Biochemistry and Cell Biology
Inhibition of brain-type glycogen phosphorylase ameliorates high glucose-induced cardiomyocyte apoptosis via Akt-HIF-1α activation
Journal: Biochemistry and Cell Biology
Manuscript ID bcb-2019-0247.R1
Manuscript Type: Article
Date Submitted by the 02-Dec-2019 Author:
Complete List of Authors: Wu, Xuehan; Nanchang University Huang, Weilu; Nanchang University Quan, Minxue; Nanchang University Chen, Yongqi;Draft Nanchang University Tu, Jiaxin; Nanchang University Zhou, Jialu; Nanchang University Xin, Hong Bo; Nanchang University Qian, Yisong; Nanchang University
Keyword:
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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1 Inhibition of brain-type glycogen phosphorylase ameliorates high glucose-induced cardiomyocyte
2 apoptosis via Akt-HIF-1α activation
3 Xuehan Wu, Weilu Huang, Minxue Quan, Yongqi Chen, Jiaxin Tu, Jialu Zhou, Hong-Bo Xin, Yisong
4 Qian
5 The National Engineering Research Center for Bioengineering Drugs and the Technologies, Institute of
6 Translational Medicine, Nanchang University, 1299 Xuefu Avenue, Nanchang 330031, P.R. China
7
8 Corresponding author: Yisong Qian (email: [email protected]).
9
10 Xuehan Wu and Weilu Huang contributedDraft equally to this study.
11
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13 Abstract
14 The brain-type glycogen phosphorylase (pygb) is one of the rate-limiting enzyme in glycogenolysis
15 that plays a crucial role in the pathogenesis of type 2 diabetes mellitus. Here we investigated the role of
16 pygb in high glucose (HG)-induced cardiomyocyte apoptosis and explored the underlying mechanisms,
17 by using the specific pygb inhibitors or pygb siRNA. Results showed that inhibition of pygb
18 significantly attenuated cell apoptosis and oxidative stress induced by HG in H9c2 cardiomyocytes.
19 Inhibition of pygb improved glucose metabolism in cardiacmyocytes, as evidenced by increased
20 glycogen content, glucose consumption and glucose transport. Mechanismly, pygb inhibition activated
21 Akt/GSK-3β signal pathway and suppressed NF-κB activation in H9c2 cells exposed to HG. 22 Additionally, pygb inhibition promoted theDraft expression and the translocation of hypoxia-inducible 23 factor-1α (HIF-1α) after HG stimulation. However, the changes in glucose metabolism and HIF-1α
24 activation mediated by pygb inhibition were significantly reversed in the presence of Akt inhibitor
25 MK2206. In conclusion, the present study suggested that inhibition of pygb prevents HG-induced
26 cardiomyocyte apoptosis via Akt-HIF-α activation.
27 Keywords: pygb; high glucose; cardiomyocyte; Akt; HIF-1α
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29
30 Introduction
31 Cardiovascular disease and its complications are the primary causes of morbidity and mortality,
32 especially in patients with diabetes mellitus (Mazzone 2010). Diabetic cardiomyopathy (DCM) is
33 characterized by impaired cardiac contractility and poor myocardial performance and can progress
34 toward overt heart failure (Trost et al. 2002). Hyperglycemia is the key initiating event in DCM that
35 triggers a series of downstream signals leading to cardiomyocyte apoptosis, oxidative stress, and
36 inflammation (Chatham and Seymour 2002; Clark et al. 2003). Therefore, the interference of
37 glycometablism in cardiomyocytes may be one of the effective strategies for DCM treatment. 38 Glycogen phosphorylase is the rate-limitingDraft enzyme in glycogenolysis which catalyzes the 39 phosphorolytic cleavage of the glucose polymer glycogen at the α-1,4-glycosidic linkage to release
40 glucose-1-phosphate from glycogen. There are three isoforms of mammalian glycogen phosphorylase,
41 liver, muscle, and brain (Henke and Sparks 2006). The brain-type glycogen phosphorylase (pygb) is
42 also highly expressed in cardiomyocytes (Kato et al. 1989) and is used as a diagnostic biomarker for
43 acute myocardial infarction (Cubranic et al. 2012; Peetz et al. 2005). Recently, the repression of
44 glycogen phosphorylase by specific inhibitors has been used for the treatment of type 2 diabetes (Nagy
45 et al. 2013; Sanae et al. 2014) and myocardial ischemia-reperfusion injury (Tracey et al. 2004) in
46 animal models. We have previously identified that naturally occurring pentacyclic triterpenes including
47 asiatic acid and maslinic acid are potent glycogen phosphorylase inhibitors, which bind at the allosteric
48 activator site, where the physiological activator AMP binds and efficiently suppress the activation of
49 glycogen phosphorylase (Guan et al. 2009; Wen et al. 2008). In addition, we also demonstrated that
50 asiatic acid protests against myocardial ischemia-reperfusion injury in in vitro and in vivo models (Dai
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51 et al. 2018; Huang et al. 2016). However, the role of pygb in high glucose-induced cardiomyocyte
52 injury and its regulatory mechanisms still remain unknown.
