Metabolic Stress and Cardiovascular Disease in Diabetes Mellitus: The role of protein O-GlcNAc Modification

Abstract

Mammalian cells metabolize glucose primarily for energy production, biomass synthesis and post-translational glycosylation; and maintaining glucose metabolic homeostasis is essential for normal physiology of cells. Impaired glucose homeostasis leads to hyperglycemia, a hallmark of diabetes. Chronically increased glucose in diabetes promotes pathological changes accompanied by impaired cellular function and tissue damage, which facilitates the development of cardiovascular complications, the major cause of morbidity and mortality of diabetic patients. Emerging roles of glucose metabolism via the hexosamine biosynthesis pathway (HBP) and increased protein modification via O-linked β-N-acetylglucosamine (O-GlcNAcylation) have been demonstrated in diabetes mellitus, and implicated in the development of diabetic cardiovascular complications. This review will discuss the biological outcomes of the glucose metabolism via the hexosamine biogenesis pathway and protein O-GlcNAcylation in regulating cellular homeostasis, and highlight the regulations and contributions of elevated O-GlcNAcylation to the pathogenesis of diabetic cardiovascular disease.

INTRODUCTION

Cardiovascular complications are the most prevalent causes of morbidity and mortality in patients with diabetes^{1, 2}. According to the American Heart Association, diabetes mellitus is rising to be one of the major risk factors for cardiovascular diseases and cardiovascular death (https://www.heart.org/en/health-topics/diabetes/why-diabetes-matters/cardiovascular-disease--diabetes) The pathogenesis of the cardiovascular diseases in diabetes is complex, regulated by numerous factors via integrated molecular signaling pathways and cellular processes³. Insulin deficiency and hyperglycemia are the characteristic features of diabetes that trigger diabetic complications, as well as other metabolic disorders, such as hyperlipidemia and obesity that contribute to pathogenesis of cardiovascular disease. Glucose is the key energy-producing molecule that is metabolized via multiple pathways, which provides carbon source for cellular biosynthesis. Maintaining glucose metabolic homeostasis is essential for normal physiological functions of cells. In diabetes, chronic increase in glucose uptake and metabolism promotes pathological transformation that results in impaired cellular function and tissue damage. The emerging roles of glucose metabolism via the hexosamine biosynthesis pathways (HBP) and increased protein modification via O-linked β-N-acetylglucosamine (O-GlcNAcylation) have been linked to the pathogenesis of diabetic cardiomyopathy and vasculopathy, including diabetic microvascular complications, such as neuropathy, nephropathy, retinopathy, as well as diabetic macrovascular complications, including hypertension, atherosclerosis, vascular calcification and stroke. The precise mechanisms responsible for the abnormal glucose metabolism-induced diabetic vasculopathy and the roles of HBP and protein O-GlcNAcylation have not been fully elucidated. This review will summarize the biological function of the HBP and protein O-GlcNAcylation in maintaining cellular homeostasis, and highlight the findings that emphasize the regulations and contributions of increased O-GlcNAcylation to the pathogenesis of diabetic cardiovascular complications.

Hyperglycemia and Cardiovascular Disease in Diabetes Mellitus

Diabetes mellitus represents a complex metabolic disorder featuring high levels of blood sugar (hyperglycemia), insulin insufficiency and excessive oxidative stress. Cardiovascular disease remains a leading cause of morbidity and mortality in diabetes mellitus⁴. The major diabetic complications in the cardiovascular system are the diabetic cardiomyopathy and diabetic vasculopathy, including microvascular and macrovascular complications. It has been well established that hyperglycemia is a major cause of cardiovascular complications and diseases in diabetes. Insulin insufficiency results in acute increase in the blood glucose and ketone concentrations that cause diabetic ketoacidosis, a metabolic disorder associated with mortality in diabetic patients. On the other hand, chronically increased glucose uptake and metabolisms lead to impaired cellular function and damages of multiple tissues, including skeletal muscle, liver, kidney, adipose tissues and blood vessels. For instance, hyperglycemia has been shown to induce the expression of cytokines, growth factors and extracellular matrix proteins in renal cells, which in turn activates the renin-angiotensin system and induces hypertension in diabetic patients⁵. Increased glucose oxidation generates acetyl-CoA that promotes fatty acid synthesis, which leads to increased production of very-low-density lipoproteins and other lipoproteins in diabetic patients, thus contributing to increased cardiovascular disease in diabetic patients. In addition, hyperglycemia activates the polyol pathway, in which glucose is converted, by the enzyme aldose reductase, to sorbitol that is further oxidized to fructose⁶. Increased activation of polyol pathway induces oxidative stress via consumption of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and generation of the advanced glycation end products (AGEs), a heterogeneous group of nonenzymatic glycation products of proteins, lipids, and nucleic acids. Hyperglycemia-induced AGEs and enhanced expression and activation of the receptor for AGEs (RAGE) have been linked to increased synthesis of diacylglycerol and activation of the protein kinase C (PKC) signaling pathway that consequently increases activation of inflammatory and oxidative signals^{4, 6–9}. Moreover, increased glucose metabolism via the hexosamine biosynthesis pathway and oxidative stress promote posttranslational protein modification via O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) in diabetes^{10, 11}, which contributes to diabetes cardiovascular disease^12–14. Altogether, hyperglycemia-induced glycolytic flux, glucose oxidation, activation of polyol pathway, HBP and AGEs/RAGE-activated PKC signaling and metabolic signals may converge to increase oxidative stress in the cardiovascular systems, which promotes diabetic cardiovascular complications, including diabetes-specific microvascular disease that causes blindness, renal failure and nerve damage, as well as diabetes-accelerated atherosclerosis and vascular calcification, increased risk of myocardial infarction, stroke and limb amputation^{6, 7, 15–19}.

