Multi-Omics after O-GlcNAc Alteration Identifies Cellular Processes Working Synergistically to Promote Aneuploidy.

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address this issue, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. Moreover, these data show how a multi-Omics platform can discern how OGT can synergistically fine-tune multiple cellular pathways.

The essential role of O-GlcNAcylation in hepatic differentiation.

BACKGROUND: O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase, which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and HCC. METHODS: Single-cell RNA-sequencing (RNA-seq) was used to investigate hepatocyte differentiation in hepatocyte-specific O-GlcNAc transferase-knockout (OGT-KO) mice with decreased hepatic O-GlcNAcylation and in O-GlcNAcase-KO mice with increased O-GlcNAcylation in hepatocytes. Patients HCC samples and the diethylnitrosamine-induced HCC model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. RESULTS: Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation, characterized by dysregulation of glycogen storage and glucose production. O-GlcNAc transferase-KO mice exacerbated diethylnitrosamine-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, O-GlcNAcase -KO mice with increased hepatic O-GlcNAcylation inhibited diethylnitrosamine-induced HCC. A progressive loss of O-GlcNAcylation was observed in patients with HCC. CONCLUSIONS: Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC.

Growing and dividing: how O-GlcNAcylation leads the way.

Cell cycle errors can lead to mutations, chromosomal instability, or death; thus, the precise control of cell cycle progression is essential for viability. The nutrient-sensing posttranslational modification, O-GlcNAc, regulates the cell cycle allowing one central control point directing progression of the cell cycle. O-GlcNAc is a single N-acetylglucosamine sugar modification to intracellular proteins that is dynamically added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. These enzymes act as a rheostat to fine-tune protein function in response to a plethora of stimuli from nutrients to hormones. O-GlcNAc modulates mitogenic growth signaling, senses nutrient flux through the hexosamine biosynthetic pathway, and coordinates with other nutrient-sensing enzymes to progress cells through Gap phase 1 (G1). At the G1/S transition, O-GlcNAc modulates checkpoint control, while in S Phase, O-GlcNAcylation coordinates the replication fork. DNA replication errors activate O-GlcNAcylation to control the function of the tumor-suppressor p53 at Gap Phase 2 (G2). Finally, in mitosis (M phase), O-GlcNAc controls M phase progression and the organization of the mitotic spindle and midbody. Critical for M phase control is the interplay between OGT and OGA with mitotic kinases. Importantly, disruptions in OGT and OGA activity induce M phase defects and aneuploidy. These data point to an essential role for the O-GlcNAc rheostat in regulating cell division. In this review, we highlight O-GlcNAc nutrient sensing regulating G1, O-GlcNAc control of DNA replication and repair, and finally, O-GlcNAc organization of mitotic progression and spindle dynamics.

Inhibition of O-GlcNAcylation Reduces Cell Viability and Autophagy and Increases Sensitivity to Chemotherapeutic Temozolomide in Glioblastoma.

Glioblastoma (GB) is the most aggressive primary malignant brain tumor and is associated with short survival. O-GlcNAcylation is an intracellular glycosylation that regulates protein function, enzymatic activity, protein stability, and subcellular localization. Aberrant O-GlcNAcylation is related to the tumorigenesis of different tumors, and mounting evidence supports O-GlcNAc transferase (OGT) as a potential therapeutic target. Here, we used two human GB cell lines alongside primary human astrocytes as a non-tumoral control to investigate the role of O-GlcNAcylation in cell proliferation, cell cycle, autophagy, and cell death. We observed that hyper O-GlcNAcylation promoted increased cellular proliferation, independent of alterations in the cell cycle, through the activation of autophagy. On the other hand, hypo O-GlcNAcylation inhibited autophagy, promoted cell death by apoptosis, and reduced cell proliferation. In addition, the decrease in O-GlcNAcylation sensitized GB cells to the chemotherapeutic temozolomide (TMZ) without affecting human astrocytes. Combined, these results indicated a role for O-GlcNAcylation in governing cell proliferation, autophagy, cell death, and TMZ response, thereby indicating possible therapeutic implications for treating GB. These findings pave the way for further research and the development of novel treatment approaches which may contribute to improved outcomes and increased survival rates for patients facing this challenging disease.

O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer's disease.