53 In the present study, a high glucose (HG)-induced cardiomyocyte injury model in human
54 cardiomyocyte cell line H9c2 was established to investigate the role of pygb in cardiomyocyte
55 apoptosis, by using the specific pygb inhibitors and pygb siRNA. In addition, the pygb-mediated signal
56 pathways were explored, with the aim to provide novel insights into the therapeutic strategies for
57 DCM.
58 Materials and methods
59 Materials
60 H9c2 cell lines were purchased fromDraft American Type Culture Collection (ATCC, CRL-1446).
61 Asiatic acid and maslinic acid were purchased from Sigma-Aldrich. MK2206 was obtained from
62 Biovision. The anti-Bcl-2, anti-Bax, anti-Akt, anti-phosphor-Akt and anti-GAPDH antibodies were
63 obtained from Santa Cruz, the anti-GLUT4, anti-HIF-1α, anti-NF-κB p65 and anti-NF-kB p65
64 (phosphor S276) antibodies were from Abcam, the anti-GSK-3β, anti-phospho-GSK-3 (Ser9) and
65 anti-caspase-9 antibodies were from Cell Signaling Technology, and the anti-pygb antibody was
66 obtained from ZenBioScience.
67 Cell culture and treatment
68 H9c2 cells were cultured in low-glucose Dulbecco’s modified essential medium (DMEM, Life
69 Technologies) containing 10% fetal bovine serum (FBS, Life Technologies), 1% streptomycin (100
70 μg/mL) and 1% penicillin (100 U/mL) at pH 7.4 in a 5% CO2 incubator at 37 ℃. The cultures were
71 incubated with 10 μM of asiatic acid or maslinic acid for 4 h, followed by the exposure of 33.3 mM of
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72 glucose (HG) for 48 h. Apoptosis was determined by Hoechst 33258 staining, cleaved caspase-9, bax
73 and bcl-2 levels. The oxidative stress was evaluated by ROS production, SOD activity and MDA
74 content, according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute).
75 Pygb knockdown by siRNA
76 The siRNA target sequence was selected in rat pygb gene (GenBank accession NM_013188.1) as
77 follows: siRNA1: GCATGTGATGAAGCCACTTAT and siRNA2:
78 GGTTCAAGTCGTCCAAGTTTG. Pygb siRNA was constructed into the lentivirus expression vector
79 PDS019_pL_shRNA_F carrying the green fluorescent protein (GFP) gene. A universal sequence was
80 used as a negative control for RNA interference. The viral particles were produced by third generation
81 packaging in 293T cells and Lentiviral stocksDraft were concentrated using ultracentrifugation. H9c2 cells
82 (5 × 104/ml) were prepared and infected at a Multiplicity of Infection (MOI) of 20 with negative
83 control (GFP) or pygb siRNA lentiviruses for 16 h at 37°C in the presence of 10 mg/ml polybrene. The
84 cultures were then washed and cultured in fresh medium for 72 h. GFP expression was detected to
85 calculate the infection efficiency.
86 Hoechst 33258 Staining
87 Cells were rinsed with phosphate-buffered saline (PBS, pH 7.4) three times and fixed with 4%
88 paraformaldehyde for 30 min at room temperature, followed by incubation with 5 μM Hoechst 33258
89 at 37 ℃ for 20 min. Fluorescence images were examined under the fluorescence microscope. Cells
90 with bright, condensed and rounded nuclei were considered as the apoptotic cells.
91 ROS measurement
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92 The production of ROS was monitored using the fluorescent probe 2’-7’-dichlorofluorescein
93 diacetate (DCFH-DA, Sigma), which is converted into highly fluorescent 2’-7’-dichlorofluorescein
94 (DCF) in the presence of ROS. Samples were rinsed with PBS, and incubated with 10 mM DCFH-DA
95 at 37 ℃ for 30 min. Fluorescence was observed using a fluorescence microscope (IX71, Olympus).
96 Western Blot
97 At the end of treatment, cells were rinsed in PBS and lysed in RIPA lysis buffer. Twenty
98 micrograms of protein were loaded into each lane, separated by 10% sodium dodecyl
99 sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes in Tris-glycine
100 buffer (48 mM Tris, 39 mM glycine, pH 9.2) containing 20% methanol. The membranes were blocked
101 with skimmed milk for 1 h, washed in TrisDraft buffered saline containing 0.1% Tween-20 (TBST) and
102 incubated overnight with the primary antibodies. After washing three times with TBST, nitrocellulose
103 membranes were incubated for 1 h at room temperature with horseradishperoxidase-conjugated goat
104 anti-rabbit or anti-mouse IgG. Bands were visualized using the SuperSignal West Pico
105 Chemiluminescent SubstrateTrial Kit (Pierce). Images were taken using the ChemiDoc XRS system
106 with Quantity One software (Bio-Rad).
