2.1 HBP

The HBP is intricately linked to O-GlcNAcylation, wherein the HBP provides the essential substrate for O-GlcNAcylation and is the primary route for producing the critical substrate, UDP-GlcNAc.^21-23 The HBP begins with the uptake of glucose, which is subsequently converted to fructose-6-phosphate through several reactions. Fructose-6-phosphate then reacts with glutamine to produce glucosamine-6-phosphate, a step catalyzed by the enzyme glutamine-fructose-6-phosphate transaminase 1. Glucosamine-6-phosphate undergoes a sequence of enzymatic reactions leading to the formation of UDP-GlcNAc. Specifically, glucosamine-6-phosphate is first N-acetylated to form N-acetylglucosamine-6-phosphate, which is then transformed into N-glucosamine-1-phosphate, culminating in the formation of UDP-GlcNAc. Once generated, UDP-GlcNAc serves as a substrate for OGT, which adds a GlcNAc monomer to the S/T residues of proteins, resulting in O-GlcNAcylation. In addition, OGA is responsible for removing GlcNAc from proteins, thereby facilitating the dynamic regulation of O-GlcNAcylation (Figure 2).

In both mice and humans, two distinct subtypes of GFAT (GFAT1 and GFAT2) exist, located on different chromosomes and encoded by separate genes. Furthermore, each subtype displays a different tissue distribution.²⁴ In the central nervous system, GFAT2 exhibits higher expression levels compared with GFAT1, whereas GFAT1 is produced at greater quantities than GFAT2 in various other bodily tissues.²⁵ Studies indicate that in mammals, GFAT exists in a tetrameric form and its functioning is regulated by the presence of glucose. Additionally, glucosamine-6-phosphate and UDP-GlcNAc have been identified as allosteric inhibitors of GFAT in mammals.²⁵ GFAT is also subject to regulation via PTMs, and over 20 phosphorylation sites have been identified to date. GFAT1 and GFAT2 can be regulated by phosphorylation at different sites by cAMP-dependent protein kinase A (PKA). Specifically, PKA phosphorylates GFAT1 at Ser-205 and GFAT2 at Ser-235 to modulate their activity.^{26, 27} In addition, the Ser-243 site of GFAT1 can be phosphorylated by AMPK and Ca ²⁺/calmodulin-dependent kinase (CaMKII; Figure 2).^{28, 29} Nonetheless, there are inconsistent findings regarding the impact of phosphorylation on GFAT's activity, potentially attributable to variations specific to different isotypes. Specifically, in the heart, it has been found that the activity of GFAT1 is significantly inhibited after phosphorylation by AMPK.²⁸ It has been observed that transcription factors can also influence the activity of GFAT and PTMs. For instance, specific protein 1 (Sp1) and activation of transcription factor 4 (ATF4) have been shown to regulate the transcriptional activity of GFAT. However, the extent of their effect on GFAT activity in different cell types remains to be investigated.^{30, 31} There are also some recognized inhibitors of GFAT.^{32, 33} Take azeserine and 6-diazo-5-oxo-l-norleucine (DON), for instance. However, a shared drawback among them is their lack of specificity and restricted applicability (Figure 2).

Glucosamine-6-phosphate N-acetyltransferase (GNPNAT or GNA1), identified as Emeg32 in mice, is involved in the conversion of glucosamine-6-phosphate to N-acetylglucosamine-6-phosphate through acetyl-CoA. The resulting product is then isomerized to N-acetylglucosamine-l-phosphate by phosphoglucosamine mutase (PGM). Subsequently, the enzyme UDP-N-acetylglucosamine pyrophosphorylase, also recognized as UDP-N-acetylglucosamine pyrophosphorylase (UAP1), facilitates the transformation of N-acetylglucosamine-1-phosphate into UDP-GlcNAc. Besides its role in a new synthesis, UDP-GlcNAc can be replenished via two other compensatory pathways.^{34, 35} The first remedial pathway involves the phosphorylation of O-GlcNAc by N-acetylglucosamine kinase, forming N-acetylglucosamine-6-phosphate. In the alternate pathway, N-acetyl-d-galactosamine is converted to N-acetylglucosamine-1-phosphate and UDP-N-acetylgalactosamine, and subsequently to UDP-GlcNAc through a series of different enzymes. Researchers have speculated about the glucose fraction of HBP metabolism in fat cells. Direct measurement of glucose flow through The study of the HBP in comparison with other glucose metabolic routes was carried out using either radioisotope or stable isotope methods in cultured cells or whole organ systems. Using ¹³C6 glucose labeling in cultured new myocytes, Gibb and colleagues³⁶ noted that the HBP metabolizes glucose more effectively than the pentose phosphate pathway, indicating that glucose processing through the HBP might be significantly greater than earlier assessments had suggested. However, this measurement was conducted under the low energy requirements of cell culture, and the energy requirements of new myocytes in reality are significantly different from those of adult hearts.³⁶ Recently, Olson et al.³⁷ developed an LC/MS technique. In isolated perfusion working hearts, the flow of HBP is measured to be 2.5 nmol/g/min, This represents merely 0.003–0.006% of the glycolytic flow, a figure that is substantially lower than prior estimates, possibly as a consequence of the heart's elevated metabolic rate. Furthermore, when the glucose concentration rises from 5 to 25 mM, it is alterations in glycolysis, not in the HBP flux rate, that lead to shifts in the relative flux of HBP compared with glycolysis. This highlights the limitations of HBP flux testing, which is evaluated as part of glycolysis. Despite the many unknowns regarding the regulation of HBP pathways, the development of new technologies has made it possible to directly measure HBP fluxes in different biological systems.

