Hexosamine Signaling Pathway Research Articles

ELT-2 promotes O-GlcNAc transferase OGT-1 expression to modulate Caenorhabditis elegans lifespan.

O-GlcNAc transferase (OGT) is the enzyme catalyzing protein O-GlcNAcylation by addition of a single O-linked-β-N-acetylglucosamine molecule (O-GlcNAc) to nuclear and cytoplasmic targets, and it uses uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a donor. As UDP-GlcNAc is the final product of the nutrient-sensing hexosamine signaling pathway, overexpression or knockout of ogt in mammals or invertebrate models influences cellular nutrient-response signals and increases susceptibility to chronic diseases of aging. Evidence shows that OGT expression levels decrease in tissues of older mice and rats. However, how OGT expression is modulated in the aging process remains poorly understood. In Caenorhabditis elegans, the exclusive mammalian OGT ortholog OGT-1 is crucial for lifespan control. Here, we observe that worm OGT-1 expression gradually reduces during aging. By combining prediction via the "MATCH" algorithm and luciferase reporter assays, GATA factor ELT-2, the homolog of human GATA4, is identified as a transcriptional factor driving OGT-1 expression. Chromatin immunoprecipitation-quantitative polymerase chain reaction and electrophoretic mobility shift assays show ELT-2 directly binds to and activates the ogt-1 promoter. Knockdown of elt-2 decreases the global O-GlcNAc modification level and reduces the lifespan of wild-type worms. The reduction in lifespan caused by elt-2 RNA interference is abrogated by the loss of ogt-1. These results imply that GATA factors are able to activate OGT expression, which could be beneficial for longevity and the development of therapeutic treatment for aging-related diseases.

GNPDA2 Gene Affects Adipogenesis and Alters the Transcriptome Profile of Human Adipose-Derived Mesenchymal Stem Cells.

Background Genome-wide association studies have found an obesity-related single-nucleotide polymorphism rs10938397 near the glucosamine-6-phosphate deaminase 2 gene (GNPDA2) encoding, an enzyme that catalyzes the deamination of the glucosamine-6-phosphate involved in the hexosamine signaling pathway, but the molecular mechanisms underlying the missing link between GNPDA2 and obesity remain elusive. Methods As obesity is accompanied by an increase in the size and the number of adipocytes, the present study investigates the possible mechanism of the GNPDA2 in adipogenesis using GeneChip® Human Transcriptome Array 2.0 in human adipose-derived mesenchymal stem cells. Results We found that overexpression of GNPDA2 enhanced accumulation of lipid droplets, and knocking down the gene decreased accumulation of lipid droplets. GO term enrichment analysis indicated that most differentially expressed genes (DEGs) affected by deficiency of GNPDA2 have functions to lipid and glucose metabolism. Further KEGG enrichment analysis showed that the greatest proportion of DEGs are involved in thermogenesis, peroxisome proliferator-activated receptor (PPAR) signaling pathway, carbon metabolism, and fatty acid metabolism including fatty acid degradation, elongation, and biosynthesis. Conclusion These findings suggest that GNPDA2 may be a critical gene for lipid and glucose metabolism, and the expression level of GNPDA2 alters the transcriptome profile of human adipose-derived mesenchymal stem cells.

Epstein-Barr virus encoded microRNA BART7 regulates radiation sensitivity of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is very sensitive to radiotherapy. To date, the underlying mechanism remains poorly understood. Here, we demonstrated that expression of EBV-encoded microRNA BART7 (ebv-miR-BART7) increases responsiveness of NPC to radiation treatment by targeting GFPT1/TGFβ1 signaling. GFPT1 is the the key rate-limiting enzyme of the hexosamine signaling pathway and governs TGFβ1 production. TGFβ1, a pleotropic cytokine with the potency to trigger self-renewal and damage-repair machinery in somatic cells. TGFβ1 can protect zebrafish embryo from the lethal effects of radiation treatment. In silico analysis showed that ebv-miR-BART7 could target GFPT1 transcript. Correlation analysis on primary NPC tissues suggested that ebv-miR-BART7 and GFPT1 have negative expression correlation. Expression of GFPT1 and TGFβ1 were inducible by radiation in NPC cell with ebv-miR-BART7 expression. Further, suppressing endogenous GFPT1 expression inhibited TGFβ1 which subsequently increased the responsiveness of NPC to radiation treatment. Taken together, our results demonstrated that ebv-miR-BART7 controls TGFβ1 production by targeting GFPT1. Detection of ebv-miR-BART7 may provide useful indicator for monitoring NPC progression and predict therapeutic outcomes.