107 Determination of glycogen and glucose
108 Cellular glycogen was measured as described previously (Verleysdonk et al. 2005). Briefly, the
109 cells were washed in ice-cold PBS and lysed by 0.1 M NaOH. The lysates were incubated at 80 °C for
110 1 h, and the glycogen was precipitated by the addition of 2.5 volumes of ethanol. After centrifugation,
111 the pellet was dried in a vacuum concentrator and resuspended in 50 mM sodium acetate buffer (pH
112 4.8). The glycogen was digested with amyloglucosidase at 37 °C for 90 min, and the resulting glucose
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113 was assayed in the microtiter plate at 492 nm using a glucose detection kit (Nanjing Jiancheng
114 Bioengineering Institute). The glucose consumption was evaluated by measuring the glucose content in
115 the supernatant of H9c2 cells, according to the manufacturer's instructions (Nanjing Jiancheng
116 Bioengineering Institute).
117 Immunocytochemistry
118 Cells were rinsed with PBS three times, fixed with 4% paraformaldehyde for 30 min at room
119 temperature and permeabilised in 0.1% Triton X-100 for 10min. An incubation in 5% bovine serum
120 albumin (BSA) in PBS for 1 h was performed to prevent the non-specific binding. The cultures were
121 incubated with the anti-HIF-1α antibody overnight at 4 °C. Then cells were incubated with fluorescein
122 isothiocyanate (FITC)-conjugated goat anti-rabbitDraft IgG (Alexa 488, Thermo Fisher) and the nuclei were
123 stained with DAPI. Immunostained cells were examined under a fluorescence microscope and digital
124 images were analyzed with Image-Pro Plus software.
125 Statistical Analysis
126 All values are expressed as the mean ± standard deviation (SD) of at least three independent
127 preparations. Differences among the groups were compared using one-way analysis of variance
128 (ANOVA) analysis followed by a Tukey post-hoc test. A difference with p < 0.05 was considered
129 statistically significant.
130 Results
131 Inhibition of pygb alleviates HG-induced myocardial apoptosis
132 The chemical structures of asiatic acid and maslinic acid, the pygb inhibitors, are shown in Fig.
133 1A. The control and pygb inhibitors-treated H9c2 cells showed weak Hoechst and DCF fluorescence,
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134 which was significantly increased after HG exposure. Both asiatic acid and maslinic acid suppressed
135 HG-induced cell apoptosis and oxidative stress in H9c2 cells (Fig. 1B). We further investigated the
136 effects of pygb on apoptotic pathways in H9c2 cells by using two pygb siRNAs. Both siRNAs reduced
137 pygb protein levels to <30% (Fig 1C and 1D). Pygb knockdown by siRNA1 caused a decrease in
138 cleaved caspase-9 and Bax/Bcl-2 ratio under normal conditions. HG induced an increase in cleaved
139 caspase-9 and Bax/Bcl-2 ratio. However, these effects were obviously reversed by both pygb siRNAs
140 (Fig. 1C, 1E and 1F). In addition, pygb knockdown prevented HG-induced inhibition of SOD activity
141 (Fig. 1G) and suppressed the accumulation of MDA (Fig. 1H). These data suggested that inhibition of
142 pygb effectively protected cardiomyocyte against HG-induced apoptosis and oxidative stress.
143 Inhibition of pygb improves glucose metabolismDraft after HG exposure in H9c2 cells
144 The effects of pygb on glucose metabolism were determined in HG-treated H9c2 cells. Glycogen
145 accumulation was significantly increased after asiatic acid or maslinic acid treatment. HG induced
146 glycogen synthesis which was further promoted in the presence of pygb inhibitors (Fig. 2A). The
147 glucose concentration in the supernatant was detected before and after HG stimulation to evaluate
148 glucose consumption. Results showed asiatic acid and maslinic acid accelerated the absorbance of
149 extracellular glucose compared with the control, and improved the uptake ability of glucose following