2.2 OGT: structural insights, regulatory mechanisms, and functional impacts

OGT was initially extracted from the liver of rats in 1992 and reached by cloning 5 years later.^38-40 The study revealed that the OGT gene is situated on the X chromosome and encodes a protein that comprises two primary domains. The initial domain includes a series of tetrapeptide repeats (TPRs), and the count of TPRs differs based on the organism.⁴⁰ TPR is a spiral-to-helical motif generally involved in regulating protein interactions.^{41, 42} In humans, the full-length OGT protein is present in both the nucleus and the cell and comprises 13 intact TPR repeats in its N-terminal domain (Figure 3). Additionally, there are two other subtypes: one subtype contains a mitochondrial localization signal and 9 intact TPRs, while the other subtype, known as sOGT, has only 2.5 TPRs. The enzymatic region of OGT is located in the C-terminus of the protein.⁴³ OGT, an soluble enzyme, is present in both the cytoplasm and nucleus of metazoan cells.^{44, 45} OGT is the exclusive enzyme tasked with catalyzing a considerable array of PTMs by attaching β-linked N-acetyl-d-glucosamine to S/T residues of nucleoproteins or cytoplasmic proteins.^46-48 However, in the case of mammals, OGT is present throughout the development of life.^49-51 Research has demonstrated that nondividing cells can survive for extended periods without OGT, but their physiology is significantly disrupted, although apoptosis does not occur.^51-53 The ablation of OGT in cardiomyocytes results in changes in cell size and increased heart failure (HF). As a result, mice with this genetic defect have a lower survival rate.⁵³ However, it is interesting that cardiomyocytes show increased apoptosis markers after mitosis.⁵⁴ Notably, the survival of the majority of mice with OGT ablation in cardiomyocytes suggests their ability to endure, albeit with the impaired performance of their typical physiological functions. It is important to recognize that phosphorylation of Ser, Thr, and Tyr residues can also regulate OGT activity and substrate recognition.⁵⁵ Research has revealed that OGT can be phosphorylated at approximately 20 distinct sites, which can be identified through proteomic techniques. It has been observed that insulin can enhance OGT phosphorylation by activating the insulin receptor (IR), resulting in elevated OGT activity.^{56, 57} OGT is also a target of phosphorylation by glycogen synthase kinase (GSK)-3β, which has been shown to enhance OGT activity.⁵⁸ The phosphorylation of OGT at Thr-444 by AMPK results in changes in its subcellular distribution and the targets it binds to for substrate interaction.⁵⁹ Furthermore, OGT Ser-20 is capable of phosphorylation by checkpoint kinase 1 (Chk1), which is necessary for cytoplasm division.⁶⁰ Ser-20 on OGT is another phosphorylation site targeted by CaMKII, and OGT's activity is enhanced following this phosphorylation.⁶¹ Apart from being phosphorylated, OGT itself can also be O-GlcNAcylated. In particular, the O-GlcNAcylation of Ser-389, situated within the TPR domain, has been demonstrated to impact OGT's subcellular localization.⁶² O-GlcNAcylation can also occur on serine 3/4 of OGT, although the specific biological implications of this modification remain to be studied (Figure 3). In addition to its intrinsic functions, OGT can also impact other proteins. Adaptor or scaffold proteins can interact with OGT and facilitate the recruitment of substrates for O-GlcNAcylation. As an example, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is O-GlcNAcylated by increased OGT activity during fasting in liver tissues. This modification leads to enhanced PGC-1α stability and the upregulation of genes involved in gluconeogenesis.⁶³ This also encompasses p38 MAPK, which is involved in directing OGT to the neurofilament heavy polypeptide,⁶⁴ and REV-ERBα inhibits the degradation of OGT.⁶⁵ Interestingly, the OGT–OGA interaction may have increased the likelihood of the “O-GlcNAcylationzyme” complex, consistent with that O-GlcNAcylation is a rapidly reversible change.⁶⁶ OGT knockout has been shown to downregulate O-GlcNAcylation, resulting in increased interaction between α-ketoglutaric acid, Hypoxia-Inducible Factor-1α (HIF-1α) hydroxylation, and von Hippel-Lindau protein, ultimately leading to HIF-1α degradation, endoplasmic reticulum stress and apoptosis mediated by C/EBP homologous protein (CHOP) are also involved in this process.⁶⁷ Furthermore, the research suggests that the PI3K–mTOR–MYC signaling pathway plays a critical role in the upregulation of OGT and the subsequent O-GlcNAcylation process. Following treatment with PI3K and mTOR inhibitors, there is a decrease in OGT protein expression and O-GlcNAcylation levels. Conversely, activation of AKT and mTOR can reverse this effect.⁶⁸ The c-MYC transcription factor, which is implicated in oncogenic properties and positioned downstream of the mTOR pathway, plays a role in governing the expression of the OGT protein.^{68, 69} Insulin exposure prompts the migration of OGT from the nucleus to the cytoplasm and plasma membrane, while variations in nutritional conditions bring about shifts in the allocation of OGT between the nucleus and cytoplasm.^{56, 70, 71} Besides functioning as a glycosyltransferase, OGT also displays protease activity. Transcriptional coregulator HCF1 as a proteolytic substrate.^{72, 73} Both UDP-GlcNAc and OGT are required for HCF1 proteolysis, which is consistent with the concept that HCF1 can undergo O-O-GlcNAcylation.⁷⁴ The Walker lab has identified several potential OGT inhibitors through high-throughput screening.⁷⁵ Two compounds TT04 and TT40, have irreversible effects.⁷⁶ However, low solubility has also been shown.⁷⁷ TT04 has been used in the laboratory as an OGT inhibitor.^{78, 79} Vocadlo and coworkers⁸⁰ has developed an O-GlcNAc substrate analog called 5-thioglucosamine (5SGlcNAc). Due to its hydrophobic characteristics, acetylated 5S-GlcNAc effortlessly penetrates cell membranes, undergoes transformation into UDP-5S-GlcNAc, and ultimately attaches to the catalytic site of OGT, resulting in the inhibition of its activity.

