP ratio and activated AMPK. (Fig.5A and Fig.S5A). Nonetheless 10mM H2O2 attenuated AMPK activation regardless of ATP depletion. Our results suggest that there are two mechanisms for H2O2-induced AMPK activation. On one hand, under lower range (between 0 to 0.1mM H2O2), AMPK could be activated by Grxs-mediated glutathionylation. On the other hand, under higher range (between 1 to 5mM H2O2), AMPK could be activated by AMP-dependent mechanism where ATP were lower. We found that an optimal AMPK activation depended on both the dose and timing. Higher concentration of H2O2 could induce robust enhancement of AMPK activation only in a short term. During prolonged time course, both AMPK phosphorylation and protein content decreased significantly. As shown in Fig.5C, after 1mM H2O2 treatment, AMPK phosphorylation reduced at 3h and was totally lost at 9h. AMPK protein level even declined earlier. This suggests that sustained high level ROS is harmful for AMPK protein and phosphorylation. During high concentration of H2O2 18

(1mM) treatment, ATP/AMP ratio decreased initially at 15min and 1h, and then increased after 3h (Fig.5D), AMPK phosphorylation was negatively associated with the ATP/AMP ratio. This result further confirmed that high concentration of ROS induced-AMPK activation is dependent on AMP. On the contrary, low ROS-induced AMPK activation could last longer. During prolonged treatment with 0.01mM H2O2, both AMPK protein and phosphorylation level were well maintained (Fig.5E), while ATP/AMP ratio were well maintained or even increased (Fig.5F). We conclude that high level ROS instantly activates AMPK in AMP-dependent manner, whereas low ROS constantly activates AMPK in a Grxs-dependent manner.

3.5 Grxs promote glycolysis, improve glucose transportation and inhibit glycogen synthesis via AMPK-dependent pathway We investigated whether Grxs could affect AMPK downstream targets. We found that Grx-1/2 silence could lead to significant downregulation of certain key glycolytic enzymes such as phosphofructokinase-1 (PFK-1) and pyruvate kinase (PK) (Fig.6A). The phospho-PFK-2/PFK-2 (p-PFK-2/PFK-2) ratio which represents glycolysis rates, decreased in Grx-1/2 siRNA-expressing cells (Fig.6B). Expression of Grx1/2 siRNA also down-regulated p-GYS (i.e. improve glycogen synthesis) (Fig.6C). Accordingly, AMPK α2 siRNA-expressing cells displayed downregulation of PFK-1, PK, and p-PFK-2/PFK-2 and p-GYS expression (Fig.6F-6H). The PFK-1, PK and lactate

19

dehydrogenase

(LDH)

activity

in

both

Grx-1/2

siRNA and

AMPK

α2

siRNA-expressing cells reduced (Fig.6D, 6E, 6I and 6J). These results indicate that similar to AMPK, Grxs could promote glycolysis, glucose transportation and inhibit glycogen synthesis. We further verified, using Seahorse, that Grxs silence could inhibit glycolysis. We found that L02 cells expressing Grxs siRNA displayed the significantly reduced extracellular acidification rate (ECAR, Fig.6K), an index of lactate production [37], but no significant difference in the oxygen consumption rate (OCR, Fig.6L). This is similar to the results in AMPK α2 siRNA-expressing cells (Fig.S6A and 6B). These results confirmed that loss of Grxs could render cells to be defective in glycolysis. Therefore, our results disclose that Grxs play an important role in glycolysis.

3.6 Elevated Grxs and antioxidant system alleviate disorders of glucose metabolism thereby ameliorating diabetes We explored the impact of elevated Grxs and antioxidant system on the metabolic alterations in diabetes. Apo and Allo intervention significantly increased Grxs as well as the expression and activity of glycolysis enzyme such as PFK-1, PK, LDH and p-PFK-2/PFK-2 ratio (Fig.7A-7E). Apo and Allo treatment raised the p-GYS/GYS 20

ratio, indicating reduced synthesis of glycogen (Fig.7F). Transmission electron microscope (TEM) images showed significantly reduced glycogen accumulation in diabetic rat liver (Fig.7G). A schematic diagram of the Apo/Allo rescuing the metabolic disorders was illustrated in Fig.7H. The results confirmed that elevated Grxs and antioxidant system caused by Apo and Allo interference improve glycolysis, glucose transportation while inhibiting glycogen synthesis. Through alleviating the disorder of glucose metabolism, we conclude that elevated Grxs and antioxidant system reduces the blood sugar levels in diabetic rats and improves diabetes symptoms (Fig.7I).