150 HG exposure (Fig. 2B). GLUT4 levels were determined for evaluation of the glucose transport capacity.
151 Both pygb inhibitors up-regulated the GLUT4 levels under normal conditions. HG resulted in an
152 increase in GLUT4 levels, but asitic acid and maslinic acid did not further promote its expression in the
153 presence of HG (Fig. 2C). Knockdown of pygb yielded the similar results with the pygb inhibitors. The
154 pygb siRNAs increased glycogen content (Fig. 2D) and enhanced glucose consumption (Fig. 2E) in
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155 H9c2 cells under normal conditions and with HG exposure. The pygb siRNAs induced a slight increase
156 in GLUT4 protein levels in the absence of HG, but remarkably up-regulated GLUT4 expression after
157 HG exposure (Fig. 2F). These data indicated that inhibition of pygb improved glucose metabolism in
158 H9c2 cells
159 Inhibition of pygb activates Akt/GSK-3β signaling pathway and prevents NF-κB activation
160 Akt/GSK-3β signaling pathway play a vital role in the regulation of glycogen synthesis in skeletal
161 muscle including heart (Markou et al. 2008; Patel et al. 2008). Our data revealed that the levels of
162 phosphor-Akt increased with the treatment of pygb inhibitor and siRNAs, leading to the inactivation of
163 GSK-3β, as evidenced by the enhanced phosphorylation at Ser9. Inhibition of pygb further activated
164 Akt/GSK-3β signaling pathway followingDraft HG stimulation (Fig. 3A-E). NF-κB signaling is involved in
165 oxidative stress and inflammation response triggered by HG (Panahi et al. 2018). Asiatic acid and
166 maslinic acid reduced the levels of phosphor-NF-κB p65 whereas pygb siRNAs did not obviously
167 affect the phosphorylation of NF-κB p65 without HG stimulation. Both pygb inhibitors and siRNAs
168 effectively suppressed p65 phosphorylation following HG treatment (Fig. 3A, 3F and 3G). Inhibition of
169 pygb also decreased the total p65 levels in H9c2 cells after 24 h of HG exposure (Fig. 3A, 3H and 3I).
170 It has been reported that HIF-1α is a key mediator of glycolysis in cardiomyocytes (Gao et al. 2015;
171 Malhotra et al. 2002). Therefore, whether pygb-mediated glucose metabolism is associated with
172 HIF-1α was examined. The result demonstrated that pygb inhibition by the inhibitors and siRNAs
173 significantly up-regulated HIF-1α levels in the absence and presence of HG treatment (Fig. 3A, 3J and
174 3K).
175 Inhibition of pygb activates HIF-1α and promotes glycogen synthesis through Akt
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176 The function of Akt in pygb-mediated HIF-1α activation and glucose metabolism were further
177 tested. The specific Akt inhibitor MK2206 was used in the experiment and was added to the cultures 1
178 h before asiatic acid treatment. HG caused an obvious nuclear translocation of HIF-1α, which was
179 further promoted in the presence of asiatic acid. However, this effect was abolished by the addition of
180 MK2206 (Fig. 4A). In addition, the increased glycogen synthesis mediated by pygb inhibition was also
181 blocked by MK2206 (Fig. 4B and 4C).
182 Discussion
183 Here we demonstrated that inhibition of pygb remarkably attenuated HG-evoked oxidative stress
184 and apoptosis in cardiomyocytes. In addition, pygb inhibition activated Akt/GSK-3β signaling pathway
185 and modulated HIF-1α activation, which Draftmay contribute to the improvement of glycometabolism in
186 cardiomyocytes during HG exposure.
187 Hyperglycemia-induced myocardial injury has been associated with cardiac inflammation,
188 oxidative stress and apoptosis (Brownlee 2005; Goldberg 2009). Hyperglycemia causes increased
189 mitochondrial production of ROS, which triggers the breakage of nuclear DNA strands and
190 subsequently the activation of mitochondria-dependent apoptosis. In the present study we showed that
191 pygb inhibition significantly reduced ROS production, alleviated oxidative stress and prevented
192 myocardial apoptosis exposed to HG, indicating that pygb play a crucial role in HG-induced cellular
193 damage.
194 Pygb is the key enzyme in glycogen metabolism, which increases intracellular glucose levels by
195 promoting glycogen decomposition. Therefore, we speculated that pygb inhibition protects against
196 HG-induced apoptosis by modulation of glycometabolism. The results confirmed that both pygb
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197 inhibitors and siRNA promoted the syntheses of glycogen, accelerated the consumption of glucose and
198 increased the expression of GLUT4, thus maintained the extracellular glucose at a lower level to
199 effectively reduced HG-induced cell damage. Akt and GSK-3 are involved in the activation of
200 glycogen synthase and stimulation of glycogen synthesis (Lavoie et al. 1999). We found here that
201 Akt/GSK-3β was remarkably activated by pygb inhibition. This result was in agreement with the
202 previous study that the pygb inhibitor maslinic acid significantly enhanced the phosphorylation levels
203 of Akt and GSK3β to modulate the hepatic glycogen metabolism (Liu et al. 2014). The activation of
204 NF-κB and its cascade genes can be regulated by PI3K/Akt signaling pathway, which plays a crucial
205 role in regulating various biological functions such as cell proliferation, cell differentiation, and
206 apoptosis (Liu et al. 2010). The present resultsDraft suggested that inhibition of pygb suppressed
207 HG-induced NF-κB activation, which in turn attenuated the NF-κB-dependent gene transcription and
208 protected cardiomyocytes from HG-induced oxidative stress and apoptosis at least partly through Akt
209 signaling.
210 Additionally, we have previously showed that maslinic acid did not reduce fasting blood glucose
211 but significantly increased liver glycogen contents in normal mice (Tang et al. 2008), which is
212 coincided with the current results that pygb inhibitors promotes glycogen synthesis whereas they have
213 no effects on oxidative stress and apoptosis under normal conditions in vitro. Thus we speculate that
214 the modulation of Akt and NF-κB pathways may have few effects on physiological parameters
215 including fasting blood glucose under normal conditions, indicating a minor side effect mediated by
216 pygb inhibitors.