Controlling glycemic levels remains the main treatment strategy for diabetes mellitus and diabetic cardiovascular disease. The long-term Diabetes Control and Complications Trial (DCCT) and the Epidemiology of Diabetes Interventions and Complications (EDIC) follow-up study have demonstrated that intensive control of glucose delays the onset and slows the progression of diabetic retinopathy, nephropathy and neuropathy in type 1 diabetes mellitus^{2, 5}. Other studies did not seem to exclusively support the effects of aggressive blood glucose-lowering regimens on cardiovascular outcome in diabetes mellitus, particularly in patients with type 2 diabetes^20–24. The United Kingdom Prospective Diabetes Study (UKPDS) has shown that glycemic control reduces diabetes-related complications in newly diagnosed patients with type 2 diabetes²⁰. However, in the large randomized control trials, ADVANCE and ACCORD trials, aggressive glucose control on type 2 diabetes patients with a history of major macrovascular or microvascular disease only resulted in a small reduction in microvascular events, but did not reduce the risk of myocardial infarct and cardiovascular mortality and increased incidence of hypoglycemia^{2, 21, 22}. Similarly, the Veterans Affairs Diabetes Trial (VADT) reported that intensive glucose lowering, compared with standard therapy, in patients with poorly controlled type 2 diabetes had no significant effect on the rates of major cardiovascular events, death, or microvascular complications among 1791 military veterans (median follow-up of 5.6 years)²³. A recent report further demonstrated that intensive glucose control in patients with type 2 diabetes did not reduce the risks of major cardiovascular events or death, compared with those in the standard-therapy group, over a period of 15 years of follow-up of the VADT²⁴. Accordingly, chronic hyperglycemia and metabolic disorder in diabetic conditions, particularly in type 2 diabetes, may cause pathologic changes in micro- and macro-vascular that cannot be reversed by further glucose lowering therapies.

It is encouraging that the 21-years follow-up studies of the randomized controlled multifactorial Steno-2 (a small study with a big heart) trial of type 2 diabetes mellitus have demonstrated increased lifespan, reduced cardiovascular events, mortality and microvascular complications^{25, 26}. The beneficial effects in the Steno-2 trial were achieved with multifactorial and target-driven treatment including glucose control combining with individually targeting diet, exercise, blood pressure and lipid lowering^{25, 26}, suggesting that it is critical to understand the regulation of the glucose metabolism and its interactions with other metabolic abnormalities in the development of cardiovascular diseases in diabetes.

Glucose Metabolism and Protein O-GlcNAcylation

Cellular metabolisms encompass chemical reactions that generate energy and produce essential metabolic molecules, such as lipids, amino acids, nucleic acids and carbohydrates, required for cellular function, growth and reproduction. Under normal conditions, intracellular metabolic pathways cross talk and coordinate to maintain cellular homeostasis in response to environmental cues²⁷. Glucose is the major intracellular energy-producing molecule and provides a carbon source for cellular biosynthesis. Therefore, maintaining glucose metabolic homeostasis is essential for normal physiological functions and survival of cells. Upon entering cells via the glucose transporter, the key first step of intracellular glucose metabolism is the conversion of glucose into glucose 6-phosphate (glucose-6-P, Figure 1), which is catalyzed by the hexokinase (HK) that uses adenosine triphosphate (ATP) as the phosphoryl donor²⁸. Glucose-6-P is the substrate for the synthesis of glycogen, a storage form of glucose polymers, which can be broken down to generate glucose when needed. Glucose-6-P can also be utilized via the pentose phosphate pathway (PPP) that generates ribose-5-phosphate for DNA and RNA synthesis and produces NADPH, which is involved in regulating the cellular redox status and the synthesis of lipids and fatty acids²⁹. As the oxidative reactions of the PPP are inhibited by NADPH, increased use of NADPH by aldose reductase-driven polyol pathway may promote the glucose flux via the PPP.

Figure 1. Glucose metabolism via hexosamine biosynthesis pathway links amino acid, fatty acid and nucleotide metabolisms to regulate cell physiology and pathology via protein O-GlcNAcylation.

In addition to glycolysis, glycogen synthesis and pentose phosphate pathway, glucose metabolism generates UDP-GlcNAc via the hexosamine biosynthesis pathway (HBP). The HBP utilizes a series of enzymes and substrates, which integrates glucose metabolism to amino acid metabolism, fatty acid metabolism and nucleotide metabolism. Dynamically regulated by the two enzymes, OGT and OGA, that catalyze the addition and removal of GlcNAc moiety on proteins respectively, O-GlcNAcylation affects a variety of cellular functions under physiological and pathological conditions. (HK: Hexokinase. GPI: Glucose-6-phosphate isomerase. GFAT: Glucosamine-fructose-6-phosphate aminotransferase. GNA: Glucosamine 6-phosphate N-acetyltransferase. PGM: Phospho-acetylglucosamine mutase, UAP: UDP-N-acetylglucosamine pyrophosphorylase. OGT: O-GlcNAc transferase. OGA: O-GlcNAcase)

Glycolysis is the major glucose metabolism pathway, which converts fructose-6-P, derived from glucose-6-P by the glucose-6-phosphate isomerase (GPI), into pyruvate in the cytoplasm and produces ATP. Oxidation of pyruvate produces acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle to generate more ATPs in the mitochondria. Pyruvate-derived acetyl-CoA can also be used to produce fatty acids, and acetyl-glucosamine in the hexosamine biosynthesis pathway. Glucose metabolism via the HBP is catalyzed by a series of enzymes, which metabolize glucose to generate uridine-diphosphate-β-D-N-acetylglucosamine (UDP-GlcNAc), an active sugar donor for glycosylation, including the modification by O-linked β-N-acetylglucosamine (O-GlcNAcylation)³⁰. The HBP pathways utilize a variety of metabolites that integrate glucose metabolism with metabolisms of amino acids, fatty acids and nucleotides (Figure 1). Glucose metabolism through the HBP is required to sustain sufficient growth factor signaling and glutamine uptake to support cell growth and survival³¹.

O-GlcNAcylation is a posttranslational protein modification that is different from the classic N-glycosylation and O-glycosylation, as it is a dynamic modification on serine or threonine residues of proteins by a single sugar moiety GlcNAc. O-GlcNAcylation is functionally analogous to phosphorylation, a common posttranslational modification that regulates many cellular signals, as O-GlcNAcylation and phosphorylation may occur on the same or adjacent serine or threonine residues on many proteins³². While phosphorylation is regulated by a large number of kinases and phosphatases, O-GlcNAcylation is tightly controlled by only two specific enzymes. The O-GlcNAc transferase (OGT) catalyzes the addition of the GlcNAc moiety to the hydroxyl group of the serine or threonine residues on the target proteins; and the O-GlcNAcase (OGA) removes the O-GlcNAc from the modified targets.

