O-GlycNacylation Remission Retards the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease spectrum associated with insulin resistance (IR), from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). O-GlcNAcylation is a posttranslational modification, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Abnormal O-GlcNAcylation plays a key role in IR, fat deposition, inflammatory injury, fibrosis, and tumorigenesis. However, the specific mechanisms and clinical treatments of O-GlcNAcylation and NAFLD are yet to be elucidated. The modification contributes to understanding the pathogenesis and development of NAFLD, thus clarifying the protective effect of O-GlcNAcylation inhibition on liver injury. In this review, the crucial role of O-GlcNAcylation in NAFLD (from NAFL to HCC) is discussed, and the effect of therapeutics on O-GlcNAcylation and its potential mechanisms on NAFLD have been highlighted. These inferences present novel insights into the pathogenesis and treatments of NAFLD.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome with excessive fat deposition in the hepatocytes [1]. It is closely associated with metabolic syndrome, obesity, insulin resistance (IR), and dyslipidemia. Due to the lack of obvious symptoms and starting with simple steatosis in most NAFLD patients, the disease is missed. However, a subset of NAFLD can develop into non-alcoholic steatohepatitis (NASH), and 20% of NASH patients progress to hepatic fibrosis. Once fibrosis occurs, a poor prognosis is developed, such as liver cirrhosis or hepatocellular carcinoma (HCC), the second-most common cause of cancer-related deaths [2]. Nowadays, with the abrupt rising of obesity and diabetes worldwide, the incidence of NAFLD has escalated rapidly [3], with a global prevalence of 25% [4]. Metabolic abnormalities are closely associated with NAFLD, and it was, hence, renamed "metabolic dysfunction-associated fatty liver disease" (MAFLD) in 2020 [5]. (For the convenience of its description, this article has used NAFLD). Moreover, NAFLD is associated with a metabolic imbalance in glucose, lipids, amino acids, bile acids, and iron [6]. Several recent studies have focused on the role of glucose or other metabolisms in NAFLD. Among these, hyperglycemia is a major influencing factor on NAFLD and stimulates insulin secretion and increases the synthesis of triglycerides in the liver. The excessive triglycerides accumulate gradually in the liver and are exported to generate hypertriglyceridemia [7]. In addition, long-term and chronic hyperglycemia-induced peroxisome proliferator-activated receptor-γ co-activator1α (PGC1α) and FoxO1, key gluconeogenic regulators, is regulated by O-GlcNAcylation [32][33][34]. PGC1α, an essential coactivator of the transcriptional stimulation of gluconeogenic genes [35][36][37], further stimulates the expression of gluconeogenic genes. OGT affects PGC1α-mediated gluconeogenesis gene expression by targeting PGC1α via the host cell factor C1 (HCF-1) [34,35]. O-GlcNAcylation also stabilizes PGC1α by recruiting BAP1 for deubiquitination to promote gluconeogenesis [34]. PGC1α promotes OGT to effectuate O-GlcNAcylation and activate FoxO1 and increases the expression of Pepck and G6pc and the transcription of ROS detoxification enzymes, manganese superoxide dismutase (MnSOD) and catalase (CAT), further promoting hepatic glucose production [32]. OGT also increases the expression of Pepck and G6pc, which induces hepatic gluconeogenesis by the O-GlcNAcylation of the cAMP-regulated transcriptional co-activator 2 (CRTC2), a co-activator of the cyclic AMPresponsive element-binding protein (CREB) [38]. It has also been suggested that OGT is involved in glucocorticoid-induced gluconeogenesis [39]. p53 is usually recognized as a tumor suppressor [40]. A recent study reported that insulin sensitivity and liver glucose homeostasis are regulated by integrating the p53 signaling pathways, which depend on p53 O-GlcNAcylation. Subsequently, O-GlcNAcylated p53 binds to the PCK1 promoter to activate the gluconeogenic effect [41].  , and CRTC2 increases the expression of gluconeogenic genes and induces hepatic gluconeogenesis. O-GlcNAcylated p53 bound to the PCK1 promoter regulated the PCK1 levels and increased glucose synthesis. LXR, AMPK, ChREBP, and SREBP-1 were directly or indirectly regulated by O-GlcNAcylation, and subsequently, the transcriptional activity of target glycolysis and lipogenic genes was increased. HBP, hexosamine biosynthetic pathway; GFAT, glutamine fructose-6-phosphate amidotransferase; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; CRTC2, cAMP-regulated transcriptional co-activator 2; CREB, cyclic AMP-responsive element-binding protein; PGC1α, peroxisome proliferator-activated receptor-γ co-activator1α; ChREBP, carbohydrate-responsive element-binding protein; AMPK, AMP-activated protein kinase; SREBP-1, sterol regulatory element-binding protein 1; LXR, liver X receptors; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase1; MnSOD, manganese superoxide dismutase. Furthermore, whether glucose flux promotes fat production through O-GlcNAcylation needed to be clarified. liver X receptors (LXRs) are lipid metabolism, glucose stability, and inflammation sensors. O-GlcNAcylation of the hepatic LXR was observed in refed mice and streptozotocin-induced diabetic mice [42]. High glucose increases the O-GlcNAcylation of the LXR and the transcriptional activity of the sterol regulatory element-binding protein 1 (SREBP-1) promoter. SREBP-1 is a master transcriptional regulator of hepatic lipogenesis [42], and the O-GlcNAcylation of the LXR upregulates the expression of SREBP-1 in the liver [42]. OGT regulates the phosphorylation and stability of SREBP-1 by increasing AMP-activated protein kinase (AMPK) O-GlcNAcylation in breast cancer [43], followed by the transcriptional activity of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). The carbohydrate-responsive element-binding protein (ChREBP) plays a vital role in glycolysis and lipogenesis. In hepatocytes, the O-GlcNAcylation of the ChREBP stabilizes the protein and increases its transcriptional activity on the target glycolysis [liver pyruvate kinase (L-PK)] and lipidogenic genes [ACC, FAS, and stearoyl-CoA desaturase1 (SCD1)] [44]. Therefore, exploring the mechanistic and kinetic characterization of O-GlcNAcylation on key signaling proteins is promising for an in-depth understanding of normal hepatic metabolism. Finally, some studies have shown that multiple nodes of the insulin signaling pathway were altered by OGT. Under normal physiological conditions, O-GlcNAcylation is responsible for insulin signaling transduction. However, it would be abnormally elevated and induce IR in the state of overnutrition.
Elevated O-GlcNAcylation is not entirely detrimental to the liver. The termination of defective liver regeneration leads to reduced hepatocyte redifferentiation, severe necroinflammation, early fibrotic changes, and the formation of dysplastic nodules leading to the development of hepatocellular carcinoma (HCC) [45]. HNF4a O-GlcNAcylation in hepatocytes plays a key role in the termination of liver regeneration and prevention of hepatic dysplasia [45]. Studies have confirmed that calcium-dependent O-GlcNAc signaling is also critical in driving hepatic autophagy to maintain a nutrient and energy balance in response to starvation [46]. In addition, O-GlcNAc maintains a normal mitochondrial function, and the long-term elevation of o-GlcNAacylation coupled with an increased OGA expression modulates the mitochondrial function and reduces antioxidant responses [47]. For other liver diseases, such as hepatitis B, O-GlcNAcylation promotes the autophagic degradation of hepatitis B virus (HBV) replication virions and proteins through the mTORC1 signaling pathway and autophagosome-lysosome fusion, resulting in reduced HBV replication [48].

