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Hepatic AMPK signaling activation in response to dynamic REDOX balance is a
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biomarker of exercise to improve blood glucose control
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Meiling Wu†1, Anda Zhao†1, Xingchen Yan†1, Hongyang Gao†2, Chunwang Zhang1,
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Xiaomin Liu1, Qiwen Luo1, Feizhou Xie3, Shanlin Liu‡1,4, Dongyun Shi‡1
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Affiliations
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1
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Fudan University, Shanghai, 200032, People's Republic of China
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences,
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2
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Shanghai, 200032, People's Republic of China
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3
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Shanghai, 200032, People's Republic of China
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4
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200032, People's Republic of China
Institute of Electronmicroscopy, School of Basic Medical Sciences, Fudan University,
Changning Maternity and Infant Health Hospital, East China Normal University,
Free Radical Regulation and Application Research Center of Fudan University, Shanghai,
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† Co-first authors contributed equally to this work
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‡ Correspondence to Prof. Dongyun Shi (Email: dyshi@fudan.edu.cn) and Prof. Shanlin
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Liu (Email: slliu@shmu.edu.cn), Department of Biochemistry and Molecular Biology,
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School of Basic Medical Sciences, Fudan University, NO.130 Dong’an Road, Shanghai,
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200032, People's Republic of China, Tel: +021 54237299 Fax: +021 54237897
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Abstract
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Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and
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alleviates hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve
31
diabetes. The exact redox mechanism of these two different approaches remains largely
32
unclear. Here, by comparing exercise and antioxidants intervention on type 2 diabetic rats,
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we found moderate exercise upregulated compensatory antioxidant capability and reached a
34
higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a
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low-level redox balance by inhibiting oxidative stress. Both of these two interventions
36
could promote glucose catabolism and inhibit gluconeogenesis through activation of
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hepatic AMPK signaling, therefore ameliorating diabetes. During exercise, different levels
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of ROS generated by exercise have differential regulations on the activity and expression of
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hepatic
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glutathionylation activation. However, excessive exercise increased oxidative damage and
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inhibited the activity and expression of AMPK. Overall, our results illustrate that both
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exercise and antioxidant intervention improve blood glucose in diabetes by promoting
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redox balance, despite different levels of redox balance. These results indicate that the
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AMPK signaling activation, combined with oxidative damage markers, could act as a
45
sensitive biomarker, reflecting the threshold of redox balance defining effective treatment
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in diabetes. These findings provide theoretical evidence for the precise treatment of
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diabetes by antioxidants and exercise.
AMPK.
Moderate
exercise-derived
ROS
promoted
hepatic
48
49
50
Key words
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Redox balance; AMPK signaling; exercise; antioxidant; glutathionylation; diabetes
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AMPK
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Introduction
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Diabetes mellitus is a chronic metabolic disorder disease, which has emerged as a global
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public health problem. According to the latest epidemiological data from the International
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Diabetes Federation, the global diabetes prevalence in 20–79 year olds was estimated to be
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10.5% (536.6 million people) in 2021, and is expected to rise to 12.2% (783.2 million) in
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2045 (1). With the development of genomics, proteomics and metabolomics, it has been
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discovered by many studies that type 2 diabetes is associated with irreversible risk factors
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such as age, genetics, race, and ethnicity and reversible factors such as diet, physical
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activity and lifestyle (2, 3).
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Aerobic metabolism in glucose oxidation, mitochondrial damage and oxidative stress have
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been considered to play a critical role in the occurrence and development of diabetes (4).
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Exercise and antioxidant supplements are often suggested as essential therapeutic strategies
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in the early stages of type 2 diabetes (5, 6), with different mechanisms. It has been reported
68
that chronic exercise training can alleviate oxidative stress and diabetic symptoms by
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improving cellular mitochondrial function and biogenesis in the diabetic state (7).
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Contradictorily, exercise also increases ROS production, while prolonged or high-intensity
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exercise could result in mitochondrial functional impairment to aggravate complications of
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diabetes (8). Since 1970s, studies have demonstrated that 1 hour of moderate endurance
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exercise can increase lipid peroxidation in humans (9, 10). In 1998, Ashton directly
74
detected increasing free radical levels in exercising humans using electron paramagnetic
75
resonance spectroscopy (EPR) and spin capture (11). These results led to a great deal of
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interest in the role of ROS in physical exercise (12-14). Regarding the contradiction of
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exercise on ROS scavenging or production, James D Watson also hypothesized that type 2
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diabetes is accelerated by insufficient oxidative stress rather than oxidative stress (15),
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based on the effect of exercise on diabetes treatment. Although Watson’s opinions
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supported that exercise could treat diabetes by producing ROS, whether exercise-induced
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ROS production is beneficial or detrimental to diabetes is still being debated. The specific
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regulation of ROS produced by exercise on diabetic blood glucose in vivo is unclear. In
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contrast, the general view of the antioxidant treatment for diabetes is that antioxidants
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reduce cytotoxic ROS and oxidation products, thus alleviating diabetes and achieving
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glycemic control (16). Our previous study also found that hepatic mitochondrial ROS
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scavengers and antioxidant substances inhibited the oxidative products such as MDA and 4-
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HNE in diabetic animals and favored glycemic control (17, 18). Exercise-induced oxidation
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and antioxidant administration, as two opposite approaches, could achieve the regulation of
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diabetes, respectively. However, the differences in redox mechanisms between these two
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approaches to diabetes treatment have not been fully understood.
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It is well established that the increase of skeletal muscle glucose uptake during exercise is
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crucial in glycemic control (19-21). Considering that liver is another vital organ for
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maintaining blood glucose homeostasis, including storing, utilizing and producing glucose,
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exercise-induced hepatic redox metabolism is also significant. The activation of hepatic
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AMP-activated protein kinase (AMPK), which acts as a ‘metabolic master switch’,
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alleviates diabetes symptoms by reducing glycogen synthesis, increasing glycolysis, and
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promoting glucose absorption in surrounding tissues (22). Therefore, the activation of
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AMPK in the liver is significant for regulating glucose and lipid metabolism in the blood.
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Zmijewski et al. found that AMPK could be activated by hydrogen peroxide stimulation
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through direct oxidative modification (23). In contrast, other studies suggested that
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oxidative stress could disrupt the activation of the AMPK signaling pathway (24, 25). Our
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previous study explored the mechanism by which redox status contributes to hepatic
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AMPK dynamic activation. Under a low ROS microenvironment, GRXs mediated S-
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glutathione modification activates AMPK to improve glucose utilization. In contrast, under
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an excessive ROS microenvironment, sustained high level ROS might cause loss of AMPK
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protein (26). These studies indicate that oxidative modification can directly regulate AMPK
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activity in liver cells, thus activating downstream signaling pathways to regulate glucose
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and lipid metabolism. However, it is unclear why two seemingly contradictory
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phenomenon of antioxidant intervention and exercise-induce ROS can promote AMPK
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activation. Moderate exercise has been proved significantly elevate systemic oxidative
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stress. At the same time, endogenous antioxidant defences also increased to counteract
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increased levels of ROS induced by exercise (27). Thus, we hypothesized that both
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antioxidants and exercise could reach either high-level or low-level redox balance in
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diabetic individuals. Moreover, the activity and expression of AMPK might be a marker of
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redox balance in vivo (Fig. 1).
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Hence, the present study was designed to understand the different mechanisms of exercise
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and antioxidant intervention in diabetes and verify the activation of hepatic AMPK as a
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hallmark of dynamic redox balance. Firstly, we utilized the streptozotocin-high fat diet
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(STZ-HFD) induced type 2 diabetic (T2DM) model in rats to clarify the hepatic redox
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status in T2DM rats after the exercise or antioxidant intervention. Then, according to the
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exercise intensity and mode, we divided the exercise groups into three modes and found
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that AMPK activation could serve as a biomarker of redox balance and moderate exercise
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in diabetic treatment. In this study, we found that AMPK activation and its downstream
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pathways could reflect the threshold of exercise or antioxidant administration for diabetes
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treatment. This study provides clues for the personalized treatment of diabetes by
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antioxidants and exercise.
