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Published in final edited form as: J Neurochem. 2016 Mar 17;137(3):371–383. doi: 10.1111/jnc.13587

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects

Jingwen Yu *, Yanqing Wu *, Peixin Yang *,†
PMCID: PMC4837015  NIHMSID: NIHMS761701  PMID: 26896748

Abstract

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) overexpression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. DMNQ, a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9 and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 overexpression or tempol treatment. SIRT2 or SIRT6 overexpression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs.

Keywords: maternal diabetes, neural tube defects, epigenetic mechanism, sirtuin deacetylase, histone acetylation, oxidative stress

INTRODUCTION

Diabetes is a significant risk factor for birth defects (Correa et al. 2008, Correa et al. 2012). Nearly 60 million women of reproductive age (18–44 years) worldwide, and 3 million in the United States alone, have diabetes, and the number is continuing to increase. A recent large-scale study shows the number of pregnant women with pre-existing diabetes has more than doubled in the past seven years, a troubling trend that engenders health risks for both mothers and newborns (Lawrence et al. 2008). Unfortunately, euglycemic control by insulin administration is difficult to achieve, as even transient exposure to maternal hyperglycemia causes embryonic malformations (Reece et al. 1996). A recent multi-center study demonstrated that diabetic women under modern preconceptional care are still three to four times more likely to have a child with birth defects than nondiabetic women (Correa et al. 2008, Correa et al. 2012). Thus, maternal diabetes-induced birth defects remain a significant health problem and the development of accessible, convenient and effective prevention strategies is urgently needed. To achieve this goal, seeking a better understanding of the mechanisms underlying maternal diabetes-induced malformations is an essential first step. Cellular stress and gene dysregulation has been observed in embryos exposed to maternal diabetes (Yang & Li 2010, Yang et al. 2013, Yang & Reece 2011, Yang et al. 2014, Yang et al. 2007, Yang et al. 2008a, Yang et al. 2008b, Xu et al. 2013, Gu et al. 2015b, Wang et al. 2013, Wang et al. 2015e, Li et al. 2013, Dong et al. 2015a, Dong et al. 2015b, Dong et al. 2015c); however, the underlying mechanism is still murky.

Although aberrant epigenetic modifications are implicated in maternal diabetes-induced NTDs (Salbaum & Kappen 2011), prior studies in our field have never addressed the epigenetic mechanism behind this disease process despite the fact that hyperglycemia is strongly associated with epigenetic modifications in the etiology of diabetic complications. One significant barrier to research in this arena is the difficulty in ascribing specific epigenetic modifications to specific diabetes-induced complications and subsequently determining whether such modifications can directly cause hyperglycemia-induced organ damages. By using our unique diabetic animal models to study maternal diabetes-induced NTDs, we planned to assess the changes of mammalian sirtuin (SIRT) expression and, thus, attempted to delineate how the changes of these two deacetylases contribute to this disease.

The idea was inspired by observations that in humans medications with deacetylase inhibitory activity induce NTDs (Robert & Guibaud 1982, Gurvich et al. 2005) as does maternal diabetes (Yang et al. 2013). Additionally, a recent study demonstrated that SIRT is required for neurulation and a sirtuin inhibitor causes NTDs in Xenopus laevis embryos (Ohata et al. 2014).

Sirtuin was first discovered in yeast as Sir2, which is essential for the maintenance of heterochromatin and prolongs lifespan (Kaeberlein et al. 1999). There are seven mammalian sirtuins, SIRT1-7 (Herskovits & Guarente 2013). Alterations of sirtuin expression and function have been linked to the pathogenesis of many human diseases including diabetes, cancer and neurological diseases (Herskovits & Guarente 2013). Thus, sirtuins, their activators and inhibitors are regarded as novel therapeutic targets for human diseases (Schemies et al. 2010). Although sirtuins also deacetylate non-histone proteins, they are typically classified as class III histone deacetylases (HDACs) (Gregoretti et al. 2004). Histone hypoacetylation creates a heterochromatic state that prevents transcription factor access to the DNA regulatory regions and, thus, suppresses gene transcription. Therefore, sirtuins are transcription suppressors. Sirtuins protect cells and organisms from aging by regulating stress responses, apoptosis and DNA repair (Herskovits & Guarente 2013), all of which are critical for the induction of diabetic embryopathy. We hypothesize that maternal diabetes and high glucose in vitro alter sirtuin expression in the developing embryo and altered sirtuin expression may play important roles in diabetic embryopathy.

