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β-hydroxybutyrate antagonizes aortic endothelial injury by promoting generation of VEGF in diabetic rats
Tissue and Cell 64 (2020) 101345
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β-hydroxybutyrate antagonizes aortic endothelial injury by promoting generation of VEGF in diabetic rats
T
Xingliang Wua, Dazhuang Miaob, Zijing Liua, Kun Liua, Boning Zhanga, Jialin Lia, Yanning Lib,*,1, Jinsheng Qia,**,1 a b
Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, Shijiazhuang, China Department of Molecular Biology, Hebei Key Lab of Laboratory Animal Science, Hebei Medical University, Shijiazhuang, China
A R T I C LE I N FO
A B S T R A C T
Keywords: β-Hydroxybutyrate Vascular endothelial growth factor Histone H3K9 β-hydroxybutyrylation Diabetic endothelial injury
Endothelial injury is regarded as the initial pathological process in diabetic vascular diseases, but effective therapy has not yet been identified. Although β-hydroxybutyrate plays various protective roles in the cardiovascular system, its ability to antagonize diabetic endothelial injury is unclear. β-hydroxybutyrate reportedly causes histone H3K9 β-hydroxybutyrylation (H3K9bhb), which activates gene expression; however, there has been no report regarding the role of H3K9bhb in up-regulation of vascular endothelial growth factor (VEGF), a crucial factor in endothelial integrity and function. Here, male Sprague-Dawley rats were intraperitoneally injected with streptozotocin to induce diabetes, and then treated with different concentrations of β-hydroxybutyrate. After 10 weeks, body weight, blood glucose, morphological changes and serum nitric oxide concentration were examined. Moreover, the mRNA expression level, protein content and distribution of VEGF in the aorta were investigated, as were total protein β-hydroxybutyrylation and H3K9bhb contents. The results showed injury of aortic endothelium, along with reductions of the concentration of nitric oxide and generation of VEGF in diabetic rats. However, β-hydroxybutyrate treatment attenuated diabetic injury of the endothelium and up-regulated the generation of VEGF. Furthermore, β-hydroxybutyrate treatment caused marked total protein βhydroxybutyrylation and significant elevation of H3K9bhb content in the aorta of diabetic rats. The ability of βhydroxybutyrate to protect against diabetic injury of the aortic endothelium was greatest for its intermediate concentration. In conclusion, moderately elevated β-hydroxybutyrate could antagonize aortic endothelial injury, potentially by causing H3K9bhb to promote generation of VEGF in diabetic rats.
1. Introduction Macrovascular complications are major causes of disability and death in patients with diabetes (Fisher et al., 2018; Pan et al., 2018). Endothelial dysfunction represents the initial pathological process and is an early manifestation of diabetic vascular disease (Khaled et al., 2018; Caradu et al., 2018). However, effective therapy for diabetic endothelial injury has not been established. Recently, it has been reported that β-hydroxybutyrate (a major component of ketone body), has various protective roles, especially in nerve and cardiovascular systems (Achanta et al., 2017; Uchihashi et al., 2017). When glucose supply is insufficient, such as in starvation
conditions, ketone bodies are generated (Sabari et al., 2017). Although a sharp increase in ketone bodies may cause ketoacidosis in patients with diabetes, moderate increases have been reported to exhibit beneficial effects (Mizuno et al., 2017; Min et al., 2018). With respect to other ketone bodies, a physical concentration of β-hydroxybutyrate below 10 mM typically has protective effects, especially for endothelial cells (Obokata et al., 2017; Stubbs et al., 2017; Rains et al., 2015). However, it is unknown whether β-hydroxybutyrate can antagonize aortic endothelial injury in diabetes. In addition to potential effects on energy supply, several mechanisms have been proposed for the protective effects of β-hydroxybutyrate (Newman et al., 2014, 2017; Rojas-Morales et al., 2016). Notably, the
⁎ Corresponding author at: Department of Molecular Biology, Hebei Key Lab of Laboratory Animal, Hebei Medical University, No. 361 East Zhongshan Road, Shijiazhuang, 050017, Hebei, PR China. ⁎⁎ Corresponding author at: Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, No. 361 East Zhongshan Road, Shijiazhuang, 050017, Hebei, PR China. E-mail addresses: [email protected] (Y. Li), [email protected] (J. Qi). 1 Present/permanent address: No. 361 East Zhongshan Road, Shijiazhuang 050017, Hebei, PR China.
https://doi.org/10.1016/j.tice.2020.101345 Received 26 September 2019; Received in revised form 14 February 2020; Accepted 14 February 2020 Available online 15 February 2020 0040-8166/ © 2020 Elsevier Ltd. All rights reserved.