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Results
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1. Exercise promotes antioxidant levels through producing ROS, leading to a high
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level of REDOX balance in the liver.
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To investigate the hepatic redox regulation in diabetes after exercise intervention, we
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established the T2DM rat model by feeding HFD followed by a low dose of STZ injection
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(35 mg/kg). The exercise intervention was started from Day 0 to Day 28 (Fig. 2A).
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According to previous studies, the initial speed of exercise was 15 m/min, and the speed
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was increased by 3 m/min every 5 min. After the speed reached 20 m/min, the speed was
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maintained for another 60 min with slope of 5%. The exercise intensity was 64%-76%
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VO2max (28). The low-intensity continuous exercise (CE) can be regarded as aerobic
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exercise.
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The ROS-generating NADPH oxidases (NOXs) have been recognized as one of the main
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sources of ROS production in cells (29). Cyclooxygenase 2 (COX2) activity could also act
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as a stimulus for ROS production (30). The expressions of NOX4 and COX2 in the liver
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were increased in the diabetic group. After exercise intervention, NOX4 and COX2 level
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were further up-regulated compared with the diabetic group (Fig. 2B-D). These results
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indicate the exercise intervention up-regulated ROS production.
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Next, we detected the expression of antioxidant enzymes in liver tissue. Nrf2 is the central
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regulator of the threshold mechanisms of oxidative stress and ROS generation (31). With
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the increase of ROS level in the development of diabetes, Nrf2 was activated to induce the
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transcription of several antioxidant enzymes (32, 33). We found an increase in Nrf2
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expression in diabetic rats (Fig. 2E-F). After CE intervention, the level of Nrf2 levels
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further increased, indicating that exercise intervention could activate antioxidant system
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(Fig. 2E-F). Under stress conditions, Nrf2 translocates to the nucleus and binds to
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antioxidant response elements (AREs), which results in the expression of diverse
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antioxidant and metabolic genes, such as thioredoxin (Trx), to relieve oxidative damage
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(34). Thioredoxin-1 (Trx-1), a type of cytosolic isoform of Trx, has been widely accepted
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to regulate glutathione metabolism with GRX and PRX. After CE intervention, we found
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the protein expression of GRX1 and TRX1 were up-regulated (Fig. 2H, 2J-K). Notably, the
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PRX expression also showed a trend of increase (Fig. 2H-1I). Sestrin2 is a cysteine sulfinyl
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reductase that plays crucial roles in regulation of antioxidant actions (35). As an
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endogenous antioxidant, the hepatic sestrin2 level also showed a significant increase in the
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CE group (Fig. 2E,2G).
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Since excess ROS can cause the increase of oxidative damage (36), we further detected the
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protein damage and lipid peroxidation to determine the redox status. 3-Nitrotyrosine (3-NT)
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and protein carbonylation are biomarkers of reactive nitrogen species (RNS) and ROS
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modified proteins (37, 38). We found that the CE intervention reduced the 3-Nitrotyrosine
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level and did not further decrease the protein carbonylation level (Fig. 2L-N). MDA, a
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biomarker of lipid peroxidation, was also significantly up-regulated in the diabetic group
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but decreased in exercise group (Fig. 2O). These results indicate that the high ROS
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production in the CE group could, instead, increase the antioxidant status to avoid oxidative
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damage. It suggests that CE can promote redox to reach a high level of balance. Therefore,
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even if exercise increases the ROS-generating enzymes NOX4 and COX2, the increase in
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ROS production does not lead to oxidative damage.
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2. Antioxidant intervention alleviates blood glucose through reducing oxidative stress,
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leading to a low level of REDOX balance in the liver.
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Recent studies have suggested that NADPH oxidase is one of the primary sources of ROS
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(29, 39). Apocynin has already been characterized as an NADPH oxidase inhibitor in the
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early 1980s, and it can also act as an antioxidant (40). Our previous study showed that
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apocynin intervention alleviated blood glucose by inhibiting oxidative products. In this
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study, the antioxidant supplement was also started from Day 0 to Day 28 in this study (Fig.
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3A). We found that apocynin supplement decreased the protein carbonylation level and
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MDA level in the liver (Fig. 3B-C). Also, the TAOC level was increased after apocynin
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treatment, indicating the decrease of oxidation level (Fig. 3D). Moreover, as endogenous
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antioxidant, the Sestrin2 and Nrf2 expression decreased after apocynin intervention (Fig.
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3E-G). These results indicate that the antioxidant intervention reduced the ROS in diabetic
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hepatocytes, thereby decreasing the ROS-induced compensatory upregulation of Sestrin2
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and Nrf2.
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Consistently, Glut2, a glucose sensor in the liver, was increased in diabetic liver and
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decreased after the apocynin supplement (Fig. 3E and 3H). The postprandial blood glucose
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, fasting blood glucose and oral glucose tolerance (2 h after oral glucose, OGTT) were
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decreased in the apocynin intervention group compared with the diabetic rat group (Fig. 3I-
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K). Consistent with the apocynin intervention group, the exercise group also showed lower
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postprandial blood glucose and fasting blood glucose levels and OGTT (Fig. 3I-K). These
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studies indicate that the apocynin treatment improved the diabetes through inhibiting ROS
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level and protein oxidative damage to achieve a low-level redox balance.
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3.
Moderate
exercise-generated
ROS
promotes
activation
of
AMPK
by
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phosphorylation and reduces blood glucose level, while excessive exercise- generated
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oxidative stress reduces AMPK expression and exacerbates diabetes.
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In order to find out the biomarkers that could reflect moderate exercise to improve blood
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glucose control, diabetic rats were divided into short-term continuous exercise (CE),
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intermittent exercise (IE), and excessive exercise (EE) according to the exercise intensity
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and mode (28). We found that the random blood glucose and 2h OGTT in CE and IE
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treated diabetic rats were decreased (Fig. 4A-B). In contrast, EE intervention did not
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improve blood glucose but slightly increased random and 2h OGTT (Fig. 4A-B).
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We detected the increase of ROS production-related enzymes, such as NOX4 and COX2 in
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the EE group, indicating the highest oxidation level (Fig. 4C-E). Next, we detected the
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expression of antioxidant enzymes and oxidative damage in the liver tissue of exercise-
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treated T2D rats. As shown in Fig. 4F-G, IE intervention increased the activity of MnSOD
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as shown by decreased level of acetylation compared with the diabetic rats. The expression
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of GRX and TRX were up-regulated after CE intervention (Fig. 4F, 4H-I). Furthermore, we
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detected the oxidative damage in these three modes. The results showed that the CE and IE
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group did not obviously change the protein carbonylation level. However, the EE
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intervention promoted the protein carbonylation in the liver, indicating this mode of action
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is not due to free radical scavenging but oxidative damage (Fig. 4J). In addition to the
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protein damage, hepatic MDA concentration showed significant up-regulation in the
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diabetic group but decreased in CE and IE group (Fig. 4K), while increased MDA in the EE
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group indicates oxidative damage. Among these three exercise modes, the IE group showed
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the lowest level of oxidation (the minor increase in NOX4 and a slight decrease in
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carbonylation). Although the levels of antioxidant enzymes such as GRX and TRX did not
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increase, the activity of MnSOD also increased significantly (Fig. 4F-4G). The reduction of
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MDA level also indicates IE group did not form oxidative damage (Fig. 4K), indicating the
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IE group could also maintain a relatively high level of redox balance. Nevertheless, the
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decrease of antioxidant enzymes and increase of oxidative damage in the EE group
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indicates that the REDOX balance in the EE group was disrupted.