Three forms of OGT proteins generated from alternative RNA splicing activity are identified: the nucleocytoplasmic OGT (ncOGT), mitochondrial OGT (mOGT), and a shorter form of OGT (sOGT)³³. While the mitochondrial-targeting domain determines the specific subcellular localization of the mOGT, the different numbers of tetratricopeptide-repeat (TPR) domains on OGT mediate interactions of OGT with other proteins and their complex formation for specificity and temporal activation of the enzyme^34–36. Two OGA alternative splicing variants, a larger cytoplasmic isoform and a smaller nuclear isoform³⁷, have been implicated in spatial distribution and function of OGA variants. The interplays of the OGT and OGA, in different cells and subcellular compartments, dynamically modulate O-GlcNAcylation homeostasis in response to changes in nutrients and environmental cues, thus regulating multiple cellular signals and functions in different tissues^{13, 14, 38}. Protein O-GlcNAcylation plays crucial roles in maintaining normal cellular functions; whereas disrupted O-GlcNAcylation homeostasis contribute to the pathogenesis of diseases, especially chronic diseases such as diabetes and cardiovascular disease^39–41.

O-GlcNAcylation and Diabetic Cardiovascular Disease

Emerging evidence has linked the glucose metabolism via HBP and protein modification by O-GlcNAcylation to the pathogenesis of diabetic cardiovascular disease. Protein O-GlcNAcylation, serving as an environmental nutrient and stress sensor, modulates stability, cellular localization and function of many nuclear and cytoplasmic proteins in response to glucose levels³⁸ and other environmental signals, such as ischemia, hypoxia, oxidative stress, osmotic pressure, and ultraviolet light⁴². Thus, hyperglycemia and other abnormal metabolisms in diabetes may synergistically induce elevation of O-GlcNAcylation via increased HBP flux, thus promoting pathogenesis of cardiovascular disease.

• O-GlcNAcylation and diabetic cardiomyopathy

Hyperglycemia and impaired insulin signaling pathways in the heart promote diabetic cardiomyopathy, the diastolic dysfunction of the heart due to structural and functional abnormality in myocardium in the absence of vascular dysfunction, such as hypertension, coronary arterial disease and hyperlipidemia^{43, 44}. Clinical and experimental studies have established the function of HBP and protein O-GlcNAcylation in regulating diabetes cardiomyopathy^12–14. Increased O-GlcNAcylation via inhibition of OGA reduces hypoxic-induced cell death of cardiomyocytes in vitro; whereas reduced O-GlcNAcylation by overexpression of OGA significantly increased hypoxic-induced cell death¹². In ischemia-reperfusion model of heart failure, acute increase in O-GlcNAcylation protects cardiomyocytes from oxidative stress-induced calcium overload and structural damage^45–47. In contrast to the protective effects of the acute increase in O-GlcNAcylation on cardiac injury, chronic elevation of O-GlcNAcylation in diabetes is associated with impaired calcium handling and contractile properties, as well as mitochondria dysfunction and cardiac hypertrophy. For example, increased OGT and decreased mitochondrial-specific OGA observed in isolated heart mitochondria from diabetic rats, are accompanied with increased protein O-GlcNAcylation⁴⁸. Inhibition of OGT or OGA activity in rat cardiomyocytes affects energy production, mitochondrial membrane potential, and mitochondrial oxygen consumption, suggesting that dysregulation of O-GlcNAcylation promotes mitochondrial dysfunction thus contributing to cardiomyopathy in diabetes⁴⁸. Additionally, increase in nuclear O-GlcNAcylation impairs cardiomyocyte calcium cycling and cardiomyocyte function, which can be rescued by overexpression of OGA that reduces O-GlcNAcylation⁴⁹. These data support that the O-GlcNAcylation-regulated calcium flux is critical for cardiomyocyte function and the development of diabetic cardiomyopathy. Consistently, studies with diabetic mouse models have shown that inhibition of O-GlcNAcylation with adenovirus-mediated OGA overexpression improves calcium handling and contractile function of the diabetic heart⁵⁰. As HBP integrates the glucose metabolism into metabolism of fatty acids, amino acids and nucleotides, chronic elevation of O-GlcNAcylation not only disrupts the glucose homeostasis but also induces other metabolic disorders, which further promotes oxidative stress and generation of advanced glycation end products, leading to diabetic cardiomyopathy.

Collectively, these studies support the concept that acute increase in protein O-GlcNAcylation may serve as a stress sensor that triggers a protective mechanism for stress-induced cardiac injury, whereas chronically elevated O-GlcNAcylation in diabetes impairs normal function of cardiomyocytes, which leads to diabetic cardiomyopthy⁵¹.

• O-GlcNAcylation and diabetic vasculopathy

Hyperglycemia is the major cause of the pathogenesis and severity of diabetic vascularopathy, the complications in the blood vessels that affect multiple organ systems, including microvascular diseases and macrovascular diseases⁷. Microvascular complications are the damaging of small blood vessels in the eye (retinopathy), the kidney (nephropathy) and the peripheral neural system (neuropathy)^{3, 52}. Macrovascular complications include coronary artery disease, peripheral arterial disease, and stroke. Diabetic vasculopathy leads to blindness, limb amputation, kidney failure, atherosclerosis, stroke and heart failure, which increases the incidence of morbidity and mortality of diabetic patients^{4, 53, 54}.