4. Discussion In this study we investigated the role of redox-mediated activation of AMPK signaling in glucose metabolism in diabetic rats. We observed that optimal ROS concomitant with Grxs could activate AMPK to promote glucose degradation and improve glucose transportation while inhibiting glycogen synthesis and keeping redox balance. Moreover we demonstrated that cellular ROS level could determine the way AMPK to be activated - either by Grx-dependent glutathionylation on AMPK-α catalytic subunit at low ROS or by AMP-dependent allosteric activation of AMPK- regulatory subunit at high level ROS, albeit sustained high level ROS could degrade AMPK (Fig.5C). Our findings not only provide personal therapeutic strategies on how to effectively activate AMPK in vivo through regulating individual ROS level but also offer Grx as

21

a potential new target for T2DM treatment. AMPK dysregulation contributes to the onset and development of T2DM [38]. However, recent research has revealed that ROS may activate AMPK [11, 25], a protein normally inactivated in diabetic subjects. These findings appear to contradict the general effect of antioxidant that is thought to improve AMPK activity therefore benefiting for treatment diabetes [6, 7]. Therefore, it is necessary to investigate the underlying mechanism pertaining to such contradictory results. We recently reported that ROS could result in insulin resistance through reprogramming the glucose metabolic pathway [13], which highlights the importance of ROS in the onset of T2DM. In present study, we confirmed that oxidative stress plays an important role in the occurrence and development of diabetes, meanwhile inhibiting oxidative stress could alleviate diabetes. Interestingly, we showed both oxidative stress and ROS inhibition can activate AMPK. STZ-HFD-induced diabetic rats displayed upregulated phospho-AMPK while AMPK-2 protein was decreased. We observed that the enhanced antioxidant system by Apo and Allo inte8rvention contributed enormously not only to AMPK activation but also to protein upregulation in diabetic rats. Although oxidative stress and antioxidant interference both activate AMPK in our diabetic model, they exhibit different effects on AMPK protein expression. Our results demonstrated the increased antioxidant system rescued mitochondrial aconitase activity, which confirmed our recent study that elevated antioxidant system could accelerate ROS conversion and protect mitochondria from ROS damage [39]. We

22

propose that enhanced antioxidant system accounts for AMPK protein upregulation by preventing protein from ROS-induced protein degradation. We further explored the mechanism by which antioxidant system contributes to AMPK activation. Studies have shown that AMPK can be regulated by either increase in cellular AMP via allosteric mechanisms [40] or by ROS [33]. Our study showed that AMPK activation caused by Apo and Allo intervention was not due to lack of ATP, but was related to an increase of antioxidant system. Our results demonstrated that AMPK phosphorylation and Grxs expression are closely related. Grxs up-regulation is accompanied by an increased phosphorylation of AMPK, whereas Grxs silence leads to the loss of AMPK phosphorylation. This suggests that Grxs can directly improve AMPK activation. Furthermore, we found that ROS was involved in regulating Grxs. ROS could regulate Grxs in a bidirectional manner. ROS and antioxidant treatment can both achieve optimal ROS level. At a given level, both oxidative stress and antioxidant interference can improve Grxs and activate AMPK. We speculate that optimal ROS concomitant with Grxs are involved in AMPK activation. Grxs are small oxidoreductases of the thioredoxin family of proteins regulating the thiol redox state of many proteins [41-43]. Grxs have a unique function of catalyzing glutathionylation. This post-translational modification can lead to activation or inhibition of proteins [44, 45]. ROS has been suggested to glutathionylation and activating AMPK[25], however no literature has been reported the role of Grxs in regulating AMPK activation. Our results show that optimal H2O2 concomitant with 23

Grxs could activate AMPK through forming the S-glutathionylation cysteine residues in AMPK α subunit. We speculated that AMPK may be glutathionylated to the AMPK-S-SG active form via sulfenic acid formation (R-SOH). This glutathionylation process will not occur if AMPK is over-oxidized to R-SO2H or R-SO3H emphasizing the optimal ROS for AMPK activation(Fig.S4). Such a scenario not only protects the thiols in AMPK against further irreversible oxidation, but also promotes AMPK’s phosphorylation. Grxs

mediated-glutathionylation

is

reversible.