Introduction: Aberrant activation of Extracellular Signal-Regulated Kinase (ERK) signaling is associated with Alzheimer's disease (AD) pathogenesis. For example, enhanced ERK signal activation mediated by Apolipoprotein E4 (APOE4), which is a critical genetic risk factor for AD, increases the transcription of amyloid precursor protein (APP). We hypothesize that O-linked N-acetylglucosamine (O-GlcNAc) regulates the phosphorylation and activation of ERK. O-GlcNAc is a single sugar post-translational modification that dynamically cycles on and off proteins in response to nutrient changes by the action of the enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, O-GlcNAc quickly returns to a baseline level after stimulus removal (called O-GlcNAc homeostasis). Methods: We did a serum reactivation time-course followed by western blot in SH-SY5Y neuroblastoma cells after long-term O-GlcNAcase (OGA) inhibition by Thiamet-G (TMG) treatment, O-GlcNAc transferase (OGT) knock-down (KD) and OGA KD. Brain tissues of C57BL6/J mice and 5XFAD Alzheimer's disease mice intra-peritoneally injected with TMG for 1 month and C57BL6/J mice intra-peritoneally injected with TMG for 6 months were also used for western blot. Results: We found that ERK1/2 phosphorylation at Thr 202/Tyr204 and Thr183/Tyr185 (p-ERK) are amplified and hence ERK1/2 are activated after long-term OGA inhibition in SH-SY5Y cells. In addition to pharmacological treatment, genetic disruption of O-GlcNAc by OGT KD and OGA KD also increased p-ERK in SH-SY5Y cells suggesting O-GlcNAc homeostasis controls ERK signaling. To determine how O-GlcNAc regulates p-ERK, we probed the expression of phosphorylated mitogen-activated protein kinase-kinase (p-MEK) which phosphorylates and activates ERK and Dual specificity phosphatase-4 (DUSP4) which dephosphorylates and inactivates ERK in SH-SY5Y cells. p-MEK increases in TMG treated and OGT KD cells whereas total DUSP4 decreases in OGT KD and OGA KD cells with serum reactivation time course. Next, we probed the role of OGA inhibition in regulating ERK activation using mice brain-tissue samples. Interestingly, 6-month intra-peritoneal TMG injection in C57BL/6J mice showed an increase in amplitude of p-ERK and APP protein levels, indicating long-term OGA inhibition potentially contributes to AD progression. Furthermore, 1-month TMG injection was sufficient to increase the amplitude of p-ERK in 5XFAD AD mice brains suggesting AD phenotype contributes to the acceleration of ERK activation mediated by OGA inhibition. Conclusion: Together, these results indicate that disruptions to O-GlcNAc homeostasis amplify ERK signal activation in AD.

AMEND: active module identification using experimental data and network diffusion.

BACKGROUND: Molecular interaction networks have become an important tool in providing context to the results of various omics experiments. For example, by integrating transcriptomic data and protein-protein interaction (PPI) networks, one can better understand how the altered expression of several genes are related with one another. The challenge then becomes how to determine, in the context of the interaction network, the subset(s) of genes that best captures the main mechanisms underlying the experimental conditions. Different algorithms have been developed to address this challenge, each with specific biological questions in mind. One emerging area of interest is to determine which genes are equivalently or inversely changed between different experiments. The equivalent change index (ECI) is a recently proposed metric that measures the extent to which a gene is equivalently or inversely regulated between two experiments. The goal of this work is to develop an algorithm that makes use of the ECI and powerful network analysis techniques to identify a connected subset of genes that are highly relevant to the experimental conditions. RESULTS: To address the above goal, we developed a method called Active Module identification using Experimental data and Network Diffusion (AMEND). The AMEND algorithm is designed to find a subset of connected genes in a PPI network that have large experimental values. It makes use of random walk with restart to create gene weights, and a heuristic solution to the Maximum-weight Connected Subgraph problem using these weights. This is performed iteratively until an optimal subnetwork (i.e., active module) is found. AMEND was compared to two current methods, NetCore and DOMINO, using two gene expression datasets. CONCLUSION: The AMEND algorithm is an effective, fast, and easy-to-use method for identifying network-based active modules. It returned connected subnetworks with the largest median ECI by magnitude, capturing distinct but related functional groups of genes. Code is freely available at https://github.com/samboyd0/AMEND .

The Essential Role of O-GlcNAcylation in Hepatic Differentiation.