In the present study, we evaluated the effect of maternal diabetes in vivo or high glucose in vitro on sirtuin expression. Using a transgenic mouse model and an in vitro whole-embryo culture system coupled with neural stem cell cultures, we demonstrated the involvement of oxidative stress and the critical role of SIRT2 and SIRT6 down-regulation in the induction of NTDs under maternal diabetic or high glucose conditions.

Materials and Methods

Animals and reagents

The procedures for animal use were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. Wild-type (WT) C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). SOD1-Transgenic (Tg) mice in C57BL/6J background were revived from frozen embryos by the Jackson Laboratory (Stock number: 002298). Streptozotocin (STZ) from Sigma (St. Louis, MO) was dissolved in sterile 0.1 M citrate buffer (pH4.5). PEGSOD, the cell permeable SOD, and DMNQ, a superoxide generating agent were both purchased from Sigma-Aldrich (St. Louis, MO).

Mouse models of diabetic embryopathy

Our mouse model of diabetic embryopathy has been described previously (Yang et al. 2013). Briefly, ten-week old WT female mice were intravenously injected daily with 75 mg/kg STZ over two days to induce diabetes. Using STZ to induce diabetes is not a complicating factor because STZ is cleared from the bloodstream rapidly (STZ serum half-life, 15 minutes), and pregnancy is not established until one-to-two weeks after STZ injection. Diabetes was defined as a 12-hour fasting blood glucose level of ≥ 14 mM. To generate SOD1 embryos, we crossed SOD1-Tg male mice with nondiabetic or diabetic WT female mice. Male and female mice were paired at 3:00 P.M., and the next morning pregnancy was established by the presence of the vaginal plug, and noon of that day was designated as day 0.5 (E0.5). WT female mice were treated with vehicle injections to serve as nondiabetic controls. On E8.75 (at 6:00 P.M.), mice were euthanized and conceptuses were dissected out of the uteri, embryos with the yolk sacs were removed from the deciduas and then yolk sacs were removed from the embryos. The embryos were used for analysis.

Whole-embryo culture

The procedure of whole-embryo culture has been previously described (Wu et al. 2015b, Yang & Li 2010, Yang et al. 2007, Zhong et al. 2015). Mouse embryos at E8.5 were dissected out of the uteri in PBS (Invitrogen, La Jolla, CA). The parietal yolk sac was removed using a pair of fine forceps, and the visceral yolk sac was left intact. Embryos (four per bottle) were cultured in 25% Tyrode’s salt solution and 75% rat serum that was freshly prepared from male rats. The embryos were cultured at 37°C in 30 revolutions/min rotation in the roller bottle system. The culture bottles were gassed 5% O2/ 5% CO2/ 90% N2 for the first 24 h and 20% O2/ 5% CO2/ 75% N2 for the last 12 h.

Embryos were cultured for 36 h with 100 mg/dl glucose, a value close to the blood glucose level of nondiabetic mice, or 300 mg/dl glucose, which is equivalent to the blood glucose level of diabetic mice, in the presence of the SIRT activator, SRT1720 (Selleckchem, Houston, TX), or the combination of a SIRT2 inhibitor AGK2 and a pan sirtuin inhibitor, Sirtinol (both were purchased from Enzo Life Sciences, Farmingdale, NY). At the end of 36 h, embryos were dissected from the yolk sac and examined under a Leica MZ16F stereomicroscope (Leica Microsystems, Bannockburn, IL) to identify embryonic malformations in a blinded fashion.

Images of the embryos were captured by a DFC420 5 MPix digital camera (Leica Microsystems). Normal embryos were classified as possessing a completely closed neural tube and no evidence of other malformations. Malformed embryos were classified as showing evidence of failed neural tube closure or of an NTD. NTDs were verified by histological sections.