Nutrient-driven O-GlcNAc cycling – think globally but act locally

Proper cellular functioning requires that cellular machinery behave in a spatiotemporally regulated manner in response to global changes in nutrient availability. Mounting evidence suggests that one way this is achieved is through the establishment of physically defined gradients of O-GlcNAcylation (O-linked addition of N-acetylglucosamine to serine and threonine residues) and O-GlcNAc turnover. Because O-GlcNAcylation levels are dependent on the nutrient-responsive hexosamine signaling pathway, this modification is uniquely poised to inform upon the nutritive state of an organism. The enzymes responsible for O-GlcNAc addition and removal are encoded by a single pair of genes: both the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA, also known as MGEA5) genes are alternatively spliced, producing protein variants that are targeted to discrete cellular locations where they must selectively recognize hundreds of protein substrates. Recent reports suggest that in addition to their catalytic functions, OGT and OGA use their multifunctional domains to anchor O-GlcNAc cycling to discrete intracellular sites, thus allowing them to establish gradients of deacetylase, kinase and phosphatase signaling activities. The localized signaling gradients established by targeted O-GlcNAc cycling influence many important cellular processes, including lipid droplet remodeling, mitochondrial functioning, epigenetic control of gene expression and proteostasis. As such, the tethering of the enzymes of O-GlcNAc cycling appears to play a role in ensuring proper spatiotemporal responses to global alterations in nutrient supply.

Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry

The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.

IMPAIRED REGULATORY MECHANISMS OF THE mTOR SIGNALING PATHWAY IN OSTEOARTHROSIS

Objective: to study the pattern of impaired regulatory mechanisms of the mammalian target of rapamycin (TOR) signaling pathway, by monitoring gene expression in the blood of patients with osteoarthrosis (OA) at different stages of the disease. Subjects and methods. The study covered 33 outpatients with OA, 14 patients with this condition prior to knee joint endoprosthesis, and 27 healthy individuals (controls) (mean age 58.0+7.4, 56.5+8.9, and 55.0+8.3 years, respectively). Total RNA was isolated from their blood and used to determine the level of gene expression by a real-time polymerase chain reaction for AMP-activated protein kinase (AMPK), hypoxia-inducible factor-1α (HIF1α), the rate-limiting proteins of the hexosamine signaling pathway — glutamine-fructose-6-phosphate amidotransferase and acetylglucosaminyltransferase, as well as the glucose transporter GLUT1 and steps 6 and 7 glycolytic pathway components — glucose-6-phosphate dehydrogenase and phosphoglycerate kinase-1, respectively; the lipogenesis-related genes — fatty acid synthase (FAS) and the activity of the pentose phosphate pathway — glucose-6-phosphate dehydrogenase in the blood of patients with OA at different stages of the disease. Results. Analysis of gene expressions showed that in the OA patients with a low expression of the mTOR gene (a LOW subgroup), the expression of AGT and GLUT1 genes proved to be significantly lower and that of the AMPK gene was higher than in the healthy individuals. In the OA patients with a high expression of the mTOR gene (a HIGH subgroup), the expression of all the genes under study was much higher, except for the FAS gene; moreover, the greatest expression excess as compared to the controls was observed for the AMPK and HIFlα genes. In the patients with endstage disease (an ES subgroup), the expression of all the study genes, including the FAS gene, turned out to be higher than in the healthy individuals. Conclusion. The development of OA is accompanied by a considerable decrease in the efficiency of energy metabolism. At the same time, in the patients with a low mTOR gene expression, energy deficiency may be due to decreased cellular metabolite transport. It may be caused by the deficiency of the end electron acceptor oxygen in the patients with a high mTOR gene expression and the pathological redistribution of energy substrate in favor of lipogenesis cannot be ruled out in those with end-stage disease.

