Diabetes and Early Development: Epigenetics, Biological Stress, and Aging

Abstract

Pregestational diabetes, either type 1 or type 2 diabetes, induces structural birth defects including neural tube defects and congenital heart defects in human fetuses. Rodent models of type 1 and type 2 diabetic embryopathy have been established and faithfully mimic human conditions. Hyperglycemia of maternal diabetes triggers oxidative stress in the developing neuroepithelium and the embryonic heart leading to the activation of proapoptotic kinases and excessive cell death. Oxidative stress also activates the unfolded protein response and endoplasmic reticulum stress. Hyperglycemia alters epigenetic landscapes by suppressing histone deacetylation, perturbing microRNA (miRNA) expression, and increasing DNA methylation. At cellular levels, besides the induction of cell apoptosis, hyperglycemia suppresses cell proliferation and induces premature senescence. Stress signaling elicited by maternal diabetes disrupts cellular organelle homeostasis leading to mitochondrial dysfunction, mitochondrial dynamic alteration, and autophagy impairment. Blocking oxidative stress, kinase activation, and cellular senescence ameliorates diabetic embryopathy. Deleting the mir200c gene or restoring mir322 expression abolishes maternal diabetes hyperglycemia-induced senescence and cellular stress, respectively. Both the autophagy activator trehalose and the senomorphic rapamycin can alleviate diabetic embryopathy. Thus, targeting cellular stress, miRNAs, senescence, or restoring autophagy or mitochondrial fusion is a promising approach to prevent poorly controlled maternal diabetes-induced structural birth defects. In this review, we summarize the causal events in diabetic embryopathy and propose preventions for this pathological condition.

Key Points

• Maternal diabetes induces structural birth defects.

• Kinase signaling and cellular organelle stress are critically involved in neural tube defects.

• Maternal diabetes increases DNA methylation and suppresses developmental gene expression.

• Cellular apoptosis and senescence are induced by maternal diabetes in the neuroepithelium.

• microRNAs disrupt mitochondrial fusion leading to congenital heart diseases in diabetic pregnancy.

Keywords

pregestational diabetes - neural tube defects - congenital heart defects - DNA methylation - histone acetylation - ER stress - autophagy - mitochondrial dynamics - microRNA - senescence

Infant mortality in the United States is among the highest in developed countries.[1] The major cause of infant deaths is structural birth defects.[1] Human epidemiological studies have shown that nongenetic factors such as maternal diabetes are predominant causal factors for structural birth defects.[2] Pregestational maternal diabetes, including type 1 and type 2 diabetes, complicated by hyperglycemia from poor glycemic control induces neural tube defects (NTDs) and congenital heart defects (CHDs).[3] Many factors are associated with diabetes, but hyperglycemia is the major teratogenic factor associated with diabetes mellitus. An early study showed that increased hemoglobin A1C (HbA1c) during pregnancy is linearly associated with increased incidences of birth defects.[4] This positive relationship between hyperglycemia and birth defect rates is recapitulated in a murine embryo culture study, where increased glucose concentrations from 150 to 905 mg/dL translated into a linear increase of birth defect rates up to 100%.[5] Even transient hyperglycemia exposure results in birth defect formation.[5] Thus, controlling the levels of hyperglycemia and its downstream cellular and epigenetic events is the key to effectively preventing or reducing the rate of birth defects in diabetic pregnancy. Therefore, knowing the adverse effect of hyperglycemia in embryogenesis is essential in understanding the biology of the conditions and also important for developing effective preventive measures.

Hyperglycemia of maternal diabetes does not universally affect each cell of the developing embryo. Certain organ systems are impacted to a greater extent than others. Some of these include the embryonic vascular system, the neuroepithelium, and the cardiac epithelium.[6] [7] The organs affected by hyperglycemia contain highly proliferating, differentiating, and migrating cells.[6] [7] Hyperglycemia appears to halt cell proliferation, differentiation, and migration, and induce cell death and premature cellular aging.[8] [9] Not all embryos exposed to maternal diabetes hyperglycemia exhibit birth defects since a number of altered developmental and metabolic events need to occur simultaneously. For example, we have shown that hyperglycemia from maternal diabetes achieves its teratogenic effect beyond a glycemic threshold. Likewise, a threshold of adverse cellular effects must be achieved for the manifestation of birth defects. Embryos without birth defects exhibit lower levels of adverse cellular effects than the threshold but higher than those of embryos under nondiabetic conditions. Additionally, embryos, that do not have structural birth defects, still can exhibit functional defects such as behavioral defects after birth.[10] Since human fetal tissues are inaccessible, animal models such as the mouse, which consistently mimics human fetal development, are best suited for studying diabetic embryopathy. Both type 1- and type 2-associated diabetic embryopathy mouse models have been established,[8] [11] and these models confirm the hypothesis that hyperglycemia is the major factor in mediating the teratogenic effects of maternal diabetes hyperglycemia. Studies have consistently used mouse type 1 diabetic embryopathy models in revealing the molecular and epigenetic alterations leading to embryonic anomalies in diabetic pregnancy.[12] [13] [14]