Cell culture and transfection

C17.2 mouse neural stem cells, originally obtained from ECACC (European Collection of Cell Culture), were maintained in DMEM (5 mM glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. The C17.2 neural stem cells are newborn mouse cerebellar progenitor cells transformed with retroviral v-myc (Snyder et al. 1992). Lipofectamine 2000 (Invitrogen, Grand Island, NY) was used according to the manufacturer’s protocol for transfection of plasmid into the cells using 1% fetal bovine serum culture conditions. The SIRT2 and SIRT6 open reading frame plasmids were purchased from Origene (Fredrick, MD).

Immunoblotting

Embryos or cells were lysed by sonication in lysis buffer (Cell Signaling Technology, #9803) with a Protease inhibitor cocktail (Sigma) (Wang et al. 2015e, Yang et al. 2013, Gu et al. 2015a, Wang et al. 2015a, Wang et al. 2015d). Equal amounts of protein from embryos or cells were resolved by SDS-PAGE gel electrophoresis and transferred onto Immunobilon-P membranes (Millipore, Billerica, MA). 2 μg Precision Plus Protein Standards (Bio-Rad, Hercules, CA) were loaded into one lane of the gel. Membranes were incubated in 5% nonfat milk for 45 minutes and then incubated for 18 hours at 4°C with the following primary antibodies at dilutions of 1:1000 to 1:2000 in 5% nonfat milk: SIRT2, SIRT6 from Sigma-Aldrich (St. Louis, MO), acetylated H3K56, H4K16, H4K9, and H3K27 from Cell Signal Technology (Danvers, MA). Membranes were then exposed to goat anti-rabbit or anti-mouse secondary antibodies. To ensure that equivalent amounts of protein were loaded among samples, membranes were stripped and probed with a mouse antibody against β-actin (Abcam, Cambridge, MA). Signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Scientific, Rockford, IL). All experiments were repeated three times.

RNA extraction and Real-time PCR (RT-PCR)

Total RNA was isolated from embryos or cultured cells using the mirVana miRNA isolation kit (Ambion, Life Technologies, Grand Island, NY) and reverse transcribed using the NCode VILO miRNA cDNA synthesis kit (Invitrogen, Life Technologies, Grand Island, NY). RT-PCR for SIRT1-7 and β-actin were performed using the Maxima SYBR Green/ROX qPCR Master Mix assay (Thermo Scientific). RT-PCR and subsequent calculations were performed by a StepOnePlus™ Real-Time PCR System (Applied Biosystem, Foster City, CA).

Statistical Analysis

Data are presented as means ± SE (standard errors). Three embryos from three different dams were used for immunoblotting and six embryos from six different dams were used for RT-PCR. Cell cultures experiments were repeated three times. One-way ANOVA was performed using the SigmaStat 3.5 software, a Tukey’s multiple-comparison test was used to estimate the significance. Statistical significance was accepted when P < 0.05.

Results

Maternal diabetes in vivo and high glucose in vitro down-regulate SIRT expression

To determine whether SIRT expression is affected by high glucose, mRNA levels of the seven SIRT members were examined in embryos from nondiabetic and diabetic dams, and in neural stem cells exposed to different glucose concentrations. Maternal diabetes significantly down-regulated the expression of SIRT1, SIRT2, SIRT6 and SIRT7 (Fig. 1A), whereas the expression of SIRT3, SIRT4 and SIRT5 was not affected by maternal diabetes (Fig. 1A). High glucose in vitro only decreased SIRT2 and SIRT6 expression in a dose-dependent manner but did not affect other SIRT expression (Fig. 1B). At the glucose concentration of 25 mM, the down-regulation of SIRT2 and SIRT6 expression reached a plateau (Fig. 1B). Because both maternal diabetes in vivo and high glucose in vitro suppressed SIRT2 and SIRT6 expression, subsequent studies focused on these two SIRTs.

Figure 1.

Figure 1

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Maternal diabetes in vivo and high glucose in vitro down-regulate SIRT expression. (A) SIRT1-7 mRNA levels in embryos from nondiabetic (ND) dams and diabetes mellitus (DM) dams were determined by RT-PCR and normalized by β-actin mRNA. (B) SIRT1-7 mRNA levels in C17.2 neural stem cells cultured under normal glucose (5 mM glucose) or high glucose (16.7, 25.0 and 33.3 mM glucose) for 48 hours. Values are mean ± SE of the data from three separate experiments. * indicate significant differences (P < 0.05) compared with the ND group or the 5 mM glucose group.