Abstract 218: The hexosamine biosyntheic pathway and O-linked glycosylation for targeted therapy in diffuse large B-cell lymphoma

Abstract Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Both nutrients are required for the synthesis of glucosamine, the precursor substrate of the hexosamine biosynthetic pathway. The hexosamine signaling pathway terminating in O-linked N-acetyl glucosamine (O-GlcNAc) cycling has been implicated in cellular signaling cascades and regulation of transcription factors involved in cancer biology. Biological functions of the hexosamine biosynthetic signaling pathways need elucidation, to determine whether altered O-GlcNAc metabolism plays a significant role in hematologic tumors such as diffuse large B-cell lymphoma (DLBCL), and utilize this bifunctional pathway as a targeted therapeutic strategy in DLBCL. We have identified a key enzyme of the hexosamine biosynthetic pathways to be highly-expressed in DLBCL cell lines and patient tumor cells. In contrast to normal circulating and tonsillar B cells, DLBCL cells expressed high levels of the terminating enzyme O-GlcNAc transferase (OGT). OGT mRNA expression is highly expressed in DLBCL in comparaison to other cancers. We discovered that several key growth and survival transcription factors, such as NF-kB and NFAT, known to be highly-activated in DLBCL, are linked to the hexoasmine biosynthetic pathway. We demonstrated that both NF-kB (p65) and NFATc1 directly associated with OGT, and down-regulation of OGT by siRNA inhibits these transcription factors activation, suggesting that both NF-kB-p65 and NFATc1 require O-GlcNAc glycosylation by OGT for their activation. These results suggest that the hexosamine pathway is highly active and utilized in DLBCL, and that exploiting this bi-functional pathway(s) as a therapeutic approach is feasible. We have previously developed an imaging agent, 99mTc-ethylenedicysteine-glucosamine (99mTc-EC-G) because EC-G mimics phosphorylated N-acetylglucosamine. ECG treatment in DLBCL cells enhances p65 and NFATc1 nuclear translocation. For therapeutic strategies, we developed metallic unlabeled Platinum (Pt) derivatives-EC-G as potential therapeutic agents. Pre-clinical in vitro studies have shown that our two lead compounds, Pt-9 and Pt-DACH-EC-G effectively inhibit lymphoma cell growth and induce apoptosis. These lead compounds can also induce DNA damage in DLBCL cells, through the up-regulation of phosphorylated histone 2AX (pH2AX), leading to the disruption of p65 and NFATc1 binding to DNA. This data importantly demonstrates that the hexosamine biosynthetic pathway is linked to key growth and survival pathways involved in the pathophysiology of DLBCL. Targeting these pathways with novel platinum EC-G compounds as a theranostic approach should lead to new, more effective treatments and diagnosis for DLBCL, particularly for relapsed/refractory DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 218. doi:1538-7445.AM2012-218

