ß-Catenin-Driven Differentiation Is a Tissue-Specific Epigenetic Vulnerability in Adrenal Cancer.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.

Identification of HDAC4 as a potential therapeutic target and prognostic biomarker for ZFTA-fused ependymomas.

Members of the HDAC family are predictive biomarkers and regulate the tumorigenesis in several cancers. However, the role of these genes in the biology of intracranial ependymomas (EPNs) remains unexplored. Here, an analysis of eighteen HDACs genes in an EPN transcriptomic dataset, revealed significantly higher levels of HDAC4 in supratentorial ZFTA fusion (ST-ZFTA) compared with ST-YAP1 fusion and posterior fossa EPNs, while HDAC7 and SIRT2 were downregulated in ST-ZFTA. HDAC4 was also overexpressed in ST-ZFTA as measured by single-cell RNA-Seq, quantitative real time-polymerase chain reaction, and immunohistochemistry. Survival analyses showed a significantly worse outcome for EPNs with higher HDAC4 and SIRT1 mRNA levels. Ontology enrichment analysis showed an HDAC4-high signature consistent with viral processes while collagen-containing extracellular matrix and cell-cell junction were enriched in those with an HDAC4-low signature. Immune gene analysis demonstrated a correlation between HDAC4 expression and low levels of NK resting cells. Several small molecules compounds targeting HDAC4 and ABCG2, were predicted by in silico analysis to be effective against HDAC4-high ZFTA. Our results provide novel insights into the biology of the HDAC family in intracranial ependymomas and reveal HDAC4 as a prognostic marker and potential therapeutic target in ST-ZFTA.

Orbicularis Oculi Muscle Immunohistochemical, Metabolic, and Morphometric Differences in Affected and Nonaffected Sides in Hemifacial Spasm vs Healthy Subjects.

BACKGROUND: Subtle morphological alterations have been reported even in the nonaffected side of the orbicularis oculi muscle in patients with hemifacial spasm. However, no previous study assessed immunohistochemical, metabolic, and morphometric alterations in orbicularis oculi muscle fibers in affected and nonaffected sides in patients with this condition, compared with samples obtained from healthy subjects. The purpose of this study is to objectively assess and compare orbicularis oculi muscle (OOM) samples of hemifacial spasm affected and nonaffected sides and healthy subjects. METHODS: Orbicularis oculi samples from 8 patients with hemifacial spasm who had not been previously treated and 6 healthy subjects were prepared using hematoxylin and eosin, nicotinamide adenine dinucleotide tetrazolium reductase, cytochrome oxidase, succinate dehydrogenase, Gomori staining, and monoclonal antibodies against myosin slow and myosin fast. A digital image analysis software was used for objective analysis. RESULTS: OOM fiber area was significantly greater in both affected ( P = 0.0379) and nonaffected sides ( P = 0.0012) of HFS samples when compared with control subjects' fibers. A significantly greater number of oxidative fibers were observed in both affected and nonaffected sides of patients with HFS when compared with control subjects ( P < 0.001 for both). A significantly greater percentage of slow fibers was observed in the affected side of HFS patients ( P = 0.0012) compared with control subjects. CONCLUSIONS: This study's findings suggest that repeated contractions might lead to OOM fiber hypertrophy, increased mitochondrial metabolism, and possible conversion of fast-twitch orbicularis oculi muscle fibers into slow-twitch fibers in patients with HFS. Alterations were observed in affected and nonaffected sides, confirming initial findings that the nonaffected side is not normal in this unique condition.

Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles.

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG-a human glioblastoma-derived cell lineage-reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/ß-catenin signaling pathways, in addition to upregulation of genes related to epithelial-mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

The chromatin remodeler complex ATRX-DAXX-H3.3 and telomere length in meningiomas.

ATRX-DAXX-H3.3 chromatin remodeler complex is a well known epigenetic factor responsible for the heterochromatin maintenance and control. ATRX is an important nucleosome controller, especially in tandem repeat regions, and DAXX is a multi-function protein with particular role in histone H3.3 deposition due to its chaperone characteristic. Abnormalities in this complex have been associated with telomere dysfunction and consequently with activation of alternative lengthening of telomeres mechanism, genomic instability, and tumor progression in different types of cancer. However, the characterization of this complex is still incomplete in meningioma. We analyzed ATRX, DAXX and H3.3 expressions and the telomere length in a cohort of meningioma of different malignant grades. We observed ATRX upregulation at gene and protein levels in grade II/III meningiomas. A low variability of telomere length was observed in meningiomas across different ages and malignant grades, in contrast to the shortening of telomere length with aging in normal controls.