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217 HIF-1 is a heterodimeric transcription factor consisting of a constitutively expressed β-subunit
218 and an oxygen-regulated α-subunit (Gu et al. 2013). Under normoxic conditions, HIF-1α is constantly
219 degraded via the ubiquitin-proteasome pathway. However, under hypoxic conditions, the HIF-1α
220 subunits are no longer degraded and translocated into the nuclei for binding to hypoxic responsive
221 elements and up-regulates several hypoxia-responsive genes and glycolytic genes (Kim et al. 2006;
222 Taylor 2008). It has been demonstrated that glucose activates HIF1 and HIF2 in beta cells and that both
223 HIF isoforms play distinct roles in the glucose stimulation of expression of glycolytic enzymes
224 (Bensellam et al. 2012). In another report, high glucose induces oxidative stress and glucose transport
225 alterations in kidneys, resulting in HIF-1α stability and more HIF-1 production. Activation of HIF-1α
226 may not only improve tubular transport efficiencyDraft but also prevent diabetes-induced alteration in
227 kidney oxygen metabolism (Nordquist et al. 2015). The Akt/GSK-3β signal pathway has been
228 implicated in the modulation of HIF-1α degradation. Exposure of cells to the PI3K inhibitor caused
229 Akt inhibition, hypophosphorylation of GSK-3β, and depletion of HIF-1α, while treatment with the
230 GSK-3β inhibitor led to a partial recovery of the HIF-1α protein depletion (Li et al. 2018). Our findings
231 are consistent with the reported roles of Akt and GSK-3β in the regulation of HIF-1α protein stability.
232 An obvious nuclear localization of HIF-1α induced by HG in H9c2 cells was observed and treatment
233 with asiatic acid further increased the fluorescence intensity in HG exposure. However, this effect was
234 strongly blocked by the Akt inhibitor MK2206, suggesting that pygb regulated HIF-1α activation
235 through Akt signaling. In addition, the increased glycogen synthesis induced by pygb inhibition was
236 abolished in the presence of MK2206. Therefore, we may concluded that Akt/GSK-3β is involved in
237 pygb-mediated HIF-1α activation and glycogen metabolism in cardiomyocytes. However, more
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238 regulators, possibly mTOR (Masoud and Li 2015), that medicate HIF-1α translocation and stabilization
239 in our model remain unknown and warrant further investigation.
240 Previous studies revealed the physical and functional interactions between HIF and NF-κB. On
241 the one hand, NF-κB activation results in HIF stabilization and activation in response to infection and
242 inflammation. On the other hand, HIF-1α is required to restrain the transcriptional activity of NF-κB
243 (Bandarra et al. 2015). It has been reported that inhibition of HIF-1α by 2-MeOE2 results in
244 suppression of NF-κB signaling pathway (Liu et al. 2019). Taken together, the suppression of NF-κB
245 mediated by pygb inhibition in our experiment is possibly attributed to direct Akt or Akt-HIF-1α
246 signaling.In conclusion, pygb inhibition prevents cardiomyocytes apoptosis through the activation of
247 Akt-HIF-1α signal pathway, which leads Draftto ameliorated glycometabolism and attenuated oxidative
248 stress. The study suggested that pygb is an important target involved in DCM and advanced the
249 understanding of the molecular mechanisms underlying the cardioprotection of pygb inhibitors.