Emerging studies have linked increased O-GlcNAcylation to diabetic vasculopathy, including microvascular and macrovascular diseases. In chronic hyperglycemia conditions, elevation of O-GlcNAcylation promotes diabetic retinopathy, via blocking the neuroprotective effect of insulin and inducing apoptosis in retinal neurons and retinal pericytes^{55, 56}. High glucose-induced O-GlcNAcylation stimulates mesangial cell lipogenesis and fibrosis, which contributes to diabetic nephropathy⁵⁷. The detrimental effects of elevation of O-GlcNAcylation have also been documented in the macrovasculature, although acute increase in O-GlcNAcylation appears to be protective for injury-induced vascular inflammatory and neointimal formation⁵⁸. In the hypertension rat models, increased vascular O-GlcNAcylation was associated with impaired vasodilator activity and hypertension⁵⁹. In addition, increased HBP flux, OGT and O-GlcNAcylation are identified in patients of idiopathic pulmonary arterial hypertension⁶⁰. Inhibition of O-GlcNAcylation by OGT knockdown in the culture pulmonary smooth muscle cells (PASMC) blocks cell proliferation, supporting the contribution of the increased OGT and O-GlcNAcylation-induced PASMC proliferation in the development of PAH⁶⁰. Nonetheless, a causative role of O-GlcNAcylation in regulating the major diabetic macrovascular diseases, such as atherosclerosis, has yet to be determined.

• O-GlcNAcylation and diabetic vascular calcification

Increased incidence of atherosclerosis is observed in human subjects with diabetes, which is associated with higher levels of arterial calcification^{61, 62}. In addition to the calcification identified in the atherosclerotic intimal lesions, medial calcification is also prevalent in the peripheral arteries in diabetic patients without coronary arterial disease^63–65. Importantly, vascular calcification promotes coronary arterial disease and peripheral arterial disease, which greatly increases the risk of cardiovascular disease and cardiovascular death in diabetes^{63, 66}.

Hyperglycemia induces oxidative stress, AGEs/RAGE and activation of inflammation signaling in the vascularture^{4, 8, 9}, which all have been shown to promote vascular calcification in vitro and in vivo^{9, 67, 68}. We and others have reported that increased oxidative stress is a major cause of vascular calcification^67–71. The mechanisms underlying redox signaling-regulated normal vascular function and oxidative stress-induced vascular cell dysfunction that promotes vascular calcification have been discussed in our recent review⁷². As both high glucose and oxidative stress increase O-GlcNAcylaton^{10, 11}, it is likely that increased O-GlcNAcylation may mediate the effects of hyperglycemia and oxidative stress-induced vascular calcification. Consistently, elevation of O-GlcNAcylation and increased vascular calcification are evident in human and mouse diabetic vascular lesions^{40, 63, 73–75}, suggesting a close correlation of increased O-GlcNAcylation with vascular calcification in diabetes.

Our recent studies have uncovered a novel function of O-GlcNAcylation in promoting diabetic vascular calcification⁷⁵. With the low-dose streptozocin (STZ)-induced mouse model of diabetes, we have demonstrated a causative link of increased O-GlcNAcylation with vascular calcification in the diabetic vasculature⁷⁵. Diabetic vascular calcification promoted by elevated O-GlcNAcylation is associated with increased vascular stiffness in mice⁷⁵. By inhibition of OGA, we have determined a direct effect of increased O-GlcNAcylation on inducing calcification of vascular smooth muscle cells (VSMC) in vitro. Furthermore, knockdown of OGT inhibits high glucose and oxidative stress-induced O-GlcNAcylation and concurrently attenuates VSMC calcification. VSMC are the key vascular cells that undergo calcification in the atherosclerosis-related intimal calcification or atherosclerosis-independent medial calcification, both commonly observed in the diabetic arteries^63–65. Accordingly, targeting OGT and O-GlcNAcylation in VSMC can be an effective approach to inhibit vascular calcification in diabetes. Further studies with smooth muscle cell-specific OGT ablation animal models are warranted to determine a definitive role of OGT-mediated protein O-GlcNAcylation in the development of vascular calcification and other cardiovascular complications in diabetes in vivo.

Regulation of O-GlcNAcylation in Diabetes

Multifaceted regulatory mechanisms increase O-GlcNAcylation in diabetes. Upregulation of OGT and downregulation of OGA are observed in response to chronic hyperglycemia in diabetes, suggesting that the dysregulation of OGT and OGA expression contributes to the increased O-GlcNAcylation in diabetes^{34, 76}. Insulin signaling has been implicated in upregulating OGT via activation of the PI3K signaling pathway⁷⁷. It has been reported that the regulation of OGT and OGA occurs at the transcriptional level. Jones and colleagues have demonstrated that E2F1 negatively regulates Ogt and Mgea5 (OGA) gene expression in HEK293 and mouse embryonic fibroblasts⁷⁸. In addition, E2F1 has recently been shown to promote hepatic gluconeogenesis and contributes to hyperglycemia during diabetes⁷⁹. It is likely that E2F1 may regulate OGA/OGT protein expression via directly modulating the OGA/OGT transcription, as well as indirectly via hyperglycemia-activated signals in diabetes. As O-GlcNAc levels change rapidly in cells in response to extracellular environmental fluctuations, the dynamic and coordinated regulation O- GlcNAcylation by OGT and OGA may integrate multiple pathways via different mechanisms in addition to the transcriptional regulation³⁴. Studies that support this later concept include the observations that insulin signaling also promotes OGT translocation from the nucleus to the cytoplasm, and its localization and activation in the lipid rafts in the plasma membrane^{77, 80, 81}. In addition, phosphorylation of OGT by calcium and calmodulin-dependent protein kinase IV (CaMK IV) is critical for activation of OGT⁸². Furthermore, a regulatory feedback loop of OGT and OGA has been suggested in maintaining O-GlcNAcylation homeostasis, thus the overall O-GlcNAcylation levels can be determined by mutual regulation and direct interactions between OGA and OGT in response to glucose and other stimuli³⁴. Taken together, these studies suggest that multiple mechanisms may regulate the transcription, expression, posttranslational modification, spatial distribution of OGT and OGA as well as their close interaction in response to metabolic stress in diabetes, thus increasing the intracellular O-GlcNAcylation levels.