Extracellular Matrix Proteome Remodeling in Human Glioblastoma and Medulloblastoma.

Medulloblastomas (MBs) and glioblastomas (GBMs) are high-incidence central nervous system tumors. Different origin sites and changes in the tissue microenvironment have been associated with the onset and progression. Here, we describe differences between the extracellular matrix (ECM) signatures of these tumors. We compared the proteomic profiles of MB and GBM decellularized tumor samples between each other and their normal decellularized brain site counterparts. Our analysis revealed that 19, 28, and 11 ECM proteins were differentially expressed in MBs, GBMs, and in both MBs and GBMs, respectively. Next, we validated key findings by using a protein tissue array with 53 MB and 55 GBM cases and evaluated the clinical relevance of the identified differentially expressed proteins through their analysis on publicly available datasets, 763 MB samples from the GSE50161 and GSE85217 studies, and 115 GBM samples from RNAseq-TCGA. We report a shift toward a denser fibrillary ECM as well as a clear alteration in the glycoprotein signature, which influences the tumor pathophysiology. MS data have been submitted to the PRIDE repository, project accession: PXD023350.

LOXL3 Silencing Affected Cell Adhesion and Invasion in U87MG Glioma Cells.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.

Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes.

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. Here, we analyzed TLR4 expression in different grades of astrocytoma, and observed increased expression in tumors, mainly in GBM, compared to non-neoplastic brain tissue. TLR4 role was investigated in U87MG, a GBM mesenchymal subtype cell line, upon LPS stimulation. p65 nuclear translocation was observed in late phase, suggesting TLR4-non-canonical pathway activation. In fact, components of ripoptosome and inflammasome cascades were upregulated and they were significantly correlated in GBMs of the TCGA-RNASeq dataset. Moreover, an increased apoptotic rate was observed when the GBM-derived U87MG cells were co-treated with LPS and Temozolomide (TMZ) in comparison to TMZ alone. Increased TLR4 immunostaining was detected in nuclei of U87MG cells 12 h after LPS treatment, concomitant to activation of DNA repair genes. Time-dependent increased RAD51, FEN1 and UNG expression levels were confirmed after LPS stimulation, which may contribute to tumor cell fitness. Moreover, the combined treatment with the RAD51 inhibitor, Amuvatinib in combination with, TMZ after LPS stimulation reduced tumor cell viability more than with each treatment alone. In conclusion, our results suggest that stimulation of TLR4 combined with pharmacological inhibition of the DNA repair pathway may be an alternative treatment for GBM patients.

The effects of postmortem delay on mouse and human microglia gene expression.

Microglia are specialized macrophages of the central nervous system (CNS) and first to react to pathogens or injury. Over the last decade, transcriptional profiling of microglia significantly contributed to our understanding of their functions. In the case of human CNS samples, either potential CNS pathology in the case of surgery samples, or a postmortem delay (PMD) due to the time needed for tissue access and collection, are potential factors that affect gene expression profiles. To determine the effect of PMD on the microglia transcriptome, we first analyzed mouse microglia, where genotype, antemortem conditions and PMD can be controlled. Microglia were isolated from mice after different PMDs (0, 4, 6, 12, and 24 hr) using fluorescence-activated cell sorting (FACS). The number of viable microglia significantly decreased with increasing PMD, but even after a 12 hr PMD, high-quality RNA could be obtained. PMD had very limited effect on mouse microglia gene expression, only 50 genes were differentially expressed between different PMDs. These genes were related to mitochondrial, ribosomal, and protein binding functions. In human microglia transcriptomes we previously generated, 31 of the 50 PMD-associated mouse genes had human homologs, and their relative expression was also affected by PMD. This study provides a set of genes that shows relative expression changes in relation to PMD, both in mouse and human microglia. Although the gene expression changes detected are subtle, these genes need to be accounted for when analyzing microglia transcriptomes generated from samples with variable PMDs.