O‐GlcNAc cycling and Epigenetics

The nutrient‐sensing hexosamine signaling pathway terminating in O‐GlcNAc cycling is emerging as a key epigenetic regulator of gene expression in mammals. Mounting evidence suggests that OGlcNAc cycling sits atop a robust regulatory network maintaining higher‐order chromatin structure and epigenetic memory. OGlcNAc cycling occurs on many cellular targets associated with signaling, morphogenesis and transcriptional regulation. In addition, it has now been added to the multifaceted ‘histone‐code’. In model systems ranging from C. elegans to man, O‐GlcNAc cycling has been shown play an important role in modulating developmental plasticity. We have focused on the influence of O‐GlcNAc cycling upon the molecular modifications of the RNA Polymerase II carboxyl‐terminal tail. In C. elegans, O‐GlcNAc marks the promoters of over 800 developmental, metabolic, and stress‐related genes. In response to starvation or feeding, levels of O‐GlcNAc at promoters remain nearly constant due to dynamic cycling mediated by the transferase OGT‐1 and the O‐GlcNAcase OGA‐1. Blocked O‐GlcNAc cycling leads to a striking nutrient dependent accumulation of O‐GlcNAc on RNA Pol II. O‐GlcNAc cycling mutants also show an exaggerated, nutrient‐responsive redistribution of promoter‐proximal RNA Pol II isoforms and extensive transcriptional deregulation. These studies in the nematode have been extended to the fly and vertebrate RNA Pol II CTD. Our findings suggest a complex interplay between the O‐GlcNAc modification at promoters and the kinase dependent ‘CTD‐code’ regulating RNA Pol II dynamics. Nutrient‐responsive O‐GlcNAc cycling may buffer the transcriptional apparatus from dramatic swings in nutrient availability by sustaining a 5′ pool of RNA Pol II poised for rapid reactivation to meet metabolic and developmental fate decisions. These findings have important implications for the inheritance of susceptibility to the diseases of aging including diabetes, cancer and neurodegeneration.

The Hexosamine Biosyntheic Pathway and Platinum-Ethylenedicysteine-Glucosamine (Pt-ECG) for Targeted Therapy in Diffuse Large B-Cell Lymphoma

Abstract 587Aggressive non-Hodgkin lymphomas (NHL), such as diffuse large B cell lymphomas (DLBCL), are very common in the US with increasing incidences. Although these lymphomas are now potentially curable, almost half the treated patients still develop relapsed/refractory disease with poor survival outcomes, indicating an urgent need for better therapeutic approaches with improved efficacy.The hexosamine signaling pathway terminating in O-linked N-acetyl glucosamine (O-GlcNAc) cycling has been implicated in cellular signaling cascades and regulation of transcription factors involved in cancer biology. Biological functions of the hexosamine biosynthetic signaling pathways need elucidation, to determine whether altered O-GlcNAc metabolism plays a significant role in hematologic tumors such as DLBCL, and utilize this bifunctional pathway as a targeted therapeutic strategy in DLBCL. We have identified key enzymes of the hexosamine biosynthetic pathways to be highly-expressed in DLBCL cell lines and patient tumor cells. In contrast to normal circulating and tonsillar B cells, DLBCL cells expressed high levels of the rate limiting enzyme glutamine: fructose-6-phosphate amidotransferase (GFAT) as well as terminating enzyme O-GlcNAc transferase (OGT). We discovered that several key growth and survival transcription factors, such as NF-kB and NFAT, known to be highly-activated in DLBCL, are linked to the hexoasmine biosynthetic pathway. We demonstrated that both NF-kB (p65) and NFATc1 directly associated with OGT, and down-regulation of OGT by siRNA inhibits these transcription factors activation, suggesting that both NF-kB-p65 and NFATc1 require O-GlcNAc glycosylation by OGT for their activation. ChiP on Chip analysis on NFATc1 indicated that this transcription factor regulates a set of genes involved in glucose metabolism, including hexokinase and GFAT. These results suggest that the hexosamine pathway is highly active and utilized in DLBCL, and that exploiting this bi-functional pathway(s) as a therapeutic approach is feasible. We have previously developed an imaging agent, 99mTc-ethylenedicysteine-glucosamine (99mTc-EC-G) because EC-G mimics phosphorylated N-acetylglucosamine. ECG treatment in DLBCL cells enhances p65 and NFATc1 nuclear translocation. For therapeutic strategies, we developed metallic unlabeled Platinum (Pt) derivatives-EC-G as potential therapeutic agents. Pre-clinical in vitro studies have shown that our two lead compounds, Pt- and Pt-(DACH)-EC-G effectively inhibit lymphoma cell growth and induce apoptosis. These lead compounds can also induce DNA damage in DLBCL cells, through the up-regulation of phosphorylated histone 2AX (pH2AX), leading to the disruption of p65 and NFATc1 binding to DNA.This data importantly demonstrates that the hexosamine biosynthetic pathway is linked to key growth and survival pathways involved in the pathophysiology of DLBCL. Targeting these pathways with novel platinum EC-G compounds as a theranostic approach should lead to new, more effective treatments and diagnosis for DLBCL, particularly for relapsed/refractory DLBCL. Disclosures:Rollo:Cell Point: Employment.