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Notably, the phosphorylation of AMPK showed different patterns in three kinds of
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exercise, among which both CE and IE intervention could promote the phosphorylation of
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AMPK compared to the diabetic rats (Fig. 4M-N). EE intervention did not increase the
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content of AMPK phosphorylation, which might be caused by the reduction of AMPK
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level. Meanwhile, the ratio of AMP to ATP was detected, and exercise-activated AMPK
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did not exhibit AMP-dependent characteristics at this time (Fig. 4L). These results suggest
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that moderate exercise-generated ROS may directly promote AMPK activation by
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phosphorylation without AMP upregulation and reduce blood and liver glucose levels.
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However, excessive exercise-generated oxidative stress reduces AMPK expression and
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exacerbates diabetes.
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4. Moderate exercise promoted glycolysis and mitochondrial tricarboxylic acid cycle
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and inhibited the gluconeogenesis in the liver of diabetic rats.
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Next, we further explored the mechanism by which inhibiting blood glucose during CE and
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IE intervention. Fructose-2,6-diphosphate (F-2,6-P2; also known as F-2,6-BP), which is a
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product of the bifunctional enzyme 6-phosphofructose 2-kinase/fructose 2,6-diphosphatase
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2 (PFK/FBPase 2, also known as PFKFB2), is a potent regulator of glycolytic and
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gluconeogenic flux. The phospho-PFKFB2 to PFKFB2 ratio represents the glycolytic rate.
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A high ratio of phospho-PFKFB2:PFKFB2 leads to an increase in the F-2,6-P2 level and
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the allosteric activation of phosphor-fructose kinase 1 (PFK1), while a low ratio leads to a
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decrease in F-2,6-P2 and an increase in gluconeogenesis (41). The overexpression of
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bifunctional enzymes in mouse liver can reduce blood glucose levels by inhibiting hepatic
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glucose production (42). Therefore, bifunctional enzymes are also a potential target for
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reducing hepatic glucose production. In our study, the p-PFK2:PFK2 ratio decreased in the
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diabetic rats but was enhanced by CE and IE intervention (Fig. 5A-C), suggesting that CE
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and IE could reverse gluconeogenesis to glycolysis by enhancing PFK/FBPase. Meanwhile,
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the substrates of the glycolytic pathway (such as DHAP, Fig. 5D) and the tricarboxylic acid
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cycle (such as citrate, succinate and malate, Fig. 5D) showed an upward trend.
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Besides glycolysis, gluconeogenesis are critical in maintaining liver and blood glucose
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homeostasis. FoxO1 has been tightly linked with hepatic gluconeogenesis through
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inhibiting the transcription of gluconeogenesis-related PEPCK and G6Pase expression (43,
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44). Herein, we found the expression of FoxO1 was increased in the diabetic group but
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reduced in the CE and IE groups (Fig. 5E-F). Meanwhile, the mRNA level of Pepck and
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G6PC also decreased in the CE and IE groups (Fig. 5H-I). These results indicate that
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moderate exercise (CE and IE) inhibited gluconeogenesis through down-regulating FoxO1.
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For the glucose uptake, we detected the protein expression of GLUT2 in the liver tissue,
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which helps in the uptake of glucose by the hepatocytes for glycolysis and glycogenesis.
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We found the level of GLUT2 was increased in diabetic mice, but down-regulated by the
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CE and IE intervention (Fig. 5E and 5G). Taken together, these results illustrated that
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moderate exercise promoted glucose transport, glucose catabolism and inhibited the
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gluconeogenesis in the liver of diabetic rats (Fig. 5J).
269
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5. Moderate exercise inhibited hepatic mitophagy, while excessive exercise exhibited
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opposite effect and inhibited the mitochondrial biogenesis.
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The electron transport associated with the mitochondrial function is considered the major
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process leading to ROS production during exercise (45). To further explore the downstream
274
signal of AMPK activation in moderate and excessive exercise, we detected the protein
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expression of mitochondrial dynamic and mitochondrial biogenesis. According to the result
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in Fig. 6A-E, we found that the mitochondrial fusion protein MFN was significantly
277
decreased in the liver of the excessive exercise group, and the mitochondrial fission protein
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(Fis) and autophagy-related protein ATG5 and LC3B did not change, compared with the
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diabetic group. Notably, the ATG5 and LC3B levels decreased in the CE and IE group,
280
compared with the diabetic group (Fig. 6A-E). Since PGC-1α is a transcriptional
281
coactivator, a central inducer of mitochondrial biogenesis in cells (46), we measured the
282
expression of PGC-1α and found that its expression was increased in CE and IE group, but
283
not in the EE group (Fig. 6F).
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These results indicates that moderate exercise promoted mitochondrial biogenesis and
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ameliorated autophagy in the liver. However, excessive exercise aggravated mitochondrial
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fission and did not alleviate autophagy. Similarly, the mitochondria structure of the live
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tissue in the EE group was fragmented and showed greatly diminished cristae and swelling
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matrix under transmission electron microscopy, reflecting a defect in oxidative
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phosphorylation. However, the CE and IE group showed increased numbers of cristae and a
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clear structure of mitochondrial cristae (Fig. 6H). These results all showed that the in vivo
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mitochondrial ROS burst caused by excessive exercise inhibited the expression of AMPK
292
and promoted mitophagy. The damage to the mitochondrial dynamics and structure in liver
293
tissue led to abnormal aerobic oxidation, thereby aggravating diabetes.
294
6. Moderate ROS activates AMPK through GRX-mediated glutathionylation
295
To further illustrate the effect of moderate ROS on the activation of AMPK, primary
296
hepatocytes were intervened with H2O2 to mimic ROS production in vivo. H2DCFDA and
297
dihydroethidium dyes were used to assess intracellular ROS levels in the H2O2-treated
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primary hepatocytes. As shown in Fig. 7A-7B, the levels of ROS (DCF), such as H2O2, and
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O2•− accumulation (dihydroethidium) were dose-dependently increased in the primary
300
hepatocytes treated with 50-200 μM H2O2. Consistent with our previous study (26), we
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found moderate ROS would activate AMPK through GRX-mediated S-glutathionylation.
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As shown in Fig. 7C, exposure to 50 and 100 μmol/l H2O2 led to an increase of GSS-
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protein adduct, concomitant with the AMPK phosphorylation (Fig. 7C-F), suggesting that
304
the ROS level within redox balance threshold could induce glutathionylation and
305
phosphorylation of AMPK to activate AMPK. However, when the concentration of ROS
306
was higher (200 μmol/l H2O2), both of AMPK glutathionylation and phosphorylation were
307
decreased (Fig. 7E-7H). These results indicates that activation of AMPK by moderate ROS
308
might be mediated through GRX-mediated S-glutathionylation.
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Meanwhile, we also detected the substrates of glycolysis and aerobic oxidation at different
310
concentrations of H2O2. We found the exposure to 20-100 μmol/l H2O2, which made cells
311
within the redox balance threshold, showed a trend of increase on glycolysis and aerobic
312
oxidation substrates, indicating the increase of hepatic glucose catabolism (data not shown).
313
314
Discussion
315
316
It has been shown that both antioxidants and exercise can be beneficial in substantially
317
ameliorating hyperglycaemia through ROS-mediated mechanisms in diabetes patients.
318
However, antioxidant intervention reduces oxidative stress, while exercise produces ROS.
319
It is imperative to explore the mechanisms underlying these seemingly paradoxical
320
approaches for effective diabetes treatment.