Background & Aims: O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase (OGT), which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and hepatocellular carcinoma. Methods: Single-cell RNA-sequencing was used to investigate hepatocyte differentiation in hepatocyte-specific OGT-KO mice with increased hepatic O-GlcNAcylation and in OGA-KO mice with decreased O-GlcNAcylation in hepatocytes. HCC patient samples and the DEN-induced hepatocellular carcinoma (HCC) model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. Results: Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation characterized by dysregulation of glycogen storage and glucose production. OGT-KO mice exacerbated DEN-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, OGA-KO mice with increased hepatic O-GlcNAcylation inhibited DEN-induced HCC. A progressive loss of O-GlcNAcylation was observed in HCC patients. Conclusions: Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC.

O-GlcNAc regulates the mitochondrial integrated stress response by regulating ATF4.

Background: Accumulation of mitochondrial dysfunctional is a hallmark of age-related neurodegeneration including Alzheimer's disease (AD). Impairment of mitochondrial quality control mechanisms leading to the accumulation of damaged mitochondria and increasing neuronal stress. Therefore, investigating the basic mechanisms of how mitochondrial homeostasis is regulated is essential. Herein, we investigate the role of O-GlcNAcylation, a single sugar post-translational modification, in controlling mitochondrial stress-induced transcription factor Activating Transcription Factor 4 (ATF4). Mitochondrial dysfunction triggers the integrated stress response (ISRmt), in which the phosphorylation of eukaryotic translation initiation factor 2α results in the translation of ATF4. Methods: We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma and HeLa cell-lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) on ISRmt using biochemical analyses. Results: We show that TMG elevates ATF4 protein levels upon mitochondrial stress in SH-SY5Y neuroblastoma and HeLa cell-lines. An indirect downstream target of ATF4 mitochondrial chaperone glucose-regulated protein 75 (GRP75) is significantly elevated. Interestingly, knock-down of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, in SH-SY5Y increases ATF4 protein and mRNA expression. Additionally, ATF4 target gene Activating Transcription Factor 5 (ATF5) is significantly elevated at both the protein and mRNA level. Brains isolated from TMG treated mice show elevated levels of ATF4 and GRP75. Importantly, ATF4 occupancy increases at the ATF5 promoter site in brains isolated from TMG treated mice suggesting that O-GlcNAc is regulating ATF4 targeted gene expression. Interestingly, ATF4 and GRP75 are not induced in TMG treated familial Alzheimer's Disease mice model. The same results are seen in a human in vitro model of AD. Conclusion: Together, these results indicate that in healthy conditions, O-GlcNAc regulates the ISRmt through regulating ATF4, while manipulating O-GlcNAc in AD has no effect on ISRmt.

Mapping the O-GlcNAc Modified Proteome: Applications for Health and Disease.

O-GlcNAc is a pleotropic, enigmatic post-translational modification (PTM). This PTM modifies thousands of proteins differentially across tissue types and regulates diverse cellular signaling processes. O-GlcNAc is implicated in numerous diseases, and the advent of O-GlcNAc perturbation as a novel class of therapeutic underscores the importance of identifying and quantifying the O-GlcNAc modified proteome. Here, we review recent advances in mass spectrometry-based proteomics that will be critical in elucidating the role of this unique glycosylation system in health and disease.

Ttc21b deficiency attenuates autosomal dominant polycystic kidney disease in a kidney tubular- and maturation-dependent manner.

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked ß-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.

Regulation of Liver Regeneration by Hepatocyte O-GlcNAcylation in Mice.

BACKGROUND & AIMS: The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here, we report that O-GlcNAcylation, an intracellular post-translational modification regulated by 2 enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration following partial hepatectomy (PHX). METHODS: We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively. RESULTS: OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia, and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-sequencing studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and immunohistochemistry analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes. CONCLUSIONS: These studies show that O-GlcNAcylation plays a critical role in the termination of liver regeneration via regulation of HNF4α in hepatocytes.

O-GlcNAc cycling mediates energy balance by regulating caloric memory.

Caloric need has long been thought a major driver of appetite. However, it is unclear whether caloric need regulates appetite in environments offered by many societies today where there is no shortage of food. Here we observed that wildtype mice with free access to food did not match calorie intake to calorie expenditure. While the size of a meal affected subsequent intake, there was no compensation for earlier under- or over-consumption. To test how spontaneous eating is subject to caloric control, we manipulated O-linked ß-N-acetylglucosamine (O-GlcNAc), an energy signal inside cells dependent on nutrient access and metabolic hormones. Genetic and pharmacological manipulation in mice increasing or decreasing O-GlcNAcylation regulated daily intake by controlling meal size. Meal size was affected at least in part due to faster eating speed. Without affecting meal frequency, O-GlcNAc disrupted the effect of caloric consumption on future intake. Across days, energy balance was improved upon increased O-GlcNAc levels and impaired upon removal of O-GlcNAcylation. Rather than affecting a perceived need for calories, O-GlcNAc regulates how a meal affects future intake, suggesting that O-GlcNAc mediates a caloric memory and subsequently energy balance.