In this review, we will summarize the signaling pathways triggered by the hyperglycemia of maternal diabetes, the epigenetic modifications initiated by hyperglycemia, and the altered gene expression in embryos of diabetic pregnancy. We will link these molecular and epigenetic alterations with cellular organelle stress and cell function. Our goal is to use the knowledge gained from our mechanistic and translational studies to design effective preventions. Folate supplementation is an effective way to prevent NTDs. Besides folate, we have found that trehalose, a naturally occurring disaccharide, that activates autophagy and quenches cellular organelle stress, can prevent maternal hyperglycemia of diabetes-induced NTDs in mice. A U.S. Food and Drug Administration-approved drug, teriflunomide, and a naturally occurring compound, echinacoside, can effectively reduce CHDs in the mouse model of diabetic pregnancy, and those two compounds activate mitochondrial fusion and restore mitochondrial function. However, the beneficial effects of these compounds need to be tested in human diabetic pregnancy. Further, other mechanisms underlying diabetic embryopathy are still elusive, and further research to uncover these mechanisms is still needed.

Hyperglycemia Activates Oxidative Stress-Responsive Kinases

The hyperglycemia of maternal diabetes increases the production of reactive oxygen species (ROS) and decreases the endogenous antioxidant capacity in the developing embryo, leading to oxidative stress.[15] [16] [17] ROS can oxidize the two key cysteine residues of thioredoxin leading to the formation of an intramolecular disulfide bond which causes thioredoxin to dissociate from apoptosis signal-regulating kinase 1 (ASK1), an oxidative stress-responsive kinase, resulting in ASK1 oligomerization and autophosphorylation.[8] [18] Deleting the Ask1 gene significantly reduces the NTD incidence in diabetic embryopathy.[8] Under nondiabetic conditions, ASK1 is usually nonphosphorylated and bound to its endogenous inhibitor, thioredoxin, which prevents the above developmental anomalies ([Fig. 1]). ASK1 is an upstream kinase that activates downstream kinases including c-Jun N-terminal kinase 1/2 (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK).[8] Maternal diabetes hyperglycemia only activates JNK1/2 but not p38MAPK.[8] Deleting either the Jnk1 or the Jnk2 gene also ameliorates hyperglycemia-induced NTDs.[19] [20] [21] ASK1-JNK1/2 signaling transduces proapoptotic signals to trigger caspase activation, which results in excessive cell death in the developing neuroepithelium.[8] The caspases involved in diabetic embryopathy have been identified. The effector caspase 8 initiates neuroepithelial cell apoptosis and the executor caspase 3 accomplishes the process of cell death.[8] Deleting the caspase 8 gene specifically in the neuroepithelium using the Cre-lox recombination approach significantly decreases the incidence of NTD in diabetic embryopathy.[8] Therefore, the proapoptotic kinase ASK1-JNK1/2 pathway mediates the teratogenic effects of oxidative stress in diabetic embryopathy. This is further affirmed by rat embryo culture studies where treating neurulation stage embryos with a JNK1/2-specific inhibitor SP600125 blocks high glucose-induced embryonic vasculopathy and embryopathy.[19] [20] [21] [22]

Fig. 1 Hyperglycemia of maternal diabetes activates oxidative stress-responsive kinases. Hyperglycemia causes oxidative stress, which leads to enhanced reactive oxygen species (ROS). ROS oxidizes thioredoxin, the inhibitor of apoptosis signal-regulating kinase 1 (ASK1), leading its dissociation and phosphorylation and activation of ASK1. ASK1 activation leads to dephosphorylation of the transcription factor forkhead box O 3a (FoxO3a), which in turn upregulates proapoptosis genes including TRADD, which induces the caspase 8 cleavage. ASK1 also activates JNK1/2 signaling which upregulates proapoptotic signals to trigger caspase activation. Apoptosis results from ASK1 acting through both FoxO3a and JNK1/2. ASK1 does not activate p38MAPK. JNK1/2, c-Jun N-terminal kinases 1/2; p38MAPK, p38 mitogen-activated protein kinase; TRADD, type 1-associated DEATH domain protein.