321
The remission of diabetes by antioxidant intervention has been well documented. Some
322
compounds in food that have substantial antioxidant activities or inhibit NADPH oxidase,
323
such as polyphenols and flavonoids (47), have been shown to improve blood glucose and
324
relieve type 2 diabetes in animal experiments. Several clinical trials also demonstrated the
325
relief of diabetic hyperglycaemia by antioxidants (48, 49). Our previous study found that
326
hepatic mitochondrial ROS scavenger and antioxidant substances inhibited the oxidative
327
products such as MDA and 4-HNE in diabetic mice and rats and improved blood glucose
328
control (18). These results indicate that reducing the oxidative level of diabetic animals
329
could treat diabetes. Hepatic AMPK regulates cellular and whole-body energy homeostasis,
330
signals to stimulate glucose uptake in skeletal muscles, fatty acid oxidation in adipose and
331
other tissues, and reduces hepatic glucose production (50, 51). Numerous pharmacological
332
agents, including the first-line oral drug metformin, natural compounds, and hormones are
333
known to activate AMPK (52-55). Moreover, our previous study found that antioxidant
334
intervention in diabetic rats could promote the phosphorylation and activation of AMPK
335
protein, thereby regulating hepatic glucose metabolism (56). Taken together, we found that
336
the activation of AMPK by antioxidant intervention was accompanied by a decrease in
337
oxidative stress level in diabetic rats, resulting in a low level of redox balance to benefit
338
diabetic hyperglycaemia.
339
In the meaning time, regular exercise have been recommended to mitigate symptoms of
340
many diseases, including psychiatric, neurological, metabolic, cardiovascular, pulmonary,
341
musculoskeletal, and even cancer (57). John Holloszy’s studies found that exercise
342
improved insulin sensitivity in patients with type 2 diabetes and provided a better
343
understanding of how muscle adapts to endurance exercise (19-21, 58-60). Although the
344
benefits of exercise are irrefutable, excessive exercise is harmful (8), suggesting the
345
importance of the amount and intensity of exercise. Recently, Chrysovalantou et al. found
346
that NADPH oxidase 4 (NOX4) is a crucial exercise-related protein to regulate adaptive
347
responses and prevent insulin resistance (61). We found that exercise could indeed increase
348
NOX4 expression, but NOX4 was also upregulated in excessive exercise. Although
349
Chrysovalantou's study highlights the role of the redox environment in exercise, the
350
biomarkers of effective intervention of moderate exercise on diabetes remain unclear.
351
352
The mechanisms of exercise for the treatment of diabetes have previously been studied
353
mainly around the skeletal muscle, as the activation of AMPK in skeletal muscle during
354
exercise is considered mainly caused by the increase of intracellular AMP:ATP ratio and
355
phosphorylation of Thr172 on the “activation loop”7 of the α-subunit (62). The activation
356
of AMPK leads to the inhibition of mTORC1 activity and activation of PGC-1α, which
357
enhances mitochondrial biogenesis and further increases muscle uptake of glucose from the
358
blood (63). However, liver energy state also plays an essential role in the activation of
359
AMPK (64) and liver is known to be critical in whole-body glucose tolerance (65). In
360
addition, AMPK is also considered as a redox-sensitive protein, and its cysteine 299 and
361
304 sites are likely to be regulated by the oxidation of hydrogen peroxide (23, 66, 67).
362
Thus, AMPK might be activated not only by the increase of AMP, but also by
363
phosphorylation through ROS regulation during exercise.
364
365
Currently there is no appropriate biomarkers to differentiate moderate and excessive
366
exercises. According to the exercise intensity and mode, we divided the exercise groups
367
into three modes: CE, IE for moderate exercises and EE for excessive exercise. Our study,
368
for the first time, found that hepatic AMPK activation could act as a biomarker of dynamic
369
redox balance during exercise to improve glycaemic control in diabetic rats. ROS generated
370
by different exercise intensities could profoundly alter the cellular redox microenvironment
371
and directly regulate the activity and expression of hepatic AMPK through a redox-related
372
mechanism. Moderate exercise produced optimal ROS directly promoted AMPK activation
373
via glutathionylation in hepatocytes. The activated AMPK signaling pathway can
374
phosphorylate and activate PFK-2 to promote glycolysis and aerobic oxidation (Fig. 8A).
375
Furthermore, AMPK activation can phosphorylate and inhibit the CRCT2 and class IIa
376
HDACs pathways in the liver, thus affecting the binding of class IIa HDACs to the FOXO-
377
family of transcription factors (68, 69). Since the gluconeogenesis related mRNA
378
expression of PEPCK and G6Pase was mainly transcribed by FOXO1, the reduction of
379
FOXO transcriptional activity induced by AMPK indicates the inhibition of
380
gluconeogenesis. Therefore, under moderate exercise, the metabolic balance between
381
glycolysis and gluconeogenesis is regulated through the activation of AMPK
382
phosphorylation, which promotes glucose catabolism and inhibits gluconeogenesis.
383
However, excessive ROS inhibits the activity and expression of AMPK in hepatocytes
384
cells, which might be related with the oxidative stress induced protein degradation (Fig.
385
8A). In addition, the number of mitochondria and the function of aerobic oxidation in the
386
EE group was significantly lower than those in the moderate exercise group. In the EE
387
group, the autophagy and fission of liver mitochondria were also up-regulated as compared
388
with the moderate exercise，accompanied by the increase of MDA, an indicator of lipid
389
peroxidation damage. These results indicate that AMPK signaling and oxidative damage-
390
related index could be investigated as sensitive biomarkers for redox status changes during
391
exercise intervention in diabetic rats. This finding can be applied to analyze the thresholds
392
of redox balance that discriminates moderate and excessive exercise.
393
394
Exercise is considered to improve blood glucose by promoting ROS levels, which seems to
395
be contradictory to antioxidant interventions of inhibiting ROS. Zsolt Radak et al proposed
396
a bell-shaped dose-response curve between normal physiological function and level of ROS
397
in healthy individuals, and suggested that moderate exercise can extend or stretch the levels
398
of ROS while increases the physiological function (70). Our results validated this
399
hypothesis and further proposed that moderate exercise could produce ROS meanwhile
400
increase antioxidant enzyme activity to maintain high level redox balance according to the
401
bell-shaped curve, whereas excessive exercise would generate a higher level of ROS,
402
leading to reduced physiological function. In this study, we found the state of diabetic
403
individuals is more applicable to the description of a S-shaped curve, due to the high level
404
of oxidative stress and decreased reduction level in diabetic individuals (Fig.8B). With the
405
increase of ROS, the physiological function of diabetic individuals gradually decreases.
406
Moderate exercise shifts the S-shaped curve into a bell-shaped dose-response curve, thus
407
reducing the sensitivity to oxidative stress in diabetic individuals and restoring redox
408
homeostasis. However, with excessive exercise, ROS production increases beyond the
409
threshold range of redox balance, resulting in decreased physiological function (Fig.8B, see
410
the decreasing portion of the bell curve to the right of the apex).
411
412
Nevertheless, the antioxidant intervention increased physiological activity by reducing ROS
413
levels in diabetic individuals, restoring a bell-shaped dose-response curve at low level of
414
ROS (Fig.8B). Therefore, redox balance could be achieved either at low level of ROS
415
mediated by antioxidant intervention or at high level of ROS mediated by moderate
416
exercise, both of which were regulated by AMPK activation. Therefore, both high and low
417
levels of redox balance can lead to high physiological function as long as they are in the
418
redox balance threshold range. Then, the activation of AMPK is an important sign of
419
exercise or antioxidant intervention to obtain redox dynamic balance which helps restore
420
physiological function. Accordingly, we speculate that the antioxidant intervention based
421
on moderate exercise might offset the effect of exercise, but antioxidants could be
422
beneficial during excessive exercise. The human study also supports that supplementation
423
with antioxidants may preclude the health-promoting effects of exercise (71). Therefore,
424
personalized intervention with respect to redox balance will be crucial for the effective
425
treatment of diabetes patients.
426
427
Together, our study revealed the mechanisms underlying seemingly contradictory effects of
428
level of ROS by either antioxidant intervention or moderate exercise for the treatment of
429
diabetes. Moderate exercise promoted hepatic ROS production and up-regulated
430
antioxidant capability, achieving a high-level balance of redox state. In contrast, antioxidant
431
intervention scavenged the hepatic free radical to form a delicate low-level balance of
432
redox state. Moreover, excessive exercise led to redox imbalance due to excess ROS levels.