2.3 OGA: structural insights and regulatory mechanisms

OGA, first identified by Dong and Hart in 1994, is a hexosaminidase.⁸¹ After isolation from the rat spleen, it is also called NCOAT, nuclear plasma O-GlcNAcylationase, and acetyltransferase.^82-84 The gene for human OGA is situated on chromosome 10 and encodes a protein consisting of an NH2-terminal glycosylhydrolase domain and a COOH-terminal domain. Short amino acid motifs or single amino acids are repeated on both sides of the glycosylhydrolase domain. After the extensive region of low complexity is the stem domain, that interacts with the COOH-terminal domain.^{85, 86} Studies on the OGA structure have revealed that the human OGA (hOGA) absence of the necessary residues to bind with acetyl-CoA. Therefore, this region has been described as a “pseudo-AT domain with no catalytic activity.”⁸⁷ Selective splicing gives rise to short OGA (sOGA), which, however, does not possess a pseudo-AT region and features a distinct amino acid COOH-terminal region.⁸⁸ In 2017, the crystal structure of hOGA was deciphered.^89-91 Recent studies have demonstrated that hOGA forms an obligatory homodimer indicating that OGA functions biologically as a dimer. The structural examination of OGA implies its potential to selectively eliminate O-GlcNAcylation from specific sites, suggesting a possible involvement in regulating transitions in O-GlcNAcylation in collaboration with OGT.⁹² Like to OGT, phosphorylation and O-GlcNAcylation serve as regulatory mechanisms for OGA as well. Mass spectrometry mapping of proteins has identified at least 20 different phosphorylation sites on OGA at Ser, Thr, and Tyr residues; however, the impact of these modifications on OGA remains unclear. Interestingly, the OGA Ser-405 site, that functions as the site where OGA interacts with OGT, is a site for O-GlcNAcylation (Figure 3).⁸⁶

3.1 Transcriptional regulation

While both OGT and OGA can exist in both the nucleus and cytoplasm, studies have shown that OGT is predominantly located in the nucleus. At the same time, OGA is mainly localized in the cytoplasm.⁹³ As the gene pool of O-GlcNAcylation transcription factors and cofactors continues to expand, recognition of their biochemical processes is growing. As a demonstration, within immune cells, O-GlcNAcylation of OGT catalyzes the activation of key transcription factors of T cells, namely nuclear factor 1 (NFA-Tc1) and nuclear factor κB (NF-κB), which plays a crucial role in initiating the activity of white blood cells. In liver cells, O-GlcNAcylation of CRTC2, FOXO1, and PGC-1α can modulate the expression of gluconeogenic genes (Table 1).^{63, 94, 95} Sp1 is a zinc-finger transcription factor that can bind to the GC-rich motifs of various promoters. A study conducted by Jackson revealed that an increase in O-GlcNAcylation on human cells' Sp1 resulted in elevated transcriptional activity.⁹⁶ The O-GlcNAcylation modification protects Sp1 from proteasome degradation.^{97, 98} In addition, the O-GlcNAcylation of Sp1 could disrupt interactions among transcription factors, suggesting that the process of O-GlcNAcylation in transcription factors contributes to the intricate mechanisms governing gene expression.^{100, 101, 99} Regarding NF-κB, O-GlcNAcylation diminishes its binding to IκBα, this leads to an increased nuclear translocation and enhanced gene expression activity. On the other hand, the O-GlcNAcylation of c-Rel is essential for its ability to bind to DNA and carry out transcription functions.¹⁰² RNA polymerase II (Pol II) is recognized to undergo modification through O-GlcNAcylation.¹⁰³ The C-terminal domain (CTD) of Pol II undergoes concurrent O-GlcNAcylation and phosphorylation at Ser-2/5 (Figure 4).