Maternal diabetes in vivo and high glucose in vitro suppress SIRT2 and SIRT6 expression through oxidative stress

Our previous studies have demonstrated that oxidative stress mediates the adverse effects of maternal diabetes and SOD1 overexpression in SOD1 Tg mice blocks maternal diabetes-induced oxidative stress (Wang et al. 2015b, Wang et al. 2015c, Wang et al. 2013, Weng et al. 2012, Li et al. 2011, Li et al. 2012). To test whether oxidative stress is involved in SIRT down-regulation, wild-type (WT) and SOD1 overexpressing embryos from nondiabetic and diabetic dams were analyzed. Maternal diabetes suppressed SIRT2 and SIRT6 expression in WT embryos but not in SOD1 overexpressing embryos (Fig. 2A). Consistent with the down-regulation of SIRT2 and SIRT6 mRNA expression (Fig. 1B), high glucose suppressed SIRT2 and SIRT6 protein expression in a dose-dependent manner (Fig. 2B). The osmotic control of glucose, mannitol, did not induce any changes in SIRT2 and SIRT6 mRNA and protein expression (Fig. 2C, D). The SOD mimetic, tempol, abrogated high glucose-induced SIRT2 and SIRT6 down-regulation (Fig. 2E). These findings support the hypothesis that oxidative stress is responsible for high glucose-suppressed SIRT2 and SIRT6 expression.

Figure 2.

Figure 2

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Maternal diabetes in vivo and high glucose in vitro suppress SIRT2 and SIRT6 expression through oxidative stress. (A) SIRT2 (37 kD), SIRT6 (40 kD) protein expression levels analyzed by Western blotting in WT and SOD1 (16 kD) overexpressing embryos from nondiabetic (ND) dams or diabetes mellitus (DM) dams mated with SOD1 transgenic males. Experiments were conducted using six embryos from six different dams (N = 6) per group. (B) SIRT2 and SIRT6 protein levels in C17.2 neural stem cells cultured under normal glucose (5 mM glucose) or high glucose (16.7, 25.0 and 33.3 mM glucose) for 48 hours. SIRT2 (C) and SIRT6 (D) mRNA and protein levels in cells cultured in 5 mM glucose with or without high mannitol (11.7, 20 and 28.3 mM mannitol) for 48 h. E) SIRT2 and SIRT6 protein levels in cells cultured under 5 mM glucose or 25 mM glucose conditions in the absence or presence of tempol (100 μM) for 48 h. In B, C, D and E, experiments were repeated three times (N = 3) and the quantification of the data were shown in the bar graph. * indicate significant differences (P < 0.05) compared with the other three groups (A and E) or the 5 mM glucose control group (B).

Oxidative stress suppresses SIRT2 and SIRT6 expression in vitro

To examine whether oxidative stress directly modulates SIRT2 and SIRT6 expression, DMNQ, a superoxide generating agent (Diers et al. 2013). DMNQ mimicked high glucose to suppress both SIRT2 and SIRT6 expression (Fig. 3A). PEGSOD, a cell permeable SOD, was used to determine whether mitigating high glucose-induced oxidative stress would restore SIRT2 and SIRT6 expression. PEGSOD treatment indeed restored high glucose-suppressed SIRT2 and SIRT6 expression (Fig. 3B).

Figure 3.

Figure 3

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Oxidative stress represses SIRT2 and SIRT6 expression. (A) SIRT2 and SIRT6 protein levels in cells cultured under 5 mM glucose or 25 mM glucose conditions in the absence or presence of DMNQ (20 μM). (B) SIRT2 and SIRT6 protein levels in cells cultured under 5 mM glucose or 25 mM glucose conditions in the absence or presence of PEGSOD (100U/ml) for 48 h. Experiments were repeated three times (N = 3) and the quantification of data was shown in the bar graphs. * indicates significant difference (P < 0.05) compared with the other three groups.