ASK1 activation also leads to the activation by dephosphorylation of the transcription factor forkhead box O 3a (FoxO3a), which once activated can enter cell nuclei and induce gene transcription.[8] The hyperglycemia of maternal diabetes decreases FoxO3a phosphorylation and increases its protein abundance in the cell nuclei.[8] Both Foxo3a germline deletion and neuroepithelium-specific deletion alleviate hyperglycemia-induced NTD formation.[8] In neurulation stage embryos, FoxO3a activation specifically upregulates a proapoptotic factor, tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD).[8] TRADD binds to another DEATH domain protein, Fas cell surface death receptor (FAS)-associated DEATH domain protein (FADD), and the TRADD/FADD complex specifically cleaves caspase 8 but not other effector caspases.[8] In a transgenic (Tg) mouse model expressing the dominant negative form of FADD, maternal diabetes hyperglycemia-induced caspase 8 cleavage and neuroepithelial cell apoptosis are abrogated, and thus the NTD incidence is significantly lower than that of their wild-type littermates.[23] Thus, hyperglycemia-induced oxidative stress transmits its proapoptotic signal via FoxO3a translocation to the nucleus to activate TRADD gene expression. This oxidative stress-ASK1-JNK1/2/FoxO3a pathway and caspase 8 cascade are also recapitulated in human NTD tissues,[8] suggesting a conserved mechanism in humans ([Fig. 1]).

Unfolded Protein Response and Endoplasmic Reticulum Stress

Maternal diabetes hyperglycemia-induced ASK1 activation triggers prolonged unfolded protein response (UPR) leading to endoplasmic reticulum (ER) stress.[24] The UPR consists of three arms: the inositol requiring enzyme 1α (IRE1α)-X-box-binding protein 1 (XBP1) arm, the double strand RNA-dependent protein kinase (PKR)-like ER kinase (PERK)-eukaryotic translation initiation factor-2α (eIF2α) arm, and the activating transcription factor 6α cleavage arm. Transient UPR activation by the accumulation of unfolded or misfolded proteins in the ER lumen increases targeted gene expression that enhances protein folding capacity, protein degradation, and protein transport, leading to the restoration of cellular homeostasis. In contrast, prolonged UPR leads to ER stress which induces cell death. Swollen/enlarged ER is present in neuroepithelial cells of neurulation-stage embryos exposed to maternal diabetes.[21] The hyperglycemia of maternal diabetes activates IRE1α and PERK through phosphorylation leading to the cleavage of XBP1 mRNA and eIF2α phosphorylation.[21] ER stress markers, the proapoptotic factor CCAAT/enhancer binding protein (C/EBP) homologous protein and other ER chaperones, are all upregulated by hyperglycemia in the developing neuroepithelium and heart.[21] Activated IRE1α cleaves miR-322 through its endonuclease activity.[25] Tg expression of mir322 specifically in the neuroepithelium inhibits hyperglycemia-induced caspase cascade activation and cell apoptosis, leading to the reduction of NTDs.[25] Deletion of the Ask1 gene, the Jnk1 gene, or the Jnk2 gene abolishes hyperglycemia-induced prolonged UPR and ER stress.[21] [24] Hyperglycemia-activated ASK1 and JNK1/2 phosphorylate and thus activate IRE1α, resulting in prolonged UPR and ER stress.[21] Reciprocally, UPR can further enhance the activation of the ASK1-JNK1/2 pathway,[21] and thus these two causal events form a vicious cycle in transmitting the proapoptotic signal to the nucleus and stimulating proapoptotic gene expression to induce diabetic embryopathy ([Fig. 2]).

Fig. 2 Hyperglycemia of maternal diabetes induces endoplasmic reticulum (ER) stress. Hyperglycemia of maternal diabetes causes oxidative stress, which activates inositol requiring enzyme 1α (IRE1α) and PKR-like ER kinase (PERK) through phosphorylation. Active IRE1α cleaves X-box-binding protein 1 (XBP1) mRNA, miR-322, and also activates ASK1-JNK1/2 signaling pathway, leading to cell apoptosis. Activated PERK phosphorylates eukaryotic translation initiation factor-2α (eIF2α), which further activates proapoptotic factor C/EBP homologous protein (CHOP). Antioxidant enzyme superoxide dismutase 1 (SOD1) overexpression or 4-phenyl butyric acid (4-PBA) treatment can alleviate ER stress and prevent diabetes-induced structural birth defects. CHD, congenital heart defect; JNK1/2, c-Jun N-terminal kinases 1/2; NTD, neural tube defect.