3.2 Calcium signaling

The interplay between Ca2+ signaling and protein O-GlcNAcylation has gained considerable attention. In the cardiac system, O-GlcNAcylation of CaMKII at Ser-279 results in CaMKII initiation and heightened arrhythmias in diabetic patients.¹³⁰ In the liver, CaMKII phosphorylates OGT, leads to elevated levels of O-GlcNAcylation, thereby activating autophagy. PLB is also subject to O-GlcNAcylation, with Ser-16 being the most probable site, which diminishes PKA-mediated PLB Ser-16 phosphorylation by inhibiting OGA or increasing OGA PLB O-GlcNAcylation levels under hyperglycemic conditions. Increased O-GlcNAcylation levels correlate with reduced SERCA activity and enhanced SERCA-PLB binding.¹³¹ The rise in PLB O-GlcNAcylation might contribute to the delayed reuptake of Ca2+ by cardiac ER/SR in diabetic patients. Additionally, it is becoming more apparent that O-GlcNAcylation levels are controlled in a manner dependent on Ca2+, like through the CaMKII/IV-mediated phosphorylation of OGT, which leads to increased activity and higher levels of O-GlcNAcylation.¹³²

3.3 Cell survival/ferroptosis/autophagy

Enhanced levels of O-GlcNAcylation in tumor cells have been linked to cell death in pharmacological investigations of OGT or OGA inhibition. As such, it is increasingly crucial to comprehend the correlation between cell death and glycosylation. Siderozosis is a regulatory iron-dependent cell death marked by the accumulation of lipid peroxidation. Experimental analyses have established that protein O-GlcNAcylation, the primary glucose flux nutrient sensor, coordinates ferritin phage and mitochondrial autophagic ferroptosis.^{133, 134} Inhibition of O-GlcNAcylation results in mitochondrial fragmentation and enhanced mitophagy, generating an additional pool of labile iron and rendering cells more susceptible to ferroptosis. De-O-GlcNAcylation of the ferritin heavy chain at Ser-179 promotes its interaction with the ferritin phage receptor NCOA4, leading to iron accumulation (Figure 5).¹³⁵ Under high glucose conditions, the O-GlcNAcylation level in mesenchymal pancreatic cancer cells was linked to ferroptosis. O-GlcNAcylation at the Ser-555 site of the zinc finger E-box-binding protein 1 (ZEB1) increases its stability and nuclear translocation amplifies the transcriptional activity of adipogenesis-related genes and eventually leads to nephrosis in cells (Figure 5).¹³⁶ The investigation discovered that O-GlcNAcylation heightens the sensitivity of HCC cells to ferroptosis through Yes-associated protein (YAP). Furthermore, YAP O-GlcNAcylation stimulates increased transcription of TFRC, resulting in elevated iron concentrations in HCC cells (Figure 5).¹³⁷ Erastin suppresses the malignant phenotype of hepatoma cells by hindering the O-GlcNAcylation of c-Jun, which further represses protein expression, transcriptional activity, and nuclear accumulation of c-Jun. The authors also noted that c-Jun's O-GlcNAcylation regulates GSH synthesis, which displayed a positive correlation with GSH in clinical samples. In summary, O-GlcNAcyated c-Jun acts as an obstacle to ferroptosis (Figure 5).¹³⁸

Autophagy is a cellular recycling pathway activated by stress and nutrient deprivation signals. In recent times, an expanding collection of studies has investigated the relationship between autophagy and glycosylation, making it imperative to comprehend the interplay between the two. UNC-like kinase 1 (ULK1) plays a pivotal role in the initiation of autophagy during starvation. Specifically, during glucose deprivation, ULK1 is O-GlcNAcylated at the Thr-754 site by OGT. This O-GlcNAcylation of ULK1 is critical for the proper activation of VPS34 through ATG14L, which is an essential step in PI(3)P production, phagophore formation, and subsequent initiation of autophagy.¹³⁹ Research indicated that the O-GlcNAcylation of AMPK diminishes its activity, which in turn inhibits ULK1 activity and autophagy.¹⁴⁰ OGT may influence the onset of autophagy in cardiomyocytes by enhancing ULK1's activity through O-GlcNAcylation.¹⁴¹

4.1 Phosphorylation

As the kinetic properties of O-GlcNAcylation have come to light, it has been suggested that this modification may have a regulatory role in protein function. O-GlcNAcylation can modify Ser and Thr residues, which are also sites of phosphorylation, leading to the increasing belief that there may be dynamic mutual regulation between O-GlcNAcylation and phosphorylation.^{142, 22} Protein phosphorylation is a common biological process that regulates the function and activity of proteins by adding phosphate groups to specific amino acid residues on protein molecules. This modification typically involves transferring phosphate from adenosine triphosphate (ATP) or guanosine triphosphate to specific amino acid residues such as serine, threonine, and tyrosine. Protein phosphorylation plays a crucial role in cell signaling, regulating various biological processes. It is one of the most fundamental and prevalent protein regulation mechanisms within cells and is essential for maintaining normal biological functions in organisms.^143-145 One of the earliest concepts was called the “yin-yang” hypothesis.¹⁴⁶ There is a potential for interaction between O-GlcNAcylation and phosphorylation, indicating the modification of specific residues on proteins by either O-GlcNAcylation or phosphorylation. Numerous proteins reinforce this idea, including c-Myc at the Thr-58 position¹⁴⁷; ER-β at Ser-16, and eNOS at Ser-1177.¹⁴⁸ Modifications at proximate sites can also have antagonistic interactions; for instance, histone deacetylase 4 (HDAC4) undergoes O-GlcNAcylation at Ser-642, which action impedes the phosphorylation of Ser-632 mediated by CaMKII.¹⁴⁹ Certain proteins undergo modifications at distinct sites through both O-GlcNAcylation and phosphorylation. For example, an increase in Thr-200 phosphorylation of CaMKIV can reduce O-GlcNAcylation levels across multiple sites.¹⁵⁰ There exists an intricate regulatory interplay between kinases and O-GlcNAcylation. For instance, AMPK can regulate O-GlcNAcylation levels and can itself be influenced by O-GlcNAcylation.¹⁵¹ GFAT is phosphorylated by AMPK at Ser-243, reducing GFAT activity and decreasing O-GlcNAcylation levels.^{152, 29} The activation of AMPK leads to enhanced phosphorylation of GFAT and a reduction in O-GlcNAcylation levels.¹⁵³ AMPK additionally focuses on OGT by phosphorylating it at the Thr-444 site, which affects only OGT's cellular localization but not its activity. All subunits of AMPK can be targeted by O-GlcNAcylation.⁵⁹ Therefore, understanding the underlying relationship and interactions between O-GlcNAcylation and phosphorylation is a crucial