Urinary Sediment Transcriptomic and Longitudinal Data to Investigate Renal Function Decline in Type 1 Diabetes.

Introduction: Using a discovery/validation approach we investigated associations between a panel of genes selected from a transcriptomic study and the estimated glomerular filtration rate (eGFR) decline across time in a cohort of type 1 diabetes (T1D) patients. Experimental: Urinary sediment transcriptomic was performed to select highly modulated genes in T1D patients with rapid eGFR decline (decliners) vs. patients with stable eGFR (non-decliners). The selected genes were validated in samples from a T1D cohort (n = 54, mean diabetes duration of 21 years, 61% women) followed longitudinally for a median of 12 years in a Diabetes Outpatient Clinic. Results: In the discovery phase, the transcriptomic study revealed 158 genes significantly different between decliners and non-decliners. Ten genes increasingly up or down-regulated according to renal function worsening were selected for validation by qRT-PCR; the genes CYP4F22, and PMP22 were confirmed as differentially expressed comparing decliners vs. non-decliners after adjustment for potential confounders. CYP4F22, LYPD3, PMP22, MAP1LC3C, HS3ST2, GPNMB, CDH6, and PKD2L1 significantly modified the slope of eGFR in T1D patients across time. Conclusions: Eight genes identified as differentially expressed in the urinary sediment of T1D patients presenting different eGFR decline rates significantly increased the accuracy of predicted renal function across time in the studied cohort. These genes may be a promising way of unveiling novel mechanisms associated with diabetic kidney disease progression.

Orbicularis Oculi Morphological Alterations in Affected and Nonaffected Sides in Hemifacial Spasm.

BACKGROUND: Although the nonaffected side appears to be clinically normal in hemifacial spasm (HFS), it is not known whether this side can be considered normal regarding histopathological findings. The purpose of this study was to objectively evaluate and compare orbicularis oculi samples of patients with HFS (not previously treated with botulinum toxin) and control patients undergoing cosmetic upper eyelid blepharoplasty. METHODS: Orbicularis oculi samples from 22 eyelids were evaluated. There were 7 samples from the affected and 7 samples from the nonaffected sides of patients with HFS who had not been previously treated with botulinum toxin, and 8 samples from normal control patients. Muscle samples were prepared using hematoxylin and eosin staining, and a digital image analysis software was used for objective analyses. RESULTS: When compared with normal controls, endomysial and perimysial connective tissue areas were significantly increased (P = 0.015) on the affected side in HFS, suggesting that this disorder is associated with chronic alterations that lead to muscle degeneration. Cell density was significantly reduced on the affected (P = 0.028) and also on the nonaffected sides in HFS (P = 0.003) compared with normal controls. This was observed, although, clinically, there were no signs or symptoms of increased muscular contraction on the nonaffected sides in any of the patients with HFS studied. CONCLUSIONS: Significant morphological differences in the orbicularis oculi muscle in patients with HFS were observed on both the affected and nonaffected sides. Our findings suggest a potential role for muscle homeostasis disturbances on both sides for patients with HFS. Affected sides in patients with HFS did, however, demonstrate muscle degeneration that was not present on the nonaffected sides.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

ATRX-DAXX Complex Expression Levels and Telomere Length in Normal Young and Elder Autopsy Human Brains.

The chromatin-remodeling complex ATRX/DAXX is one of the major epigenetic factors that controls heterochromatin maintenance due to its role in histone deposition. ATRX is involved in nucleosome configuration and maintenance of higher order chromatin structure, and DAXX is a specific histone chaperone for H3.3 deposition. Dysfunctions in this complex have been associated with telomere shortening, which influences cell senescence. However, data about this complex in brain tissue related to aging are still scarce. Therefore, in the present study, we analyzed ATRX and DAXX expressions in autopsied human brain specimens and the telomere length. A significant decrease in gene and protein expressions was observed in the brain tissues from the elderly compared with those from the young, which were related to short telomeres. These findings may motivate further functional analysis to confirm the ATRX-DAXX complex involvement in telomere maintenance and brain aging.

CD99 Expression in Glioblastoma Molecular Subtypes and Role in Migration and Invasion.