The causal relationship of ER stress-inducing NTDs in diabetic pregnancy is affirmed by a study using the ER stress inhibitor, 4-phenyl butyric acid (4-PBA), an FDA-approved drug for treating urea cycle disorders and hyperammonemia.[26] [27] Treating diabetic mouse dams with 4-PBA significantly reduces NTD incidences in embryos,[26] supporting the hypothesis that ER stress mediates the teratogenicity of maternal diabetes hyperglycemia leading to failure of neural tube closure. 4-PBA is an ER chemical chaperone that increases the protein folding capacity of the ER by interacting with hydrophobic regions of unfolded proteins. In humans, 4-PBA can be orally administrated and does not cause severe adverse effects in nonpregnant individuals. The first step in repurposing 4-PBA for the prevention of hyperglycemia-induced structural birth defects is to test its safety in human pregnancy. 4-PBA could be especially valuable in preventing structural birth defects in diabetic pregnancy because there are no known preventive measures for these types of birth defects. In mice, Tg expression of the antioxidant enzyme superoxide dismutase 1 (SOD1) abrogates maternal diabetes hyperglycemia-induced ER stress in neurulation stage embryos,[28] indicating that quenching oxidative stress is also an effective means of preventing diabetic embryopathy ([Fig. 2]). However, human antioxidant trials do not show solid beneficial effects against human diseases,[29] questioning the efficacy of general antioxidants in mitigating intracellular oxidative stress. A new generation of cellular organelle-specific antioxidants such as MitoQ may be effective for preventing diabetic embryopathy. The combination of antioxidants and other nutrients such as myoinositol, a cell membrane stabilizing lipid, which shows preventive effects diabetic embryopathy,[30] may be another choice for human diabetic pregnancy.

Altered Mitochondrial Dynamics

Defective mitochondria are present in neuroepithelial cells of embryos exposed to maternal diabetes hyperglycemia.[31] Two proapoptotic B-cell lymphoma-2 (Bcl-2) family members, Bcl-2-associated death promoter (Bad) and Bcl-2 homology domain 3 (BH3)-interacting domain death agonist (BID), show increased activity in neurulation stage embryos exposed to hyperglycemia.[31] Specifically, hyperglycemia dephosphorylates and thus activates Bad, and induces the cleavage of BID.[31] Cleaved BID induces oligomerization of Bcl-2 antagonist killer (BAK), which damages the mitochondrial membrane leading to the release of cytochrome c into the cytosol, activating the intrinsic mitochondrial apoptosis pathway.[32] Dephosphorylated Bad also activates the mitochondrial apoptosis pathway in a similar manner to cleaved BID.[32] Excessive apoptotic cells are present in the developing neuroepithelium and heart in diabetic pregnancy.[8] [9]

In the embryonic heart exposed to hyperglycemia, there is increased presence of small mitochondria.[33] Mitochondrial morphology is dynamic due to two counteractive processes: mitochondrial fusion and fission. Mitochondrial fusion is enabled by two mitochondrial tether proteins, Mitofusin 1 and 2 (Mfn1 and 2), which support the fusion of two small mitochondria into a larger one. Conversely, mitochondrial fission occurs when a large mitochondrion splits into two smaller ones and this process is mainly regulated by another mitochondrial tether protein, dynamin-like protein 1 (Drp1). Maternal diabetes hyperglycemia suppresses the expression of Mfn1 and Mfn2 but does not affect Drp1 expression in the developing heart.[33] Hyperglycemia upregulates through the transcription factor FoxO3a,[33] two microRNAs (miRNAs), miR-140 and miR-195. These two miRNAs degrade Mfn1 and Mfn2 mRNAs and thus block mitochondrial fusion. Deletion of the Foxo3a gene either in cardiac progenitors or cardiomyocytes rescues mitochondrial fusion and function, leading to the prevention of CHDs in diabetic pregnancy by restoring cardiac cell apoptosis and cell proliferation.[33] Either germline deletion of mir140 or cardiomyocyte-specific deletion of mir195 also restores mitochondrial fusion and significantly reduces CHD incidences in diabetic pregnancy.[33] Restoring either Mfn1 or Mfn2 in Tg mouse models effectively rescues mitochondrial fusion and alleviates CHD formation in diabetic pregnancy,[33] strongly supporting the hypothesis that impaired mitochondrial fusion causes cellular dysfunction leading to CHD formation ([Fig. 3]). The study also points towards a potential preventive measure for maternal diabetes hyperglycemia-induced CHDs. When maternal mice with induced diabetes are treated with two compounds, teriflunomide and echinacoside, Mfn1 and Mfn2 expression is restored, along with mitochondrial fusion, in diabetic pregnancy.[33]

Fig. 3 Hyperglycemia of maternal diabetes impairs mitochondrial fusion. Hyperglycemia-activated FoxO3a increases transcription of miR-140 and miR-195, which in turn represses Mfn1 and Mfn2, leading to mitochondrial fusion defects and CHD. Two mitochondrial fusion activators, teriflunomide (TERI) and echinacoside (ECH), can restore mitochondrial fusion and function and reduce CHD incidences in diabetic pregnancy. CHD, congenital heart defect; FoxO3a, forkhead box O 3a; Mfn1/2, Mitofusin 1 and 2.