4.2 Ubiquitination

O-GlcNAcylation also interacts with other PTMs.⁶ For instance, O-GlcNAcylation of OGT and OGA can inhibit protein ubiquitination, thereby suppressing their degradation.^{154, 155} O-GlcNAcylation controls protein ubiquitination via phosphorylation, as it can impact phosphorylation processes,²² phosphorylation, in turn, can govern the process of ubiquitination,¹⁵⁶ it is plausible that O-GlcNAcylation controls protein ubiquitin and stability through interaction with phosphorylation. Yet, the impact of O-GlcNAcylation on protein ubiquitination is bidirectional and some studies have found that proteins modified by O-GlcNAcylation are more stable and not easy to be degraded by ubiquitination. In contrast, some proteins are more likely to be degraded by ubiquitin proteasomes and the specific mechanism remains to be studied. Tumor suppressor protein p53 is intricately regulated, with low levels maintained under normal conditions and rapid accumulation occurring in response to DNA damage through proteasomal degradation.¹⁵⁷ Administering OGA inhibitors to MCF-7 cells increases p53 O-GlcNAcylation, reducing cell survival. O-GlcNAcylation hinders p53 phosphorylation at Thr-155 (Ser-149), decreasing ubiquitination and subsequent degradation. Δ-lactoferrin acts as a transcription factor, inducing cell cycle arrest by upregulating Skp1 and Bax. Lactoferrin undergoes O-GlcNAcylation and phosphorylation at Ser-10.^{158, 159} O-GlcNAcylation enhances lactoferrin stability, maintaining its crucial transcriptional activity. Following activation, lactoferrin undergoes phosphorylation at the Ser-10 site, promoting transcription, and ultimately undergoes degradation via K-379 polyubiquitination.¹⁶⁰ Casein kinase 2 (CK2) is a Ser/Thr PKA linked to cellular proliferation, and DNA repair.¹⁶¹ The phosphorylation of the CK2 subunit Thr-344 enhances protein stability through facilitating interaction with Pin1.¹⁶² Additionally, O-GlcNAcylation modifies proximal Ser-347, counteracting the phosphorylation at Thr-344 and facilitating its degradation. CRTC2, a transcriptional coactivator regulated by the cAMP response element-binding protein, serves as a coactivator for the cAMP response molecular binding protein. In the fasting state, glucagon induces the dephosphorylation of CRTC2 at Ser-171, promoting its translocation into the nucleus to initiate the transcription of genes involved in glycogenesis. Conversely, during feeding, insulin activates the Ser/Thr kinase SIK2, leading to the phosphorylation of CRTC2 at Ser-171. This phosphorylation causes the relocation of CRTC2 to the cytoplasm, where it undergoes degradation through ubiquitination.¹⁶³ However, whether O-GlcNAcylation directly affects CRTC2 ubiquitination and protein stability needs to be further studied. A20-Zinc finger protein inhibits apoptosis and inflammation. Hyperglycemia promotes O-GlcNAcylation, ubiquitination, and degradation of A20, thereby accelerating atherosclerosis (AS) in diabetic mice.¹⁶⁴ A20 is a protein that displays both ubiquitin ligase and deubiquitinase activity. Therefore, a plausible hypothesis is that the regulation of A20 protein levels by O-GlcNAcylation could constitute the tipping point for the subsequent ubiquitination of its target proteins. Keratin pairs 8/18 are expressed in specific combinations during the development and differentiation of tissues. Both keratins are extensively involved in regulating protein interactions, ubiquitination, and filament formation, largely via phosphorylation. Interestingly, keratin 8 and 18 are also highly O-GlcNAcylated. This modification increases the soluble pool of keratin 8/18, enhances their ubiquitination, and facilitates proteasome-mediated degradation. Conversely, the deletion of O-GlcNAcylation sites from keratin 18 makes it more stable. Notably, in vivo, studies have not provided evidence of a correlation between O-GlcNAcylation and phosphorylation of keratin 8/18.¹⁶⁵ The molecular chaperone Hsp90 plays a critical role in stabilizing and activating its client proteins. The function of Hsp90 is stringently regulated by cochaperones and various PTMs, which in turn facilitate the activation of its client proteins. The main negative regulator of NRF2 and KEAP was identified as an OGT substrate. The study uncovered that efficient ubiquitination and subsequent degradation of NRF2 require the essential O-GlcNAcylation of KEAP1 at Ser-104. Interestingly, variations in glucose levels were observed to parallel changes in both O-GlcNAcylation levels and NRF2 activation. This connection suggests that O-GlcNAcylation plays a pivotal role as a crucial link between nutritional sensing and the activation of downstream stress resistance pathways.¹⁶⁶ In 2021, Huang H found that O-GlcNAcylation, a newly discovered modification of FOXA2, might boost HCC cell migration by destabilizing FOXA2 and inhibiting the transcriptional activity of its downstream target gene E-cadherin.¹⁶⁷