The UDP-GlcNAc concentrations modulate the OGT activities³⁶, suggesting that increased O-GlcNAcylation in diabetes may be attributed, at least in part, to the increased HBP influx. Glucosamine has been shown to increase glucose production and expression of gluconeogenic enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase. Glucosamine increases the O-GlcNAc transferase (OGT)-dependent protein O-GlcNAc level. Moreover, inhibition of OGT (by ST045849) decreases glucose production⁸³. Of note, reduced UDP-GlcNAc levels due to glucose deprivation have also been associated with increased O-GlcNAcylation; and acute response to glucose starvation-induced O-GlcNAcylation may be linked to increased binding of OGT to its modified proteins^{84, 85}. Given that the enzymatic activity of OGT can be regulated by its interacting proteins, OGT binding partners can modulate O-GlcNAcylation via regulating OGT activity. For example, OGT binds to the unconventiaonl prefolding RPB5 interactor (URI) and protein phosphatase 1γ, which regulates OGT activity⁸⁶. Phosphorylation of URI inhibits OGT activity; while protein phosphatase1γ dephosphorylates URI and thus maintaining OGT activity (Figure 2). The spatiotemporal distribution of the adaptor proteins in different cellular compartments and the amount of the target proteins may determine biological outcomes of O-GlcNAc modification in a context-dependent manner. Consequently, cells with abnormal O-GlcNAc modification may discriminatively promote the signaling pathways that cause impaired cardiovascular functions and the development of cardiovascular diseases in diabetes.

Figure 2. OGT interaction with various targeting proteins.

1) regulates its enzymatic activity (top center); and 2) forms complexes that lead to O-GlcNAcylation of a variety of proteins that modulate molecular and cellular functions. The OGT-interacted and O-GlcNAc-regulated molecules involved in epigenetic gene regulation, transcription, circadian clock and autophagy, as well as intracellular signaling pathways, including the insulin signaling pathways, are shown and described in the text.

O-GlcNAcylation-regulated Molecular and Cellular Signals

The distinct effects of acute and chronic elevation of O-GlcNAcylation on cardiovascular injury and the development of cardiovascular disease support the spatiotemporal control of O-GlcNAcylation on important molecules that mediate cellular physiology and pathology. As a key ubiquitous nutrient and stress sensor, the protein O-GlcNAcylation via HBP regulates multiple cellular pathways in all cell types, in a cell autonomous manner as well as through cell-cell cross talks. The impact of O-GlcNAcylation on insulin resistance serves as a good model to illustrate how the elevation of O-GlcNAcylation in pancreatic β cells, hepatic cells and adipose cells not only interferes with normal cellular function but also contributes to systemic metabolic dysregulation^{76, 87}, which further enhances diabetic cardiovascular complications^{88, 89}.

• O-GlcNAc regulation of insulin resistance

Hyperglycemia is positively associated with elevated HBP and O-GlcNAcylation in diabetic animals, which have also been found associated with insulin resistance, another hallmark of diabetes^{75, 88–90}. The impacts of spatiotemporal regulation of protein O-GlcNAcylation on insulin resistance in diabetes have been documented at multiple levels. Increased HBP flux by overexpression of GFAT (glutamine: fructose-6-phosphate amidotransferase, Figure 1) causes insulin resistance in mice⁹¹ and decreases insulin sensitivity in rat fibroblasts⁹². In contrast, inhibition of the HBP flux by GFAT inactivation blocks hyperglycemia-induced insulin resistance⁹³. Consistent with the observations that increased O-GlcNAcylation promotes insulin resistance^{88, 89}, inhibition of O-GlcNAcylation by overexpression of OGA improves glucose tolerance and insulin sensitivity in diabetic mice⁸⁷. These studies support the important role of the HBP and O-GlcNAcylation in regulating glucose intolerance and insulin resistance in diabetes.

Increased O-GlcNAcylation is associated with hyperglycemia-induced pancreatic β cell death⁷⁶. Pancreatic β cells are the primary regulator of glucose flux by sensing the plasma glucose concentration and secreting appropriate amounts of insulin to direct glucose uptake and storage. Inhibition of the HBP flux and O-GlcNAcylation in pancreatic β cells attenuates hyperglycemia-induced apoptosis of the pancreatic β cells in vivo⁷⁶, supporting the role of O-GlcNAcylation in mediating hyperglycemia-induced β cell death and insulin resistance.

O-GlcNAcylation can trigger hepatic gluconeogenesis, via direct O-GlcNAc modification on the central transcriptional regulator of gluconeogenic genes, the transducer of regulated cyclic adenosine monophosphate response element–binding protein 2, which inhibits its phosphorylation and promotes its cytoplasm to nuclear translocation⁸⁷. Furthermore, increased HBP flux alters the activity and translocation of protein kinase C isoforms. In hepatocytes, O-GlcNAc modifications are identified on serine or threonine residuals at similar positions in several PKC isozyme; and increased O-GlcNAcylation of PKC alpha correlates negatively with PKCalpha activity⁹⁴. Considering that activation of PKC family of enzymes is an important regulator of insulin signaling pathway and oxidative status, O-GlcNAcylation of PKC isoforms should modulate their activity in a cell-dependent manner.

In addition, direct O-GlcNAc modification has been identified on several key cellular mediators in the insulin signaling pathways, including the insulin receptor substrate (IRS) and the insulin-regulated signals, such as protein kinase B/Akt, protein-tyrosine phosphatase 1B (PTP 1B), forkhead box protein O1 (FOXO1) and phosphoinositide-dependent kinase-1 (PDK-1)^{89, 95–97}. Insulin also induces the recruitment of OGT from the nucleus to the plasma membrane through its interaction with the phosphatidylinositol 3,4,5-trisphosphate (PIP3), thus catalyzing O-GlcNAc modification of the insulin receptor and insulin signaling molecules⁸⁰. O-GlcNAc modification on these key insulin signals inhibits insulin-activated phosphorylation, translocation and activity of these molecules, leading to insulin resistance.