Impaired Autophagy Perturbs Cellular Homeostasis

Autophagy is a process that removes from the dysfunctional cellular cargo including damaged mitochondria, ER, other cellular organelles, and protein aggregations, via the double membrane autophagosome. Deficiency of the autophagy gene, autophagy and beclin 1 regulator 1 (Ambra1), results in NTD formation,[34] and its rare mutations are associated with human NTDs.[35] This evidence suggests that impaired autophagy could be a molecular event in the NTD formation of diabetic pregnancy. Indeed, maternal diabetes hyperglycemia inhibits autophagy by blocking the lipidation of microtubule-associated protein 1A/1B-light chain 3, inhibiting autophagy-related genes and thus reducing the number of autophagosomes in neuroepithelial cells.[31] Trehalose, a naturally occurring disaccharide, reactivates autophagy in neuroepithelial cells, resolves mitochondrial dysfunction and ER stress, and thus significantly reduces NTD formation in diabetes-affected embryos ([Fig. 4]).[31] Hyperglycemia of diabetes also induces the phosphorylation of protein kinase C α (PKCα), which increases the expression of an miRNA, miR-129-2.[12] miR-129-2 degrades the mRNA of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α), an autophagy activator, leading to autophagy impairment in neuroepithelial cells.[12] Deleting the Pkca gene or Tg expression of PGC1α restores autophagy and cellular homeostasis in neuroepithelial cells and thus reduces NTD formation in diabetic pregnancy with hyperglycemia,[12] supporting autophagy impairment as a causal event in diabetic embryopathy.

Fig. 4 Impaired autophagy perturbs cellular homeostasis. Hyperglycemia of maternal diabetes inhibits autophagy by blocking the lipidation of microtubule-associated protein 1A/1B-light chain 3 (LC3). This inhibits autophagy-related genes (ATGs) and thus reduces the numbers of autophagosomes, leading to cellular organelle stress including mitochondrial dysfunction and ER stress. Hyperglycemia induces myristoylated alanine-rich C kinase substrate (MARCKS) acetylation at lysine 165, a prerequisite for its phosphorylation. Phosphorylated MARCKS dissociates from the mitochondria and ER, leading to the cellular organelle stress and defects. CHD, congenital heart defect; ER, endoplasmic reticulum; NTD, neural tube defect.

Maternal diabetes hyperglycemia-activated mammalian target of rapamycin (mTOR) signaling is also involved in autophagy reduction in diabetic embryopathy.[36] Deletion of the Rps6kb1 gene that encodes 70-kDa ribosomal protein S6 kinase 1, a major downstream effector of mTOR, rescues autophagy and inhibits cellular organelle stress, and reduces diabetic embryopathy.[36] The oxidative stress kinase-activated transcription factor, FoxO3a, is also essential for blocking autophagy in diabetic embryopathy.[37] Deleting the Foxo3a gene or Tg expression of a dominant negative form of FoxO3a lacking the DNA transactivation activity, reactivates the autophagy pathway and reduces NTD incidences in diabetic pregnancy.[37] This evidence suggests that the oxidative stress-responsive kinase-FoxO3a pathway is crucial for hyperglycemia-inhibiting autophagy. Functional autophagy, including mitophagy and reticulophagy, is required for the removal of cellular dysfunctional organelles. A cell membrane protein, myristoylated alanine-rich C kinase substrate (MARCKS), protects mitochondria and ER from stresses.[13] Maternal diabetes hyperglycemia induces MARCKS acetylation at lysine 165, a prerequisite for its phosphorylation.[13] Phosphorylated MARCKS dissociates from the mitochondria and ER[13] inactivating its protective effects. Tg expression of a phosphorylation dead MARCKS mutation retains its protective effects and activates mitophagy and reticulophagy, thus restoring cellular homeostasis ([Fig. 4]).[13] Thus, poorly controlled maternal diabetes disables functional autophagy.

DNA Hypermethylation

Epigenetics refers to heritable genetic changes that do not involve alterations in DNA structure or sequence and can influence gene function via transcription or translation.[38] [39] Epigenetics mainly comprises DNA methylation, histone modification, chromatin remodeling, and miRNAs. Epigenetics is widely implicated in various physiological and pathological processes.[40] [41] DNA methylation, namely the addition of a methyl group into 5′ carbon of cytosine, primarily occurs on 5'-cytosine-phosphodiester bond-guanine-3' (CpG) dinucleotides.[38] CpG islands are the regions that have a higher density of CpG sites than the rest of the genome. Elevated methylation of CpG islands in gene promoters can impede the binding of transcription factors and recruit repressive methyl-binding proteins, leading to the silence of gene expression.[42] [43] [44] DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs) that includes DNMT1, DNMT3A, and DNMT3B. The maintenance of DNA methylation patterns during DNA replication is conducted by DNMT1, while the newly established DNA methylation (known as de novo DNA methylation) is catalyzed by DNMT3A and DNMT3B.[41] [45] Demethylation of DNA is mediated by the active ten-eleven-translocases enzymes that can convert 5-methylcytosine to the intermediate 5-hydroxymethylcytosine.[46]