5.1 Cardiovascular diseases

The prevalence of CVD has become a substantial menace to human health and welfare. Astonishingly, there has been an almost twofold increase in CVD cases, rising from 271 million in 1990 to 523 million in 2019. Concurrently, cardiovascular-related fatalities have exhibited a consistent ascent, escalating from 12.1 million to 18.6 million during the same timeframe.^168-170 As a metabolically demanding organ, the heart relies on an adequate energy supply to support its contractile and diastolic functions during both the relaxation and contraction phases. To achieve this goal, the heart requires a significant supply of ATP-based energy, and CVD results in changes in cellular energy metabolism, stress response, and signaling. However, a common denominator in all of these diseases is the dysregulation of O-GlcNAcylation signaling. Existing research suggests that the role of O-GlcNAcylation in CVD is inconsistent. The study primarily focuses on two aspects: on the one hand, O-GlcNAcylation is believed to have adverse effects on chronic CVD; on the other hand, O-GlcNAcylation plays a protective role in acute CVD. Therefore, the benefits or drawbacks of O-GlcNAcylation depend on the particular disease environment.

5.1.1 HF

The American Heart Association characterizes HF, also referred to as congestive HF, as a syndrome. This is a collection of signs and symptoms that arise due to the heart's inability to pump effectively. HF is described as a chronic, progressively worsening condition where the heart muscle cannot pump sufficient blood to fulfill the body's requirements for blood and oxygen.¹⁷¹ In animal models and patients, the hallmark of central muscle failure is an increase in protein O-GlcNAcylation.¹⁷² However, it is unclear whether too much O-GlcNAcylation is a cause or consequence of cardiomyopathy. The researchers found that the hearts of OGT transgenic mice showed increased levels of O-GlcNAcylation and the potential to develop severe dilated cardiomyopathy (DCM), ventricular arrhythmias, and premature death. In contrast, the O-GlcNAcylation levels of the hearts of OGA transgenic mice were lower, but the heart function of transgenic mice did not change compared with littermate-type mice. Even the hearts of OGA transgenic mice have a certain resistance to pathological stress caused by stress overload, and after stress, myocardial O-GlcNAcylation levels are reduced and pathological hypertrophy is reduced. The experiment also found that although the amount of OGT in the heart muscle of mice is increasing, if OGT is crossed with OGA transgenic mice, cardiomyopathy and premature death can be saved. Transcriptomic and functional studies have also shown damage to the mitochondria of the heart of OGT transgenic mice. OGA transgenic hybridization improves heart damage caused by mitochondrial damage by restoring the activity of complex I. Excess O-GlcNAcylation can lead to cardiomyopathy, in part because of energy deficiencies. Weakening of O-GlcNAcylation is beneficial in combating pathological remodeling and HF caused by stress overload (Figure 6).¹⁷³ The study utilized gene knockout techniques to eliminate the OGT gene in cardiomyocytes, resulting in the absence of O-GlcNAcylation. The findings revealed a notable decrease in autophagy, particularly under fasting conditions. The lack of OGT impacted the early stages of autophagy. The researchers observed O-GlcNAcylation of Unc-51-like autophagy-activated kinase 1 (ULK1), a crucial kinase initiating autophagy, in cardiomyocytes, with diminished levels in cells where OGT was knocked out. In summary, the study underscores the significance of O-GlcNAcylation levels in triggering autophagy in cardiomyocytes (Figure 6A; Table 2).¹⁴¹

TABLE 2. List of selected O-GlcNAcylation proteins in disease (2010–2023).

Disease	Protein	Protein function	References
Accelerated atherosclerosis and diabetic heart	A20	Decreased A20 expression.	164
Heart development	Angiopoietin-1	Angiopoietin-1 expression was markedly reduced in OGT cKO hearts. The absence of angiopoietin-1 in cardiomyocytes resulted in embryonic lethality.	189
Tumor-associated macrophages	Cathepsin B	OGT deletion in macrophages reduces O-GlcNAcylation and mature cathepsin B levels in the tumor microenvironment (TME), impeding cancer metastasis progression and chemotherapy resistance.	190
Hepatocellular carcinoma	YTHDF2 (Ser-263)	Improved the protein's stability and its cancer-promoting activity by preventing its ubiquitination.	191
Macrophage inflammation	S6K1	Suppress S6K1 phosphorylation and mTORC1 signaling.	192
Autoimmune disorders	Foxp3 Stat5	Foxp3 and Stat5 regulate the lineage stability and functionality of regulatory T (Treg) cells, thereby fostering the maintenance of immune homeostasis.	193
Alzheimer's disease	Tau	Suppress O-GlcNAcylation of tau and increase the phosphorylation of tau at specific sites.	194

• The proteins listed in this table were primarily chosen based on their incorporation within the main content of the article.