A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome

Protein-O-linked N-Acetyl-β-D-glucosaminidase (O-GlcNAcase, OGA; also known as hexosaminidase C) participates in a nutrient-sensing, hexosamine signaling pathway by removing O-linked N-acetylglucosamine (O-GlcNAc) from key target proteins. Perturbations in O-GlcNAc signaling have been linked to Alzheimer's disease, diabetes and cancer. Mammalian O-GlcNAcase exists as two major spliced isoforms differing only by the presence (OGA-L) or absence (OGA-S) of a histone-acetyltransferase domain. Here we demonstrate that OGA-S accumulates on the surface of nascent lipid droplets with perilipin-2; both of these proteins are stabilized by proteasome inhibition. We show that selective downregulation of OGA-S results in global proteasome inhibition and the striking accumulation of ubiquitinylated proteins. OGA-S knockdown increased levels of perilipin-2 and perilipin-3 suggesting that O-GlcNAc-dependent regulation of proteasomes might occur on the surface of lipid droplets. By locally activating proteasomes during maturation of the nascent lipid droplet, OGA-S could participate in an O-GlcNAc-dependent feedback loop regulating lipid droplet surface remodeling. Our findings therefore suggest a mechanistic link between hexosamine signaling and lipid droplet assembly and mobilization.

O-Linked-N-Acetylglucosamine Cycling and Insulin Signaling Are Required for the Glucose Stress Response inCaenorhabditis elegans

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose-insulin homeostasis.

59 Dual targeting of mTOR and HSP90 for therapy of pancreato-biliary carcinomas

The enzymes of O-GlcNAc cycling couple the nutrient-dependent synthesis of UDP-GlcNAc to O-GlcNAc modification of Ser/Thr residues of key nuclear and cytoplasmic targets. This series of reactions culminating in O-GlcNAcylation of targets has been termed the hexosamine signaling pathway (HSP). The evolutionarily ancient enzymes of O-GlcNAc cycling have co-evolved with other signaling effecter molecules; they are recruited to their targets by many of the same mechanisms used to organize canonic kinase-dependent signaling pathways. This co-recruitment of the enzymes of O-GlcNAc cycling drives a binary switch impacting pathways of anabolism and growth (nutrient uptake) and catabolic pathways (nutrient sparing and salvage). The hexosamine signaling pathway (HSP) has thus emerged as a versatile cellular regulator modulating numerous cellular signaling cascades influencing growth, metabolism, cellular stress, circadian rhythm, and host–pathogen interactions. In mammals, the nutrient-sensing HSP has been harnessed to regulate such cell-specific functions as neutrophil migration, and activation of B-cells and T-cells. This review summarizes the diverse approaches being used to examine O-GlcNAc cycling. It will emphasize the impact O-GlcNAcylation has upon signaling pathways that may be become deregulated in diseases of the immune system, diabetes mellitus, cancer, cardiovascular disease, and neurodegenerative diseases.

Toxic Effects of Hyperglycemia Are Mediated by the Hexosamine Signaling Pathway and O-Linked Glycosylation in Early Mouse Embryos1