• O-GlcNAc regulation of cellular functions

Cellular microenvironment plays a key role in regulating the O-GlcNAc levels in a specific cell type, thus determining the unique cellular responses. Peripheral circadian clocks modulate cell homeostasis by coupling the autonomous intracellular rhythms with changes in extracellular environment^{98, 99}. Direct modification by O-GlcNAc has been identified on several key transcriptional regulators of the circadian rhythm, including BMAL1, CLOCK and PERIOD, which prevents ubiquitination and hence prolongs the stability of these transcriptional regulators^100–102. Increased O-GlcNAcylation of these key cellular circadian regulators disrupts peripheral circadian clocks^{98, 99}. In addition, a recent study uncovers an important role of the clock regulator REV-ERBα as an OGT binding partner that protects cytoplasmic OGT from degradation and facilitates O-GlcNAcylation of cytosolic and nuclear proteins¹⁰³. Accordingly, increased O-GlcNAcylation coupled with cellular circadian clock may upregulate signaling pathways that promote cardiovascular disease in diabetes.

Similarly, O-GlcNAcylation regulates autophagy^{104, 105}, an important cellular process that contribute to maintain cellular homeostasis in response to changes of nutrients, via recycling intracellular energy or removing cytotoxic proteins and damaged organelles. Inhibition of O-GlcNAcylation on an autophagy vesicle protein SNAP-29 promotes autophagosomes and endosomes/lysosomes fusion and autophagic flux, and facilitates autophagic degradation of protein aggregates¹⁰⁴. In addition, OGT/O-GlcNAcylation regulates expression of autophagy genes and shapes autophagy structures via Akt/FOXO signaling¹⁰⁶. It remains to be determined whether direct O-GlcNAcylation of AKT and FOXO transcription factors affects autophagy activation, flux, and lysosomal fusion, thereby contributing to diabetic cardiomyopathies^107–109. Of note, the expression of the major autophagy transcription factors, C/EBPβ and FOXO1, are highly rhythmically regulated in a cell-autonomous manner^{110, 111}. Coincidently, these transcriptional factors are also modified by O-GlcNAcylation^{96, 112}. Therefore, O-GlcNAcylation may represent a common regulation of the nutrient sensing cellular process, such as autonomous circadian rhythm and autophagy, which contributes to the temporal compartmentalization of cellular signaling in response to metabolic abnormality in diabetes.

• GlcNAc and epigenetic regulation of gene expression

O-GlcNAc modification on targeting proteins is regulated by OGT binding to its adaptors selectively, via its tetratricopeptide repeat domains. The binding of OGT to a variety of partners enables the formation of distinct functional complexes. The binding of OGT with ubinuclein1 (UBN1), for example, mediates O-GlcNAcylation of histone proteins that directly modulates chromatin structure and hence cell survival¹¹³. UBN1 binds with the calcineurin binding protein 1 and the histone cell cycle regulator (HIRA) to form the histone chaperone HIRA complex that deposits histone variant H3.3 on chromatin in a replication-independent manner¹¹⁴. Active genes often have histone H3.3 deposited¹¹⁵. Ablation of HIRA-mediated chromatin assembly and histone H3.3 nucleosome causes hypertrophy and increased susceptibility to oxidative stress in the HIRA deficiency skeletal muscle cells and cardiomyocytes^{116, 117}. The binding of OGT to UBN1 recruits OGT to the HIRA complex and thus modifies HIRA on serine 231, which promotes the assembly of the HIRA-H3.3 complex and H3.3 nucleosome. OGT inhibition or expression of the HIRA serine 231 mutant delays premature cellular senescence in primary human fibroblasts, whereas overexpression of OGT accelerates senescence, supporting an important role of OGT-mediated O-GlcNAcylation in modulating chromatin structures that regulate cellular function and survival ¹¹³.

In addition, OGT has been found in the histone H3K4 methyltransferase complexes via binding to one of the key components, the host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression¹¹⁸. HCF-1 undergoes proteolytic maturation in which any of six repeated sequences is cleaved. The tetratricopeptide-repeat domain of OGT binds to a proteolytic repeat at the carboxyl-terminal of HCF-1, which results in cleavage and proteolytic maturation of HCF-1 that directly promotes histone H3K4 trimethylaton¹¹⁸, an indicator for active transcription.

Although the precise mechanisms and the biological significance of the crosstalk between O-GlcNAcylation and DNA demethylation remains elusive, OGT has been reported to bind to the three known members in the ten-eleven translocation methylcytosine dioxygenase family (TET), which converts 5-methylcytosine to 5-hydroxymethylcytosine and regulates gene transcription. OGT binds to the C-terminal region of TET1 that stimulates TET1 demethylation activity and thus controlling the developmental fate of embryonic stem cells¹¹⁹. Recent studies have shown that TET1 binds a significant proportion of polycomb group target genes, supporting a role of TET1 in transcriptional repression¹²⁰. Accordingly, OGT may repress gene transcription via TET1. In contrast, TET2 recruits OGT to the transcriptional start site, and thus modifying histone H2B by O-GlcNAcylation, the dual epigenetic modifications on both DNA and histones by TET2 and OGT coordinate the regulation of gene transcription¹²¹. On the other hand, OGT binding and O-GlcNAcylation of TET3 promotes its nuclear export, and thus inhibiting the formation of 5-hydroxymethylcytosine catalyzed by TET3¹²². Taken together, OGT binding and differential regulation of TET family proteins link glucose metabolism to DNA epigenetic modification. Considering the important role of TET family, particularly TET2 in cardiovascular disease¹²³, it is likely that O-GlcNAcylation of these epigenetic regulators contributes to diabetic cardiovascular disease.

• O-GlcNAc regulation of signaling molecules in cardiovascular cells

As reviewed by Hart and colleagues^{14, 124}, the function of O-GlcNAcylation in regulating gene transcription is documented in every step during transcription process, via direct O-GlcNAc modification on RNA polymerase II, transcription factors such as Sp1, FOXOs and RUNX2, and proteins involved in multiple essential transcriptional processes, including ubiquitination and methylation of histones, DNA demethylation by the TET proteins, and the binding timing of TATA-binding protein on promoters. Thus, increased O-GlcNAcylation may affect the overall transcriptional process as well as regulate specific transcription factors to modulate the transcription of disease-related signals. In addition, the finding of O-GlcNAcylation of ribosome proteins suggest a role of O-GlcNAcylation in regulating protein translation¹²⁵. Nonetheless, posttranslational modification by O-GlcNAcylation represents an important molecular mechanism that affects stability, cellular localization, protein-protein interaction, and function of proteins. For instance, O-GlcNAcylation of transcription factor Sp1 affects Sp1 interaction with other transcription factors, thus regulating the transcriptional activity of Sp1¹²⁶, including the intercellular adhesion molecule-1¹²⁷ and the key angiogenic growth factor vascular endothelial growth factor A¹²⁸. Therefore, dysregulation of multiple signaling pathways by increased O-GlcNAcylation at transcription, translation and posttranslational levels promotes insulin resistance and diabetic cardiovascular disease.