Compensatory myocardial hypertrophy may evolve into HF if under conditions of constant stress. It has been reported that protein O-GlcNAcylation signaling is also associated with myocardial hypertrophy. Peroxisome proliferation activator-γ-coactivator-1α (PGC-1α) is a transcriptional coactivator that interferes with mitochondrial biosynthesis and is abundantly expressed in myocyte radium.¹⁷⁴ Moreover, PGC-1α activity is affected by O-GlcNAcylation levels. However, a relative decrease in PGC-1α expression levels has been observed in myocardial hypertrophy, possibly by the fact that PGC-1β can be directly affected by O-GlcNAcylation, and its activity and expression are subsequently inhibited, ultimately inhibiting fatty acid oxidation/mitochondrial gene expression, leading to metabolic dysregulation (Figure 6A).¹⁷⁵ Maintaining cardiac balance involves protein arginine methyltransferase 5 (PRMT5) regulation of reduced protein O-GlcNAcylation in human DCM hearts. Mice with cardiomyocyte-specific PRMT5 deletion exhibit DCM and HF, accompanied by increased O-GlcNAcylation as revealed through transcriptomic and metabolomic analysis. PRMT5 deletion regulates transcription and induces abnormal splicing, inhibiting OGA expression. Remarkably, a positive correlation between PRMT5 and OGA expression was observed in human DCM hearts (Figure 6A).¹⁷⁶ A variety of research has demonstrated that acute, pharmacologically induced increases in O-GlcNAcylation levels are effective in reducing cellular and tissue damage. Additionally, these elevations have been shown to promote functional recovery in cardiac tissues.^177-179 Several studies have established a correlation between reduced O-GlcNAcylation levels and an increased vulnerability of cardiomyocytes to oxidative stress. This suggests that lower levels of O-GlcNAcylation may compromise the heart cells' ability to withstand oxidative damage.¹⁸⁰ These studies collectively indicate a growing association between protein O-GlcNAcylation and cardiovascular events.

5.2 Cancer

Cancer is the second leading cause of death after CVD worldwide, representing a remarkable threat to humans’ lives and health. According to the latest data from the International Agency for Research, nearly 19.3 million new cancer diagnoses and over 10.0 million deaths occurred in 2020.²⁰⁹ Thus, it is urgent to clarify the underlying mechanisms involved in cancer progression and to identify novel therapeutic targets for cancer clinical treatment.

Many types of tumor cells exhibit elevated levels of O-GlcNAcylation, potentially due to increased nutrient flux and metabolic alterations in these cells. Additionally, O-GlcNAcylation can modulate key proteins associated with cell cycle and proliferation, such as p53, c-Myc, and AKT, affecting their stability, activity, and subcellular localization. It also regulates pathways related to cell apoptosis and survival, influencing tumor cell viability. In summary, the relationship between O-GlcNAcylation and cancer is intricate, with this PTM playing a pivotal role in tumor onset and progression.²¹⁰ Therapeutic strategies targeting O-GlcNAcylation may offer novel opportunities for cancer treatment. The study explores the role of glucose metabolism in enhancing the protumor functions of tumor-associated macrophages (TAMs). Increased glucose uptake in M2-type TAMs activates O-GlcNAcylation, promoting cancer metastasis and resistance to chemotherapy. This process increases the levels of the enzyme cathepsin B in the tumor environment, facilitated by OGT. A lack of OGT in macrophages reduces cancer spread and chemoresistance. In humans, high OGT levels in TAMs are linked to cathepsin B expression, predicting chemotherapy results and cancer outcomes. This research highlights the importance of glucose metabolism in tumor-promoting TAMs and provides insights into the mechanisms involved (Table 2).¹⁹⁰ The study shows that breast cancer reduces O-GlcNAc protein modification in skeletal muscles through miR-122 contained in extracellular vesicles. This affects muscle protein balance and function. O-GlcNAcylation interacts with NEK10-mediated phosphorylation of RYR1, causing its increased degradation. Breast cancer-driven miR-122 reduces OGT, raising RYR1 levels. Muscle O-GlcNAcylation is also influenced by hypoxia and lactate, especially after exercise. In cancer, reduced O-GlcNAcylation boosts cytosolic Ca²⁺ leading to muscle damage. This is linked to decreased muscle mass and function in mice with tumors, highlighting O-GlcNAcylation's importance in muscle health.²¹¹ Research reveals that under high glucose conditions, O-GlcNAcylation levels and ferroptosis significantly increase in mesenchymal pancreatic cancer cells. Specifically, ZEB1's O-GlcNAcylation plays a pivotal role. Mechanistically, glucose-induced O-GlcNAcylation of ZEB1 at Ser-555 enhances its stability and nuclear translocation, subsequently upregulating lipid synthesis genes FASN and FADS2, leading to lipid peroxidation-dependent ferroptosis (Table 2). These findings highlight a novel role of glucose metabolism and O-GlcNAcylation in ferroptosis sensitivity, offering potential therapeutic avenues for refractory tumors.¹³⁶ The study reveals that HBV infection notably elevates O-GlcNAcylation of the RNA m6A reader YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). O-GlcNAcylation of YTHDF2 at Ser-263 enhances its protein stability and cancer-promoting activity (Table 2). In terms of mechanism, YTHDF2 stabilizes the transcription of MCM2 and MCM5, facilitating the progression of the cell cycle and contributing to tumorigenesis in HBV-associated HCC.¹⁹¹ Aberrant O-GlcNAc modifications correlate with cancer. The study emphasizes O-GlcNAcylation's role in cancer metabolism and epigenetics, impacting signaling proteins, metabolic enzymes, and transcription factors, particularly in chromatin interactions, underscoring its regulatory significance in cancer progression.