Aberrant DNA methylation has been demonstrated to be involved in the pathogenesis of diabetic embryopathy.[47] [48] [49] One study used a Tg rat model of type 2 diabetes to test whether diabetic pregnancy causes aberrant DNA methylation in the promoter of sterol regulatory element-binding transcription factor 2 (Srebf2), a gene critical for cholesterol metabolism. Hypermethylation of CpG sites in the promoter of Srebf2 was observed in fetal liver and brain, which negatively correlated with the Srebf2 gene expression, suggesting their possible roles in inducing metabolic dysfunction in offspring.[47] Another study explored the potential of aberrant DNA methylation as a diagnostic biomarker for diabetic embryopathy. Researchers identified a methylation signature including 237 differentially methylated loci capable of distinguishing infants with diabetic embryopathy from controls. These loci were found proximal to genes related to Mendelian syndromes that overlap the diabetic embryopathy phenotype (e.g., CACNA1C, ANKRD11, and TRIO) or genes known to affect embryonic development (e.g., BRAX1 and RASA3).[48] Pax3, a gene expressed in embryonic neuroepithelium, is required for neural tube closure. The methylation level of a Pax3 CpG island was decreased upon neurulation of embryos and the formation of neuronal precursors from embryonic stem cells (ESCs). However, when ESCs were exposed to hyperglycemia-induced oxidative stress, methylation of the Pax3 CpG island was increased. Further study in ESCs showed that DNMT3B was responsible for methylation and silencing of Pax3 before differentiation. This study provided a molecular mechanism for birth defects induced by Pax3 insufficiency in diabetic pregnancy ([Fig. 5]).[49]

Fig. 5 Hyperglycemia of maternal diabetes induces DNA hypermethylation. Hyperglycemia upregulates DNA methyltransferase 3A and 3B (DNMT3A/3B), which causes the hypermethylation of CpG island in the promoters of essential genes for neural tube closure or heart development, leading to reduced expression of these genes and defects. CHD, congenital heart defect; NTD, neural tube defect, 5mC, 5-methylcytosine.

Increased Histone Acetylation

Histone modification is another type of epigenetic regulation. Histone is the main protein component of chromatin, consisting of four core members H2A, H2B, H3, and H4, around which the DNA molecule is wrapped.[50] Histone modification alters chromatin architecture and thereby affects the accessibility of transcription factors and initiation complexes, inducing gene activation or silencing.[51] Histone modification primarily includes well-studied methylation and acetylation, and ubiquitination, phosphorylation, and sumoylation.[52] The nomenclature of histone modifications relies on the types of histone protein and amino acid, and the location and type of modification.[53] For example, H3K4me, indicates a methylation at lysine 4 residue of histone H3. The maintenance of histone modification usually needs three factors: “writer” (including methyltransferase, acetyltransferase, etc.), “eraser” (including demethylase, deacetylase, etc.), and “reader” (effector proteins recognizing specific banding sites).[54] [55] Mutation or abnormal expression of these modifiers can cause aberrant histone modification and subsequently, aberrant target gene expression.

Histone modification is implicated in early embryonic development[56] [57] and is closely associated with congenital diseases.[58] [59] [60] We previously demonstrated that aberrant histone modifications are implicated in maternal diabetes hyperglycemia-induced NTDs.[61] We focused on stress-resistant sirtuin (SIRT) family histone deacetylases because of the causal role of cellular stress in diabetic embryopathy. The acetylation of histone (H) 3 lysine (K) 56, H3K9, H3K14, and H3K27 (putative substrates of SIRT2 and SIRT6) was increased by maternal diabetes in vivo or high glucose in vitro, which was reversed by SOD1 overexpression or Tempol treatment. The overexpression of SIRT2 or SIRT6 prevented the increase in acetylation of their histone substrates. Application of SIRT activators or inhibitors altered histone acetylation and NTD formation. This study suggested that SIRT downregulation-induced histone acetylation may be involved in diabetes-induced NTDs.[61] Another study tested whether maternal hyperglycemia could disrupt early pancreas development through histone modification.[62] Hyperglycemic conditions significantly decreased the levels of definitive endoderm marker SRY-box transcription factor 17 (SOX17), C-X-C chemokine receptor type 4 (CXCR4), Forkhead box protein A2 (FOXA2), and Eomesodermin homolog (EOMES) during differentiation, which was associated with the retention of repressive histone methylation mark histone 3 lysine 9 trimethlyation (H3K9me3) on their promoters. Treatment with wingless-related integrated (Wnt)/β-catenin signaling activator restored expression levels and histone methylation status in hyperglycemic conditions. The disruption of definitive endoderm differentiation triggered subsequent impairment in the formation of pancreatic progenitor cells. This study suggests that histone modification may be involved in the impairment of early pancreas specification and development.