Glioblastoma (GBM) is the most aggressive type of brain tumor, with an overall survival of 17 months under the current standard of care therapy. CD99, an over-expressed transmembrane protein in several malignancies, has been considered a potential target for immunotherapy. To further understand this potentiality, we analyzed the differential expression of its two isoforms in human astrocytoma specimens, and the CD99 involved signaling pathways in glioma model U87MG cell line. CD99 was also analyzed in GBM molecular subtypes. Whole transcriptomes by RNA-Seq of CD99-siRNA, and functional in vitro assays in CD99-shRNA, that are found in U87MG cells, were performed. Astrocytoma of different malignant grades and U87MG cells only expressed CD99 isoform 1, which was higher in mesenchymal and classical than in proneural GBM subtypes. Genes related to actin dynamics, predominantly to focal adhesion, and lamellipodia/filopodia formation were down-regulated in the transcriptome analysis, when CD99 was silenced. A decrease in tumor cell migration/invasion, and dysfunction of focal adhesion, were observed in functional assays. In addition, a striking morphological change was detected in CD99-silenced U87MG cells, further corroborating CD99 involvement in actin cytoskeleton rearrangement. Inhibiting the overexpressed CD99 may improve resectability and decrease the recurrence rate of GBM by decreasing tumor cells migration and invasion.

Targeted Assessment of G0S2 Methylation Identifies a Rapidly Recurrent, Routinely Fatal Molecular Subtype of Adrenocortical Carcinoma.

PURPOSE: Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with few therapies; however, patients with locoregional disease have variable outcomes. The Cancer Genome Atlas project on ACC (ACC-TCGA) identified that cancers of patients with homogeneously rapidly recurrent or fatal disease bear a unique CpG island hypermethylation phenotype, "CIMP-high." We sought to identify a biomarker that faithfully captures this subgroup.Experimental Design: We analyzed ACC-TCGA data to characterize differentially regulated biological processes, and identify a biomarker that is methylated and silenced exclusively in CIMP-high ACC. In an independent cohort of 114 adrenocortical tumors (80 treatment-naive primary ACC, 22 adrenocortical adenomas, and 12 non-naive/nonprimary ACC), we evaluated biomarker methylation by a restriction digest/qPCR-based approach, validated by targeted bisulfite sequencing. We evaluated expression of this biomarker and additional prognostic markers by qPCR. RESULTS: We show that CIMP-high ACC is characterized by upregulation of cell cycle and DNA damage response programs, and identify that hypermethylation and silencing of G0S2 distinguishes this subgroup. We confirmed G0S2 hypermethylation and silencing is exclusive to 40% of ACC, and independently predicts shorter disease-free and overall survival (median 14 and 17 months, respectively). Finally, G0S2 methylation combined with validated molecular markers (BUB1B-PINK1) stratifies ACC into three groups, with uniformly favorable, intermediate, and uniformly dismal outcomes. CONCLUSIONS: G0S2 hypermethylation is a hallmark of rapidly recurrent or fatal ACC, amenable to targeted assessment using routine molecular diagnostics. Assessing G0S2 methylation is straightforward, feasible for clinical decision-making, and will enable the direction of efficacious adjuvant therapies for patients with aggressive ACC.

Serum interleukin-17A level is associated with disease activity of adult patients with dermatomyositis and polymyositis.

OBJECTIVES: To assess serum interleukin (IL)-17A levels in patients with dermatomyositis (DM) and polymyositis (PM) and correlate them with the demographic, clinical, laboratory and therapeutic data of these diseases. METHODS: This was a cross-sectional, single-centre study that included defined DM and PM patients who were age-, gender- and ethnicity-matched to healthy individuals. Serum IL-17A analysis, as well as analysis for other cytokines (IL-6, TNFα and IFNγ), was performed by multiplex immunoassay. The disease status parameters were based on the International Myositis Assessment and Clinical Studies Group (IMACS) set scores. RESULTS: Eighty DM, 32 PM patients and 104 healthy individuals were enrolled. Mean age of patients with DM and PM was 46.0 and 47.7, respectively, with a predominance of women and white ethnicity in both groups. Overall, clinical, laboratory, therapeutic, and current disease status were similar among patients with DM and PM. Median serum IL-17A level was higher in patients with PM and DM than the control group (0.73 vs. 0.49 vs. 0.35 pg/mL, respectively; p<0.050) and higher in PM when compared to DM (p<0.001). In DM, serum IL-17A levels were associated with cumulative cutaneous lesions, IMACS parameters, and serum IL-6 and IFNγ levels. In PM, serum IL-17A levels correlated with patients' current age, IMACS parameters and serum TNFα and IFNγ levels. CONCLUSIONS: Serum IL-17A levels are not only increased, but also associated with disease activity in patients with DM and PM. Our data strongly suggest that IL-17A may be a biomarker of disease activity for these systemic autoimmune myopathies.

