OGT suppresses S6K1-mediated macrophage inflammation and metabolic disturbance.

Enhanced inflammation is believed to contribute to overnutrition-induced metabolic disturbance. Nutrient flux has also been shown to be essential for immune cell activation. Here, we report an unexpected role of nutrient-sensing O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling in suppressing macrophage proinflammatory activation and preventing diet-induced metabolic dysfunction. Overnutrition stimulates an increase in O-GlcNAc signaling in macrophages. O-GlcNAc signaling is down-regulated during macrophage proinflammatory activation. Suppressing O-GlcNAc signaling by O-GlcNAc transferase (OGT) knockout enhances macrophage proinflammatory polarization, promotes adipose tissue inflammation and lipolysis, increases lipid accumulation in peripheral tissues, and exacerbates tissue-specific and whole-body insulin resistance in high-fat-diet-induced obese mice. OGT inhibits macrophage proinflammatory activation by catalyzing ribosomal protein S6 kinase beta-1 (S6K1) O-GlcNAcylation and suppressing S6K1 phosphorylation and mTORC1 signaling. These findings thus identify macrophage O-GlcNAc signaling as a homeostatic mechanism maintaining whole-body metabolism under overnutrition.

O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity.

Excessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.

O-GlcNAcase targets pyruvate kinase M2 to regulate tumor growth.

Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked ß-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, which facilitates O-GlcNAcylation of PKM2 by O-GlcNAc transferase (OGT). O-GlcNAcylation inhibits PKM2 catalytic activity and thereby promotes aerobic glycolysis and tumor growth. These studies define a causative role for OGA in tumor progression and reveal PKM2 O-GlcNAcylation as a metabolic rheostat that mediates exquisite control of aerobic glycolysis.

Adipocyte OGT governs diet-induced hyperphagia and obesity.

Palatable foods (fat and sweet) induce hyperphagia, and facilitate the development of obesity. Whether and how overnutrition increases appetite through the adipose-to-brain axis is unclear. O-linked beta-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) couples nutrient cues to O-GlcNAcylation of intracellular proteins at serine/threonine residues. Chronic dysregulation of O-GlcNAc signaling contributes to metabolic diseases. Here we show that adipocyte OGT is essential for high fat diet-induced hyperphagia, but is dispensable for baseline food intake. Adipocyte OGT stimulates hyperphagia by transcriptional activation of de novo lipid desaturation and accumulation of N-arachidonyl ethanolamine (AEA), an endogenous appetite-inducing cannabinoid (CB). Pharmacological manipulation of peripheral CB1 signaling regulates hyperphagia in an adipocyte OGT-dependent manner. These findings define adipocyte OGT as a fat sensor that regulates peripheral lipid signals, and uncover an unexpected adipose-to-brain axis to induce hyperphagia and obesity.

Transcriptional regulation of O-GlcNAc homeostasis is disrupted in pancreatic cancer.

Many intracellular proteins are reversibly modified by O-linked GlcNAc (O-GlcNAc), a post-translational modification that dynamically regulates fundamental cellular processes in response to diverse environmental cues. Accumulating evidence indicates that both excess and deficiency of protein O-GlcNAcylation can have deleterious effects on the cell, suggesting that maintenance of O-GlcNAc homeostasis is essential for proper cellular function. However, the mechanisms through which O-GlcNAc homeostasis is maintained in the physiologic state and altered in the disease state have not yet been investigated. Here, we demonstrate the existence of a homeostatic mechanism involving mutual regulation of the O-GlcNAc-cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) at the transcriptional level. Specifically, we found that OGA promotes Ogt transcription through cooperation with the histone acetyltransferase p300 and transcription factor CCAAT/enhancer-binding protein ß (C/EBPß). To examine the role of mutual regulation of OGT and OGA in the disease state, we analyzed gene expression data from human cancer data sets, which revealed that OGT and OGA expression levels are highly correlated in numerous human cancers, particularly in pancreatic adenocarcinoma. Using a KrasG12D -driven primary mouse pancreatic ductal adenocarcinoma (PDAC) cell line, we found that inhibition of extracellular signal-regulated kinase (ERK) signaling decreases OGA glycosidase activity and reduces OGT mRNA and protein levels, suggesting that ERK signaling may alter O-GlcNAc homeostasis in PDAC by modulating OGA-mediated Ogt transcription. Our study elucidates a transcriptional mechanism that regulates cellular O-GlcNAc homeostasis, which may lay a foundation for exploring O-GlcNAc signaling as a therapeutic target for human disease.