O-GlcNAcylation Contributes to IR
The liver is an insulin-sensitive organ and essential for maintaining blood glucose. IR also plays a vital role in the occurrence and development of type 2 diabetes mellitus (T2DM) and NAFLD. Strikingly, NAFLD occurs in 70-80% of T2DM and obesity patients, and most NAFLD patients, develop hepatic IR [49]. The pathogenesis of NAFLD is closely related to IR as it is one of the components of the pathogenesis of NAFLD [50]. Additionally, IR is characterized by decreased glucose uptake and utilization in tissues, including liver tissue, adipose tissue, and muscle tissue [51]. IR increases the circulating free fatty acids through dysregulated lipolysis, resulting in an impaired insulin signal, a reduced clearance rate of glucose metabolism, and the dysregulation of lipid aggregation and decomposition [52]. In addition, the body increases lipid synthesis for energy by breaking down fat. Insulin increases lipase activity, thereby elevating the uptake of triglycerides by the adipose tissue and fat storage in the liver [53]. Lipid deposition in the liver further exacerbates IR. Preview studies demonstrated a critical role of O-GlcNAcylation in attenuating insulin signaling [49,50] (Figure 2). clearance rate of glucose metabolism, and the dysregulation of lipid aggregation and decomposition [52]. In addition, the body increases lipid synthesis for energy by breaking down fat. Insulin increases lipase activity, thereby elevating the uptake of triglycerides by the adipose tissue and fat storage in the liver [53]. Lipid deposition in the liver further exacerbates IR. Preview studies demonstrated a critical role of O-GlcNAcylation in attenuating insulin signaling [49,50] (Figure 2). . Insulin binding to the insulin receptor (IR) leads to the recruitment of IRS-1 and activates PI3K, producing PIP3 and activating PDK1 and AKT. The PI3K/AKT pathway induces the expression of GLUT4 and its transport from intracellular vesicles to cell membranes to promote glucose uptake. In addition, the PI3K/AKT pathway activates GSK3β and GS to promote glycogen synthesis. Insulin signaling was inhibited by O-GlcNAcylation (Right). OGT inactivated key insulin signaling proteins, including IRS-1, PI3K, PDK1, AKT, and PTP1B, and attenuated insulin signaling and insulin resistance. PI3K, phosphatidylinositol-3-kinase; PDK1, phosphoinositide-dependent protein kinase 1; AKT, serine/threonine-protein kinase B; GSK3β, glycogen synthase kinase 3 beta; GS, glycogen synthase; PTP1B, protein tyrosine phosphatase 1B; OGT, O-GlcNAc transferase.
A major mechanism for terminating insulin signaling is the inactivation of insulin receptor substrates. OGT inactivates the insulin signaling proteins, including insulin receptor substrate 1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), phosphoinositide-dependent protein kinase 1 (PDK1), serine/threonine-protein kinase B (AKT), and protein tyrosine phosphatase 1B (PTP1B), promoting the attenuation of insulin signaling [50,51]. Interestingly, OGT uses IRS-1 as its direct substrate [54]. In 3T3-L1 adipocytes, the Tyr608 phosphorylation of IRS-1 is inhibited by elevated O-GlcNAcylation, thereby reducing AKT activity [54]. In addition, OGT also uses PDK1 and PI3K as direct substrates in insulin signal attenuation [54,55]. Decreased AKT activity is vital for terminating insulin signaling, and O-GlcNAcylation plays a key role in the regulation of AKT activity. Normal O-GlcNAcylation is valuable for AKT signal transduction, while the O-GlcNAcylation of Thr305/Thr312 disrupts the interaction between AKT and PDK1, resulting in the downregulation of AKT activity [56]. Then, the decreased AKT activity reduces glycogen synthesis via glycogen synthase kinase 3 beta (GSK3β) phosphorylation. GSKβ is modified by O-GlcNAcylation, after the inhibition of GSK3β by lithium, and the overall O-GlcNAcylation level is significantly increased [57]. However, the function of GSK3β O-GlcNAcylation needs further exploration. In addition, PTP1B controls hepatic insulin signaling by inhibiting PTP1B O-GlcNAcylation, improving insulin sensitivity, and reducing liver lipid deposition [58]. Another study found that the O-GlcNAcylation level of glycogen synthase (GS) is increased, and the activity of GS is decreased after high glucose or glucosamine treatment, thereby leading to IR [59]. Therefore, abnormal glycogenogenesis and gluconeogenesis are closely related to O-GlcNAcylation during the development of hepatic IR.
signal attenuation [54,55]. Decreased AKT activity is vital for terminating insulin signaling, and O-GlcNAcylation plays a key role in the regulation of AKT activity. Normal O-GlcNAcylation is valuable for AKT signal transduction, while the O-GlcNAcylation of Thr305/Thr312 disrupts the interaction between AKT and PDK1, resulting in the downregulation of AKT activity [56]. Then, the decreased AKT activity reduces glycogen synthesis via glycogen synthase kinase 3 beta (GSK3β) phosphorylation. GSKβ is modified by O-GlcNAcylation, after the inhibition of GSK3β by lithium, and the overall O-Glc-NAcylation level is significantly increased [57]. However, the function of GSK3β O-Glc-NAcylation needs further exploration. In addition, PTP1B controls hepatic insulin signaling by inhibiting PTP1B O-GlcNAcylation, improving insulin sensitivity, and reducing liver lipid deposition [58]. Another study found that the O-GlcNAcylation level of glycogen synthase (GS) is increased, and the activity of GS is decreased after high glucose or glucosamine treatment, thereby leading to IR [59]. Therefore, abnormal glycogenogenesis and gluconeogenesis are closely related to O-GlcNAcylation during the development of hepatic IR.