250
251 Conflict of interest statement
252 The authors declare that there is no conflict of interest associated with this work.
253
254 Funding statement
255 This work was supported by grants to Y.-S. Qian from the National Natural Science Foundation of
256 China (grants 81400220 and 81860020).
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258 References 259 260 Bandarra, D., Biddlestone, J., Mudie, S., Muller, H.A., and Rocha, S. 2015. 261 HIF-1alpha restricts NF-kappaB-dependent gene expression to control innate 262 immunity signals. Disease models & mechanisms 8(2): 169-181. 263 doi:10.1242/dmm.017285. 264 265 Bensellam, M., Duvillie, B., Rybachuk, G., Laybutt, D.R., Magnan, C., Guiot, Y., et 266 al. 2012. Glucose-induced O(2) consumption activates hypoxia inducible 267 factors 1 and 2 in rat insulin-secreting pancreatic beta-cells. PloS one 7(1): 268 e29807. doi:10.1371/journal.pone.0029807. 269 270 Brownlee, M. 2005. The pathobiology of diabetic complications: a unifying 271 mechanism. Diabetes 54(6): 1615-1625. doi:10.2337/diabetes.54.6.1615. 272 273 Chatham, J.C., and Seymour, A.M. 2002. Cardiac carbohydrate metabolism in Zucker 274 diabetic fatty rats. Cardiovascular research 55(1): 104-112. 275 doi:10.1016/s0008-6363(02)00399-1. 276 Draft 277 Clark, R.J., McDonough, P.M., Swanson, E., Trost, S.U., Suzuki, M., Fukuda, M., et 278 al. 2003. Diabetes and the accompanying hyperglycemia impairs 279 cardiomyocyte calcium cycling through increased nuclear O-GlcNAcylation. 280 The Journal of biological chemistry 278(45): 44230-44237. 281 doi:10.1074/jbc.M303810200. 282 283 Cubranic, Z., Madzar, Z., Matijevic, S., Dvornik, S., Fisic, E., Tomulic, V., et al. 2012. 284 Diagnostic accuracy of heart fatty acid binding protein (H-FABP) and 285 glycogen phosphorylase isoenzyme BB (GPBB) in diagnosis of acute 286 myocardial infarction in patients with acute coronary syndrome. Biochemia 287 medica 22(2): 225-236. doi:10.11613/bm.2012.025. 288 289 Dai, Y., Wang, Z., Quan, M., Lv, Y., Li, Y., Xin, H.B., et al. 2018. Asiatic acid 290 protests against myocardial ischemia/reperfusion injury via modulation of 291 glycometabolism in rat cardiomyocyte. Drug design, development and therapy 292 12: 3573-3582. doi:10.2147/DDDT.S175116. 293 294 Gao, Q., Guan, L., Hu, S., Yao, Y., Ren, X., Zhang, Z., et al. 2015. Study on the 295 mechanism of HIF1a-SOX9 in glucose-induced cardiomyocyte hypertrophy. 296 Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 74: 297 57-62. doi:10.1016/j.biopha.2015.07.009. 298
14
https://mc06.manuscriptcentral.com/bcb-pubs Page 15 of 24 Biochemistry and Cell Biology
299 Goldberg, R.B. 2009. Cytokine and cytokine-like inflammation markers, endothelial 300 dysfunction, and imbalanced coagulation in development of diabetes and its 301 complications. The Journal of clinical endocrinology and metabolism 94(9): 302 3171-3182. doi:10.1210/jc.2008-2534. 303 304 Gu, H.F., Zheng, X., Abu Seman, N., Gu, T., Botusan, I.R., Sunkari, V.G., et al. 2013. 305 Impact of the hypoxia-inducible factor-1 alpha (HIF1A) Pro582Ser 306 polymorphism on diabetes nephropathy. Diabetes care 36(2): 415-421. 307 doi:10.2337/dc12-1125. 308 309 Guan, T., Li, Y., Sun, H., Tang, X., and Qian, Y. 2009. Effects of maslinic acid, a 310 natural triterpene, on glycogen metabolism in cultured cortical astrocytes. 311 Planta medica 75(10): 1141-1143. doi:10.1055/s-0029-1185481. 312 313 Henke, B.R., and Sparks, S.M. 2006. Glycogen phosphorylase inhibitors. Mini 314 reviews in medicinal chemistry 6(8): 845-857. 315 doi:10.2174/138955706777934991. 316 317 Huang, X., Zuo, L., Lv, Y., Chen, DraftC., Yang, Y., Xin, H., et al. 2016. Asiatic Acid 318 Attenuates Myocardial Ischemia/Reperfusion Injury via 319 Akt/GSK-3beta/HIF-1alpha Signaling in Rat H9c2 Cardiomyocytes. 320 Molecules 21(9). doi:10.3390/molecules21091248. 321 322 Kato, K., Shimizu, A., Kurobe, N., Takashi, M., and Koshikawa, T. 1989. Human 323 brain-type glycogen phosphorylase: quantitative localization in human tissues 324 determined with an immunoassay system. Journal of neurochemistry 52(5): 325 1425-1432. doi:10.1111/j.1471-4159.1989.tb09189.x. 326 327 Kim, J.W., Tchernyshyov, I., Semenza, G.L., and Dang, C.V. 2006. HIF-1-mediated 328 expression of pyruvate dehydrogenase kinase: a metabolic switch required for 329 cellular adaptation to hypoxia. Cell metabolism 3(3): 177-185. 330 doi:10.1016/j.cmet.2006.02.002. 331 332 Lavoie, L., Band, C.J., Kong, M., Bergeron, J.J., and Posner, B.I. 1999. Regulation of 333 glycogen synthase in rat hepatocytes. Evidence for multiple signaling 334 pathways. The Journal of biological chemistry 274(40): 28279-28285. 335 doi:10.1074/jbc.274.40.28279. 336 337 Li, A.G., Murphy, E.C., Culhane, A.C., Powell, E., Wang, H., Bronson, R.T., et al. 338 2018. BRCA1-IRIS promotes human tumor progression through PTEN 339 blockade and HIF-1alpha activation. Proceedings of the National Academy of