Tissue and Cell 64 (2020) 101345
X. Wu, et al.
protective effects of β-hydroxybutyrate are reportedly mediated through a membrane receptor, interactions with other proteins, or inhibition of the NRLP3 inflammasome (Rahman et al., 2014; Fu et al., 2014; Youm et al., 2015). In addition, β-hydroxybutyrate has been shown to enter the nucleus and inhibit histone deacetylase, thereby activating expression of protective genes (Wang et al., 2017; Sleiman et al., 2016). β-hydroxybutyrate also directly causes histone H3K9 βhydroxybutyrylation (H3K9bhb), which then activates gene expression, independent of acetylation (Xie et al., 2016). Physiological levels of vascular endothelial growth factor (VEGF) are required for the maintenance of normal endothelial integrity and function (Laakkonen et al., 2018; Logue et al., 2017). Although excess VEGF may exacerbate angiogenesis, insufficient levels of VEGF also lead to endothelial dysfunction (Zafar et al., 2017). With respect to alleviation of endothelial injury in the context of diabetes, there has been no report regarding whether β-hydroxybutyrate can up-regulate the generation of VEGF via H3K9bhb. In this study, diabetes was induced in male Sprague-Dawley rats, which were then treated with different concentrations of β-hydroxybutyrate. Morphological changes and serum nitric oxide (NO) concentration were assessed to evaluate the ability of β-hydroxybutyrate to antagonize diabetic injury of the aortic endothelium. Moreover, VEGF generation, total protein β-hydroxybutyrylation and H3K9bhb content were determined to investigate the underlying mechanism by which βhydroxybutyrate promoted generation of aortic VEGF in diabetic rats.
2.3. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) The mRNA expression levels of VEGF were quantified by real-time RT-PCR. Total RNA was isolated using TRIzol reagent (Takara, Dalian, China) and reverse transcribed to cDNA using RevertAid First Strand cDNA synthesis Kit (Fermentas, Shanghai, China), followed by real-time PCR amplification. The comparative Ct method was used to calculate the relative abundance of mRNA, compared with the abundance of 18S rRNA. Specific primers used were as follows: Rat VEGF (forward) 5′-AGGCGAGGCAGCTTGAGTTA-3′, (reverse) 5′-CTGTCGACGGTGACG ATGGT); and 18S rRNA (forward) 5′-AAGTTTCAGCACATCCTGCGA GTA-3′, (reverse) 5′-TTGGTGAGGTCAATGTCTGCTTTC-3′. 2.4. Western blotting VEGF protein contents, as well as the levels of β-hydroxybutyrylation and H3K9bhb, were measured by western blotting, using a previously described protocol (Li et al., 2018). Briefly, anti-VEGF (Sangon Biotech, Shanghai, China), anti-pan β-hydroxybutyrylation (Jingjie Biotech, hangzhou, China) and anti-H3K9bhb antibodies (Jingjie Biotech, hangzhou, China) were used. Band intensity was quantified and calculated. An antibody to β-actin (Sangon Biotech, Shanghai, China) served as a loading control routinely. 2.5. Immunohistochemistry
2. Material and methods
The distribution of endothelial VEGF in aortic tissue was detected by immunohistochemistry. The procedures were as follows: aorta samples were embedded in paraffin, then was cut and dewaxed. After sections had been incubated with H2O2, they were incubated with goat serum (Zhongshan, Beijing, China). Sections were incubated with antiVEGF antibody (diluted 1:50, Sangon Biotech, Shanghai, China) and then the secondary antibody (diluted 1:200, Zhongshan, Beijing, China). 3, 3 -Diaminobenzidine solution was used for color emergence.
2.1. Animals and experiment design Seventy healthy male Sprague-Dawley rats (246.45 ± 29.37 g) were provided by the Department of Experimental Animals of Hebei Medical University. Animal care and use were performed in accordance with the procedures outlined in the National Institutes of Health Guidelines. The experimental protocol was approved by the Institutional Ethics Committee of Hebei Medical University. Rats were randomly divided into five groups: diabetes mellitus (DM) (n = 15), DM + low concentration β-hydroxybutyrate (BHB1) (n = 15), DM + intermediate concentration β-hydroxybutyrate (BHB2) (n = 15), DM + high concentration β-hydroxybutyrate (BHB3) (n = 15), and control (Con) (n = 10). All four DM groups were intraperitoneally injected were injected intraperitoneally with streptozotocin (40 mg/kg, dissolved in 0.1 mol/L citrate buffer, pH 4.4, freshly made, 10 mg/mL), to induce diabetes, as previously reported (Li et al., 2018). Rats in DM + BHB1, DM + BHB2, and DM + BHB3 groups were injected subcutaneously with 160, 200, and 240 mg/kg/day β-hydroxybutyrate (Sigma, Steinheim, Germany), respectively (Bae et al., 2016; Orhan et al., 2016); rats in the DM and Con groups were administered an equivalent volume of saline. Body weight and fasting blood glucose were measured at 3 days and 10 weeks after diabetes induction. At the end of the experiment, aorta tissues were collected for hematoxylineosin (HE) staining, while serum samples were collected for measurement of NO concentrations. A segment of aorta was used for detections of VEGF mRNA expression level, protein content, and immunohistochemistry staining, as well as for measurement of protein βhydroxybutyrylation and H3K9bhb contents.