Deglutathionylation

or

glutathionylation in vivo has been reported to be dependent on intracellular redox state [46]. As oxidoreductases, Grxs act as GSH-dependent reductases under reducing state and as GSSG-dependent oxidases under oxidative state. Hence Grxs could maintain the redox homeostasis [47]. We believe that upregulated Grxs not only promotes AMPK’s phosphorylation but also defends against oxidative stress and maintaining cellular redox balance. This study also discovered an important role of Grxs in regulating glucose metabolism. Grxs1/2 silencing inhibited AMPK phosphorylation as well as PFK-1, PK, p-PFK2/PFK2 and phospho-glycogen synthase, indicating that Grxs regulate glycolysis,

glucose

transportation

and

glycogen

synthesis

through

an

AMPK-dependent pathway. Apo/Allo interference elevated Grx and rescued the glucose metabolism, ameliorating hyperglycemia in diabetic rats, which further confirmed the important function of Grxs in diabetes.

24

ROS has already been disclosed to contribute to AMPK activation. However the distinct mechanisms for AMPK activation by different concentrations for ROS have not addressed yet. In our present investigation, we discovered two different AMPK activation mechanisms. Except the Grxs-mediated AMPK activation under low ROS microenvironment, AMPK could be activated by AMP-dependent mechanism under higher ROS microenvironment as shown in cell experiments (Fig.5A and 5B). When cells were exposed to 1 mM H2O2, decreased ATP level significantly increased AMPK phosphorylation in a short term, whereas AMPK degradation was observed upon prolonged treatment of H2O2 (Fig.5C). This mechanism was further confirmed in vivo. As shown in STZ-induced diabetic rats (Fig.2C), STZ administration induced oxidative stress as indicated by the reduction of mitochondrial aconitase activity. Such oxidative stress impaired mitochondria and caused less ATP production, where the loss of ATP (i.e. increased AMP) activates AMPK. Therefore AMPK was activated on a STZ dose-dependent manner in the diabetic rats (Fig.1A). However accumulating ROS over a threshold level could cause protein damage. We assumed that significantly decreased AMPK 2 protein expression could be due to high level of ROS induced protein degradation in vivo. Therefore this could explain the contrary results from STZ-induced rats: Although AMPK underwent AMP-dependent activation, the overall AMPK activity was reduced due to AMPK degradation in diabetic rats. On the contrary, low ROS-induced AMPK activation is constant in parallel with the persistently expressed AMPK protein (Fig.5E). Our findings

25

highlighted that high level of ROS could instantly activate AMPK through AMP-dependent pathway. However sustained high level ROS could be harmful to both AMPK protein and activation. Our study showed that cellular ROS level dictated the way of AMPK activation either by

Grx-dependent

or

by

AMP-dependent

activation.

An

optimal

ROS

microenvironment achieved either by low dose of ROS treatment or appropriate antioxidant interference is of critical importance for AMPK activation-based diabetes treatment. As we recently disclosed that different cells held different redox homeostasis threshold [23]. To achieve maximal treatment effectiveness, it is important to administer optimal dose of antioxidant or oxidant (exercise) based on individual redox state. Our results provide insights that it is important to analyze cellular redox state before treatment, in order to secure optimal dose of interference and guide individual-based treatment for T2DM. Since Grxs-dependent AMPK activation could achieve both AMPK protein expression and activation, it could be utilized as optimal strategy in clinic for T2DM treatment to avoid the side effect derived from loss of ATP and AMPK protein. Moreover, the novel function of Grxs in regulating AMPK and glucose metabolism may provide new targets for T2DM treatment. AMPK dysregulation contributes to the onset and development of T2DM. AMPK could be activated either by Grx-dependent redox modification or by AMP-dependent allosteric activation. Grxs concomitant with optimal ROS act as an essential mediator

26

for AMPK activation. Under low ROS microenvironment, Grxs-mediated S-glutathionylation on AMPK-α catalytic subunit activates AMPK to improve glucose transportation and degradation while inhibiting glycogen synthesis. While, under high ROS microenvironment, AMPK is activated by an AMP-dependent mechanism, however sustained high level ROS also causes loss of AMPK protein.

