Intracellular Metabolism in Diabetic Embryopathy

Diabetes mellitus in early pregnancy can cause congenital birth defects in infants, a complication known as diabetic embryopathy. Formation of structural abnormalities, commonly seen in the central nervous and cardiovascular systems, is associated with increased programmed cell death (apoptosis) and decreased cell proliferation in the developing organs. Under maternal hyperglycemic conditions, influx of glucose into cells of the embryos disturbs intracellular metabolic homeostasis. Disturbed glycolysis generates factors through side pathways to perturb cell signaling and organelle functions. Perturbed phospholipid metabolism produces signaling metabolites and peroxidation products to suppress cell survival and induce apoptosis. Targeting the key processes and factors in the metabolism of glucose and phospholipids is a potential intervention strategy to prevent birth defects in diabetic pregnancies.


Introduction
Diabetes mellitus in early pregnancy imposes a risk of birth defects in infants [1][2][3][4]. This type of diabetic complication, known as diabetic embryopathy, account for birth defect rates three times higher than those in the general population [3][4][5][6][7]. Unfortunately, the number of women of childbearing age with type 1 and type 2 diabetes is increasing [8][9][10], imposing tremendous challenges to perinatal management and health care at large [11,12]. Although aggressive glycemic control and specialized antenatal care, including dietary supplementation of vitamins and folic acid, are widely available in the United States and other developed countries, the birth defect rates in diabetic pregnancies have not been significantly reduced for decades [2,13,14]. Therefore, it is urgent to understand the mechanism of diabetic embryopathy to develop interventions to prevent birth defects in diabetic pregnancies.

Glucose Metabolism
Glucose is an important source of energy for cells to survive and function. Cells uptake glucose through glucose transporters In the conversion of glucose to sorbitol, nicotinamide adenine dinucleotide phosphate (NADPH), a co-factor of aldose reductase, is oxidized into NADP+ [50,51]. NADPH is an important antioxidant in the cytosol and mitochondria. Reduction of NADPH leads to a diminishing of antioxidative capacity of the cell and a disturbance of mitochondrial respiratory activity which causes the production of ROS [60,61]. NADPH is also a co-factor of glutathione reductase in the conversion of oxidized GSH (GSSG) into reduced GSH [50].
In the pentose phosphate pathway, glucose-6-phosphate is converted into 6-phosphogluconolactone by glucose-6-phosphate dehydrogenase [62,63]. In this reaction, NADP+ is reduced to NADPH, which potentially facilitates the conversion of GSSG into GSH to increase antioxidative capacity [62,63]. However, it has been shown that this pathway is inhibited in embryos cultured in high glucose [64].
Overall, the levels of GSH are reduced in the embryos exposed to hyperglycemia [33, 65,66]. The depletion of antioxidants further augments oxidative stress, leading to increases in apoptosis in hyperglycemia-induced embryonic malformations. In the embryos of diabetic mice, global protein O-GlcNAcylation is increased [75]. Administration of glucosamine, a substrate for generating GlcNAc, to normal pregnant mice mimics the effect of high glucose and leads to NTD formation in the embryos [64]. The elevation of protein O-GlcNAcylation is associated with increased activation of OGT. Inhibition of OGT results in reduction of embryonic malformations [75].

Phospholipid Metabolism and Peroxidation
Phospholipids are major components of the plasma membrane [87]. The metabolism of phospholipids gives a rise to a large variety of metabolites, contributing to intracellular signaling [88,89].
Disturbance of lipid metabolism has been characterized in the embryos of diabetic animals or embryos cultured in high glucose [29,[90][91][92].
In the embryos of diabetic animals, phospholipid metabolism increases the levels of ceramide [29]. Ceramide has been shown to perturb intracellular signaling and organelle functions to induce apoptosis [93][94][95]. In contrast, the levels of phosphatidylglycerolphosphate (PGP) are decreased [29]. Two PGPs form a diphosphatidylglycerol, known as cardiolipin [96].
Deficiency of this molecule is associated with disorders in the CNS and CVS, including neurodegenerations and Barth syndrome with cardiac anomalies [97][98][99].
Arachidonic acid is another major component of the plasma membrane. Arachidonic acid can be released from the membrane by cytosolic phospholipase A2 (cPLA2) [109]. In the cytoplasm, arachidonic acid can be converted into prostaglandin E2 (PGE2) by cyclooxygenase-2 (COX-2) [110,111], or become PGE2-like isoprostanes, such as 8-iso-prostagladin F2 (8-iso-PGF2) and 8-iso-PGF2, by non-COX-mediated peroxidation, involving ROS ( Figure   6) [112,113]. In the cells of embryos from diabetic animals, decreases in arachidonic acid levels have been observed, indicating an active metabolism of the phospholipid [90,114]. Associated with the changes in arachidonic acid, the levels of PGE2 are decreased [115][116][117][118][119][120][121]. The reduction in PGE2 in the embryo may be due to decreased COX-2 enzymatic activity and downregulation of COX-2 expression [115,121,122]. Experiments show that blocking Cox-2 in the embryos cultured in normal glucose mimics the effect of high glucose on embryonic development [122]. Administration of PGE2 to embryo cultures in high glucose reduces neural tube defects [123,124]. These suggest that PGE2 may protect embryos from hyperglycemic insult.
In contrast, the levels of 8-iso-PGF2 are dramatically elevated in embryos under hyperglycemic conditions [112,120,125,126].
Isoprostanes have been shown to exert cytotoxic effects in cells [127]. Treatment of embryos in culture with 8-iso-PGF2α causes malformations and growth retardation [125]. These studies indicate a shifted metabolism of arachidonic acid toward producing cytotoxic isoprostanes to exacerbate the perturbed intracellular homeostasis to induce apoptosis.

Potential Interventions Targeting Glucose and Phospholipid Metabolism
Delineation of the molecular mechanisms of diabetic embryopathy provides crucial data for developing interventions to reduce fetal abnormalities. However, limited efforts have been made to target glucose and phospholipid metabolism in diabetic embryopathy.
Inhibition of aldose reductase to reduce sorbitol generation have been tested to ameliorate complications in diabetic patients [128][129][130]. The similar approach was applied to embryos cultured in high glucose; however, exerted no effect on neural malformations, although reduced sorbitol level [56]. Further exploration of the approach in diabetic pregnancies in vivo is warranted. Inhibition of OGT to suppress protein O-GlcNAcylation has been shown to decrease of malformations in the embryos of diabetic mice [75].
Replenishing arachidonic acid to restore membrane integrity and/or inhibit lipid peroxidation has been explored. In the embryos cultured in high glucose [122], or the embryos in diabetic pregnant animals [131][132][133], treatment with arachidonic acid decreases malformation rates. These findings indicate that a potential strategy to use certain species of phospholipids, such as fish oil to replenish cardiolipin, to reduce embryonic malformations in diabetic pregnancies.

Concluding Remarks
The mechanisms of diabetic embryopathy is complex, involving various cellular and molecular processes. The influx of glucose and associated glucose and lipid metabolism generate an aberrant intracellular environment, disturb intracellular signaling, and ultimately induce apoptosis in developing embryos and malformations. Interventions to suppress the production of toxic metabolites of glucose and phospholipids or restore normal metabolic balances may be a promising strategy to prevent birth defects in diabetic pregnancies.