5.3 Inflammation

Inflammation is a reaction of the body to harm or infection, involving a variety of cells and molecules. Prolonged, mild, chronic inflammation characterizes the progression of obesity and type 2 diabetes. This inflammatory condition is associated with many subsequent health issues, including AS, insulin resistance, and an increased risk of autoimmunity. All these problems are linked to overnutrition and elevated body fat.^212-215 Under high-glucose conditions, O-GlcNAcylation can enhance the transcriptional activity of NF-κB, leading to the development of inflammation-related complications in diabetes. Conversely, in situations like acute cardiovascular injury, O-GlcNAc can inhibit NF-κB transcriptional activity, exerting anti-inflammatory functions. This indicates that O-GlcNAcylation is a double-edged sword in inflammatory diseases. In other literature, O-GlcNAcylation has also been shown to alter the function of key transcription factor regulatory elements, such as STAT and others.^{189, 216, 217} O-GlcNAcylation regulates these factors, impacting their stability, function, and movement into the nucleus, and thus influencing their capacity to manage the inflammatory response. The research emphasizes that while increased glucose metabolism in immune cells is a characteristic feature of many inflammatory conditions, the precise role of glucose metabolic pathways in inflammation remains somewhat unclear. The study identified an anti-inflammatory effect associated with O-GlcNAc signaling in connection to the HBP. In LPS-activated macrophages, despite increased glycolysis, HBP activity and protein O-GlcNAcylation were reduced. Depletion of OGT, a key enzyme for protein O-GlcNAcylation, heightened the immune response and worsened inflammation related to sepsis. At the molecular level, OGT-induced O-GlcNAcylation on the RIPK3 kinase restricted specific RIPK3 interactions, thus dampening innate immune and necroptotic signaling. Overall, the study underscores the crucial interplay between glucose metabolism and the activation of immune cells in the context of sepsis-related inflammation (Table 2).²¹⁸ The study uncovers that an excess of nutrients triggers an upsurge in O-GlcNAc pathway within macrophages. During the proinflammatory activation of macrophages, O-GlcNAc pathway is decreased. Disrupting O-GlcNAc pathway by eliminating OGT boosts the proinflammatory polarization of macrophages, induces inflammation and lipolysis in adipose tissue, raises lipid accumulation in peripheral tissues, and worsens tissue-specific and systemic insulin resistance in mice subjected to a high-fat diet. OGT influences macrophage proinflammatory activation by facilitating the O-GlcNAcylation of ribosomal protein S6 kinase beta-1 (S6K1), hindering its phosphorylation, and suppressing mTORC1 signaling (Table 2).¹⁹² The study reveals that O-GlcNAc posttranscriptional modifications upon T-cell receptor activation stabilize Forkhead box protein P3 (FOXP3) protein and activate signal transducer and activator of transcription 5 (STAT5), integrating these crucial pathways. While Treg cells with O-GlcNAc deficiencies develop normally, they exhibit slightly reduced FOXP3 expression, compromised lineage stability, and functional efficacy, leading to lethal autoimmune diseases in mice. Additionally, the absence of protein O-GlcNAcylation weakens the IL-2/STAT5 signaling. Overall, these findings underscore the essential role of protein O-GlcNAcylation in the stability and functional efficacy of Treg cells.¹⁹³ The study delved into the function of enzymes related to O-GlcNAcylation in the gut microbiome. It found that bacterial OGAs and OGTs, especially in the Bacteroidetes and Firmicutes phyla, are abundant in healthy guts but decrease in those with ulcerative colitis. In laboratory tests, bacterial OGAs were found to break down host O-GlcNAcylated proteins, including a key component for NF-κB activation. In animal tests, these gut-derived OGAs protected mice from chemically induced colitis. The research uncovers a new enzymatic activity in the gut microbiome and suggests bacterial OGAs as a potential treatment for colitis.²¹⁹ The aforementioned studies reveal the pivotal role of O-GlcNAcylation in various physiological and pathological processes, including inflammation, immune cell activation, obesity-induced insulin resistance, and gut microbiome functions. These findings emphasize the significance of O-GlcNAcylation in regulating inflammatory responses, immune homeostasis, and gut health, offering novel therapeutic strategies for related diseases.

5.4 Nervous system

The brain, as one of the organs in the body with a high energy demand, requires a significant amount of glucose. However, the brain is unable to store glucose. Consequently, the levels of glucose in peripheral blood can potentially affect the normal functioning of the brain.^{220, 221} Studies have already demonstrated that when there is an abnormal uptake of glucose in the brain, brain diseases may ensue.²²² Therefore, the HBP, acting as a nutritional sensor, has become a key focus in the research on the development of brain diseases.