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340 Sciences of the United States of America 115(41): E9600-E9609. 341 doi:10.1073/pnas.1807112115. 342 343 Liu, J., Wang, X., Chen, Y.P., Mao, L.F., Shang, J., Sun, H.B., et al. 2014. Maslinic 344 acid modulates glycogen metabolism by enhancing the insulin signaling 345 pathway and inhibiting glycogen phosphorylase. Chinese journal of natural 346 medicines 12(4): 259-265. doi:10.1016/S1875-5364(14)60052-2. 347 348 Liu, S., Shen, H., Xu, M., Liu, O., Zhao, L., Liu, S., et al. 2010. FRP inhibits 349 ox-LDL-induced endothelial cell apoptosis through an 350 Akt-NF-{kappa}B-Bcl-2 pathway and inhibits endothelial cell apoptosis in an 351 apoE-knockout mouse model. American journal of physiology. Endocrinology 352 and metabolism 299(3): E351-363. doi:10.1152/ajpendo.00005.2010. 353 354 Liu, Y., Zou, X., Ou, M., Ye, X., Zhang, B., Wu, T., et al. 2019. Toxoplasma gondii 355 Cathepsin C1 inhibits NF-kappaB signalling through the positive regulation of 356 the HIF-1alpha/EPO axis. Acta tropica 195: 35-43. 357 doi:10.1016/j.actatropica.2019.04.018. 358 Draft 359 Malhotra, R., Tyson, D.G., Sone, H., Aoki, K., Kumagai, A.K., and Brosius, F.C., 3rd. 360 2002. Glucose uptake and adenoviral mediated GLUT1 infection decrease 361 hypoxia-induced HIF-1alpha levels in cardiac myocytes. Journal of molecular 362 and cellular cardiology 34(8): 1063-1073. doi:10.1006/jmcc.2002.2047. 363 364 Markou, T., Cullingford, T.E., Giraldo, A., Weiss, S.C., Alsafi, A., Fuller, S.J., et al. 365 2008. Glycogen synthase kinases 3alpha and 3beta in cardiac myocytes: 366 regulation and consequences of their inhibition. Cellular signalling 20(1): 367 206-218. doi:10.1016/j.cellsig.2007.10.004. 368 369 Masoud, G.N., and Li, W. 2015. HIF-1alpha pathway: role, regulation and 370 intervention for cancer therapy. Acta pharmaceutica Sinica. B 5(5): 378-389. 371 doi:10.1016/j.apsb.2015.05.007. 372 373 Mazzone, T. 2010. Intensive glucose lowering and cardiovascular disease prevention 374 in diabetes: reconciling the recent clinical trial data. Circulation 122(21): 375 2201-2211. doi:10.1161/CIRCULATIONAHA.109.913350. 376 377 Nagy, L., Docsa, T., Szanto, M., Brunyanszki, A., Hegedus, C., Marton, J., et al. 2013. 378 Glycogen phosphorylase inhibitor 379 N-(3,5-dimethyl-Benzoyl)-N'-(beta-D-glucopyranosyl)urea improves glucose 380 tolerance under normoglycemic and diabetic conditions and rearranges hepatic 381 metabolism. PloS one 8(7): e69420. doi:10.1371/journal.pone.0069420.
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382 383 Nordquist, L., Friederich-Persson, M., Fasching, A., Liss, P., Shoji, K., Nangaku, M., 384 et al. 2015. Activation of hypoxia-inducible factors prevents diabetic 385 nephropathy. Journal of the American Society of Nephrology : JASN 26(2): 386 328-338. doi:10.1681/ASN.2013090990. 387 388 Panahi, G., Pasalar, P., Zare, M., Rizzuto, R., and Meshkani, R. 2018. High glucose 389 induces inflammatory responses in HepG2 cells via the oxidative 390 stress-mediated activation of NF-kappaB, and MAPK pathways in HepG2 391 cells. Archives of physiology and biochemistry 124(5): 468-474. 392 doi:10.1080/13813455.2018.1427764. 393 394 Patel, S., Doble, B.W., MacAulay, K., Sinclair, E.M., Drucker, D.J., and Woodgett, 395 J.R. 2008. Tissue-specific role of glycogen synthase kinase 3beta in glucose 396 homeostasis and insulin action. Molecular and cellular biology 28(20): 397 6314-6328. doi:10.1128/MCB.00763-08. 398 399 Peetz, D., Post, F., Schinzel, H., Schweigert, R., Schollmayer, C., Steinbach, K., et al. 400 2005. Glycogen phosphorylaseDraft BB in acute coronary syndromes. Clinical 401 chemistry and laboratory medicine 43(12): 1351-1358. 402 doi:10.1515/CCLM.2005.231. 