Author contributions K.D. and M.W. carried out the major experiments; X.L. assisted with the establishment of diabetic animal models; D.Z. and Y.H. carried out the HPLC and GSH/GSSG ratio measurement; Y.W. assisted with cell culture; L.Y. contributed to discussion and reviewed the manuscript; D.S. designed and supervised experiments and wrote the manuscript. Author Disclosure Statement No competing financial interests exist for any of the authors.

Acknowledgments The authors are sincerely grateful to Dr. Xiaodong Zhang from Chengdu Vero Biotech, Miss Ava Nasrollahzadeh, from Harvard University, and Mr Mike Lin, from Medical College of Fudan University, for their proof reading. This work was supported by National Natural Science Foundation of China (31270901, 30970684). 27

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Figure legends Figure 1. Enhanced antioxidant system contributes to dramatic AMPK upregulation and activation in AMP-independent manner in diabetic rats. A and B: Protein expression of AMPK α1, AMPK α2 and p-AMPK α1/α2 in diabetic and Apo or Allo intervention rat livers. C and D: Analysis of ATP, AMP and ATP/AMP in diabetic and Apo or Allo intervention rats lives by HPLC. n=7-8 per group. *P<0.05, **P<0.01, ***P<0.001 compared with Chow group (A and C) or HFD+STZ (35mg/kg) group (B and D).

Figure 2. Inhibition of ROS production can regulate AMPK activation by 32

changing the redox state in diabetic rats . A: Protein expression of redox related proteins including GR, Grx, Trx, GPx-1, Prx-1, CAT, Mn-SOD, acetyl-Mn-SOD and Cu-Zn-SOD. B: Measurement of GSH/GSSG ratio in rat livers. C: Aconitase activity analysis in rat livers. D: A schematic diagram of the Apo/Allo elevating antioxidant system and protecting cells system. Apo and Allo treatment not only suppress the sources of superoxide anion, but also increase the antioxidant systems, thereby decreasing the hydroxyl free radical and preventing the inactivation of aconitase, and other ROS-sensitive proteins such as AMPK. n=7-8 per group. *P<0.05, **P<0.01, ***P<0.001 compared with HFD+STZ (35mg/kg) group, except in (B and C, Chow group or HFD+STZ (35mg/kg) group).

Figure 3. Grxs were involved in promoting AMPK activation. A: L02 cells were treated with doses of diamide for 1h and total proteins were extracted for AMPK α2, p-AMPK α1/α2 and Grx-1 expressions. B: L02 cells were treated with doses of H2O2 for 15 min and total proteins were extracted for Grx-1 expressions. C: L02 cells were pretreated with doses of NAC for 1h and then exposed to H2O2 (10μmol/L; 1mmol/L; respectively) for 15min and total proteins were extracted for the expressions of Grx-1 and Grx-2. D: L02 cells were pretreated with doses of DTT for 1h then exposed to diamide (1mmol/L) for 1h and total proteins were extracted for the expressions of Grx-1 and Grx-2. E: L02 cells were treated with doses of diamide for 1h and total proteins were extracted for AMPK α2, p-AMPK α1/α2 expressions. F: L02 cells were

33

pretreated with DTT (5mmol/L) for 1h then exposed to diamide (1mmol/L) for 1h and total proteins were extracted for the expressions of p-AMPK α1/α2, AMPK α2. G: Grx-1/2 siRNA were transfected to L02 cells for 48h and total proteins were extracted for protein expressions of p-AMPK α1/α2, AMPK α2, p-ACC, ACC, Grx-1 and Grx-2. H: AMPK α2 siRNA was transfected to L02 cells for 48h and total proteins were extracted for protein expressions of p-AMPK α1/α2, AMPK α2, p-ACC, ACC, Grx-1 and Grx-2. *P<0.05, **P<0.01, ***P<0.001 compared with control group (white column) in all figures.