Altered Micro-RNA Expression

miRNAs are small, noncoding RNAs of approximately 22 bp, that can regulate gene expression at the posttranscription level.[50] miRNAs have a genomic origin and are named primary miRNAs after transcription and precursor miRNAs after cleavage by RNase II enzyme Drosha.[63] Mature single-stranded miRNAs are incorporated into an RNA-induced silencing complex where they bind to the 3′UTR of target mRNAs in a base-pairing manner.[64] Perfect base pairing triggers the degradation of target mRNAs while imperfect pairing causes translational repression.[65] [66] Studies have revealed that miRNAs are essential for the development of embryogenesis.[67] [68] Many cellular biological processes such as proliferation, differentiation, and apoptosis are also regulated by miRNAs.[69] [70] [71]

Our group extensively investigated the potential roles of miRNAs in the pathogenesis of diabetic embryopathy.[12] [25] [33] [72] [73] [74] [75] We performed a global miRNA profiling study and found a total of 149 miRNAs mapped to embryonic hearts that were significantly altered by maternal pregestational diabetes. Bioinformatics analysis indicated that the majority of potential miRNA target genes were linked to cardiac development-related pathways, including STAT3 and IGF-1, as well as transcription factors (Cited2, Zeb2, Mef2c, Smad4, and Ets1). In addition, overexpression of SOD1 reversed the change of miRNAs induced by maternal diabetes hyperglycemia, suggesting that oxidative stress is responsible for the dysregulation of miRNAs.[72] Nuclear factor-erythroid 2-related factor 2 (Nrf2) is the master regulator of the cellular antioxidant system. We tried to determine whether hyperglycemia represses the expression of Nrf2 and Nrf2-regulated antioxidant genes through the redox-sensitive miR-27a. Maternal diabetes in vivo or high glucose in vitro both significantly upregulated miR-27a and downregulated Nrf2 expression. An miR-27a inhibitor alleviated the Inhibitory effect of high glucose on Nrf2 expression, and an miR-27a mimic inhibited Nrf2 expression in Neural Stem Cells (NSCs). SOD2 overexpression reversed the hyperglycemia-induced upregulation of miR-27a and downregulation of Nrf2 and Nrf2-regulated antioxidant enzymes. This study suggested that maternal diabetes hyperglycemia-induced oxidative stress upregulates miR-27a, which subsequently represses Nrf2 and its responsive antioxidant enzymes, leading to diabetic embryopathy.[73] In a study investigating the mechanism underlying high glucose-induced ASK1 activation, we found high glucose suppressed miR-17 expression and increased the expression of its target gene, thioredoxin-interacting protein (Txnip). The upregulated Txnip enhanced its binding to thioredoxin (ASK1 inhibitor), thereby sequestering Trx from the Trx–ASK1 complex and activating the ASK1 pathway.[74] We also investigated another high glucose-induced apoptosis pathway where miR-322 is involved. Maternal diabetes in vivo and high glucose in vitro significantly decreased the expression of miR-322 and increased the level of TNF receptor-associated factor 3 (TRAF3) protein, which can be abolished by SOD1 or treatment with the SOD1 mimetic Tempol. Further study confirmed that miR-322 regulates apoptosis by targeting TRAF3 mRNA and repressing its translation. This study demonstrates the involvement of oxidative stress and the miR-322-TRAF3 pathway in high glucose-induced caspase activation and apoptosis.[25] [75] In another study we focused on autophagy regulators modulated by diabetes in the murine developing neuroepithelium. The deletion of the Prkcα gene restores diabetes-induced autophagy impairment, cellular organelle stress, and apoptosis, reducing proper NTD formation. PKCα enhances the expression of miR-129-2, a negative regulator of autophagy, which suppresses autophagy by directly targeting PGC1α, a positive regulator for mitochondrial function. These findings identified, PKCα and miR-129-2, as negative autophagy regulators involved in the pathogenesis of hyperglycemia-inducing NTDs.[12]

It stands to reason that maternal diabetes hyperglycemia might alter the expression of miRNAs that regulate genes critical for neural tube development and closure. miRNA-30 family members that are predicted to target genes implicated in brain development, were upregulated in NSCs from embryos of diabetic pregnancy compared to control. Among them, the upregulation of miRNA-30b coincided with the downregulation of its target gene, Sirtuin 1 (Sirt1). Further overexpression and knockdown experiments suggest that miRNA-30b alters cell lineage specification via Sirt1.[76] In another study analyzing epigenetic factors in mice embryonic NSCs exposed to hyperglycemia, authors reported that hyperglycemia increased the protein levels of Doublecortin (Dcx) and platelet-activating factor acetyl hydrolase, isoform 1b, subunit 1 (Pafah1b1), concomitant with decreased expression of miR-200a, miR-466a-3p, miR-200b, and miR-466d-3p, which are predicted to target these genes. Knockdown of these specific miRNAs in NSCs caused increased expression of Dcx and Pafah1b1 proteins confirming their target specificity.[77]

Further studies are still needed to comprehensively elucidate the etiologic roles of epigenetics in diabetic embryopathy, which will hopefully uncover additional approaches for the prevention and treatment of these defects in humans.