403 404 Sanae, F., Kamiyama, O., Ikeda-Obatake, K., Higashi, Y., Asano, N., Adachi, I., et al. 405 2014. Effects of eugenol-reduced clove extract on glycogen phosphorylase b 406 and the development of diabetes in db/db mice. Food & function 5(2): 407 214-219. doi:10.1039/c3fo60514k. 408 409 Tang, X.Z., Guan, T., Qian, Y.S., Li, Y.M., Sun, H.B., Huang, J.H., et al. 2008. 410 Effects of maslinic acid as a novel glycogen phosphorylase inhibitor on blood 411 glucose and hepatic glycogen in mice. Chinese Journal of Natural Medicines 412 6(1): 53-56. doi: 10.1016/S1875-5364(09)60005-4. 413 414 Taylor, C.T. 2008. Mitochondria and cellular oxygen sensing in the HIF pathway. The 415 Biochemical journal 409(1): 19-26. doi:10.1042/BJ20071249. 416 417 Tracey, W.R., Treadway, J.L., Magee, W.P., Sutt, J.C., McPherson, R.K., Levy, C.B., 418 et al. 2004. Cardioprotective effects of ingliforib, a novel glycogen 419 phosphorylase inhibitor. American journal of physiology. Heart and 420 circulatory physiology 286(3): H1177-1184. doi:10.1152/ajpheart.00652.2003. 421 422 Trost, S.U., Belke, D.D., Bluhm, W.F., Meyer, M., Swanson, E., and Dillmann, W.H. 423 2002. Overexpression of the sarcoplasmic reticulum Ca(2+)-ATPase improves
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424 myocardial contractility in diabetic cardiomyopathy. Diabetes 51(4): 425 1166-1171. doi:10.2337/diabetes.51.4.1166. 426 427 Verleysdonk, S., Kistner, S., Pfeiffer-Guglielmi, B., Wellard, J., Lupescu, A., Laske, 428 J., et al. 2005. Glycogen metabolism in rat ependymal primary cultures: 429 regulation by serotonin. Brain research 1060(1-2): 89-99. 430 doi:10.1016/j.brainres.2005.08.045. 431 432 Wen, X., Sun, H., Liu, J., Cheng, K., Zhang, P., Zhang, L., et al. 2008. Naturally 433 occurring pentacyclic triterpenes as inhibitors of glycogen phosphorylase: 434 synthesis, structure-activity relationships, and X-ray crystallographic studies. 435 Journal of medicinal chemistry 51(12): 3540-3554. doi:10.1021/jm8000949. 436 437
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439 Figure captions
440 Fig. 1. Inhibition of pygb attenuates apoptosis in H9c2 cardiomyocytes after high glucose exposure. (A)
441 Chemical structures of asiatic acid and maslinic acid. (B) Nuclear condensation and morphology was
442 visualized by Hoechest 33258 staining (up), and ROS production was detected by DCF fluorescence
443 (down). (C) Protein levels of pygb, cleaved caspase-9, Bax, Bcl-2 and GAPDH were detected by
444 Western blot. (D-F) Semiquantitative analyses of pygb, cleaved caspase-9 and Bax/Bcl-2 ratio were
445 performed by calculating the density of the Western blot bands. (G) The SOD activity and (H) MDA
446 content were determined with the treatment of pygb siRNAs. Values presented are the mean ± SD. *P
447 < 0.05; **P < 0.01. 448 Fig. 2. Inhibition of pygb improves glucoseDraft metabolism in H9c2 cells. The effect of pygb inhibitors 449 asiatic acid (AA) and maslinic acid (MA) on (A) glycogen content, (B) glucose consumption and (C)
450 GLUT4 protein levels were measured under normal and high glucose conditions. The effect of pygb
451 siRNAs on (D) glycogen content, (E) glucose consumption and (F) GLUT4 protein levels were
452 measured under normal and high glucose conditions. Values presented are the mean ± SD. *P < 0.05;
453 **P < 0.01.
454 Fig. 3. Effects of pygb on Akt/GSK-3β, NF-κB and HIF-1α signal pathways in H9c2 cells. (A) Protein
455 levels of phosphor-Akt (pAkt), total Akt (tAkt), phosphor-GSK-3β (pGSK-3β), total GSK-3β
456 (tGSK-3β), phosphor-NF-κB p65 (pNF-κB p65), total NF-κB p65 (tNF-κB p65), HIF-1α and GAPDH
457 were detected by Western blot under normal and high glucose conditions. Semiquantitative analyses of
458 (B and C) pAkt/tAkt, (D and E) pGSK--3β/tGSK-3β, (F and G) pNF-κB p65, (H and I) NF-κB p65 and
459 (J and K) HIF-1α were performed by calculating the density of the Western blot bands. Values
460 presented are the mean ± SD. *P < 0.05; **P < 0.01.
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461 Fig. 4. Inhibition of pygb activates HIF-1α and promotes glycogen synthesis through Akt. (A) The
462 nuclear translocation of HIF-1α was determined by immunocytochemistry. The glycogen content was
463 measured in (B) asiatic acid-treated and (C) pygb siRNA-treated H9c2 cells in the absence or presence
464 of Akt inhibitor MK2206 under high glucose conditions. Values presented are the mean ± SD. *P <
465 0.05; **P < 0.01. *P < 0.05; **P < 0.01.
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