2.6. Statistical analysis Statistical analysis was performed using SPSS Statistics software and the data were presented as means ± standard deviations (SD). Oneway analysis of variance was used to analyze the differences among the groups, and the differences between two groups were evaluated by Fisher’s Least Significant Difference. Differences with P < 0.05 were considered statistically significant. 3. Results 3.1. β-hydroxybutyrate attenuated diabetic injury of the endothelium Body weight and blood glucose were measured at 3 days and 10 weeks after diabetes induction. Body weight was significantly lower at 10 weeks in the DM group than in the Con group, but did not significantly differ between BHB groups and the DM group (Fig. 1a). Compared with the Con group, blood glucose increased significantly in the DM and BHB groups at 3 days after diabetes induction, indicating that the diabetic rat model had been successfully established (Fig. 1b). After 10 weeks of treatment, the high blood glucose was not alleviated by any concentrations of β-hydroxybutyrate. Morphological changes of the aortic endothelium were evaluated by HE staining (Fig. 2). Intact endothelium was observed in the Con group, but the endothelium was deteriorating in the DM group. The deteriorating endothelium was similar in the BHB1 and DM groups; however, the endothelium was nearly intact in the BHB2 group and only exhibited partial deterioration in the BHB3 group. Furthermore, serum NO concentrations were assessed to determine endothelial cell function (Fig. 3a). Compared with the Con group, NO
2.2. Detection of serum NO concentration Serum NO concentrations were analyzed by a detection kit (Jiancheng, Nanjing, China). Briefly, after serum and reaction agents had been added, NO was first changed to nitrite; it was then reacted with the chromogenic agent to generate a pink azo compound, which was quantified by a microplate Reader at 550 nm. 2
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real time RT-PCR (Fig. 3b). Compared with the Con group, the mRNA expression level of VEGF was lower in the DM group. Compared with the DM group, the mRNA expression levels of VEGF were significantly greater in the BHB2 and BHB3 groups. Furthermore, western blotting assessment of VEGF protein content showed changes consistent with the mRNA expression levels (Fig. 3c). VEGF protein content was lower in the DM group than in the Con group; this reduced level of VEGF was significantly increased in the BHB2 and BHB3 groups. Immunohistochemistry staining revealed the distribution of endothelial VEGF (Fig. 4). Notably, endothelial VEGF staining was clear in the Con group, but was nearly absent in the DM group. However, endothelial VEGF staining could be detected in the BHB groups, similar to the changes in its mRNA expression level and protein content. These results indicated that β-hydroxybutyrate promoted the generation of endothelial VEGF, with the most distinctive effects from its intermediate concentration. 3.3. β-hydroxybutyrate induced H3K9bhb of diabetic aorta Fig. 1. Changes in general indicators in the experimental rats. Body weight (A) and blood glucose (B) were measured at 3 days and 10 weeks after diabetes induction. Data were shown as means ± SD (n = 9). **P < 0.01 vs control (Con) group. DM, diabetes mellitus; BHB, β-hydroxybutyrate.
The total protein β-hydroxybutyrylation in aorta was detected by western blotting. Total protein β-hydroxybutyrylation revealed extensive unique bands (Fig. 5a). Compared with the Con group, the total protein β-hydroxybutyrylation in aorta was slightly elevated in the DM group, but was markedly increased in the BHB2 and BHB3 groups. Then H3K9bhb content was also evaluated (Fig. 5b). Compared with the Con group, H3K9bhb content increased in the DM group, and was even higher in the BHB groups. Notably, the increase in H3K9bhb content in the BHB2 and BHB3 groups significantly differed from the H3K9bhb content in the DM group; the highest H3K9bhb content was detected in the BHB2 group. These results demonstrated that β-hydroxybutyrate could cause significant increases in β-hydroxybutyrylation and H3K9bhb contents, which presumably promoted generation of VEGF in diabetic aorta. 4. Discussion Although endothelial injury is the initial pathological insult involved in diabetes-related vascular diseases, there remains a lack of effective therapies. Recently, β-hydroxybutyrate was reported to have various protective roles in the cardiovascular system, but its ability to antagonize diabetic injury of the endothelium was not elucidated. The present study showed that moderately elevated β-hydroxybutyrate could antagonize aortic endothelial injury via H3K9bhb, thereby promoting the generation of VEGF in diabetic rats. In addition to their barrier function, endothelial cells secrete cytoactive factors, such as NO, which are crucial in vascular homeostasis. Endothelial dysfunction is regarded as the initial pathological manifestation of diabetes-related vascular complications (Khaled et al., 2018; Caradu et al., 2018; Goligorsky et al., 2017). This study also confirmed demonstrated reductions in endothelial cells and serum NO concentrations in diabetic rats, indicative of endothelial injury. Although excess VEGF may exacerbate angiogenesis, insufficient levels also lead to endothelial dysfunction (Zafar et al., 2017). In this study, the generation of aortic VEGF was reduced in diabetic rats, contributing to endothelial injury. The major component of ketone bodies, β-hydroxybutyrate, has various protective roles in the cardiovascular system (Uchihashi et al., 2017; Mizuno et al., 2017; Yu et al., 2018). Although a sudden increase in ketone bodies may cause ketoacidosis, the physical presence of βhydroxybutyrate has been reported to exhibit beneficial effects in patients with diabetes (Mizuno et al., 2017; Min et al., 2018). With respect to other ketone bodies, β-hydroxybutyrate has protective effects on endothelial cells (Rains et al., 2015; Han et al., 2018). Although β-hydroxybutyrate has been reported to protect against low glucose-induced endothelial cell damage, it has been unclear whether β-hydroxybutyrate can antagonize aortic endothelial injury in patients with
Fig. 2. Pathological changes of aortic endothelium. Morphological changes of aortic endothelium were evaluated by hematoxylin-eosin staining (×400). Scala bar: 50 μm. Intact endothelium was observed in the control (Con) group, whereas deterioration of endothelium was seen in the diabetes mellitus (DM) group. Only partial deterioration of the endothelium was observed in the BHB2 and BHB3 groups. BHB, β-hydroxybutyrate.