Maternal diabetes in vivo and high glucose in vitro-induced oxidative stress enhances histone acetylation that is subjected to SIRT2 and SIRT6 regulation

Studies have revealed the histone targets of SIRT2 and SIRT6. H4K16 and H3K27 are deacetylated by SIRT2 (Vaquero et al. 2006, Wirth et al. 2009), whereas H3K9 and H3K56 are deacetylation targets of SIRT6 (Michishita et al. 2008, Yang et al. 2009). To explore whether high glucose-suppressed SIRT2 and SIRT6 expression leads to enhanced acetylation of these histone targets and whether oxidative stress is responsible for these enhance histone acetylation. Specific histone lysine acetylation was assessed in WT and SOD1 overexpressing embryos from nondiabetic and diabetic dams. Maternal diabetes significantly increased the levels of acetylated H4K16 and H3K27 in WT embryos but this increase was diminished in SOD1 overexpressing embryos (Fig. 4A). Likewise, the levels of acetylated H3K9 and H3K56 were significantly higher in WT embryos from diabetic dams than in their counterparts from nondiabetic dams (Fig. 3B). The increase in H3K9 and H3K56 acetylation by maternal diabetes was not detected in SOD1 overexpressing embryos (Fig. 4B). Tempol effectively suppressed high glucose-increased H4K16, H3K27, H3K9 and H3K56 acetylation in neural stem cells (Fig. 4C, D). These observations support the hypothesis that reduced SIRT2 and SIRT6 expression by high glucose results in enhanced histone acetylation in specific lysine residues through oxidative stress.

Figure 4.

Figure 4

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Maternal diabetes- or high glucose in vitro-induced oxidative stress enhances histone acetylation that is subjected to SIRT2 and SIRT6 regulation. Acetylated H4K16 (11 kD), H3K27 (17 kD) (A) and H3K9 (17 kD), H3K56 (17 kD) (B) protein levels in WT and SOD1 overexpressing embryos from nondiabetic (ND) dams or diabetes mellitus (DM) dams mated with SOD1 transgenic males. Acetylated H4K16, H3K27 (C) and H3K9, H3K56 (D) protein levels in cells cultured under 5 mM glucose or 25 mM glucose conditions in the absence or presence of tempol (100 μM) for 48 h. Experiments were repeated three times (N = 3) and the quantification of data was shown in the bar graphs. * indicate significant difference (P < 0.05) compared with the other three groups.

SIRT2 and/or SIRT6 overexpression abolishes high glucose-increased histone acetylation

To ascertain whether enhanced histone acetylation under high glucose conditions is due to SIRT2 and SIRT6 down-regulation, we utilized SIRT2 and SIRT6 overexpression. SIRT2 or SIRT6 overexpression abrogated high glucose-reduced SIRT2 and SIRT6 expression (Fig. 5A, B). SIRT2 or SIRT6 overexpression under normal glucose conditions slightly decreased the acetylation of H4K16/H3K27, and H3K9/H3K56, respectively (Fig. 5A, B). SIRT2 overexpression abolished high glucose-increased acetylation of H4K16/H3K27 (Fig. 5A). Similarly, SIRT6 overexpression blocked high glucose-increased acetylation of H3K9/H3K56 (Fig. 5B). Thus, high glucose-enhanced acetylation of specific histone lysine residues is likely due to the reduction of SIRT2 and SIRT6.

Figure 5.

Figure 5

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SIRT2 or SIRT6 overexpression abolishes high glucose-increased histone acetylation. Levels of SIRT2, acetylated H4K16 and H3K27 (A), and SIRT6, acetylated H3K9 and H3K56 (B) protein levels in cells treated cultured under 5 mM glucose or 25 mM glucose conditions with the transfection of the SIRT2 (A) or the SIRT6 (B) plasmid. Experiments were repeated three times (N = 3) and the quantification of data was shown in the bar graphs. * indicate significant differences (P < 0.05) compared with the other three groups.

Sirtuin activator, SRT1720, blocks high glucose-induced histone acetylation and ameliorates high glucose-induced NTD formation

Maternal diabetes in vivo and high glucose in vitro adversely affect the developing embryo leading to NTDs. Next, we sought to determine the functionality of SIRT down-regulation in high glucose-induced NTD formation. The novel and potent sirtuin activator, SRT1720, was used in C17.2 neural stem cell culture and whole-embryo culture studies. SRT1720 abolished high glucose-increased histone acetylation (Fig. 6A), restored SIRT2 and SIRT6 expression (Fig. 6A), and increased the expression of two SIRT1 responsive genes, Nrf2 and SOD2 (Fig. 6B). Under high glucose conditions, eight out of twelve embryos exhibited NTDs (Table 1); however, with the presence of 100 μM SRT1720, only two out of twelve embryos had NTDs (Table 1). Under normal glucose conditions, no embryo had NTDs and SRT1720 treatment did not adversely impact embryonic development (Table 1).