433
Hepatic AMPK signaling activation could act as a sign and hallmark of moderate exercise
434
and dynamic redox balance to guide appropriate exercise or antioxidant intervention. These
435
results illustrate that it is necessary to develop a moderate exercise program according to
436
the REDOX microenvironment of diabetes patients and provide theoretical evidence for the
437
precise treatment of diabetes by antioxidants and exercise.
438
439
Materials and Methods
440
441
1. Materials
442
T-AOC kit was supplied by Changzhou Redox Biological Technology Corporation
443
(Jiangsu , CN). Antibodies against Actin, Acetylated-Lysine, P-PFK2, Ace-SOD2, ATG5,
444
LC3A/B, GAPDH, MFN1 and IgG-HRP were purchased from Cell Signaling Technology
445
(USA). Antibodies against CAT, PRX1, AMPKa1, GRX1, GRX2, SOD2, HSP90, COX1,
446
COX2 and PFK2 were purchased from ProteinTech (Wuhan, CN). Antibodies against 3-
447
NT, 4HNE, NOX4 and PGC-1α were purchased from Abcam. Antibodies against p-
448
AMPKα1/α2 were purchased from SAB (Signalway Antibody, USA). The detailed
449
antibody information is shown in Supplementary file 1a. High-fat diet (HFD) were
450
purchased from Shanghai SLRC laboratory animal Company Ltd (Shanghai, China) and the
451
nutritional composition is shown in Supplementary Table S2.
452
453
2. Animal
454
Male SD rats (150–160 g body weight, 6-8 weeks) were purchased from Fudan University
455
Animal Center (Shanghai, China). Normal chow and high-fat diet (HFD) were purchased
456
from Shanghai SLRC laboratory animal Company Ltd (Shanghai, China) and the
457
nutritional composition was shown in Supplementary file 1b. All animal care and
458
experimental procedures were approved by the Fudan University Institutional Laboratory
459
Animal Ethics Committee (NO. 20170223-123). Animals were housed in a pathogen free
460
environment with 12 h dark/light cycles.
461
462
3. Establishment of diabetic rat model
463
Rats were divided into six groups in a non-blinded, randomized manner: Control (Ctl),
464
STZ+HFD diabetic rat (T2D), Continuous exercise+STZ+HFD diabetic rat (T2D+CE),
465
intermittent exercise+STZ+HFD diabetic rat (T2D+IE), excessive exercise+STZ+HFD
466
diabetic rat (T2D+EE) and Apocynin+STZ+HFD diabetic rat (T2D+APO) (n=8 per group).
467
The sample size was calculated according to the Power Curve. The diabetic rats model was
468
established by 12hr-fasting followed by intraperitoneal injection of 0.1 M streptozotocin
469
(STZ) citrate solution (pH 4.5) at a dose of 35 mg/kg for Day1, and 35 mg/kg for Day 2 at
470
the 5th week. The HFD was started from the 1st week to the 8th week. After 8 weeks of
471
intervention, the mice were sacrificed. The tissues and plasma were collected and preserved
472
at −80 °C for further analysis.
473
Rat were acclimated to treadmill running for 3 days before the initiation of the experiments
474
and the exercise training intervention was continued for 4 weeks (5 times per week). All
475
animals were randomized before the initiation of exercise tests.
476
Continuous exercise:
477
The initial speed was 15 m/min, and the speed was increased by 3 m/min every 5 min.
478
After the speed reached 20 m/min, the speed was maintained for another 60 min with slope
479
of 5%. The exercise intensity was 64%-76% VO2max (28, 72, 73).
480
Intermittent exercise:
481
The initial speed was 15 m/min, and the speed was increased by 3 m/min every 5 min.
482
After the speed reached 20 m/min, the speed was maintained for 20 min and then 5 min rest
483
at 5 m/min. The training was continued for 3 times, and the total running time is 60 min
484
with two 5 min rest with slope of 5%.
485
Excessive exercise:
486
The initial speed was 15 m/min, and the speed was increased by 3 m/min every 5 min.
487
After the speed reached 50 m/min, the speed was maintained for another 60 min with slope
488
of 5%. The exercise intensity was higher than 80% VO2max.
489
OGTT was performed in the fasting mice with intraperitoneal injection of glucose at 1 g/kg
490
of body weight, and glucose was measured at 15min, 30 min, 60 min and 120 min,
491
respectively. Blood glucose was determined by glucometer (Roche, Switzerland).
492
493
4. Cell culture
494
Normal Human Hepatic Cell Line L02 cells (Cell Bank of Chinese Academy of Sciences)
495
were grown in DMEM supplemented with 10% FBS (GIBCO, USA) in a humidified
496
incubator (Forma Scientific) at 37 °C and 5% CO2 as described previously. The medium
497
were supplemented with 10% FBS (GIBCO, USA), 2 mmol/l glutamine, 1 mmol/l sodium
498
pyruvate, 10 mmol/l HEPES, 50 μmol/l β-mercaptoethanol, 105 U/l penicillin and
499
streptomycin. Glutamine and sodium pyruvate were purchased from Sinopharm Chemical
500
Reagent Co., Ltd, HEPES were purchased from Beyotime Biotechnology (Shanghai, CN).
501
All cell lines used in the study were tested for mycoplasma and were STR profiled.
502
503
5. Flow cytometry
504
For measurement of intracellular Superoxide, primary hepatocytes were stained with 5 μM
505
hydroethidine (superoxide indicator) (Thermo Fisher Scientific, USA). Stained cells were
506
analyzed with NovoCyte Quanteon flow cytometer (Agilent Technologies, Inc.), and
507
acquired data were analyzed with NovoExpress software (Agilent Technologies, Inc.) and
508
FlowJo software (TreeStar, Ashland, OR).
509
510
6. ATP and AMP content analysis
511
Liver tissue (20–30 mg) were homogenized on ice by perchloric acid. Homogenized
512
samples were centrifuged for 12,000 rpm at 4 °C (30 min). Supernatant was then
513
neutralized with 4 M K2CO3, followed by further centrifugation for 12,000 rpm at 4 °C for
514
20 min. Supernatant was obtained for the determination of ATP and AMP content by high
515
performance liquid chromatography (HPLC). The detection wavelength was 254 nm.
516
517
7. SOD activity assay
518
SOD activity assay was carried out using a chemiluminometric detector (Lumat LB9507,
519
Berthold). Superoxide anions were generated by adding xanthine oxidase (XO) into the
520
reaction system consisting of xanthine and Lucigenin. The drop of luminescence within
521
2 min was recorded as the relative SOD activity.
522
523
8. MDA content assay
524
MDA from the oxidative polyunsaturated fatty acids (PUFA) degradation is determined by
525
the reaction of thiobarbituric acid (TBA) with MDA to generate the stable end product of
526
MDA-TBA adduct. Liver tissue lysis was reacted with TBA to form a red product which
527
can be detected using fluorometric (Ex/Em: 532/553 nm) plate reader.
528
529
9. S-Glutathionylation of AMPK and detection of GSS-AMPK adduct formation
530
GSS-AMPK adduct were measured as described previously (23, 26). Primary hepatocytes
531
(2×106 cell/well) were incubated with ethyl ester GSH-biotin (6 mM) for 1 h. Cells were
532
then washed twice with culture buffer to remove the excess of GSH and treated with H2O2
533
for 30 min. Cell lysates were prepared in the presence of N-ethylmaleimide (5 mM) and
534
then passed through Bio-Gel P10 to remove free GSH-biotin and N-ethylmaleimide. The
535
level of GSS-protein conjugates was determined using non-reducing Western blot analysis
536
with streptavidin-HRP, whereas GSS-AMPK subunit levels were measured after pull-down
537
with streptavidin-agarose (60 min at 4 °C), followed by reducing SDS-PAGE and Western
538
blot analysis with antibodies against AMPK α subunit.