concentration was significantly reduced in the DM group. However, the reduced NO concentration was alleviated by β-hydroxybutyrate treatments; the NO concentration was significantly greater in the BHB2 group than in the DM group. These results indicated that β-hydroxybutyrate, especially at an intermediate concentration, could attenuate diabetic injury of the endothelium, without affecting body weight and blood glucose in diabetic rats. 3.2. β-hydroxybutyrate promoted generation of endothelial VEGF At first, mRNA expression levels of aortic VEGF were evaluated by 3
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Fig. 3. Nitric oxide (NO) concentration and vascular endothelial growth factor (VEGF) generation in diabetic rat aorta. (A) Changes in serum NO concentration (n = 9). VEGF mRNA expression level (B) and protein content (C) were measured by real-time RT-PCR and western blotting, respectively. Data were repeated three times and shown as means ± SD. **P < 0.01 vs control (Con) group; ## P < 0.01 or #P < 0.05 vs diabetes mellitus (DM) group. BHB, β-hydroxybutyrate.
inflammasome (Rahman et al., 2014; Fu et al., 2014; Youm et al., 2015). Furthermore, β-hydroxybutyrate may inhibit histone deacetylase, thereby elevating histone acetylation and activating expression of protective genes, such as superoxide dismutase (Wang et al., 2017; Sleiman et al., 2016; Huang et al., 2018). Recently, β-hydroxybutyrate was found to directly cause H3K9bhb, which activates gene expression, independent of acetylation (Xie et al., 2016). This histone modification directly links metabolite and gene expression regulation to the effects of β-hydroxybutyrate. H3K9bhb can also elevate brain-derived neurotrophic factor levels and alleviate depressive behaviors in mice (Chen et al., 2017). However, with respect to alleviation of endothelial injury within the context of diabetes, there has been no report of whether βhydroxybutyrate up-regulates generation of VEGF via production of H3K9bhb. The results of the present study showed that, along with causing total protein β-hydroxybutyrylation, β-hydroxybutyrate treatment could cause marked elevation of H3K9bhb, despite slight elevation in diabetic aorta. The β-hydroxybutyrate-induced increase in H3K9bhb was concentration-dependent, with the most marked effects at its intermediate concentration, consistent with its ability to protect against endothelial injury and cause up-regulation of VEGF. These results indicated that β-hydroxybutyrate-induced H3K9bhb might lead to increased generation of VEGF. Nevertheless, reduced generation of endothelial VEGF in the diabetic aorta, may involve intracellular oxidative stress and other mechanisms (Zafar et al., 2017), which would potentially be targeted by exogenous β-hydroxybutyrate. Further studies are needed to test these hypotheses and clarify the intrinsic mechanism by which β-hydroxybutyrate protects against diabetic endothelial injury. In conclusion, moderately elevated β-hydroxybutyrate could antagonize aortic endothelial injury, potentially by causing H3K9bhb to promote generation of VEGF in diabetic rats.
Fig. 4. Distribution of endothelial vascular endothelial growth factor (VEGF). The distribution of endothelial VEGF was illustrated by immunohistochemistry (×400). Scala bar: 50 μm. VEGF staining was obvious in the control (Con) group, nearly absent in the diabetes mellitus (DM) group, and returned in the βhydroxybutyrate (BHB)-treated groups.
diabetes (Soejima et al., 2018). In this study, the results demonstrated that β-hydroxybutyrate could attenuate injury of the endothelium and promote generation of endothelial VEGF in diabetic rats. Moreover, the protective effects of β-hydroxybutyrate were concentration-dependent, such that the intermediate concentration exhibited the most conspicuous protective effects; this demonstrated a unique protective effect of β-hydroxybutyrate against diabetic endothelial injury. A series of mechanisms have been proposed for the protective effects of β-hydroxybutyrate (Newman et al., 2014, 2017; Rojas-Morales et al., 2016). In particular, β-hydroxybutyrate may bind a membrane receptor, interact with other proteins, or inhibit the NRLP3
CRediT authorship contribution statement Xingliang Wu: Investigation, Writing - original draft preparation. Dazhuang Miao: Investigation, Methodology. Zijing Liu: Investigation, Data curation. Kun Liu: Methodology. Boning Zhang: Data curation. Jialin Li: Visualization. Yanning Li: Conceptualization, Writing - reviewing & editing. Jinsheng Qi: Supervision, Project administration. 4
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Fig. 5. Total protein β-hydroxybutyrylation and H3K9bhb contents in diabetic rat aorta. (A) Total protein β-hydroxybutyrylation was detected by western blotting. (B) H3K9bhb content was determined by western blotting. Data were repeated three times and shown as means ± SD. **P < 0.01, vs control (Con) group; ## P < 0.01 or #P < 0.05, vs diabetes mellitus (DM) group. BHB, β-hydroxybutyrate.