The Role of O-GlcNAcylation in Immune Cell Activation.

O-GlcNAcylation is a dynamic post-translational modification where the sugar, O-linked ß-N-acetylglucosamine (O-GlcNAc) is added to or removed from various cytoplasmic, nuclear, and mitochondrial proteins. This modification is regulated by only two enzymes: O-GlcNAc transferase (OGT), which adds O-GlcNAc, and O-GlcNAcase (OGA), which removes the sugar from proteins. O-GlcNAcylation is integral to maintaining normal cellular function, especially in processes such as nutrient sensing, metabolism, transcription, and growth and development of the cell. Aberrant O-GlcNAcylation has been associated with a number of pathological conditions, including, neurodegenerative diseases, cancer, diabetes, and obesity. However, the role of O-GlcNAcylation in immune cell growth/proliferation, or other immune responses, is currently incompletely understood. In this review, we highlight the effects of O-GlcNAcylation on certain cells of the immune system, especially those involved in pro-inflammatory responses associated with diabetes and obesity.

O-GlcNAcylation and O-GlcNAc Cycling Regulate Gene Transcription: Emerging Roles in Cancer.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a single sugar post-translational modification (PTM) of intracellular proteins linking nutrient flux through the Hexosamine Biosynthetic Pathway (HBP) to the control of cis-regulatory elements in the genome. Aberrant O-GlcNAcylation is associated with the development, progression, and alterations in gene expression in cancer. O-GlcNAc cycling is defined as the addition and subsequent removal of the modification by O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) provides a novel method for cells to regulate various aspects of gene expression, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. We will focus on the complex relationship between phosphorylation and O-GlcNAcylation in the regulation of the RNA Polymerase II (RNAP II) pre-initiation complex and the regulation of the carboxyl-terminal domain of RNAP II via the synchronous actions of OGT, OGA, and kinases. Additionally, we discuss how O-GlcNAcylation of TATA-box binding protein (TBP) alters cellular metabolism. Next, in a non-exhaustive manner, we will discuss the current literature on how O-GlcNAcylation drives gene transcription in cancer through changes in transcription factor or chromatin remodeling complex functions. We conclude with a discussion of the challenges associated with studying O-GlcNAcylation and present several new approaches for studying O-GlcNAc regulated transcription that will advance our understanding of the role of O-GlcNAc in cancer.

Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy.

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.

OGA Inhibition Alters Energetics and Nutrient Sensing in Alzheimer's Disease Cytoplasmic Hybrids.

BACKGROUND: Alzheimer's disease (AD) features reductions in key bioenergetic fluxes and perturbed mitochondrial function. Cytoplasmic hybrids (cybrids) generated through the transfer of AD subject mitochondria to mtDNA-depleted SH-SY5Y neuroblastoma cells recapitulate some of these features in an in vitro setting. OBJECTIVE: For this study, we used the AD cybrid model to assess the impact of a nutrient-excess like-state via increasing O-GlcNAcylation on whole cell and mitochondrial homeostasis. METHODS: We induced increased O-GlcNAc by treating AD and control cybrid cell lines with Thiamet G (TMG), an inhibitor of the O-GlcNAcase enzyme that mediates removal of the nutrient-dependent O-GlcNAc modification. RESULTS: Relative to control cybrid cell lines, AD cybrid lines showed a blunted response to TMG-induced O-GlcNAcylation. At baseline, AD cybrid cell line mitochondria showed partial activation of several proteins that help maintain bioenergetic homeostasis such as AMP-Regulated Kinase suggesting that AD mitochondria initiate a state of nutrient stress promoting energetic compensation; however, this compensation reduces the capacity of cells to respond to additional nutrient-related stresses such as TMG treatment. Also, TMG caused disruptions in acetylation and Sirtuin 3 expression, while lowing total energetic output of the cell. CONCLUSION: Together, these findings suggest that modulation of O-GlcNAc is essential for proper energetic function of the mitochondria, and AD mitochondrial capacity to handle nutrient-excess is limited.