539
540
10. Metabolite profiling detection
541
Cellular metabolites were extracted and analysed by LC-MS/MS. Ferulic acid was added as
542
an internal standard to metabolite extracts, and metabolite abundance was expressed
543
relative to the internal standard and normalized to cell number. Mass isotopomer
544
distribution was determined by LC-MS/MS (AB SCIEX Triple-TOF 4600) with selective
545
reaction monitoring (SRM) in positive/negative mode
546
547
11. Western blot analysis
548
Cells and tissues were lysed in a buffer containing 1% Nonidet P-40, 0.25% sodium
549
deoxycholate, 150 mmol/l NaCl, 10 mmol/l Tris, 1 mmol/l EGTA, 1% proteinase and
550
phosphatase inhibitor cocktails (Sigma-Aldrich) at 4 °C for 30 min. Cell lysates were
551
resolved by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis,
552
transferred to polyvinylidene fluoride (PVDF) membranes, and immunoblotted with
553
primary antibodies. Membranes were incubated with HRP-conjugated secondary antibodies
554
and visualized using chemiluminescent substrate (ECL; Tanon, CN) and Tanon-5200
555
Chemiluminesent Imaging System (Tanon, CN).
556
557
12. Transmission electron microscope (TEM)
558
Rat liver tissue (1 mm*1 mm) were fixed by paraformaldehyde. The samples were
559
examined with a Jeol Jem-100SV electron microscope (Japan) which was operated at 80
560
Kv after fixed by 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) at Institute of
561
Electron microscopy, Shanghai Medical College of Fudan University.
562
563
13. Western blot analysis
564
Cells were lysed RIPA buffer with phosphatase inhibitor at 4 °C for 30 min. Cell lysates
565
were resolved by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF)
566
membranes, and probed with primary antibodies. Membranes were incubated with
567
peroxidase-conjugated secondary antibodies and visualized using a chemiluminescent
568
substrate
569
Chemiluminesent Imaging System.
(ECL;
GE
Amersham
Pharmacia,
Beijing,
China)
and
Tanon-5200
570
571
14. Statistics
572
The experimental data were expressed as mean ± SEM. One-way ANOVA was used to
573
compare among groups. Data analysis was conducted by Graphpad prism 9 statistical
574
analysis software. p < 0.05 was considered statistically significant. Data are expressed as
575
means ± SEM; n = 3 for cells experiment (n = 3 represents three times of individual
576
experiment); n = 8 for animal experiment.
577
578
Acknowledgments
579
The authors thank Dr. Xiaodong Zhang from Chengdu Brilliant Pharmaceuticals for his
580
proof reading and editing of the manuscript. The authors are also indebted to Dr. Rutan
581
Zhang and Prof. Liang Qiao from Fudan University, for analysis of LC-MS/MS data. The
582
authors thank Dr. Ziqing Tang, Mr. Qi Kang, Dr. Xiaomin Liu, Mr. Yipei He, Dr. Xiao
583
Zhang, Mrs Lihan Jiang, Mr. Kelei Dong for their assistance in animal experiments. The
584
authors are also indebted to Institute of Electronmicroscopy from Fudan University, for the
585
help on electronmicroscopy analysis.
586
This work was supported by grants from the National Natural Science Foundation of China
587
(Grants No. 31770916).
588
589
Competing interests:
590
All other authors declare they have no competing interests
591
592
Data and materials availability:
593
All data are available in the main text or the supplementary materials.
594
595
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by Thioredoxin1 during energy starvation. Cell Metab 19, 232-245 (2014).
M. M. Mihaylova, R. J. Shaw, The AMPK signalling pathway coordinates cell
growth, autophagy and metabolism. Nat Cell Biol 13, 1016-1023 (2011).
M. M. Mihaylova et al., Class IIa histone deacetylases are hormone-activated
regulators of FOXO and mammalian glucose homeostasis. Cell 145, 607-621
(2011).
Z. Radak et al., Exercise, oxidants, and antioxidants change the shape of the bellshaped hormesis curve. Redox Biol 12, 285-290 (2017).
M. Ristow et al., Antioxidants prevent health-promoting effects of physical exercise
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T. G. Bedford, C. M. Tipton, N. C. Wilson, R. A. Oppliger, C. V. Gisolfi,
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R. E. Shepherd, P. D. Gollnick, Oxygen uptake of rats at different work intensities.
Pflugers Arch 362, 219-222 (1976).
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Figure legend:
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Fig. 1 A model of the different mechanisms of exercise and antioxidant intervention in
diabetes.
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A graphical abstract of this study. Moderate exercise upregulated compensatory antioxidant
capability and reached a high-level redox balance, whereas antioxidant intervention
achieved a low-level redox balance by inhibiting oxidative stress for treating diabetes.
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Fig. 2 Moderate exercise induced ROS production in exercise group and increased the
antioxidant status.
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A. Experimental design. T2DM rats model was fed by high-fat diet plus a low dose of STZ
injection (35 mg/kg). The high-fat diet (HFD, 60% calories from fat) was started from the
1st week to the 8th week. The exercise intervention was started from 1st week to 4th week. BD. Representative protein level and quantitative analysis of NOX4 (27 kDa), COX2 (17
kDa) and Actin (45 kDa) in the rats in the Ctl, T2D and T2D + CE groups. E-G.
Representative protein level and quantitative analysis of Nrf2(97 kDa), Sestrin2 (56 kDa)
and Actin (45 kDa) in the rats in the Ctl, T2D and T2D + CE groups. H-K. Representative
protein level and quantitative analysis of PRX1 (27 kDa), Grx1 (17 kDa), Trx1 (12 kDa)
and Actin (45 kDa) in the rats in the Ctl, T2D and T2D + CE groups. The rat livers were
homogenized by 1% SDS and analyzed by Western blots with the appropriate antibodies.
L-M. Representative protein level and quantitative analysis of 3-NT and Actin (45 kDa) in
the rat in the Ctl, T2D and T2D + CE groups. N-O. Liver protein carbonylation (O) and
MDA content (P) level was detected in the rats of Ctl, T2D, T2D + CE groups. (ns: not
significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with all groups
by one-way ANOVA and Tukey’s post hoc test; data are expressed as the mean ± SEM; n =
4-8 per group).
804
Figure 2_source data_01
805
806
Figure 2_source data_02
807
808
Fig. 3 Antioxidant intervention alleviates blood glucose through promoting the
upregulation of reducing levels
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820
A. Experimental design. T2DM rats model was fed by high-fat diet plus a low dose of STZ
injection (35 mg/kg). The apocynin intervention was started from 1st week to 4th week. B-E.
Liver protein carbonylation, MDA content (C) and TAOC (D) level were detected in the
rats of Ctl, T2D and T2D + APO groups. E-H. Representative protein level and quantitative
analysis of Nrf2 (97 kDa), Sestrin2 (57 kDa), Glut2 (60-70 kDa) and HSP90 (90 kDa) in
the rats in the Ctl, T2D and T2D + APO groups. I. Postprandial blood glucose levels of Ctl,
T2D, T2D + CE and T2D + APO groups at the end of 8th week. J. Fasting blood glucose
levels of Ctl, T2D, T2D + CE and T2D + APO groups at the end of 8th week. K. Blood
glucose level after oral glucose administration (0 min, 60min and 120 min) in Ctl, T2D,
T2D + CE and T2D + APO groups at the end of 8th week (*P < 0.05, **P < 0.01, ***P <
0.001, ****P < 0.0001 compared with all groups by one-way ANOVA and Tukey’s post
hoc test; data are expressed as the mean ± SEM; n = 4-8 per group).
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Figure 3_source data_01
822
Figure 3_source data_02
823
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825
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Fig. 4 Moderate exercise-generated ROS promotes activation of AMPK by
phosphorylation and reduces blood glucose level, while excessive exercise- generated
oxidative stress reduces AMPK expression and exacerbates diabetes.
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A. Postprandial blood glucose levels of Ctl, T2D, T2D + CE, T2D + IE and T2D + EE
groups at the end of 8th week. B. Blood glucose level after oral glucose administration in
Ctl, T2D, T2D + CE, T2D + IE and T2D + EE groups at the end of 8th week. C-E.