Declaration of Competing Interest
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β-hydroxybutyrate antagonizes aortic endothelial injury by promoting generation of VEGF in diabetic rats
T
Xingliang Wua, Dazhuang Miaob, Zijing Liua, Kun Liua, Boning Zhanga, Jialin Lia, Yanning Lib,*,1, Jinsheng Qia,**,1 a b
Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, Shijiazhuang, China Department of Molecular Biology, Hebei Key Lab of Laboratory Animal Science, Hebei Medical University, Shijiazhuang, China
A R T I C LE I N FO
A B S T R A C T
Keywords: β-Hydroxybutyrate Vascular endothelial growth factor Histone H3K9 β-hydroxybutyrylation Diabetic endothelial injury
Endothelial injury is regarded as the initial pathological process in diabetic vascular diseases, but effective therapy has not yet been identified. Although β-hydroxybutyrate plays various protective roles in the cardiovascular system, its ability to antagonize diabetic endothelial injury is unclear. β-hydroxybutyrate reportedly causes histone H3K9 β-hydroxybutyrylation (H3K9bhb), which activates gene expression; however, there has been no report regarding the role of H3K9bhb in up-regulation of vascular endothelial growth factor (VEGF), a crucial factor in endothelial integrity and function. Here, male Sprague-Dawley rats were intraperitoneally injected with streptozotocin to induce diabetes, and then treated with different concentrations of β-hydroxybutyrate. After 10 weeks, body weight, blood glucose, morphological changes and serum nitric oxide concentration were examined. Moreover, the mRNA expression level, protein content and distribution of VEGF in the aorta were investigated, as were total protein β-hydroxybutyrylation and H3K9bhb contents. The results showed injury of aortic endothelium, along with reductions of the concentration of nitric oxide and generation of VEGF in diabetic rats. However, β-hydroxybutyrate treatment attenuated diabetic injury of the endothelium and up-regulated the generation of VEGF. Furthermore, β-hydroxybutyrate treatment caused marked total protein βhydroxybutyrylation and significant elevation of H3K9bhb content in the aorta of diabetic rats. The ability of βhydroxybutyrate to protect against diabetic injury of the aortic endothelium was greatest for its intermediate concentration. In conclusion, moderately elevated β-hydroxybutyrate could antagonize aortic endothelial injury, potentially by causing H3K9bhb to promote generation of VEGF in diabetic rats.
1. Introduction Macrovascular complications are major causes of disability and death in patients with diabetes (Fisher et al., 2018; Pan et al., 2018). Endothelial dysfunction represents the initial pathological process and is an early manifestation of diabetic vascular disease (Khaled et al., 2018; Caradu et al., 2018). However, effective therapy for diabetic endothelial injury has not been established. Recently, it has been reported that β-hydroxybutyrate (a major component of ketone body), has various protective roles, especially in nerve and cardiovascular systems (Achanta et al., 2017; Uchihashi et al., 2017). When glucose supply is insufficient, such as in starvation
conditions, ketone bodies are generated (Sabari et al., 2017). Although a sharp increase in ketone bodies may cause ketoacidosis in patients with diabetes, moderate increases have been reported to exhibit beneficial effects (Mizuno et al., 2017; Min et al., 2018). With respect to other ketone bodies, a physical concentration of β-hydroxybutyrate below 10 mM typically has protective effects, especially for endothelial cells (Obokata et al., 2017; Stubbs et al., 2017; Rains et al., 2015). However, it is unknown whether β-hydroxybutyrate can antagonize aortic endothelial injury in diabetes. In addition to potential effects on energy supply, several mechanisms have been proposed for the protective effects of β-hydroxybutyrate (Newman et al., 2014, 2017; Rojas-Morales et al., 2016). Notably, the
⁎ Corresponding author at: Department of Molecular Biology, Hebei Key Lab of Laboratory Animal, Hebei Medical University, No. 361 East Zhongshan Road, Shijiazhuang, 050017, Hebei, PR China. ⁎⁎ Corresponding author at: Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, No. 361 East Zhongshan Road, Shijiazhuang, 050017, Hebei, PR China. E-mail addresses: [email protected] (Y. Li), [email protected] (J. Qi). 1 Present/permanent address: No. 361 East Zhongshan Road, Shijiazhuang 050017, Hebei, PR China.
https://doi.org/10.1016/j.tice.2020.101345 Received 26 September 2019; Received in revised form 14 February 2020; Accepted 14 February 2020 Available online 15 February 2020 0040-8166/ © 2020 Elsevier Ltd. All rights reserved.
