The enzyme involved in the terminal transfer of the GlcNAc moiety to target proteins is the O-linked N-acetylglucosaminyltransferase (OGT) while the removal of GlcNAc is catalyzed by the β-selective N-acetylglucosaminidase (O-GlcNAcase or OGA- annotated as meningioma-expressed antigen mgea5 and characterized as a hyaluronidase (Heckel et al. 1998)) (Wells and Hart 2003; Zachara et al. 2004). These form a single cooperatively regulated enzyme complex whose activity is exquisitely sensitive to UDP-GlcNAc and hence nutrient supply (Kreppel and Hart 1999). Both enzymes are products of single, highly conserved genes with differentially spliced variants in mammals.

OGT maps to Xq13 on the mammalian X-chromosome and encodes three variants which differ in the length of their amino terminal tetratricopeptide (TRP) repeats (which vary from 3–12 repeats) and in their subcellular distribution: short OGT, mitochondrial OGT (mOGT) and nuclear/cytoplasmic OGT (ncOGT) (Lubas et al. 1997; Kreppel et al. 1997; Kreppel and Hart 1999; Lubas and Hanover 2000). The TRP domains produce a superhelical structure containing an asparagine ladder that is critical for protein recognition (Jinek et al. 2004). Alternate splicing of these differentially targeted variants therefore produces isoforms with varying TRPs thus allowing each isoform to modify a select subset of substrates, conferring on this single gene enzyme the ability to modify many targets (Lubas et al. 1997; Kreppel et al. 1997; Lazarus et al. 2006).

OGA exists as two isoforms: the full-length isoform incorporates a histone acetyltransferase (HAT) domain in its C-terminus and a short isoform lacking the C-terminal HAT domain. The shorter isoform is associated with lipid droplets and thought to have a role in lipid droplet assembly and mobilization, since it promotes proteasomal degradation of surface lipid droplet proteins (Keembiyehetty et al. 2011). The longer isoform, on the other hand, is present throughout the nucleus and cytoplasm. Moreover, this bifunctional nuclear/cytoplasmic OGA associates with OGT through specific domains into a single O-GlcNAczyme complex (Whisenhunt et al. 2006). Basal OGT and OGA activities are modulated by their own intrinsic O-GlcNAcylation status (Kreppel et al. 1997; Gloster and Vocadlo 2010) supporting the idea of cooperative activity between the two enzymes. Ultimately this co-operativity results in a finely balanced equilibrium whereby even small deviations in O-linked glycosylation impact on cellular signalling activities and therefore cellular homeostasis in response to a nutrient signal.

OGT and OGA appear to regulate attachment and removal of O-GlcNAc in much the same way that kinases and phosphatases regulate phosphorylation. OGT was originally believed specifically to target serine/threonine residues on a number of nucleoplasmic proteins to alter their activity and stability in response to nutrient availability (Wells et al. 2001). Indeed, for a subset of proteins, OGT competes dynamically with protein kinases leading to the idea that such modification is functionally reciprocal to phosphorylation at the same sites (Kelly et al. 1993; Comer and Hart 2001; Chou et al. 1995). Whereas protein phosphorylation is achieved by the coordinate regulation of approximately 500 protein kinases and ~ 100 phosphatases (Forrest et al. 2003; Manning et al. 2002) the regulation of O-GlcNAcylation is achieved by the concerted action of the two highly conserved enzymes, OGT and OGA, highlighting their potential significance as regulators of kinase dependent cellular pathways.