O-GlcNAc and NAFL
NAFLD is a generalized term encompassing a range of liver conditions of varying severities resulting in liver fibrosis [52]; a simple steatosis named NAFL resulted from triglyceride accumulation in the cytoplasm of hepatocytes. On the other hand, the ChREBP is a pivotal transcription factor mediating the effects of glucose on glycolysis and lipogenesis genes. A previous study showed that the ChREBP is a regulatory center of adipogenesis in vivo and plays a decisive role in developing hepatic steatosis and IR; the specific inhibition of the ChREBP significantly improves hepatic steatosis in ob/ob mice [66]. A further study demonstrated that O-GlcNAcylation stabilizes the ChREBP and increases the activity on glycolytic lipogenic genes (L-PK, ACC, FAS, and SCD1) [44] (Table 1). Importantly, the overexpression of OGT significantly increases the ChREBP in C57BL/6J mice liver, resulting in enhanced lipogenic gene expression and excess hepatic triglyceride deposition [44]. Furthermore, HCF-1 O-GlcNAcylation, in response to glucose or a high-carbohydrate diet (HCD), first recruited OGT to the ChREBP, which led to ChREBP O-GlcNAcylation and activation [67]. Whether the mechanism of O-GlcNAcylation regulates the ChREBP in HCD-induced NAFLD mice needs to be investigated further.
The level of SREBP-1, a transcription factor that activates FAS and ACC1, is elevated [68], accompanied by hepatic steatosis [69]. Mice with the liver-specific overexpression of mature human SREBP-1 develop hepatic lipid accumulation and feature a fatty liver by the age of 6 months [70]. A previous study demonstrated that excessive glucose promotes lipid accumulation by upregulating lipid genes, such as SREBP-1, FAS, and ACC1, in cultured hepatocytes and animal model liver tissues [71]. Previous studies have shown that SREBP-1 protein expression is regulated by O-GlcNAcylation [43]. Also, the overexpression of glutamine fructose-6-phosphate amidotransferase (GFAT) promotes lipid accumulation in hepatic cells as well as inflammatory pathway activation by increasing the ER stress by the HBP [72], which indicates a critical role of the HBP in thyroglobulin (TG) accumulation. However, an updated study did not observe the response of SREBP-1 O-GlcNAcylation to GFAT inhibitors [73]. The correlation between SREBP-1 and the HBP and whether SREBP-1 directly effectuates O-GlcNAcylation is yet to be elucidated.

O-GlcNAc and NASH
In the preliminary stage, most patients with NAFLD manifest as hepatic steatosis without any symptoms. As the disease progresses, a proportion of the patients show NASH with inflammatory manifestation, hepatocyte injury, and fibrosis [74]. Nevertheless, the molecular mechanisms underlying the development of NAFLD and NASH are poorly understood. Protein O-GlcNAcylation impedes insulin signaling and promotes adipogenesis [16]. A recent study showed that inositol hexakisphosphate kinases 1 (IP6K1) inhibitors improve metabolic disorders, NAFLD/NASH. and fibrosis by altering these pathways [17]. How IP6K1 stimulates the protein O-GlcNAcylation to improve NAFLD by knocking down OGT remains to be explored.