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protective effects of β-hydroxybutyrate are reportedly mediated through a membrane receptor, interactions with other proteins, or inhibition of the NRLP3 inflammasome (Rahman et al., 2014; Fu et al., 2014; Youm et al., 2015). In addition, β-hydroxybutyrate has been shown to enter the nucleus and inhibit histone deacetylase, thereby activating expression of protective genes (Wang et al., 2017; Sleiman et al., 2016). β-hydroxybutyrate also directly causes histone H3K9 βhydroxybutyrylation (H3K9bhb), which then activates gene expression, independent of acetylation (Xie et al., 2016). Physiological levels of vascular endothelial growth factor (VEGF) are required for the maintenance of normal endothelial integrity and function (Laakkonen et al., 2018; Logue et al., 2017). Although excess VEGF may exacerbate angiogenesis, insufficient levels of VEGF also lead to endothelial dysfunction (Zafar et al., 2017). With respect to alleviation of endothelial injury in the context of diabetes, there has been no report regarding whether β-hydroxybutyrate can up-regulate the generation of VEGF via H3K9bhb. In this study, diabetes was induced in male Sprague-Dawley rats, which were then treated with different concentrations of β-hydroxybutyrate. Morphological changes and serum nitric oxide (NO) concentration were assessed to evaluate the ability of β-hydroxybutyrate to antagonize diabetic injury of the aortic endothelium. Moreover, VEGF generation, total protein β-hydroxybutyrylation and H3K9bhb content were determined to investigate the underlying mechanism by which βhydroxybutyrate promoted generation of aortic VEGF in diabetic rats.
2.3. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) The mRNA expression levels of VEGF were quantified by real-time RT-PCR. Total RNA was isolated using TRIzol reagent (Takara, Dalian, China) and reverse transcribed to cDNA using RevertAid First Strand cDNA synthesis Kit (Fermentas, Shanghai, China), followed by real-time PCR amplification. The comparative Ct method was used to calculate the relative abundance of mRNA, compared with the abundance of 18S rRNA. Specific primers used were as follows: Rat VEGF (forward) 5′-AGGCGAGGCAGCTTGAGTTA-3′, (reverse) 5′-CTGTCGACGGTGACG ATGGT); and 18S rRNA (forward) 5′-AAGTTTCAGCACATCCTGCGA GTA-3′, (reverse) 5′-TTGGTGAGGTCAATGTCTGCTTTC-3′. 2.4. Western blotting VEGF protein contents, as well as the levels of β-hydroxybutyrylation and H3K9bhb, were measured by western blotting, using a previously described protocol (Li et al., 2018). Briefly, anti-VEGF (Sangon Biotech, Shanghai, China), anti-pan β-hydroxybutyrylation (Jingjie Biotech, hangzhou, China) and anti-H3K9bhb antibodies (Jingjie Biotech, hangzhou, China) were used. Band intensity was quantified and calculated. An antibody to β-actin (Sangon Biotech, Shanghai, China) served as a loading control routinely. 2.5. Immunohistochemistry
2. Material and methods
The distribution of endothelial VEGF in aortic tissue was detected by immunohistochemistry. The procedures were as follows: aorta samples were embedded in paraffin, then was cut and dewaxed. After sections had been incubated with H2O2, they were incubated with goat serum (Zhongshan, Beijing, China). Sections were incubated with antiVEGF antibody (diluted 1:50, Sangon Biotech, Shanghai, China) and then the secondary antibody (diluted 1:200, Zhongshan, Beijing, China). 3, 3 -Diaminobenzidine solution was used for color emergence.
2.1. Animals and experiment design Seventy healthy male Sprague-Dawley rats (246.45 ± 29.37 g) were provided by the Department of Experimental Animals of Hebei Medical University. Animal care and use were performed in accordance with the procedures outlined in the National Institutes of Health Guidelines. The experimental protocol was approved by the Institutional Ethics Committee of Hebei Medical University. Rats were randomly divided into five groups: diabetes mellitus (DM) (n = 15), DM + low concentration β-hydroxybutyrate (BHB1) (n = 15), DM + intermediate concentration β-hydroxybutyrate (BHB2) (n = 15), DM + high concentration β-hydroxybutyrate (BHB3) (n = 15), and control (Con) (n = 10). All four DM groups were intraperitoneally injected were injected intraperitoneally with streptozotocin (40 mg/kg, dissolved in 0.1 mol/L citrate buffer, pH 4.4, freshly made, 10 mg/mL), to induce diabetes, as previously reported (Li et al., 2018). Rats in DM + BHB1, DM + BHB2, and DM + BHB3 groups were injected subcutaneously with 160, 200, and 240 mg/kg/day β-hydroxybutyrate (Sigma, Steinheim, Germany), respectively (Bae et al., 2016; Orhan et al., 2016); rats in the DM and Con groups were administered an equivalent volume of saline. Body weight and fasting blood glucose were measured at 3 days and 10 weeks after diabetes induction. At the end of the experiment, aorta tissues were collected for hematoxylineosin (HE) staining, while serum samples were collected for measurement of NO concentrations. A segment of aorta was used for detections of VEGF mRNA expression level, protein content, and immunohistochemistry staining, as well as for measurement of protein βhydroxybutyrylation and H3K9bhb contents.
