Protein O-linked β-N-acetylglucosamine: a novel effector of cardiomyocyte metabolism and function

Abstract

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide β-N-acetyl-glucosamine (O-GlcNAc) is emerging as an important mechanism for the regulation of numerous biological processes critical for normal cell function. Active synthesis of O-GlcNAc is essential for cell viability and acute activation of pathways resulting in increased protein O-GlcNAc levels improves the tolerance of cells to a wide range of stress stimuli. Conversely sustained increases in O-GlcNAc levels have been implicated in numerous chronic disease states, especially as a pathogenic contributor to diabetic complications. There has been increasing interest in the role of O-GlcNAc in the heart and vascular system and acute activation of O-GlcNAc levels have been shown to reduce ischemia/reperfusion injury, attenuate vascular injury responses as well mediate some of the detrimental effects of diabetes and hypertension on cardiac and vascular function. Here we provide an overview of our current understanding of pathways regulating protein O-GlcNAcylation, summarize the different methodologies for identifying and characterizing O-GlcNAcylated proteins and subsequently focus on two emerging areas: 1) the role of O-GlcNAc as a potential regulator of cardiac metabolism and 2) the cross talk between O-GlcNAc and reactive oxygen species. This article is part of a Special Section entitled "Post-translational Modification."

Figure 1. The hexosamine biosynthesis pathway (HBP) and protein O-GlcNAcylation

Glucose imported into the cells is rapidly phosphorylated to glucose-6-phosphate and converted to fructose-6-phosphate, which is subsequently metabolized to glucosamine-6-phosphate by L-glutamine-D-fructose 6-phosphate amidotransferase (GFAT). Synthesis of glucosamine-6-phosphate is dependent on availability of glutamine, which is formed by glutamine synthetase (GTS). Glucosamine-6-phosphate is subsequently metabolized by glucosamine 6-phosphate N-acetyltransferase (Emeg32) to N-acetylglucosamine-6-phosphate, which is converted to N-acetylglucosamine-1-phosphate by Phosphoacetylglucosamine mutase (Agm1) and the synthesis of UDP- uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is catalyzed by UDP-N-acetylglucosamine pyrophosphorylase (Uap1). Flux through the HBP can be increased with glucosamine, which is phosphorylated by hexokinase (HK) to form glucosamine 6-phosphate thereby bypassing GFAT. UDP-GlcNAc is the obligatory substrate for OGT (uridine-diphospho-N-acetylglucosamine: polypeptide β-N-acetylglucosaminyltransferase) leading to the formation of O-linked ß-N-acetylglucosamine (O-GlcNAc)-modified proteins. ß-N-acetylglucosaminidase (OGA) catalyzes the removal of O-GlcNAc from the proteins.

Figure 2. Acute and transcriptional regulation of cardiac metabolism mediated by the hexosamine biosynthesis pathway (HBP) and O-GlcNAc

Flux through the HBP is regulated by glutamine-D-fructose 6-phosphate amidotransferase (GFAT) and is dependent on the availability of glucose and glutamine. O-GlcNAc transferase (OGT) is subject to both O-GlcNAcylation (green circles) and insulin-mediated phosphorylation (red circles) both of which have been reported to increase its activity. AMPK, which is well recognized as a key regulator of cellular metabolism has been reported to be a target for O-GlcNAcylation and that this increases its activity; furthermore, GFAT has been shown to phosphorylated by AMPK also leading to increased activity. This provides a feed forward loop leading to further increase in OGT activity and subsequent increase in O-GlcNAcylation. Increases in O-GlcNAc levels can potentially acutely regulate cardiac metabolism, not only by modification of AMPK, but also by O-GlcNAcylation of other key mediators of carbohydrate and fatty acid metabolism. A number of transcription factors, which play critical role in the longer-term regulation of metabolism, are also known to be O-GlcNAcylated.

Figure 3. The intersection between O-GlcNAcylation, ROS and mitochondrial function

In response to an acute stress, including that induced by ROS, there is a rapid increase in cellular and potentially mitochondrial O-GlcNAcylation as a result of either an increased flux through the hexosamine biosynthesis pathway (HBP) and/or decreased O-GlcNAcase activity (OGA). This acute increase in O-GlcNAc levels has been shown to improve tolerance of mitochondria to oxidative stress and levels and attenuate loss of mitochondrial membrane potential leading to increased cell survival. Strategies designed to acutely augment this response have been shown to improve cell survival; conversely decreased O-GlcNAc levels lead to decreased cell survival. Chronic increases in cellular O-GlcNAc levels are typically associated with metabolic disease such as diabetes and the resulting hyperglycemia. Sustained increases in glucose levels can increase O-GlcNAc synthesis by directly increasing flux through the HBP as well as indirectly, as a result of hyperglycemia induced ROS production, which has also been shown to stimulate O-GlcNAc synthesis. Hyperglycemia is also known to lead to impaired mitochondrial function in part due to increased O-GlcNAcylation of mitochondrial proteins, which could in turn further increase ROS production and thereby in a feed forward manner continue to increase O-GlcNAc levels with even greater decline in mitochondria function. This O-GlcNAc mediated impairment of mitochondrial function, could then increase the vulnerability to additional stress thus leading to increased cell death. Indeed, targeted overexpression of mitochondrial OGT has been shown to lead to increased apoptosis.