Previous studies have indicated that O-GlcNAcylation is upregulated in NASH mice; however, the causal correlation between the upregulation of O-GlcNAcylation and the pathology of NASH is unclear. NF-κB, a proinflammatory transcription, is related to many pathogenic liver diseases [75], and NF-κB activated by inositol requiring enzyme 1α (IRE1α) causes liver inflammation and promotes NASH [76,77]. In addition, the activity of NF-κB is regulated by O-GlcNAcylation [60], and the upregulated O-GlcNAcylation activates NF-κB and increases inflammatory damage [78]. Transcriptional activity of L-PK, ACC, FAS, and SCD1 Hepatic TG deposition [44] Cell and Animal SREBP- ChREBP, carbohydrate-responsive element-binding protein; L-PK, liver pyruvate kinase; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase1; TG, thyroglobulin; SREBP-1, sterol regulatory element-binding protein 1; AMPK, AMP-activated protein kinase; IP6K1, inositol hexakisphosphate kinases 1; NASH, non-alcoholic steatohepatitis; NF-κB, Nuclear factor-κB; HSCs, hepatic stellate cells; SRF, serum response factor; α-SMA, α-smooth muscle actin; RIPK3, receptor-interacting protein kinase 3; JNK, c-Jun N-terminal kinases; NAFLD-HCC, NAFLD-associated hepatocellular carcinoma. Up arrow represents up-regulation, Down arrow represents down-regulation. ROS accumulation and related ER stresses are caused by fat toxicity [79,80]. The transcription of GTAT is upregulated under ER stress, increasing protein O-GlcNAcylation [81]. Another study showed that O-GlcNAcylation, OGT, and GFAT levels are increased in mice with a methionine-choline deficient (MCD) diet, and the upregulated OGT and GFAT originate from the upstream target IRE1α induced via ER stress [19]. Currently, transcription factor X-box-binding protein 1 (XBP1) is the only known transcription factor downstream of IRE1α [82], and a key transcription factor is involved in hepatic adipogenesis and inflammation through ER stress [83]. These studies suggested that the upstream activator of the HBP is regulated by the transcription of XBP1 and is a positive regulatory loop for the onset of NASH. In another study, the expression of fructose-1,6-bisphosphatase (FBPase) was upregulated in NASH mice, leading to elevated F6P levels, HBP flux, and upregulated O-GlcNAcylation [18]. The increased level of protein O-GlcNAcylation by elevating the HBP flux in the liver plays a critical role in establishing a correlation between the increase in liver FBPase and NASH [84].

O-GlcNAc and Hepatic Fibrosis
Hepatic fibrosis is the most critical predictor of mortality in NAFLD, and the risk of liver-associated mortality increases exponentially with the increase in the fibrosis stage [85]. NASH patients with liver fibrosis are prone to develop cirrhosis [86]. Currently, only a few studies are related to O-GlcNAcylation and liver fibrosis. Hepatic stellate cells (HSCs) are the major source of the extracellular matrix in the liver [87]. Activated HSCs contribute to fibrogenesis. Interestingly, O-GlcNAcylation is involved in activating HSCs and collagen expression [62]. HSC activation originates from FoxO1 inactivation, leading to NAFLD fibrosis [88]. Paradoxically, the expression and activity of FoxO1 are increased in NASH patients [89]. Since FoxO1 plays a critical role in fibrosis and could be O-GlcNAcylated, it is essential to elucidate the role of FoxO1 O-GlcNAacylation on liver fibrosis through gene knockdown.
It was found that OGT-deficient hepatocytes are prone to hepatocyte ballooning, inflammation, and liver fibrosis [65]. OGT, a negative regulator of HSC activation, exerts a protective effect against hepatic fibrosis by boosting SRF O-GlcNAcylation. Therefore, the OGT expression and O-GlcNAcylation were decreased in HSCs isolated from MCD-fed mice livers [63]. In contrast, a recent study reported that OGT-deficient necroptotic hepatocytes secrete trefoil factor 2 (TFF2), which induces HSC activation, proliferation, and migration via platelet-derived growth factor receptorβ (PDGFRβ) signaling [90]. Thus, it is essential to clarify whether O-GlcNAc could be used as a biomarker for liver disease.

O-GlcNAc and NAFLD-HCC
NAFLD is becoming the leading cause of HCCs. NAFLD/NASH-HCC incidence and mortality rates are rising worldwide [91]. Furthermore, a retrospective cohort study from 2002 to 2012 indicated that NASH-related HCC increased significantly, and the number of patients undergoing liver transplantation for HCCs secondary to NASH increased by nearly four-fold, while the number of patients with HCCs secondary to chronic hepatitis C virus (HCV) increases only by two-fold [92]. NAFLD-HCC patients exhibit upregulated levels of OGT, which plays an oncogenic role by activating the oncogenic c-jun N-terminal kinases (JNK)/c-Jun/AP-1 and nuclear factor-kappa B (NF-κB) cascades [93]. Another study demonstrated that OGT is a key inhibitor of hepatocyte necroptosis in alcoholic fatty liver disease, and the lack of O-GlcNAcylation induces necroptosis in hepatocytes [65]. However, the specific pathogenesis mechanisms of NAFLD-HCC have not yet been totally revealed.