Maternal hyperglycemia is believed to be the metabolic derangement associated with both early pregnancy loss and congenital malformations in a diabetic pregnancy. Using an in vitro model of embryo exposure to hyperglycemia, this study questioned if increased flux through the hexosamine signaling pathway (HSP), which results in increased embryonic O-linked glycosylation (O-GlcNAcylation), underlies the glucotoxic effects of hyperglycemia during early embryogenesis. Mouse zygotes were randomly allocated to culture treatment groups that included no glucose (no flux through HSP), hyperglycemia (27 mM glucose, excess flux), 0.2 mM glucosamine (GlcN) in the absence of glucose (HSP flux alone), and O-GlcNAcylation levels monitored immunohistochemically. The impact of HSP manipulation on the first differentiation in development, blastocyst formation, was assessed, as were apoptosis and cell number in individual embryos. The enzymes regulating O-GlcNAcylation, and therefore hexosamine signaling, are the beta-linked-O-GlcNAc transferase (OGT) and an O-GlcNAc-selective beta-N-acetylglucosaminidase (O-GlcNAcase). Inhibition of these enzymes has a negative impact on blastocyst formation, demonstrating the importance of this signaling system to developmental potential. The ability of the OGT inhibitor benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (BADGP) to reverse the glucotoxic effects of hyperglycemia on these parameters was also sought. Excess HSP flux arising from a hyperglycemic environment or glucosamine supplementation reduced cell proliferation and blastocyst formation, confirming the criticality of this signaling pathway during early embryogenesis. Inhibition of OGT using BADGP blocked the negative impact of hyperglycemia on blastocyst formation, cell number, and apoptosis. Our results suggest that dysregulation of HSP and O-GlcNAcylation is the mechanism by which the embryotoxic effects of hyperglycemia are manifested during preimplantation development.

Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 ( O -GlcNAcase) knockout impacts O -GlcNAc cycling, metabolism, and dauer

A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. Two highly conserved enzymes (O-GlcNAc transferase and O-GlcNAcase) catalyze the final steps in this nutrient-driven "hexosamine-signaling pathway." A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes. Here, we show that Caenorhabditis elegans oga-1 encodes an active O-GlcNAcase. We also describe a knockout allele, oga-1(ok1207), that is viable and fertile yet accumulates O-GlcNAc on nuclear pores and other cellular proteins. Interfering with O-GlcNAc cycling with either oga-1(ok1207) or the O-GlcNAc transferase-null ogt-1(ok430) altered Ser- and Thr-phosphoprotein profiles and increased glycogen synthase kinase 3beta (GSK-3beta) levels. Both the oga-1(ok1207) and ogt-1(ok430) strains showed elevated stores of glycogen and trehalose, and decreased lipid storage. These striking metabolic changes prompted us to examine the insulin-like signaling pathway controlling nutrient storage, longevity, and dauer formation in the C. elegans O-GlcNAc cycling mutants. Indeed, we found that the oga-1(ok1207) knockout augmented dauer formation induced by a temperature sensitive insulin-like receptor (daf-2) mutant under conditions in which the ogt-1(ok430)-null diminished dauer formation. Our findings suggest that the enzymes of O-GlcNAc cycling "fine-tune" insulin-like signaling in response to nutrient flux. The knockout of O-GlcNAcase (oga-1) in C. elegans mimics many of the metabolic and signaling changes associated with human insulin resistance and provides a genetically amenable model of non-insulin-dependent diabetes.

Role of Insulin, Adipocyte Hormones, and Nutrient-Sensing Pathways in Regulating Fuel Metabolism and Energy Homeostasis: A Nutritional Perspective of Diabetes, Obesity, and Cancer

Traditionally, nutrients such as glucose and amino acids have been viewed as substrates for the generation of high-energy molecules and as precursors for the biosynthesis of macromolecules. However, it is now apparent that nutrients also function as signaling molecules in functionally diverse signal transduction pathways. Glucose and amino acids trigger signaling cascades that regulate various aspects of fuel and energy metabolism and control the growth, proliferation, and survival of cells. Here, we provide a functional and regulatory overview of three well-established nutrient signaling pathways-the hexosamine signaling pathway, the mTOR (mammalian target of rapamycin) signaling pathway, and the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. Nutrient signaling pathways are interconnected, coupled to insulin signaling, and linked to the release of metabolic hormones from adipose tissue. Thus, nutrient signaling pathways do not function in isolation. Rather, they appear to serve as components of a larger "metabolic regulatory network" that controls fuel and energy metabolism (at the cell, tissue, and whole-body levels) and links nutrient availability with cell growth and proliferation. Understanding the diverse roles of nutrients and delineating nutrient signaling pathways should facilitate drug discovery research and the search for novel therapeutic compounds to prevent and treat various human diseases such as diabetes, obesity, and cancer.