Figure 6.

Figure 6

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Sirtuin activator decreased, whereas sirtuin inhibitors increased histone acetylation that is subjected to SIRT2 and SIRT6 regulation. (A) Acetylated H4K16 and H3K27 protein levels in C17.2 neural stem cells cultured under 5 mM glucose or 25 mM glucose conditions in the absence or presence of SRT1720 (100 μM) for 48 h. (B) Nrf2 and SOD2 mRNA levels were detected in cells treated with or without SRT1720 (100 μM) for 48 h. (C) Acetylated H3K9 and H3K56 protein levels in cells treated with AGK2 or/and Sirtinol for 48 h. Experiments were repeated three times (N = 3) and the quantification of data was shown in the bar graphs. * indicate significant differences (P < 0.05) compared with the other three groups.

Table 1.

The sirtuin activator, SRT1720, ameliorates hyperglycemia-induced NTDs

Groups Number of normal embryos Number of NTDs embryos Total embryos
Normal glucose (100 mg/dl) 12 0 12
Normal glucose + 100 μM SRT1720 12 0 12
High glucose (300 mg/dl) 4 8* 10
High glucose + 100 μM SRT1720 10 2 12
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*

indicates significantly difference compared with other groups in Fisher Exact Test.

Pharmacological SIRT inhibitors mimic high glucose to increase histone acetylation and induce NTD formation

To reveal whether down-regulation of SIRT2 and SIRT6 exerts adverse effects similar to high glucose on neurulation leading to NTDs, the combination of a SIRT2 inhibitor, AGK2, and a pan sirtuin inhibitor, Sirtinol, was used in C17.2 neural stem cell cultures and whole-embryo cultures. The combination of AGK2 and Sirtinol significantly increased all histone acetylation downstream of SIRT2 and SIRT6 (Fig 6C), whereas AGK2 or Sirtinol alone did not elevate all histone acetylation (Fig. 6C). The combination of 5 μM AGK2 and 10 μM Sirtinol induced approximately 16.7% NTDs in cultured embryos, which was not significantly different compared with the normal glucose group (Table 2). High doses of AGK2 (10 μM) and Sirtinol (20 μM) resulted in a significantly higher rate of NTDs (75%), which was significantly different compared with the normal glucose group (Table 2).

Table 2.

Sirtuin inhibitors mimic high glucose to induce neural tube defects (NTDs)

Groups Number of normal embryos Number of NTDs embryos Total embryos
Normal Glucose (100 mg/dl) + vehicle 12 0 12
5 μM AGK2 + 10 μM Sirtinol 10 2 12
10 μM AGK2 + 20 μM Sirtinol 4 8* 12
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*

indicates significantly difference compared with the other two groups in Chi-square Test. (AGK2, Enzo Life Sciences, PA) plus a pan sirtuin inhibitor (Sirtinol, Enzo Life Sciences, PA). Vehicle is DMSO (concentrations less than 0.1%).