The mutual inhibition of caspase 8 and RIPK3 is essential for the development of NASH and hepatocarcinogenesis [64,94], and RIPK3 prevents cell proliferation from limiting the development of HCCs by inhibiting caspase 8 cleavage and JNK activation [64]. A study discovered that O-GlcNAcylation inhibits RIPK3 protein expression and stability [65]. Further investigation would analyze the molecular mechanism underlying OGT-regulated-RIPK3 gene transcription by O-GlcNAcylation. Nonetheless, only a few studies have elaborated on the role of OGA in the liver. Targeting O-GlcNAcylation is a potential therapy for NAFLD-HCC.
Furthermore, OGT overexpression in the liver increased intracellular palmitic acid levels and promoted HCC by activating ER stress-associated oncogenic signaling cascades, including the JNK/c-Jun/AP1 and NF-κB signaling pathways [93]. Typically, 2/3 of NAFLD-HCC tumors show OGT overexpression, while 1/3 of no change in OGT expression is seen, suggesting that OGT expression is associated with gene polymorphism related to the occurrence and progression of NAFLD and NASH, such as PNOLA3 p.I148M, TM6SF2 p.E167K, and MBOAT7 rs641738 [95,96]. Further studies should investigate whether OGT has a prognostic value for NAFLD-HCC.

Drugs Ameliorates NAFLD through Inhibition of O-GlcNAcylation
Metformin (MET) inhibits the proliferation of cervical cancer cells by reducing the O-GlcNAcylation of AMPK and increasing the level of phospho-AMPK [97] (Table 2). Another study indicated that MET inhibits the O-GlcNAc modification of NF-κB p65 and the ChREBP in the diabetic retina [98]. In addition, MET has been shown to have a protective effect on NAFLD, but the specific mechanism is yet unclear [99]. Furthermore, O-GlcNAcylation is activated, and AMPK/ACC pathway phosphorylation is inhibited in high-fat diet (HFD)-fed mice [100]. It has also been suggested that MET reduces hepatic TG accumulation and improves obesity-related NAFLD by inhibiting hepatic apolipoprotein A5 (ApoA5) synthesis through the AMPK/LXRα signaling pathway [101]. Therefore, it was speculated that MET promotes AMPK phosphorylation in the NAFLD liver by regulating AMPK O-GlcNAcylation and inhibiting the O-GlcNAc modification of the ChREBP, further increasing fat mobilization and reducing fat deposition in the liver. Also, inflammatory damage is alleviated by inhibiting the O-GlcNAc modification of NF-κB p65 in NAFLD patients.
The glucagon-like peptide-1 (GLP-1) receptor agonist, liraglutide, improves NASH by lowering liver enzyme levels and reducing liver fat [103]. Also, liraglutide and semaglutide improved NASH in clinical trials [114,115]. Yu et al. [116] proposed that GLP-1 inhibits the activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, and reduced the production of ROS by enhancing mitophagy in hepatocytes, eventually improving NAFLD and delaying the progression of NASH. In addition, the activity of GLP-1 was enhanced by the inhibition of proteolysis due to O-GlcNAcylation [102]. However, the mechanisms underlying the elevated protein O-GlcNAcylation induced by GLP-1 that alleviated NAFLD/NASH are yet to be elaborated.
The positive cardiovascular and metabolic effects of angiotensin (Ang)-converting enzyme inhibitors (ACEIs) are mainly dependent on the reduction of AngII formation and the increase in the negatively regulated Ang 1-7 axis of the renin-angiotensin system (RAS) [127,128]. Some studies have shown that ACE/AngII/AT1 contributes to the occurrence and progression of NAFLD [129]. The activation of the ACE2/Ang-(1-7)/Mas axis ameliorates hepatic IR through the Akt/PI3K/IRS-1/JNK insulin signaling pathway [130]. Moreover, Ang1-7 contributes to the correction of diabetic retinopathy by reducing the O-GlcNAcylation of the retinal protein in HFD-fed mice through the Mas/EPAC/Rap1/OGT signaling axis [106]. Also, ACEI therapy has been shown to reduce the incidence of liver cancer and cirrhosis in NAFLD patients [107].