2.6. Statistical analysis Statistical analysis was performed using SPSS Statistics software and the data were presented as means ± standard deviations (SD). Oneway analysis of variance was used to analyze the differences among the groups, and the differences between two groups were evaluated by Fisher’s Least Significant Difference. Differences with P < 0.05 were considered statistically significant. 3. Results 3.1. β-hydroxybutyrate attenuated diabetic injury of the endothelium Body weight and blood glucose were measured at 3 days and 10 weeks after diabetes induction. Body weight was significantly lower at 10 weeks in the DM group than in the Con group, but did not significantly differ between BHB groups and the DM group (Fig. 1a). Compared with the Con group, blood glucose increased significantly in the DM and BHB groups at 3 days after diabetes induction, indicating that the diabetic rat model had been successfully established (Fig. 1b). After 10 weeks of treatment, the high blood glucose was not alleviated by any concentrations of β-hydroxybutyrate. Morphological changes of the aortic endothelium were evaluated by HE staining (Fig. 2). Intact endothelium was observed in the Con group, but the endothelium was deteriorating in the DM group. The deteriorating endothelium was similar in the BHB1 and DM groups; however, the endothelium was nearly intact in the BHB2 group and only exhibited partial deterioration in the BHB3 group. Furthermore, serum NO concentrations were assessed to determine endothelial cell function (Fig. 3a). Compared with the Con group, NO
2.2. Detection of serum NO concentration Serum NO concentrations were analyzed by a detection kit (Jiancheng, Nanjing, China). Briefly, after serum and reaction agents had been added, NO was first changed to nitrite; it was then reacted with the chromogenic agent to generate a pink azo compound, which was quantified by a microplate Reader at 550 nm. 2
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real time RT-PCR (Fig. 3b). Compared with the Con group, the mRNA expression level of VEGF was lower in the DM group. Compared with the DM group, the mRNA expression levels of VEGF were significantly greater in the BHB2 and BHB3 groups. Furthermore, western blotting assessment of VEGF protein content showed changes consistent with the mRNA expression levels (Fig. 3c). VEGF protein content was lower in the DM group than in the Con group; this reduced level of VEGF was significantly increased in the BHB2 and BHB3 groups. Immunohistochemistry staining revealed the distribution of endothelial VEGF (Fig. 4). Notably, endothelial VEGF staining was clear in the Con group, but was nearly absent in the DM group. However, endothelial VEGF staining could be detected in the BHB groups, similar to the changes in its mRNA expression level and protein content. These results indicated that β-hydroxybutyrate promoted the generation of endothelial VEGF, with the most distinctive effects from its intermediate concentration. 3.3. β-hydroxybutyrate induced H3K9bhb of diabetic aorta Fig. 1. Changes in general indicators in the experimental rats. Body weight (A) and blood glucose (B) were measured at 3 days and 10 weeks after diabetes induction. Data were shown as means ± SD (n = 9). **P < 0.01 vs control (Con) group. DM, diabetes mellitus; BHB, β-hydroxybutyrate.
The total protein β-hydroxybutyrylation in aorta was detected by western blotting. Total protein β-hydroxybutyrylation revealed extensive unique bands (Fig. 5a). Compared with the Con group, the total protein β-hydroxybutyrylation in aorta was slightly elevated in the DM group, but was markedly increased in the BHB2 and BHB3 groups. Then H3K9bhb content was also evaluated (Fig. 5b). Compared with the Con group, H3K9bhb content increased in the DM group, and was even higher in the BHB groups. Notably, the increase in H3K9bhb content in the BHB2 and BHB3 groups significantly differed from the H3K9bhb content in the DM group; the highest H3K9bhb content was detected in the BHB2 group. These results demonstrated that β-hydroxybutyrate could cause significant increases in β-hydroxybutyrylation and H3K9bhb contents, which presumably promoted generation of VEGF in diabetic aorta. 4. Discussion Although endothelial injury is the initial pathological insult involved in diabetes-related vascular diseases, there remains a lack of effective therapies. Recently, β-hydroxybutyrate was reported to have various protective roles in the cardiovascular system, but its ability to antagonize diabetic injury of the endothelium was not elucidated. The present study showed that moderately elevated β-hydroxybutyrate could antagonize aortic endothelial injury via H3K9bhb, thereby promoting the generation of VEGF in diabetic rats. In addition to their barrier function, endothelial cells secrete cytoactive factors, such as NO, which are crucial in vascular homeostasis. Endothelial dysfunction is regarded as the initial pathological manifestation of diabetes-related vascular complications (Khaled et al., 2018; Caradu et al., 2018; Goligorsky et al., 2017). This study also confirmed demonstrated reductions in endothelial cells and serum NO concentrations in diabetic rats, indicative of endothelial injury. Although excess VEGF may exacerbate angiogenesis, insufficient levels also lead to endothelial dysfunction (Zafar et al., 2017). In this study, the generation of aortic VEGF was reduced in diabetic rats, contributing to endothelial injury. The major component of ketone bodies, β-hydroxybutyrate, has various protective roles in the cardiovascular system (Uchihashi et al., 2017; Mizuno et al., 2017; Yu et al., 2018). Although a sudden increase in ketone bodies may cause ketoacidosis, the physical presence of βhydroxybutyrate has been reported to exhibit beneficial effects in patients with diabetes (Mizuno et al., 2017; Min et al., 2018). With respect to other ketone bodies, β-hydroxybutyrate has protective effects on endothelial cells (Rains et al., 2015; Han et al., 2018). Although β-hydroxybutyrate has been reported to protect against low glucose-induced endothelial cell damage, it has been unclear whether β-hydroxybutyrate can antagonize aortic endothelial injury in patients with
Fig. 2. Pathological changes of aortic endothelium. Morphological changes of aortic endothelium were evaluated by hematoxylin-eosin staining (×400). Scala bar: 50 μm. Intact endothelium was observed in the control (Con) group, whereas deterioration of endothelium was seen in the diabetes mellitus (DM) group. Only partial deterioration of the endothelium was observed in the BHB2 and BHB3 groups. BHB, β-hydroxybutyrate.