Discussion

In this study, we revealed a possible epigenetic mechanism underlying the cause of diabetic embryopathy. Maternal diabetes and high glucose in vitro suppress SIRT2 and SIRT6 expression leading to enhanced histone acetylation in specific lysine residues. Oxidative stress is responsible for the down-regulation of SIRT2 and SIRT6 and subsequent enhanced histone acetylation. Maternal diabetes and high glucose in vitro increase ROS production and concomitantly repress endogenous antioxidant enzyme expression (Yang et al. 2014). These events collectively result in oxidative stress in the developing embryo, which induces apoptosis in neural stem cells of the developing neuroepithelium leading to NTD formation (Yang et al. 2008a, Yang et al. 2013, Wu et al. 2015a). Multiple lines of evidence support this oxidative stress hypothesis concerning the cause of diabetic embryopathy (Yang et al. 2014, Wang et al. 2015b). Overexpression of the antioxidant enzyme, SOD1, in a transgenic mouse model prevents maternal diabetes-induced NTD formation through mitigating oxidative stress (Wang et al. 2015b, Wang et al. 2015c, Wang et al. 2013, Weng et al. 2012, Li et al. 2011, Li et al. 2012), oxidative stress-triggered pro-apoptotic kinase signaling and apoptosis (Yang et al. 2013). Likewise, the SOD mimetic, tempol, suppresses high glucose-induced oxidative stress and neural stem cell apoptosis (Wang et al. 2015e). The present study utilized these two complimentary approaches, the SOD1 transgenic model and tempol in vitro treatment, in testing the oxidative stress hypothesis in SIRT regulation and histone acetylation. By supporting this hypothesis, we demonstrated that both SOD1 overexpression in vivo an tempol treatment in vitro reverted the down-regulation of SIRT2 and SIRT6, and the up-regulation of histone acetylation. Furthermore, we demonstrated that a superoxide generating agent, DMNQ, mimics high glucose to suppress SIRT2 and 6 expression, whereas a cell permeable antioxidant enzyme, PEGSOD, inhibits high glucose-induced SIRT2 and SIRT6 down-regulation. These findings collectively support our central hypothesis that high glucose suppresses SIRT2 and 6 expression, leading to increased histone acetylation via oxidative stress.

SIRTs are a class of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases whose primary substrates are histones (Nakagawa & Guarente 2011). Seven mammalian SIRT2 have been identified and they regulate an array of cellular functions including oxidative stress-resistance, energy metabolism, apoptosis and aging through the up-regulation of antioxidant enzymes and suppression of pro-apoptotic factor expression (Taylor et al. 2008). Because oxidative stress-induced apoptosis is the primary cause of diabetic embryopathy (Yang et al. 2013), we hypothesized that maternal diabetes and high glucose in vitro suppress SIRT expression. Indeed, we found that SIRT2 and SIRT6 expression were suppressed by maternal diabetes and high glucose. SIRT7 is suppressed by maternal diabetes in vivo but not down-regulated by high glucose in vitro. This is probably due to the subtle difference between the in vivo neural stem cells and the in vitro neural stem cell line. Nevertheless, we focused on SIRT2 and SIRT6 which were down-regulated both in vivo and in vitro. Studies have demonstrated that diabetes and high glucose in vitro negatively regulate SIRT expression. Hyperglycemia of diabetes causes a significant decrease in SIRT1 expression in rat retinas (Zheng et al. 2012). Both type 1 and type 2 diabetes suppress SIRT3 expression in skeletal muscles (Jing et al. 2011). High glucose coupled with oxidative stress inhibits SIRT3 expression in tenocytes (Poulsen et al. 2014). The mechanism whereby high glucose suppresses SIRT expression is little known. The poly (ADP-ribose) polymerase (PARP) activity is implicated in SIRT1 downregulation by diabetes (Zheng et al. 2012). The down-regulation of SIRT3 by high glucose may involve miR28-5p (Poulsen et al. 2014). We have recently demonstrated the alteration of miRNA expression in embryos and neural stem cells exposed to diabetes and high glucose, respectively (Gu et al. 2015b). Thus, miRNA may play a role in maternal diabetes- or high glucose-induced SIRT2 and SIRT6 downregulation.

Although other SIRTs are also repressed by maternal diabetes, SIRT2 and SIRT6 are down-regulated by both maternal diabetes and high glucose in vitro, suggesting that they are the most prominent targets in diabetic embryopathy. SIRT2 is primarily present in the cytoplasm and deacetylates H4K16 in controlling cell cycle progression (Vaquero et al. 2006). SIRT2 regulates adipocyte differentiation by modulating the activities of Forkhead transcription factor 1 (FoxO1) through deacetylation (Jing et al. 2007). Because FoxO3a is activated and contributes to NTD formation in diabetic embryopathy (Yang et al. 2013), it is of great interest to test whether reduced SIRT2 expression increases FoxO3a activities. SIRT6 predominately localizes in the nucleus and the loss of SIRT6 gene produces a premature aging phenotype associated with instability (Mostoslavsky et al. 2006). SIRT6 preferentially deacetylates H3K9 and H3K56 (Michishita et al. 2008, Yang et al. 2009). By restoring SIRT6 and SIRT2 expression, which was suppressed by high glucose, we confirmed H4K16/H3K27 and H3K9/H3K56 as SIRT6 and SIRT2 substrates, respectively. SIRT6 deletion leads to mitochondrial dysfunction by diminishing mitochondrial respiration while boosting glycolysis (Zhong et al. 2010). Reduced SIRT6 may be one of the mechanisms leading to mitochondrial dysfunction in diabetic embryopathy. The functional significance of SIRT2 and SIRT6 in the etiology of diabetic embryopathy needs be further explored.