Representative protein level and quantitative analysis of NOX4 (27 kDa), COX2 (17 kDa)
and HSP90 (90 kDa) in the rats in the Ctl, T2D, T2D + CE, T2D + IE and T2D + EE
groups. F-J. Representative protein level and quantitative analysis of Ace-SOD2 (27 kDa),
SOD2 (17 kDa), Grx1 (17 kDa), Trx1 (12 kDa) and Actin (45 kDa) in the rats in the Ctl,
T2D, T2D + CE, T2D + IE and T2D + EE groups. K-L. Liver protein carbonylation
content (k), liver MDA content (L) and AMP/ATP ratio (M) were detected in the rats of
Ctl, T2D, T2D + CE, T2D + IE and T2D + EE groups. N-O. Representative protein level
and quantitative analysis of P-AMPK (67 kDa), AMPK (67 kDa) and Actin (45 kDa) in the
rats in the Ctl, T2D, T2D + CE, T2D + IE and T2D + EE groups. (ns: not significant; *P <
0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with all groups by one-way
ANOVA and Tukey’s post hoc test; data are expressed as the mean ± SEM; n = 4-8 per
group).
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Figure 4_source data_01
844
Figure 4_source data_02
845
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Fig. 5 Moderate exercise promoted glycolysis and mitochondrial tricarboxylic acid
cycle and inhibited the gluconeogenesis in the liver of diabetic rats.
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A-B. Representative protein level and quantitative analysis of P-PFK2 (64 kDa), PFK2 (64
kDa) and GAPDH (37 kDa) in the rats in the Ctl, T2D, T2D + CE and T2D + IE groups. C.
Liver glucose level after oral glucose administration in Ctl, T2D, T2D + CE and T2D + IE
groups at the end of 8th week. D. Relative concentrations of substrates for glycolysis
(DHAP and Lactate) and the tricarboxylic acid cycle (citrate, succinate and malate) in the
rat of Ctl, T2D, T2D + CE and T2D + IE groups. The concentration of substrates was
analyzed by LC-MS/MS. E-G. Representative protein level and quantitative analysis of
FoxO1 (82 kDa), GLUT2 (60-70 kDa) and Actin (45 kDa) in the rats in the Ctl, T2D, T2D
+ CE and T2D + IE groups. H-I. Expression of hepatic Pepck and G6C mRNA in the Ctl,
T2D, T2D + CE and T2D + IE groups were evaluated by real-time PCR analysis. Values
represent mean ratios of Pepck and G6pase transcripts normalized to GAPDH transcript
levels. J. Schematic diagram illustrating the effect of CE and IE on glycolysis,
gluconeogenesis and mitochondrial tricarboxylic acid cycle (ns: not significant; *P < 0.05,
**P < 0.01, ****P < 0.0001 compared with all groups by one-way ANOVA and Tukey’s
post hoc test; data are expressed as the mean ± SEM; n = 6-8 per group).
863
Figure 5_source data_01
864
Figure 5_source data_02
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867
Fig. 6 Moderate exercise inhibited hepatic mitophagy, while excessive exercise
exhibited opposite effect and inhibited the mitochondrial biogenesis.
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A-F. Representative protein level and quantitative analysis of MFN1 (82 kDa), ATG5 (55
kDa), FIS (25 kDa), LC3A/B (14,16 kDa), PGC-1α (130 kDa) and Actin (45 kDa) in the
rats in the Ctl, T2D, T2D + CE, T2D + IE and T2D + EE groups. G. TEM analysis of the
ultrastructure of hepatocytes in the rats in the T2D, T2D + CE, T2D + IE and T2D + EE
groups (The yellow arrows point to mitochondria). (Scale bar = 2 μm; ns: not significant;
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with all groups by oneway ANOVA and Tukey’s post hoc test; data are expressed as the mean ± SEM; n = 8 per
group).
876
Figure 6_source data_01
877
Figure 6_source data_02
878
879
Fig. 7 Moderate ROS activates AMPK through GRX-mediated glutathionylation
880
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A-B. Analysis of superoxide (A) and ROS (B) generation using hydroethidine (A) and
H2DCFDA (B) probes in primary hepatocytes under H2O2 stress (50–200 μmol/l, 30 min).
The fluorescence intensity was detected by flow cytometry. C-D. Representative protein
level and quantitative analysis of GSS-adduct protein and GAPDH in primary hepatocytes
under H2O2 stress (50–200 μmol/l). Hepatocytes loaded with EE-GSH-biotin were
incubated with/without H2O2 for 30 min, and the amounts of GSS-protein adduct formation
were determined using non-reducing SDS-PAGE and Western blot analysis with
streptavidin-HRP. E-F. Representative protein level and quantitative analysis of P-AMPK
(67 kDa) and AMPK (67 kDa) in primary hepatocytes under H2O2 stress (50–200 μmol/l,
30 min). G-H. AMPK cysteine gel shift immunoblot. Cysteine dependent shifts by
incubation of AMPK protein with glutathione reductase and PEG-Mal. PEG2-mal labelled
glutathionylation modification shifts AMPK by ~10 kDa above the native molecular
weight. Representative protein level and quantitative analysis of GSS-AMPK (72 kDa),
AMPK (67 kDa) and GAPDH in primary hepatocytes under H2O2 stress.
894
Figure 7_source data_01
895
Figure 7_source data_02
896
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Fig. 8 Schematic diagram of redox balance threshold.
898
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913
914
A. The increased ROS and/or decreased antioxidative capacity (AOD) cause an imbalanced
redox state and declined AMPK activity in diabetic individuals. Moderate exercise
promotes the activity of antioxidant enzymes by generating benign ROS to reach redox
balance, and directly promotes AMPK signaling, thus reducing glucose levels in the blood
and liver. Excessive exercise causes excess ROS and exceeds the redox balance threshold,
inhibiting AMPK activity and expression, thus leading to exacerbation of diabetes. (AMPK
and P-AMPK in grey circles indicate decrease, red circles indicate increase, blue circles
indicate no significant changes). B. Dose-response curve of diabetic individuals with
exercise and antioxidant intervention. The state of diabetic individuals is applicable to the
description of a S-shaped curve, due to the high level of oxidative stress and decreased
reduction level in diabetic individuals. With the increase of ROS, the physiological function
of diabetic individuals gradually decreases. Moderate exercise shifts the S-shaped curve
upward and to the right, forming a bell-shaped dose-response curve, thus reducing the
sensitivity to oxidative stress in diabetic individuals and restoring redox homeostasis.
However, with excessive exercise, ROS production increases beyond the threshold range of
redox balance, resulting in decreased physiological function. The ROS at the peak of the
bell-shaped curve for antioxidant interventions (optimal physiological activity) is lower
915
916
917
than the ROS at the peak for moderate exercise. The intervals on either side of the peak
correspond to the range of redox balance thresholds, where antioxidant interventions are at
low levels of redox balance and exercise is at high levels of redox balance.