Figure 4. Glutaredoxins activate AMPK through S-glutathionylation. A: L02 cells loaded with or without EE-GSH-biotin were incubated with/without H2O2 for 15 min, and the amounts of GSS-protein adduct formation and Grx-1 were determined using non-reducing SDS-PAGE and Western blot analysis with streptavidin-HRP. B: Cell extracts obtained under the same conditions as in A were incubated with streptavidin-agarose, and the amounts of GSS-protein conjugates of the AMPK α and phospho-Thr172-AMPKα (p-AMPK α) were determined by probing the Western blots with specific antibodies. C: L02 cells incubated with or without H2O2 for 15min for the measurement of GSH/GSSG ratio. D: L02 cells incubated with or without H2O2 for 15min for the measurement of ATP, AMP and ATP/AMP by HPLC. E: HEK 293T cells loaded with or without EE-GSH-biotin were treated with H2O2 for 20min following transfection with control or GRX siRNA. GSS-protein were purified 34

using streptavidin pull-down and then subjected to Western blot analysis using anti-AMPKα and anti-p-AMPK α antibody. *P<0.05, **P<0.01, ***P<0.001 compared with control group (white column) in all figures.

Figure 5. AMPK was instantly activated in AMP-dependent manner under high ROS microenvironment . A: 293T cells incubated with or without H2O2 for 15min and total proteins were extracted for protein expressions of p-AMPK α1/α2. B: 293T cells incubated with or without H2O2 for 15min for the measurement of ATP and ATP/AMP by HPLC. C: 293T cells incubated with H2O2 (1mmol/L) for 15min, 1h, 3h, 6h and 9h, respectively, and total proteins were extracted for protein expressions of AMPK α2 and p-AMPK α1/α2. D: 293T cells incubated with H2O2 (1mmol/L) for 15min, 1h, 3h, 6h and 9h, respectively, and for the measurement of ATP and ATP/AMP by HPLC. E: 293T cells incubated with H2O2 (0.01mmol/L) for 15min, 1h, 3h, 6h and 9h, respectively, and total proteins were extracted for protein expressions of AMPK α2 and p-AMPK α1/α2. F: 293T cells incubated with H2O2 (0.01mmol/L) for 15min, 1h, 3h, 6h and 9h, respectively, and for the measurement of ATP and ATP/AMP by HPLC. *P<0.05, **P<0.01, ***P<0.001 compared with control group (white column) in all figures.

Figure 6. Grxs promote glycolysis, improve glucose transportation and inhibit

35

glycogen synthesis via AMPK-dependent pathway. Grx-1/2 siRNA (A-E) or AMPK α2 siRNA (F-J) were transfected to L02 cells for 48h, respectively. A and F: Protein expression of PFK-1 and PK. B and G: Glycolysis rate analysis. C and H: Protein expression of p-GYS. D and I: GK, PFK-1, PK activity analysis. E and J: LDH activity analysis. K and L: Metabolic assays of ECAR (K) and OCR (L) in Grx-siRNA L02 cells using an XF24 Extracellular Flux Analyzer. *P<0.05, **P<0.01, ***P<0.001 compared with control group (Scrambled siRNA) in all figures.

Figure 7. Elevated Grxs and antioxidant system alleviate disorders of glucose metabolism thereby ameliorating diabetes. A and B: Protein expression and activity analysis of GK, PFK-1 and PK in rat livers. C: Protein expression of LDH in rat livers. D: LDH activity analysis. E: Glycolysis rate analysis. F: Protein expressions of p-GYS in rat livers. G: TEM analysis of rat livers (White arrow pointed to glycogen particles). H: A Schematic illustrating Apo and Allo rescuing the disorders of glucose metabolism in STZ-HFD induced diabetic rats. I: Postprandial blood glucose levels. n=7-8 per group. *P<0.05, **P<0.01, ***P<0.001 compared with HFD+STZ (35mg/kg) group.

Highlights: 

Glutaredoxins concomitant with optimal ROS activate AMPK through 36

S-glutathionylation 

Grxs improve glycolysis and glucose uptake while inhibiting glycogen synthesis



Different ROS levels result in distinct mechanisms for AMPK activation



Elevated Grxs and antioxidant system ameliorate hyperglycemia in diabetic rats

37

Figure 1

38

Figure 2

39

Figure 3

40

Figure 4

Figure 5 41

42

Figure 6

43

Figure 7

44

Graphical abstract

45