concentration was significantly reduced in the DM group. However, the reduced NO concentration was alleviated by β-hydroxybutyrate treatments; the NO concentration was significantly greater in the BHB2 group than in the DM group. These results indicated that β-hydroxybutyrate, especially at an intermediate concentration, could attenuate diabetic injury of the endothelium, without affecting body weight and blood glucose in diabetic rats. 3.2. β-hydroxybutyrate promoted generation of endothelial VEGF At first, mRNA expression levels of aortic VEGF were evaluated by 3
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Fig. 3. Nitric oxide (NO) concentration and vascular endothelial growth factor (VEGF) generation in diabetic rat aorta. (A) Changes in serum NO concentration (n = 9). VEGF mRNA expression level (B) and protein content (C) were measured by real-time RT-PCR and western blotting, respectively. Data were repeated three times and shown as means ± SD. **P < 0.01 vs control (Con) group; ## P < 0.01 or #P < 0.05 vs diabetes mellitus (DM) group. BHB, β-hydroxybutyrate.
inflammasome (Rahman et al., 2014; Fu et al., 2014; Youm et al., 2015). Furthermore, β-hydroxybutyrate may inhibit histone deacetylase, thereby elevating histone acetylation and activating expression of protective genes, such as superoxide dismutase (Wang et al., 2017; Sleiman et al., 2016; Huang et al., 2018). Recently, β-hydroxybutyrate was found to directly cause H3K9bhb, which activates gene expression, independent of acetylation (Xie et al., 2016). This histone modification directly links metabolite and gene expression regulation to the effects of β-hydroxybutyrate. H3K9bhb can also elevate brain-derived neurotrophic factor levels and alleviate depressive behaviors in mice (Chen et al., 2017). However, with respect to alleviation of endothelial injury within the context of diabetes, there has been no report of whether βhydroxybutyrate up-regulates generation of VEGF via production of H3K9bhb. The results of the present study showed that, along with causing total protein β-hydroxybutyrylation, β-hydroxybutyrate treatment could cause marked elevation of H3K9bhb, despite slight elevation in diabetic aorta. The β-hydroxybutyrate-induced increase in H3K9bhb was concentration-dependent, with the most marked effects at its intermediate concentration, consistent with its ability to protect against endothelial injury and cause up-regulation of VEGF. These results indicated that β-hydroxybutyrate-induced H3K9bhb might lead to increased generation of VEGF. Nevertheless, reduced generation of endothelial VEGF in the diabetic aorta, may involve intracellular oxidative stress and other mechanisms (Zafar et al., 2017), which would potentially be targeted by exogenous β-hydroxybutyrate. Further studies are needed to test these hypotheses and clarify the intrinsic mechanism by which β-hydroxybutyrate protects against diabetic endothelial injury. In conclusion, moderately elevated β-hydroxybutyrate could antagonize aortic endothelial injury, potentially by causing H3K9bhb to promote generation of VEGF in diabetic rats.
Fig. 4. Distribution of endothelial vascular endothelial growth factor (VEGF). The distribution of endothelial VEGF was illustrated by immunohistochemistry (×400). Scala bar: 50 μm. VEGF staining was obvious in the control (Con) group, nearly absent in the diabetes mellitus (DM) group, and returned in the βhydroxybutyrate (BHB)-treated groups.
diabetes (Soejima et al., 2018). In this study, the results demonstrated that β-hydroxybutyrate could attenuate injury of the endothelium and promote generation of endothelial VEGF in diabetic rats. Moreover, the protective effects of β-hydroxybutyrate were concentration-dependent, such that the intermediate concentration exhibited the most conspicuous protective effects; this demonstrated a unique protective effect of β-hydroxybutyrate against diabetic endothelial injury. A series of mechanisms have been proposed for the protective effects of β-hydroxybutyrate (Newman et al., 2014, 2017; Rojas-Morales et al., 2016). In particular, β-hydroxybutyrate may bind a membrane receptor, interact with other proteins, or inhibit the NRLP3
CRediT authorship contribution statement Xingliang Wu: Investigation, Writing - original draft preparation. Dazhuang Miao: Investigation, Methodology. Zijing Liu: Investigation, Data curation. Kun Liu: Methodology. Boning Zhang: Data curation. Jialin Li: Visualization. Yanning Li: Conceptualization, Writing - reviewing & editing. Jinsheng Qi: Supervision, Project administration. 4
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Fig. 5. Total protein β-hydroxybutyrylation and H3K9bhb contents in diabetic rat aorta. (A) Total protein β-hydroxybutyrylation was detected by western blotting. (B) H3K9bhb content was determined by western blotting. Data were repeated three times and shown as means ± SD. **P < 0.01, vs control (Con) group; ## P < 0.01 or #P < 0.05, vs diabetes mellitus (DM) group. BHB, β-hydroxybutyrate.
Declaration of Competing Interest
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