O-GlcNAc modification on serine and threonine residues on proteins may compete with phosphorylation on the same or adjacent sites that cause conformational changes of these proteins, thus affecting the activity, stability or cellular locations. O-GlcNAc modification on Akt, for example, affects Akt phosphorylation, and Akt signaling-activated translocation of the glucose transporter and glucose uptake in cardiomyocytes, thus increasing intracellular Ca^{2+ 129}. Activation of Akt signaling promotes coronary endothelial nitric oxide synthase¹³⁰, and the increased nitric oxide mediates coronary vasodilation, myocardial substrate flexibility and energy homeostasis¹³¹. Thus, impaired Akt activation by O-GlcNAcylation may not only affect glucose handling in the cardiac cells, but also inhibit insulin-stimulated coronary endothelial nitric oxide synthase activity and nitric oxide production, leading to impaired endothelial/cardiomyocyte coupling, cardiac stiffness and diastolic dysfunction¹²⁹. Consistently, inhibition of O-GlcNAcylation by OGA overexpression in endothelial cells increases capillary density and improves endothelium-dependent relaxation in diabetes¹³².

In VSMC, O-GlcNAc modification on Akt promotes Akt phosphorylation and vascular calcification in diabetes⁷⁵. Different from the previously identified O-GlcNAc modification sites at Akt threonine (Thr) 305/312 that inhibit the phosphorylation of Akt at Thr 308 in breast cancer cells¹³³, O-GlcNAc modification on Akt at Thr 430/479 in VSMC does not affect AKT phosphorylation at Thr 308 but enhances Akt phosphorylation/activation at serine 473⁷⁵. Therefore, the regulation of O-GlcNAcylation on Akt activity appears to be cell type-specific and depend on the sites of modification. This may explain the observations that Akt activation is decreased in diabetic cardiomyocytes and myotubes^{134, 135}, while VSMC in diabetic models exhibits sustained activation of Akt after chronic hyperglycemia⁷⁵. O-GlcNAcylation-induced Akt activation in VSMC upregulates the osteogenic transcription factor Runx2, the master regulator for VSMC calcification in vitro and promotes vascular calcification in vivo^{67, 68, 136}. Chronic hyperglycemia-induced Akt and Runx2 expression in the diabetic vasculature is associated with increased O-GlcNAcylation and vascular calcification⁷⁵. O-GlcNAc modification of Runx2 has also been reported in bone marrow mesenchymal stem cells¹³⁷, but the functional consequence of such modification remains unclear. Further investigations to determine whether Runx2 is modified by O-GlcNAc in VSMC and how O-GlcNAcylation of Runx2 contributes to its osteogenic function will provide new molecular insights into the pathogenesis of diabetic vascular disease and strategies for prevention and therapy.

Taken together, elevation of O-GlcNAcylation impairs the cellular nutrient sensing process, such as circadian clock and/or autophagy, which spatiotemporally modulates the expression, stability, cellular localization and function of diverse signaling molecules that promote the development of insulin resistance and diabetic cardiovascular disease. As chronic hyperglycemia is also linked to abnormal metabolisms of lipids/fatty acids and amino acids in diabetes, the elevated protein O-GlcNAcylation likely interacts with other posttranslational modification systems, such as phosphorylation, ubiquitination, acetylation, and methylation³⁴. The disrupted interplays in a cell specific manner may impair protein stability and function, thus leading to impaired cellular function and tissue damage that promotes diabetic cardiovascular disease.

Conclusions and Prospective

Serum glucose level is the primary biomarker for diabetes, and glucose-lowering regimen remains the major treatment strategy for diabetes and diabetic cardiovascular disease. However, chronic hyperglycemia and metabolic stress in diabetic conditions, particularly in type 2 diabetes, may cause pathologic changes that cannot be reversed by simple control of glucose. Pantalone et al found that the current diagnostic marker, hemoglobin A1c (HbA1c), measured at the time of diabetes diagnosis had no apparent effects on longitudinal changes in cardiovascular complication¹³⁸. Emerging evidence suggest that impaired glucose intake and metabolism may integrate with abnormal metabolisms of lipids/fatty acids, amino acids and nucleotides in diabetes, leading to elevation of O-GlcNAcylation. Recent studies have detected increased O-GlcNAc in erythrocytes of pre-diabetic and diabetic patients^{139, 140}. In a young adult population, Myslicki JP et al identified a positive association between O-GlcNAc and HOMA-IR (insulin resistance), whereas no correlation was found between HbA1c and HOMA-IR, suggesting that O-GlcNAc may serve as a potential sensitive marker of early metabolic dysfunction, compared to HbA1c, in the young population free of clinically relevant pathologies¹⁴¹.

Glucose metabolism via HBP and elevation of O-GlcNAcylation have emerged to be an important regulating axis in promoting insulin resistance and the development of cardiovascular disease. Increased HBP flux and O-GlcNAcylation promotes glucose tolerance and insulin resistance, possibly via regulating pancreatic β cell death, hepatic gluconeogenesis, and direct O-GlcNAc modification on several key cellular mediators in the insulin signaling pathways. Acute elevation of O-GlcNAcylation appears to protect stress-induced cardiovascular inflammation and injury. In contrast, chronic elevation of O-GlcNAcylation in diabetes causes impaired calcium handling and contractile properties, as well as mitochondria dysfunction and cardiac hypertrophy. Increased O-GlcNAcylation has been associated with hyperglycemia and oxidative stress that promotes diabetic vascular calcification and vascular stiffness in mice. As a causative regulation of O-GlcNAcylation on the development of major diabetic macrovascular disease, such as atherosclerosis and vascular calcification, has yet to be determined, future investigation in this area with the use of vascular cell-specific OGT ablation animal models will provide important molecular and mechanistic insights into OGT-mediated protein O-GlcNAcylation in the development of cardiovascular complications in diabetes.

