Aerobic Exercise Training Prevents Heart Failure-Induced Skeletal Muscle Atrophy by Anti-Catabolic, but Not Anabolic Actions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resistance training with excessive training load and insufficient recovery alters skeletal muscle mass-related protein expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Células satélite e regeneração muscular

Embora os núcleos das fibras musculares esqueléticas não se dividam, o músculo tem uma pequena capacidade de reconstituição. As células satélites são as responsáveis pela regeneração do músculo esquelético e ajustes induzidos pelo exercício. Células satélites são pequenas células miogênicas mononucleadas, fusiformes, dispostas paralelamente às fibras musculares dentro da lâmina basal que envolve as fibras e só podem ser identificadas no microscópio eletrônico. Essas células contribuem para o crescimento do músculo no embrião e no período pósnatal e são quiescentes no adulto. Têm potencial para, quando ativadas, se diferenciarem em mioblastos, se duplicarem ou migrarem para região lesionada e fundirem-se às células musculares acelerando o processo regenerativo. Vários são os fatores que estimulam essas funções (IGF-I, FGF, citocinas, etc.), sendo que o exercício pode potencializar a produção deles. A regeneração é uma adaptação que ocorre no músculo esquelético em resposta ao traumatismo. Este processo tem sido descrito desde o final do século XIX, mas somente nos últimos trinta anos foi realmente compreendida a capacidade regenerativa das fibras musculares esqueléticas. O reparo tecidual é comum a todos os tecidos do organismo e envolve ações integradas entre células, matriz e mensageiros químicos, visando restaurar, no menor período de tempo possível, a integridade do tecido e o equilíbrio biológico. A regeneração pode ser divida em três fases: fase inflamatória, fase proliferativa e fase de remodelagem. Este trabalho tem como objetivo realizar uma revisão sobre as células satélite e seu papel na regeneração muscular a fim de sistematizar o que pesquisadores de diversas tem publicado sobre o tema atualmente.

Effects of rumen-protected choline supplementation on metabolic and performance responses of transition dairy cows

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estratégias para minimização do estresse oxidativo em sistemas de produção in vitro de embriões bovinos destinados à vitrificação

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glucose,cholesterol,total proteins and insulin-like growth factor typeI values in the follicular fluid of female river buffaloes (Bubalus bubalis)

Twenty six Murrah female river buffaloes, between 45 and 70 d post-partum, empty, multiparae, with an average live weight of 675 ± 56 kg, and average body condition of 3.5 points, in a 1 to 5 scale, were used to determine the concentrations of glucose, cholesterol, total protein and insulin-like growth factor type I(IGF-I) in the follicular fluid. The fluid was collected from dominant follicles, with diameters between 8 and 12 mm, by in vivo follicular aspiration. The oestrous cycle stage was not taken into account. The wave of follicular development was synchronized six days prior to the collection. Biochemical analyses of glucose and cholesterol were performed by the enzymatic colorimetric method with the utilization of commercial kits of Glicose (GOD-PAP) and Cholesterol (CHOD-PAP) (Kovalent), respectively. For the determination of total protein, the commercial kit total Protein (Kovalent), method Biuret, was employed. Readings were carried out through absorption spectrophotometry with visible light. Through the radioimmunoanalysis (RIA) technique the concentration of IGF-I was obtained using commercial kits of IRMA IGF-I (IMMUNOTECH). Descriptive statistics was used, by applying the PROC MEANS procedure of the SAS (2009) statistical package. Glucose concentrations (4.0 ± 0.75 mmol/L) and IGF-I (340 ± 129.83 ng/mL) showed higher values in female river buffaloes and dairy cows regarding those reported in other studies. However, cholesterol levels (0.51 ± 0.12 mmol/L) and total proteins (58.4 ± 4.43 g/L) were lower. Results indicate that there is a relationship between the concentration of biochemical indicators, the nutritional aspects, the diameter of the aspired follicles and the productive period.

Expressão do gene da leptina e seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras

Objetivou-se com este trabalho avaliar os efeitos de uma dieta de alto nível de energia e proteína combinada com a aplicação de bST no perfil de expressão dos genes da leptina e de seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras. Foram utilizadas amostras de parênquima mamário de 32 novilhas holandesas distribuídas aleatoriamente em quatro tratamentos (n=8): dieta com alto ou baixo teor de energia e proteína combinada ou não com a aplicação de bST. O delineamento utilizado foi em blocos casualizados com arranjo de tratamentos em esquema fatorial 2 × 2. A extração do RNA total das amostras de tecido foi feita e o nível de expressão gênica foi analisado por qRT-PCR utilizando-se o gene da glicuronidase β como controle, pelo método 2-ΔΔCt. Animais que receberam a dieta com alto conteúdo de energia e proteína apresentaram maior expressão de mRNA de leptina, com aumento de 56%, e menor expressão de mRNA do receptor Ob-Rb, com redução de 18%. Por outro lado, a aplicação de bST resultou em diminuição da expressão do mRNA de leptina e do receptor Ob-Rb em 74% e 23%, respectivamente. Não houve interação entre dieta e aplicação de bST. O aumento na expressão de leptina pode explicar, ao menos em parte, os efeitos negativos da dieta de alta energia e proteína, oferecida no período pré-púbere, sobre a produção de leite de novilhas leiteiras.