The Hexosamine Signaling Pathway: Deciphering the "O-GlcNAc Code"

A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. Like phosphorylation, protein O-GlcNAcylation dramatically alters the posttranslational fate and function of target proteins. Indeed, O-GlcNAcylation may compete with phosphorylation for certain Ser/Thr target sites. Like kinases and phosphatases, the enzymes of O-GlcNAc metabolism are highly compartmentalized and regulated. Yet, O-GlcNAc addition is subject to an additional and unique level of metabolic control. O-GlcNAc transfer is the terminal step in a "hexosamine signaling pathway" (HSP). In the HSP, levels of uridine 5'-diphosphate (UDP)-GlcNAc respond to nutrient excess to activate O-GlcNAcylation. Removal of O-GlcNAc may also be under similar metabolic regulation. Differentially targeted isoforms of the enzymes of O-GlcNAc metabolism allow the participation of O-GlcNAc in diverse intracellular functions. O-GlcNAc addition and removal are key to histone remodeling, transcription, proliferation, apoptosis, and proteasomal degradation. This nutrient-responsive signaling pathway also modulates important cellular pathways, including the insulin signaling cascade in animals and the gibberellin signaling pathway in plants. Alterations in O-GlcNAc metabolism are associated with various human diseases including diabetes mellitus and neurodegeneration. This review will focus on current approaches to deciphering the "O-GlcNAc code" in order to elucidate how O-GlcNAc participates in its diverse functions. This ongoing effort requires analysis of the enzymes of O-GlcNAc metabolism, their many targets, and how the O-GlcNAc modification may be regulated.

A Caenorhabditis elegans model of insulin resistance: Altered macronutrient storage and dauer formation in an OGT-1 knockout

O-linked N-acetylglucosamine (O-GlcNAc) is an evolutionarily conserved modification of nuclear pore proteins, signaling kinases, and transcription factors. The O-GlcNAc transferase (OGT) catalyzing O-GlcNAc addition is essential in mammals and mediates the last step in a nutrient-sensing "hexosamine-signaling pathway." This pathway may be deregulated in diabetes and neurodegenerative disease. To examine the function of O-GlcNAc in a genetically amenable organism, we describe a putative null allele of OGT in Caenorhabditis elegans that is viable and fertile. We demonstrate that, whereas nuclear pore proteins of the homozygous deletion strain are devoid of O-GlcNAc, nuclear transport of transcription factors appears normal. However, the OGT mutant exhibits striking metabolic changes manifested in a approximately 3-fold elevation in trehalose levels and glycogen stores with a concomitant approximately 3-fold decrease in triglycerides levels. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity, and dauer formation. The OGT knockout suppresses dauer larvae formation induced by a temperature-sensitive allele of the insulin-like receptor gene daf-2. Our findings demonstrate that OGT modulates macronutrient storage and dauer formation in C. elegans, providing a unique genetic model for examining the role of O-GlcNAc in cellular signaling and insulin resistance.

Turnover and characterization of UDP- N-acetylglucosaminyl transferase in a stably transfected HeLa cell line

To estimate the turnover of UDP- N-acetylglucosaminyl transferase (OGT), we exposed stably transfected HeLa cells to tetracycline for 16 h to induce OGT gene expression and increase cytosolic enzyme levels. Removal of tetracycline led to a progressive decrease in OGT activity (after a 6 h lag period), yielding an estimated OGT half-life of 13 h. A similar half-life (12 h) was obtained by measuring the loss of biosynthetically labeled OGT ([ 35S]methionine pulse-chase experiments). Since OGT turnover was relatively slow, it is unlikely that changes in OGT gene expression or protein expression play a role in the short-term regulatory actions mediated by the hexosamine signaling pathway. We also found that the overexpressed 110 kDa murine OGT subunit (recombinant enzyme) was enzymatically similar to the endogenous holoenzyme derived from rat brain tissue. Thus, stably transfected HeLa cells provide an abundant source of enzyme that can be used to study the structure, function, and regulation of OGT.