The elevation of O-GlcNAcylation in diabetes is mediated by multifaceted regulatory mechanisms. Both the HBP flux and the concentration of UDP-GlcNAc determine the levels of O-GlcNAcylation (Figure 1). As OGT/OGA is the only pair of enzymes that dynamically regulate O-GlcNAcylation, dysregulation of the expression of OGT and OGA appears to modulate the increased O-GlcNAcylation in diabetes. However, the rapid changes of O-GlcNAcylation levels in response to environmental fluctuations also support the involvement of dynamic posttranslational regulatory mechanisms, including OGT phosphorylation, translocation, OGA/OGT interaction, and most importantly OGT interaction with its targeting proteins (Figure 2).

O-GlcNAcylation-regulated molecular and cellular functions have been demonstrated in multiple levels. At the cellular level, increased O-GlcNAcylation is linked to nutrient sensing cellular process, such as peripheral cellular circadian clock, autophagy and cell death. It is reasonable to assume that O-GlcNAcylation acts as a common sensor for these cellular processes to maintain cellular homeostasis in response to extracellular changes. However, chronic elevation of O-GlcNAcylation and other metabolic abnormalities may cause epigenetic changes that are not “reversible”, thus leading to tissue damage. In fact, dysregulation of O-GlcNAc modification on many cytoplasmic and nuclear proteins has been linked to altered expression, stability, translocation, protein-protein interaction and function of key regulators that promote insulin resistance and the development of cardiovascular disease. In addition, O-GlcNAcylation may interplay with other protein posttranslational modifications, including phosphorylation, ubiquitination, acetylation and methylation, to modulate protein stability and function. Therefore, understanding the spatiotemporal regulation of O-GlcNAcylation in specific cardiovascular cells and subcellular compartments under physiological and pathological conditions is critical to uncovering the mechanisms underlying O-GlcNAcylation-regulated pathogenesis of cardiovascular diseases in diabetes.

O-GlcNAcylation appears to be the “integrated sensor” for metabolic stress in diabetes, including oxidative stress, insulin resistance and other metabolic abnormality, and a potential sensitive marker for early detection of diabetes and metabolic dysfunction. Large scale clinical studies are warranted to validate whether O-GlcNAc may represent a promising biomarker for diabetes and for prediction of the development of diabetic cardiovascular complications. Further development of sensitive, reliable and cost-effective tools and approaches for studying O-GlcNAcylation as well as their clinical applications should facilitate the translation of basic science discovery to clinic. Since chronically elevated-O-GlcNAcylation promotes the development of diabetic cardiovascular disease, it is plausible to explore O-GlcNAcylation as a target for prevention and therapy. In this regard, specifically cellular/tissue deliver of low toxic and potent antagonists for OGT/O-GlcNAcylation is critical. As we are uncovering the cell-specific O-GlcNAcylation-regulated signaling network and disease-specific O-GlcNAc modification proteins and mechanisms, new strategies and targets will emerge for precision therapies that prevent and treat cardiovascular disease in diabetes.

HIGHLIGHTS.

This review discusses glucose metabolism via the hexosamine biogenesis pathway and protein O-GlcNAcylation in regulating cellular homeostasis and highlights:

• The emerging role of chronic elevation of O-GlcNAcylation in promoting diabetic cardiovascular disease.

• The multifaceted regulatory mechanisms underlying upregulation of O-GlcNAcylation in diabetes.

• The spatiotemporal regulation of increased O-GlcNAcylation on molecular and cellular function, including transcription, translation, nutrient sensing cellular processes, such as peripheral cellular circadian clock and autophagy, and key molecular signals that promote insulin resistance and pathogenesis of cardiovascular diseases in diabetes.

• The potential of O-GlcNAcylation as an “integrated sensor” for detecting diabetes and predicting the development of diabetic cardiovascular disease.

ABBREVIATIONS

AGEs

advanced glycation end products

ATP

adenosine triphosphate

DCCT

Diabetes Control and Complications Trial

EDIC

Epidemiology of Diabetes Interventions and Complications

FOXO1

forkhead box protein O1

GFAT

glutamine: fructose-6-phosphate amidotransferase

glucose-6-P

glucose 6-phosphate

GNA

Glucosamine 6-phosphate N-acetyltransferase

GPI

Glucose-6-phosphate isomerase

HBP

hexosamine biosynthesis pathways

HIRA

histone cell cycle regulator

HK

Hexokinase

IRS

insulin receptor substrate

mOGT

mitochondral OGT

NADPH

nicotinamide adenine dinucleotide phosphate

ncOGT

nucleocytoplasmic OGT

OGA

O-GlcNAcase

O-GlcNAcylation

O-linked β-N-acetylglucosamine modification

OGT

O-GlcNAc transferase

PASMC

pulmonary arterial smooth muscle cells

PDK-1

phosphoinositide-dependent kinase-1

PGM

Phospho-acetylglucosamine mutase

PIP3

phosphatidylinositol 3,4,5-trisphosphate

PKC

Protein Kinase C

PPP

pentose phosphate pathway

PTP 1B

protein-tyrosine phosphatase 1B

RAGE

Receptor for advanced glycation end products

ROS

reactive oxygen species

sOGT

shorter form of OGT

STZ

streptozocin

TCA

tricarboxylic acid

TET

ten-of-eleven translocation

TPR

tetratricopeptide-repeat

UAP

UDP-N-acetylglucosamine pyrophosphorylase

UBN1

ubinuclein1

UDP-GlcNAc

uridine-diphosphate-β-D-N-acetylglucosamine

UKPDS

United Kingdom Prospective Diabetes Study

URI

unconventional prefolding RPB5 interactor

VADT

Veterans Affairs Diabetes Trial

VSMC

vascular smooth muscle cells