Mitochondria Transcription Factor A: A Putative Target for the Effect of Melatonin on U87MG Malignant Glioma Cell Line.

The disruption of mitochondrial activity has been associated with cancer development because it contributes to regulating apoptosis and is the main source of reactive oxygen species (ROS) production. Mitochondrial transcription factor A (TFAM) is a protein that maintains mitochondrial DNA (mtDNA) integrity, and alterations in its expression are associated with mitochondrial damage and cancer development. In addition, studies have shown that mitochondria are a known target of melatonin, the pineal gland hormone that plays an important anti-tumorigenic role. Thus, we hypothesized that melatonin decreases the expression of TFAM (RNA and protein) in the human glioblastoma cell line U87MG, which disrupts mtDNA expression and results in cell death due to increased ROS production and mitochondrial damage. Our results confirm the hypothesis, and also show that melatonin reduced the expression of other mitochondrial transcription factors mRNA (TFB1M and TFB2M) and interfered with mtDNA transcription. Moreover, melatonin delayed cell cycle progression and potentiated the reduction of cell survival due to treatment with the chemotherapeutic agent temozolomide. In conclusion, elucidating the effect of melatonin on TFAM expression should help to understand the signaling pathways involved in glioblastoma progression, and melatonin could be potentially applied in the treatment of this type of brain tumor.

N-acetylcysteine Counteracts Adipose Tissue Macrophage Infiltration and Insulin Resistance Elicited by Advanced Glycated Albumin in Healthy Rats.

Background: Advanced glycation endproducts elicit inflammation. However, their role in adipocyte macrophage infiltration and in the development of insulin resistance, especially in the absence of the deleterious biochemical pathways that coexist in diabetes mellitus, remains unknown. We investigated the effect of chronic administration of advanced glycated albumin (AGE-albumin) in healthy rats, associated or not with N-acetylcysteine (NAC) treatment, on insulin sensitivity, adipose tissue transcriptome and macrophage infiltration and polarization. Methods: Male Wistar rats were intraperitoneally injected with control (C) or AGE-albumin alone, or, together with NAC in the drinking water. Biochemical parameters, lipid peroxidation, gene expression and protein contents were, respectively, determined by enzymatic techniques, reactive thiobarbituric acid substances, RT-qPCR and immunohistochemistry or immunoblot. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/mass spectrometry (LC-MS/MS) and ELISA. Results: CML and PYR were higher in AGE-albumin as compared to C. Food consumption, body weight, systolic blood pressure, plasma lipids, glucose, hepatic and renal function, adipose tissue relative weight and adipocyte number were similar among groups. In AGE-treated animals, insulin resistance, adipose macrophage infiltration and Col12a1 mRNA were increased with no changes in M1 and M2 phenotypes as compared to C-albumin-treated rats. Total GLUT4 content was reduced by AGE-albumin as compared to C-albumin. NAC improved insulin sensitivity, reduced urine TBARS, adipose macrophage number and Itgam and Mrc mRNA and increased Slc2a4 and Ppara. CD11b, CD206, Ager, Ddost, Cd36, Nfkb1, Il6, Tnf, Adipoq, Retn, Arg, and Il12 expressions were similar among groups. Conclusions: AGE-albumin sensitizes adipose tissue to inflammation due to macrophage infiltration and reduces GLUT4, contributing to insulin resistance in healthy rats. NAC antagonizes AGE-albumin and prevents insulin resistance. Therefore, it may be a useful tool in the prevention of AGE action on insulin resistance and long-term complications of DM.