Adipocytes with increased hexosamine flux exhibit insulin resistance, increased glucose uptake, and increased synthesis and storage of lipid

The hexosamine signaling pathway has been shown to serve a nutrient-sensing function. We have previously shown that overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine-fructose-6-phosphate amidotransferase) in adipose tissue of transgenic mice results in skeletal muscle insulin resistance and altered regulation of leptin and adiponectin. To dissect the pathways by which the hexosamine pathway affects fuel storage and energy homeostasis, we have examined the characteristics of adipocytes from these animals. After 3 mo of age, epididymal fat pads from adult transgenic animals are 42% heavier (P = 0.003) and individual adipocytes are 23% larger in diameter (P < 0.05) than those from littermate wild-type controls. Isolated adipocytes from transgenic mice are insulin resistant, with a 2.5-fold increase in the ED50 for stimulation of 2-deoxy-D-glucose uptake. However, maximal insulin-stimulated glucose uptake is increased in transgenic adipocytes by 39% (P < 0.05). This upregulation of glucose uptake was associated with a 41% increase in the expression of GLUT4 mRNA and a 28% increase in GLUT4 protein in transgenics compared with controls (P < 0.05). GLUT1 mRNA and protein did not significantly differ between fasted control and transgenics. Total lipid synthesis was also increased in epididymal adipocytes from transgenic animals by 206% compared with controls (P < 0.05). Fatty acid oxidation was increased 1.6-fold in the transgenic adipocytes (P < 0.05). We conclude that the hexosamine signaling pathway upregulates fat storage in adipocytes in states of carbohydrate excess, in part by increasing GLUT4 and glucose uptake and by augmenting fatty acid synthesis.

Activation of the hexosamine signaling pathway in adipose tissue results in decreased serum adiponectin and skeletal muscle insulin resistance.

Overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine:fructose-6-phosphate amidotransferase) in muscle and adipose tissue of transgenic mice was previously shown to result in insulin resistance and hyperleptinemia. Explanted muscle from transgenic mice was not insulin resistant in vitro, suggesting that muscle insulin resistance could be mediated by soluble factors from fat tissue. To dissect the relative contributions of muscle and fat to hexosamine-induced insulin resistance, we overexpressed glutamine:fructose-6-phosphate amidotransferase 2.5-fold, specifically in fat under control of the aP2 promoter. Fasting glucose, insulin, and triglycerides were unchanged in the transgenic mice; leptin and beta-hydroxybutyrate levels were 91% and 29% higher, respectively. Fasted transgenic mice have mild glucose intolerance and skeletal muscle insulin resistance in vivo. In fasting transgenic mice, glucose disposal rates with hyperinsulinemia were decreased 27% in females and 10% in males. Uptake of 2-deoxy-D-glucose into muscle was diminished by 45% in female and 21% in male transgenics. Serum adiponectin was also lower in the fasted transgenics, by 37% in females and 22% in males. TNF alpha and resistin mRNA levels in adipose tissue were not altered in the fasted transgenics; levels of mRNA for leptin were increased and peroxisome proliferator-activated receptor gamma decreased. To further explore the relationship between adiponectin and insulin sensitivity, we examined mice that have been refed for 6 h after a 24-h fast. Refeeding wild-type mice resulted in decreased serum adiponectin and increased leptin. In transgenic mice, however, the regulation of these hormones by refeeding was lost for adiponectin and diminished for leptin. Refed transgenic female and male mice no longer exhibited decreased serum adiponectin in the refed state, and they were no longer insulin resistant as by lower or unchanged insulin and glucose levels. We conclude that increased hexosamine levels in fat, mimicking excess nutrient delivery, are sufficient to cause insulin resistance in skeletal muscle. Changes in serum adiponectin correlate with the insulin resistance of the transgenic animals.