The potent SIRT activator, SRT1720, abolished high glucose-induced NTD formation, supporting a critical role for SIRTs in diabetic embryopathy. Our findings are in agreement with those in a recent report that the naturally occurring polyphenol resveratrol, an arguable SIRT activator (Gertz et al. 2012), reduced embryonic oxidative stress and apoptosis in diabetic embryopathy (Singh et al. 2011). SRT1720 is a small molecular SIRT activator that is structurally unrelated to, and 1,000-fold more potent than, resveratrol to activate SIRT1 (Milne et al. 2007). SRT1720 ameliorates insulin resistance, increases mitochondrial mass, and prolongs life span in genetic or diet-induced obese and diabetic rodents (Milne et al. 2007). It is unknown whether SRT1720 also activates SIRTs other than SIRT1. Since studies have demonstrated that SRT1720 rescues mitochondrial function after oxidant injury (Funk et al. 2010) and SIRT6 promotes mitochondrial function (Zhong et al. 2010), SRT1720 may activate SIRT6. However, the beneficial effects of SRT1702 may be achieved by primarily activating SIRT1 to compensate for the loss of SIRT2 and SIRT6 activities in diabetic embryopathy. SRT1720 restores the expression and activities of SIRT2 and SIRT6. Because SIRT1720 also up-regulates SIRT1 responsive genes, we cannot rule out SIRT1 involvement in the beneficial effect of SRT1720. The combination of two pharmacological SIRT inhibitors mimics high glucose in NTD induction. AGK2 is a potent SIRT2 inhibitor (Outeiro et al. 2007), and Sirtinol is a cell permeable inhibitor of the sirtuin family members (Grozinger et al. 2001). Individual effects of these two inhibitors were not tested because the combination of AGK2 and Sirtinol at their low doses only induces few cases of NTDs and the effect is not statistically significant compared with that of the control group. A selective SIRT6 has recently been developed and future studies may use this inhibitor to evaluate the effect of SIRT6 inhibition on neural tube closure (Parenti et al. 2014). We expected that the combination of AGK2 and Sirtinol would inhibit the activities of both SIRT2 and SIRT6. Indeed, AGK2 or Sirtinol individually does not inhibit all histone substrates downstream SIRT2 and 6, whereas the combination of AGK2 and Sirtinol inhibits lysine acetylation of all four histones. Therefore, AGK2 and Sirtinol were used in combination to determine whether they mimicked high glucose in the induction of neural tube defects.

In summary, we found that maternal diabetes and high glucose in vitro repressed SIRT2 and SIRT6 expression leading to enhanced histone acetylation in specific lysine residues. Mitigating oxidative stress abolished the inhibitory effect of SIRT2 and SIRT6 expression by maternal diabetes and high glucose, thus reversing enhanced histone acetylation. SIRT activation ameliorated high glucose-induced NTD formation, whereas SIRT inhibition resulted in NTD formation. We revealed an epigenetic mechanism involving SIRT deacetylases and the acetylation of their histone substrates, which may play critical roles in maternal diabetes-induced NTDs.

Acknowledgments

We thank the Office of Dietary Supplements, National Institute of Health (NIH) for providing financial support. This study was supported by NIH R01DK083243, R01DK101972, R01DK103024 and the Basic Science Award (1-13-BS-220), American Diabetes Association. We are grateful to Dr. Julie Wu at the University of Maryland School of Medicine for critical reading and editing assistance.

Footnotes

Conflict of interest disclosure

The authors have no conflicts of interest to report.

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