918
919
Supplementary file 1a Antibody information
920
Supplementary file 1b Chow and HFD diet nutrition composition
Gr
aphi
cabst
r
act
Protein Carbonyl
0.25
ns
0.20
0.15
0.10
0.05
0.00
O
D
+C
E
C
TL
T2
T2 D
D
+C
E
0
T2
1
D
2
2.5
2.0
1.5
1.0
0.5
Relative protein level
GRX1
T2
0
J
TL
1
Sestrin2
T
T2 2D
D
+C
E
Actin
TL
T2
T2 D
D
+C
E
C
0
C
2
N
MDA (mmol/l)
3-NT Level
TL
3
Relative protein level
G
C
0.6
1
D
+C
E
0.9
2
T2
1.2
PRX1
GRX1
TRX1
Relative protein level
D+
CE
T2
D
T2
Ct
l
COX2
D
Nrf2
T2
0.8
D
+C
E
D
T2
1.0
3
T2
3
TL
1.2
Relative protein level
D
TL
M
C
TL
T2
T2 D
D
+C
E
C
NOX4
C
Actin
Relative protein level
Sestrin2
1.5
C
TL
T
T2 2D
D
+C
E
F
Relative protein level
E
T
T2 2D
D
+C
E
C
TL
T
T2 2D
D
+C
E
D+
CE
T2
Relative protein level
Actin
1.4
TL
L
CE
Nrf2
Relative protein level
COX2
D+
NOX4
T2
D
CE
D+
T2
D
T2
C
C
Actin
T2
Ct
l
D
T2
Ct
l
B
Relative protein level
3-NT
l
Ct
A
H
I
0.0
1.6
K
1.6
5
MDA
4
3
2
1
PRX1
1.4
ns
1.2
1.0
0.8
0.6
TRX1
1.4
1.2
1.0
0.8
0.6
A
Apocynin
Intervention
Blood glucose monitoring
4th week
STZ 35mg/kg
Day 0
8th week
Experimental day
HFD diet
0.20
6
8
D
D
T2
16
PO
+A
E
D
T2
T2
D
+C
D
tl
0
0
PO
Ctl
T2D
T2D+CE
T2D+APO
10
T2
0
T2
D
T2 +CE
D
+A
PO
4
tl
T2
D
10
OGTT
30
20
8
C
nmol/ml
20
K
T2
T2
Fasting BG
12
30
C
mmol/l
40
J
Blood glucose (mmol/L)
Postprandial BG
0.0
D
+A
+A
PO
0.4
0.5
D
0.6
1.0
D
+A
0.8
1.5
T2
1.0
2.0
tl
1.2
T2
D
T2
D
0.4
ns
GLUT2
C
0.6
Relative protein level
0.8
1.4
T2
D
1.0
H
Sestrin
C
tl
Relative protein level
1.2
PO
tl
T2
T2
1.4
C
tl
HSP90
C
tl
C
D
T2
GLUT2
Relative protein level
Sestrin2
G
Nrf2
+A
0
D
0
PO
2
+A
T2
D+
T2
D
T2
l
AP
O
F
Nrf2
4
2
PO
D
tl
C
E
6
+A
0.10
4
D
0.15
IU/ml
10
nmol/l
IU/ml
8
0.00
I
TAOC
0.25
0.05
Ct
D
MDA
T2
C
Protein Carbonyl
PO
B
0
60
120
Time (minutes)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
ns
ns
F
1.6
ns
1.0
0.8
0.6
I
AMP/ATP
P-AMPK
AMPK
Actin
TL
T
T2 2D
D
+
T2 CE
D
T2 +IE
D
+E
E
C
T2D
T2D
Ctl T2D CE IE EE
Ace-SOD2
SOD2
GRX1
TRX1
Actin
TRX1
J
1.6
1.4
0.4
1.2
0.3
1.0
0.8
0.6
M
ns
T2D
Ctl T2D CE
IE
EE
EE
NOX4
1.2
COX2
1.0
0.8
Actin
0.6
G
Relative protein level
C
Protein Carbonyl content
ns
ns
0.2
0.1
0.0
N
8
6
T2 T2D
D
+
T2 CE
D
T2 +IE
D
+E
E
COX2
TL
0
C
0
MDA (nmol/ml)
10
TL
T
T2 2D
D
+
T2 CE
D
T2 +IE
D
+E
E
C
10
Relative protein level
TL
T
T2 2D
D
+
T2 CE
D
T2 +IE
D
+E
E
20
IE
TL
T
T2 2D
D
+
T2 CE
D
T2 +IE
D
+E
E
GRX1
30
Ctl T2D CE
Relative protein level
L
40
TL
T
T2 2D
D
+
T2 CE
D
T2 +IE
D
+E
E
1.2
C
20
mmol/l
30
OGTT-2H
C
1.4
B
mmol/l
E
C
TL
T2
T2 D
D
+
T2 CE
D
T2 +IE
D
+E
E
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
Relative protein level
T2 T2D
D
+
T2 CE
D
T2 +IE
D
+E
E
TL
C
Bood glucose
ns
C
T2 T2D
D
+
T2 CE
D
T2 +IE
D
+E
E
TL
H
C
Relative protein level
TL
T
T2 2D
D
+
T2 CE
D
T2 +IE
D
+E
E
C
mmol/l
40
Ratio
C
TL
T
T2 2D
D
+
T2 CE
D
T2 +IE
D
+E
E
Relative protein level
A
D
NOX4
1.6
1.4
Aceyl-SOD/SOD
1.2
1.0
0.8
0.6
0.4
K
MDA
6
ns
5
4
3
2
P-AMPK/Actin
ns
ns
4
2
0
B
P-PFK2/PFK2
Liver glucose
ns
1.2
1.0
0.8
0.6
OAA
1.4
J
5
Fold change
4
3
2
1
C
TL
T2
T2 D
D
+C
T2 E
D
+I
E
0
I
G6PC-mRNA
2.5
Fold change
2.0
1.5
1.0
0.5
T2
T2 D
D
+C
T2 E
D
+I
E
C
TL
0.0
tl
T2
D
T2
C
E
+I
D
+C
E
0
T2
E
+I
E
PEPCK1-mRNA
D
+C
D
T2
H
T2
D
T2
C
TL
0.6
1
D
Actin
2
D
0.8
3
T2
GLUT2
α-KG
Malate
4
T2
1.0
Glutamate
Succinate
GLUT2
5
1.2
FoxO1
C
G
FoxO1
Relative protein level
Ctl
T
T2 2D
D
+C
T2 E
D
+I
E
T
T2 2D
D
+C
T2 E
D
+I
E
TL
C
F
T2D
T2D CE IE
Citrate
0
Relative protein level
E
Ace-CoA
1
TL
GAPDH
Lactate
TL
P-PFK2
DHAP
2
D
+C
T2 E
D
+I
E
Relative protein level
1.4
PFK2
1 1.5 2
3
1.6
C
T2D
T2D CE
IE
Ctl
D
C
Relative content
A
T2
G
2 μm
ns
ns
1.0
0.5
0.0
T2D
2 μm
E
LC3
2.5
2.0
1.5
1.0
0.5
0.0
T2D+IE
2 μm
TL
T2
T2 D
D
+C
T2 E
D
T2 +IE
D
+E
E
PGC-1α
2.0
1.5
1.0
Relative protein level
MFN1
C
LC3A/B
T
T2 2D
D
+C
T2 E
D
T2 +IE
D
+E
E
B
0.5
1.6
C
TL
T2
T2 D
D
+C
T2 E
D
T2 +IE
D
+E
E
TL
Fis
Relative protein level
Actin
C
ATG5
Relative protein level
MFN1
T2
D
D
+C
T2 E
D
T2 +IE
D
+E
E
2.0
T2
Fis
TL
D
Relative protein level
Ctl T2D CE IE EE
C
1.5
D
+C
T2 E
D
T2 +IE
D
+E
E
D
T2
C
TL
Relative protein level
A
T2D
C
ATG5
2.0
1.5
1.0
0.5
0.0
F
PGC-1a
ns
1.2
0.8
0.4
T2D+CE
T2D+EE
2 μm
D
C
Ctl
H2O2 (μM)
50 100 200
GSS-protein
Ctl
50 μM
GSS-protein
Relative protein level
A
2.5
2.0
1.5
1.0
0.5
GAPDH
100 μM
H2O2 (μM)
E
H2O2 (μM)
Dihydroethidium
Ctl
B
Relative protein level
F
200 μM
CTL 50 100 200
50 100 200
P-AMPK
Ctl
2.5
2.0
1.5
1.0
0.5
AMPK
P-AMPK/AMPK
CTL 50 100 200
H2O2 (μM)
G
50 μM
H2O2 (μM)
Ctl
GSS-AMPK
100 μM
AMPK
200 μM
GAPDH
DCF
50 100 200
Relative protein level
H
2.5
GSS-AMPK
2.0
1.5
1.0
0.5
CTL 50 100 200
H2O2
Redox
bal
ance