An Engineered Rare Codon Device for Optimization of Metabolic Pathways.

Rare codons generally arrest translation due to rarity of their cognate tRNAs. This property of rare codons can be utilized to regulate protein expression. In this study, a linear relationship was found between expression levels of genes and copy numbers of rare codons inserted within them. Based on this discovery, we constructed a molecular device in Escherichia coli using the rare codon AGG, its cognate tRNA (tRNA(Arg) (CCU)), modified tRNA(Asp) (GUC → CCU), and truncated aspartyl-tRNA synthetase (TDRS) to switch the expression of reporter genes on or off as well as to precisely regulate their expression to various intermediate levels. To underscore the applicability of our work, we used the rare codon device to alter the expression levels of four genes of the fatty acid synthesis II (FASII) pathway (i.e. fabZ, fabG, fabI, and tesA') in E. coli to optimize steady-state kinetics, which produced nearly two-fold increase in fatty acid yield. Thus, the proposed method has potential applications in regulating target protein expression at desired levels and optimizing metabolic pathways by precisely tuning in vivo molar ratio of relevant enzymes.

O-GlcNAc signaling in cancer metabolism and epigenetics.

The covalent attachment of ß-D-N-acetylglucosamine monosaccharides (O-GlcNAc) to serine/threonine residues of nuclear and cytoplasmic proteins is a major regulatory mechanism in cell physiology. Aberrant O-GlcNAc modification of signaling proteins, metabolic enzymes, and transcriptional and epigenetic regulators has been implicated in cancer. Relentless growth of cancer cells requires metabolic reprogramming that is intertwined with changes in the epigenetic landscape. This review highlights the emerging role of protein O-GlcNAcylation at the interface of cancer metabolism and epigenetics.

O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat.

Induction of beige cells causes the browning of white fat and improves energy metabolism. However, the central mechanism that controls adipose tissue browning and its physiological relevance are largely unknown. Here, we demonstrate that fasting and chemical-genetic activation of orexigenic AgRP neurons in the hypothalamus suppress the browning of white fat. O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates fundamental cellular processes. The levels of O-GlcNAc transferase (OGT) and O-GlcNAc modification are enriched in AgRP neurons and are elevated by fasting. Genetic ablation of OGT in AgRP neurons inhibits neuronal excitability through the voltage-dependent potassium channel, promotes white adipose tissue browning, and protects mice against diet-induced obesity and insulin resistance. These data reveal adipose tissue browning as a highly dynamic physiological process under central control, in which O-GlcNAc signaling in AgRP neurons is essential for suppressing thermogenesis to conserve energy in response to fasting.

O-GlcNAc: A Bittersweet Switch in Liver.

The liver is a vital organ responsible for maintaining nutrient homeostasis. After a meal, insulin stimulates glycogen and lipid synthesis in the liver; in the fasted state, glucagon induces gluconeogenesis and ketogenesis, which produce glucose and ketone bodies for other tissues to use as energy sources. These metabolic changes involve spatiotemporally co-ordinated signaling cascades. O-linked ß-N-acetylglucosamine (O-GlcNAc) modification has been recognized as a nutrient sensor and regulatory molecular switch. This review highlights mechanistic insights into spatiotemporal regulation of liver metabolism by O-GlcNAc modification and discusses its pathophysiological implications in insulin resistance, non-alcoholic fatty liver disease, and fibrosis.

Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. In addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30°C), MS2 exhibits a K(m) for 16:0-Acyl Carrier Protein of 23.3 ± 4.0 µm, a V(max) of 38.3 ± 4.5 nmol mg⁻¹ min⁻¹, and a catalytic efficiency/K(m) of 1,873 M⁻¹ s⁻¹. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.