Premature Aging and Developmental Senescence

Senescence is where proliferating cells cease their cell cycles and become quiescent. Senescent cells also produce a unique secretome called senescence-associated secretory phenotype consisting of cytokines, chemokines, and growth factors, which adversely affect neighboring cells. Senescence typically occurs in aged cells or organisms; however, maternal diabetes hyperglycemia triggers premature senescence in the developing neuroepithelium before neural tube closure.[14] Hyperglycemia-activated FoxO3a increases the transcription of miR-200c, which in turn represses the expression of two transcription repressors Zinc finger E box-binding homeobox 1 and 2 (ZEB1 and ZEB2).[14] ZEB1 and ZEB2 normally prevent the expression of the two cell cycle inhibitors, p21 and p27. miR-200c-inhibited ZEB1 and ZEB2 lead to the increase of p21 and p27, which induces premature senescence in the neuroepithelium.[14] Deficiency of the Foxo3a gene, the mir200c gene, the cell cycle inhibitors p21 or p27, or Tg expression of the dominant negative FoxO3a mutant abolishes maternal diabetes hyperglycemia-induced neuroepithelial cell senescence, preventing NTDs in diabetic embryopathy ([Fig. 6]).[14] This evidence suggests that targeting senescence would be an effective prevention for NTDs. Indeed, using the senomorphic rapamycin in treating diabetic dams blocks senescence and prevents NTD formation.[14]

Fig. 6 Hyperglycemia of maternal diabetes induces premature senescence. Hyperglycemia activates FoxO3a by dephosphorylation, which translocates into the nucleus and increases the expression of the miR-200c gene. miR-200c represses Zinc finger E box-binding homeobox 1/2 (ZEB1/2) expression by degrading their mRNAs, and preventing them from repressing the transcription of p21 and p27, leading to the increased expression of these senescence mediators, which ultimately triggers premature senescence. Treatment with rapamycin inhibits premature senescence and reduces NTD formation. FoxO3a, forkhead box O 3a; NTD, neural tube defect.

Conclusive Remarks and Future Directions

Maternal diabetes hyperglycemia-induced oxidative stress activates a group of kinases that mediates the teratogenicity leading to NTDs and CHDs. Kinase signaling, in turn, activates transcription factors, alters the expression of enzymes that install epigenetic modifications, and alters miRNA expression. Oxidative stress-initiated signaling triggers prolonged UPR and ER stress, impairs mitochondrial fusion and autophagy, and induces premature senescence. Future studies will aim to establish the connections between cellular organelles, RNA metabolism, and the fate of senescent cells.

Folic acid can reduce hyperglycemia-induced NTDs but its beneficial effects on CHDs are still unclear. Folic acid is the major donor of the methyl group for DNA, RNA, and protein methylation, suggesting that folic acid can enhance methylation. However, hyperglycemia-increased DNA methylation contributes to NTD formation. It is most likely that folic acid does not work through DNA methylation to elicit its preventive effect against NTDs. Further studies should focus on RNA and protein methylation to determine if folic acid selectively enhances the methylation of a specific group of mRNAs and proteins.

Kinase inhibitors to ASK1 and JNK1/2 are effective in reducing NTDs in animal models of diabetic embryopathy. The ER stress inhibitor 4-PBA, the autophagy activator trehalose, the mitochondrial fusion activators teriflunomide and echinacoside, and the senomorphic rapamycin all show efficacy in ameliorating diabetic embryopathy in the mouse model of diabetic pregnancy. The safety profiles of these chemical and naturally occurring compounds need to be carefully assessed in animal models and humans before clinical trials testing these as dietary supplements can proceed. Other embryonic anomalies observed in diabetic embryopathy such as kidney and eye defects need to be further explored in animal models. It remains unclear if the mechanisms described above are involved in all organ systems or if there are yet unidentified pathways involved.

Because human fetal tissues are relatively inaccessible, most of the studies in diabetic embryopathy have been conducted on animal models and/or in vitro experiments. This is a limitation. While these models have been extensively utilized in experimental research and exhibit similarities in gene expression and morphological development to humans, significant differences remain between species, as well as between in vivo and in vitro conditions. A recent study using human neurulation stage embryos demonstrates that apoptosis and gene expression changes including Wnt genes are involved in neural tube closure.[78] Morphological studies have shown that cardiac development sequences share a high degree of similarity in humans and mice.[79] Histone methylation and acetylation are key mechanisms in gene regulation during human heart morphogenesis.[80] Therefore, findings from animal models and in vitro studies recapitulate some key aspects of human neurulation and fetal heart development.

Conflict of Interest

None declared.

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Publication History

Received: 13 May 2024

Accepted: 26 August 2024

Accepted Manuscript online:
29 August 2024

Article published online:
27 September 2024