Whilst this reciprocity certainly exists for a subset of proteins and is well characterized for some, such as the RNA polymerase II C-terminal domain (Comer and Hart 2001) and Sp1 (Kudlow 2006), for other proteins such as p53, O-GlcNAcylation can sterically hinder nearby phosphorylation sites and impact on their activity (Yang et al. 2006). The number of proteins whose activity and stability is modified by O-linked glycosylation is extensive and increasing and includes transcription factors, nuclear pore proteins, cytoskeletal components, metabolic enzymes as well as phosphatases and kinases that are known regulators of early developmental processes. There is in fact extensive cross-talk between OGT and the canonical nutrient sensing kinase cascades such as Insulin-Akt, MAPK, mTOR and AMPK (Hanover et al. 2010). Whilst O-linked glycosylation often results in decreased protein activity this is not a generalised phenomenon since in some proteins such as the global transcription factor Sp1 GlcNAc modification can increase protein activity (Goldberg et al. 2006; Wells and Hart 2003). Moreover, studies also show that O-linked glycosylation can both prevent and increase target susceptibility to proteasomal degradation (Han and Kudlow 1997). The 26S proteasome is a proteolytic organelle present in both the cytoplasm and nucleus responsible for degradation of proteins that are marked for turnover by polyubiquitination, thus controlling protein half-life, maintenance of metabolic proteins and elimination of damaged proteins. It is present throughout preimplantation development and its function is essential for early embryonic development (McCue et al. 2008). In the case of Sp1, under conditions of inadequate nutrition, O-GlcNAcylation of this transcription factor is reduced and this correlates with increased degradation that can be inhibited by proteasomal inhibition (Han and Kudlow 1997). Conversely hyperglycosylated Sp1 is protected from proteasomal degradation leading to the suggestion that O-GlcNAcylation of Sp1 may play a role as a nutritional checkpoint by generally reducing transcription in the absence of adequate nutrition (Han and Kudlow 1997). Consistent with this hypothesis, Sp1 promoter activation is reduced in an O-GlcNAc dependent manner as a result of an interaction between OGT and the transcriptional repressor mSin3A (Yang et al. 2002). Inhibition of the proteasome itself by O-linked glycosylation also couples protein turnover to metabolic state allowing cells to control the availability of amino acids and regulatory proteins (Zhang et al. 2003).

O-GlcNAcylation and/or the enzyme OGT, can also interact with other post-translational modifications such as ubiquitination (Guinez et al. 2008), which targets cellular proteins to the proteasome, and nitrosylation (Ryu and Do 2011) although the mechanisms by which these interactions occur are not clearly defined. Moreover, the OGT/OGA complex associates with histone deacetylases (HDACs) in nuclear transcriptional co-repression complexes to regulate transcription. Disruption of the enzyme complex regulating O-GlcNAcylation in these complexes interferes with transcriptional repression (Whisenhunt et al. 2006), thus implicating the enzyme complex in still broader roles. More recently OGT and O-GlcNAcylation have also become linked to the multi-faceted “histone code” with recent findings suggesting that all four core histones are modified by O-GlcNAc (Sakabe et al. 2010). The sites of O-GlcNAc-histone modification hint at a role in chromatin remodeling and add to a mounting body of evidence linking O-GlcNAc cycling to higher-order chromatin organization and epigenetic memory (Hanover et al. 2012). The identity and function of these targets and partners would suggest that HSP plays a critical role in controlling all aspects of cellular function ranging from protein activity and stability, fuel metabolism, cytoskeletal organization, cell growth, gene expression and differentiation as well as longer-term transcriptional regulation of developmental processes in response to nutrient availability.

Significantly, the toxicity associated with exposure of embryos to a hyperglycemic environment on a number of morphological parameters of early development including blastocyst formation (Fig. 3.2a), cell number and apoptosis is either reversed or ameliorated by inhibition of OGT (Pantaleon et al. 2010). This provides conclusive evidence that the HSP does indeed represent the mechanism by which hyperglycemia impacts adversely on early embryo development and survival. Previous studies had suggested that hyperglycemia triggers cell death in the early embryo by a decrease in glucose transporter GLUT1 expression, to trigger Bax-dependent apoptosis in the mouse blastocyst (Chi et al. 2000). This event however, is more likely to be secondary to dysregulation of the HSP and increased O-GlcNAcylation given the ability of OGT inhibition to reverse the impact of a hyperglycemic insult on embryo differentiation, proliferation and survival (Pantaleon et al. 2010).