Acetaminophen (APAP) overdose is a common cause of acute liver failure (ALF) in North American and European countries [131,132]. The increase in the hepatic O-GlcNacylated protein leads to the dysregulation of the hepatic glutathione (GSH) supplement response and increases the APAP-induced hepatic injury, while reduced O-GlcNacylation causes rapid GSH replenishment and the subsequent inhibition of APAP-induced liver injury [133]. Increased hepatic O-GlcNacylation as a response to excessive APAP increases and delays JNK activation, which is correlated to pronounced liver damage [133]. Moreover, Chen et al. [108] displayed a positive correlation between O-GlcNacylated c-Jun and GSH synthesis in clinical liver cancer samples. The overexpression of O-GlcNAcylated c-Jun inhibits ferroptosis by inducing GSH synthesis and blocking c-Jun O-GlcNacylation, which is beneficial for the treatment of iron apoptosis-related HCC [108]. Also, oral GSH exhibits a therapeutic effect on NAFLD patients; however, the mechanisms are remained unknown [109].
Alpha-lipoic acid (ALA) protects the kidney from oxidative damage in diabetic rats by reducing the O-GlcNAcylation of ERK and p38 [110]. In another study, ALA slowed the development of diabetic complications and ensured the function and health of red blood cells by reducing the O-GlcNAcylation modification levels of antioxidant enzymes: CuZn-superoxide dismutase (SOD), CAT, heat shock protein (HSP) 70, and HSP 90 [111]. Furthermore, it confirmed that the O-GlcNAcylation of the thioredoxin interacting protein (TXNIP) activates the NLRP3 inflammasome by interacting with the NLRP3 [134]. In a clinical trial, ALA was demonstrated to improve IL-6 and serum adiponectin levels in NAFLD patients [135]. Recently, two studies showed that ALA attenuates hepatic triglyceride accumulation and NAFLD by inhibiting the NLRP3 inflammasome [112,113]. Whether ALA plays a crucial role in NAFLD by changing the total level of O-GlcNAcylation or directly reducing the O-GlcNAcylation of NLRP3 and the role of O-GlcNAcylation in NAFLD, although drugs such as ALA, GSH, and ACEI exert a protective effect through anti-inflammatory and antioxidant effects, are yet to be clarified.
Hitherto, the pharmacological treatment of NAFLD by directly inhibiting O-GlcNAc has rarely been studied. Lee et al., showed that curcumin regulates the expression of SIRT1 and SOD1 through O-GlcNAcylation signaling [19]. It also reduces hepatitis by blocking the HBP flux signaling pathway; the anti-inflammatory effect of curcumin was achieved by inhibiting O-GlcNAcylation and blocking the NF-κB signaling pathway [19]. Silibinin blocks the NF-κB signaling pathway by inhibiting O-GlcNAcylation and alleviates inflammation in NASH mice [18]. Therefore, additional drug studies are required to further explore the treatment of NAFLD/NASH by targeting O-GlcNAcylation.

Conclusions
In this study, elevated O-GlcNAcylation promoted the development and exacerbation of IR and was eventually involved in the progression of NAFL-NASH-cirrhosis-hepatoma tetralogy. In addition, the potential drugs targeted at O-GlcNAcylation in the NAFLD intervention were reviewed. Thus, elucidating the molecular mechanisms of O-GlcNAcylation provided additional strategies and ideas for preventing and treating NAFLD.
Author Contributions: Y.S. and P.Y.: study concept, design, methodology, and funding acquisition. Y.Z.: drafting of the manuscript, critical revision of the manuscript for important intellectual content, and validation. Z.L.: visualization, editing, and supervision. M.X., D.Z. and J.L.: acquisition of data, analysis, and interpretation of data. All authors have read and agreed to the published version of the manuscript. Acknowledgments: The graphical abstracts were created with BioRender software (BioRender.com).

Conflicts of Interest:
The authors declare no conflict of interest.