The central mechanism implicated in the regulation of embryonic cellular proliferation and survival is the PI 3 kinase/Akt pathway (Riley et al. 2006). In the early embryo this survival signaling pathway is normally activated by trophic growth factor signals (Li et al. 2007) to reduce apoptosis and enhance cellular proliferation by activating downstream pathways of cellular growth, proliferation and survival. Additionally it regulates glucose transporter recycling at the plasma membrane allowing the embryo to co-ordinate nutrient transport and protein synthetic activities with the modified metabolic requirements of stimulated proliferation (Pantaleon and Kaye 1996). Impaired PI 3-kinase activation and reduced Akt activity in response to increased O-GlcNAcylation provides an explanation for decreased survival in response to hyperglycemia, defective glucose transporter recycling and stimulated cell proliferation and hence the insulin/growth factor resistance feature of diabetes (Whelan et al. 2010).

Whilst glucosamine can overcome the block to blastocyst formation (Pantaleon et al. 2008) in the absence of glucose and act as a potent stimulator of embryonic levels of O-GlcNAcylation (Pantaleon et al. 2010) it can be misleading as a hyperglycemic mimetic tool when used in isolation in vitro in the absence of glucose.

This is because in the absence of glucose, glucosamine may activate additional stress response pathways as a result of oxidative stress (Jansen et al. 2009), independently of effects through O-GlcNAcylation. Glucosamine enters cells and is readily phosphorylated to GlcN-6-P and thus enters the HSP downstream of entry into the PPP at F-6-P as discussed earlier (Fig. 3.1). In the absence of glucose, flux through the PPP is attenuated reducing NADPH production and increasing oxidative stress (Jansen et al. 2009). Moreover, GlcN-6-P is a potent inhibitor of the first enzyme of the PPP glucose-6-P-dehydrogenase (G6PDH)(Wu et al. 2001) and this high Glcn-6-P concentration arising from glucosamine supplementation may also limit PPP flux arising from any gluconeogenic activity, should this be occuring. The result is compromised development and increased cell death which cannot be reversed by OGT inhibition in sharp contrast to the ability of BADGP to reverse the glucotoxic effects of hyperglycemia on blastocyst formation completely (Fig. 3.2; Pantaleon et al. 2010).

Although such metabolism has not previously been reported in preimplantation embryos, administration of glucosamine to pregnant (day 7.5) mice inhibits the PPP and increases oxidative stress more potently than hyperglycemia in the whole embryo within 3 h (Horal et al. 2004). Moreover, recent studies in mouse oocytes aiming to establish dose response models for glucose and glucosamine in a mouse IVM system have demonstrated that glucosamine alone during IVM in the absence of glucose is unable to support subsequent embryo development (Frank et al. 2013) suggesting that the contribution of the PPP during oocyte maturation can be as critical for subsequent development as hexosamine signalling. Not only will this limit synthesis of NADPH (Wu et al. 2001) and thus increase oxidative stress but reduce the bioavailability of pentose phosphates required for nucleic acid synthesis to impact on embryo viability.

An in vivo mouse model of periconceptional glucosamine supplementation (daily glucosamine injections (20–400 mg/kg) administered for 3–6 days before and 1 day after mating) confirms the potential impact that perturbed HSP activity specifically during this developmental period can have on post natal outcomes. (Schelbach et al. 2013). Intriguingly there appeared to be an age dependency to different outcomes with reduced litter size independent of dose in younger mothers compared with a significantly higher incidence of fetal congenital abnormalities in older mothers, similar to and consistent with pathologies associated with hyperglycemic or diabetic mice (Schelbach et al. 2013). It is difficult to know the relevant contribution of effects on embryonic development versus maternal perturbations, given that maternal systemic glucosamine supplementation may also impact on decidualisation (Tsai et al. 2013) and by implication subsequent placentation to contribute to altered fetal growth in response to an adverse environment.

Nonetheless, the available evidence strongly implicates perturbed O-GlcNAc cycling as a result of either pharmacological inhibition or a perturbed nutrient environment with perturbed developmental outcomes, specifically the glucotoxic effects of hyperglycemia during preimplantation development . The system appears to be very tightly regulated since low and high levels of O-GlcNAcylation have a negative impact on early development with the potential to alter long-term health outcomes (Fig. 3.3). Based on the available evidence it is tempting to speculate that perturbed O-GlcNAcylation may underlie the impact of environmental stress associated with embryo culture that impacts on embryo viability and post-natal outcomes (Lane and Gardner 2005).