Lyophilized bovine colostrum as a source of immunoglobulins and insulin-like growth factor for newborn goat kids

The objective of this study was to evaluate lyophilized bovine colostrum as an alternative source of passive immunity and insulin like growth factor I (IGF-I) for goat kids, considering newborns consuming non-maternal colostrum. Twenty-nine male newborns received 5% of body weight of lyophilized bovine (LBC) or goat colostrum (GC), both with 55 mg/mL of IgG, at 0, 7 and 14 h of life. Blood samples were collected at 0, 7, 14, 18, 24, 36, 48, 72 and 96 h of life to determine serum IgG, total protein (TP), IGF-I and apparent efficiency of IgG absorption at 7, 14, 18 and 24 h (AEA(7hr), AEA(14hr), AEA(18hr), AEA(total), respectively). In LBC, the values of serum IgG at 14, 18,24 and 48 h (13.1, 13.4, 14.1 and 14.6 mg/mL, respectively) were higher than the values at 0 and 7 h (0.04 and 6.9 mg/mL, respectively). In GC, the serum IgG at 18 h (9.3 mg/mL) was higher than the value at 7 h (5.5 mg/mL). AEA(7hr) and AEA(14hr) in LBC were the same (19.2 and 18.5%, respectively, P>0.05) and the values of AEA(18hr) and AEA(total), 9.3 and 9.5%, respectively, were equal and smaller than AEA(7hr), and AEA(14hr). In GC, AEA(7hr), 20.8%, was higher than AEA(14hr), 16.1% (P<0.05) and AEA(18hr), and AEA(total), 9.2 and 8.0%, respectively, were equal and smaller than AEA(7hr) and AEA(14hr). The serum TP and IGF-I were not affected by colostrum feeding. Considering the variables study in the present work, lyophilized bovine colostrum constitutes a promising alternative substitute to goat colostrum in newborn goat kids, since the supply of immunoglobulins and IGF-I was suitable for the kids. (c) 2012 Elsevier B.V. All rights reserved.

Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt

Goncalves DA, Silveira WA, Lira EC, Gra a FA, Paula-Gomes S, Zanon NM, Kettelhut IC, Navegantes LC. Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt. Am J Physiol Endocrinol Metab 302: E123-E133, 2012. First published September 27, 2011; doi:10.1152/ajpendo.00188.2011.-Although it is well known that administration of the selective beta(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 mu M), a PKA activator. The in vitro addition of triciribine (10 mu M), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

The consequences of growth hormone-releasing hormone receptor haploinsufficiency for bone quality and insulin resistance

Objective Growth hormone (GH)/insulin-like growth factor (IGF) axis and insulin are key determinants of bone remodelling. Homozygous mutations in the GH-releasing hormone receptor (GHRHR) gene (GHRHR) are a frequent cause of genetic isolated GH deficiency (IGHD). Heterozygosity for GHRHR mutation causes changes in body composition and possibly an increase in insulin sensitivity, but its effects on bone quality are still unknown. The objective of this study was to assess the bone quality and metabolism and its correlation with insulin sensitivity in subjects heterozygous for a null mutation in the GHRHR. Patients and methods A cross-sectional study was performed on 76 normal subjects (68.4% females) (N/N) and 64 individuals (64.1% females) heterozygous for a mutation in the GHRHR (MUT/N). Anthropometric features, quantitative ultrasound (QUS) of the heel, bone markers [osteocalcin (OC) and CrossLaps], IGF-I, glucose and insulin were measured, and homeostasis model assessment of insulin resistance (HOMAIR) was calculated. Results There were no differences in age or height between the two groups, but weight (P = 0.007) and BMI (P = 0.001) were lower in MUT/N. There were no differences in serum levels of IGF-I, glucose, T-score or absolute values of stiffness and OC, but insulin (P = 0.01), HOMAIR (P = 0.01) and CrossLaps (P = 0.01) were lower in MUT/N. There was no correlation between OC and glucose, OC and HOMAIR in the 140 individuals as a whole or in the separate MUT/N or N/N groups. Conclusions This study suggests that one allele mutation in the GHRHR gene has a greater impact on energy metabolism than on bone quality.

Pamidronate for the treatment of osteoporosis secondary to chronic cholestatic liver disease in Wistar rats

Osteoporosis is a major complication of chronic cholestatic liver disease (CCLD). We evaluated the efficacy of using disodium pamidronate (1.0 mg/kg body weight) for the prevention (Pr) or treatment (Tr) of cholestasis-induced osteoporosis in male Wistar rats: sham-operated (Sham = 12); bile duct-ligated (Bi = 15); bile duct-ligated animals previously treated with pamidronate before and 1 month after surgery (Pr = 9); bile duct-ligated animals treated with pamidronate 1 month after surgery (Tr = 9). Rats were sacrificed 8 weeks after surgery. Immunohistochemical expression of IGF-I and GH receptor was determined in the proximal growth plate cartilage of the left tibia. Histomorphometric analysis was performed in the right tibia and the right femur was used for biomechanical analysis. Bone material volume over tissue volume (BV/TV) was significantly affected by CCLD (Sham = 18.1 ± 3.2 vs Bi = 10.6 ± 2.2%) and pamidronate successfully increased bone volume. However, pamidronate administered in a preventive regimen presented no additional benefit on bone volume compared to secondary treatment (BV/TV: Pr = 39.4 ± 12.0; Tr = 41.2 ± 12.7%). Moreover, the force on the momentum of fracture was significantly reduced in Pr rats (Sham = 116.6 ± 23.0; Bi = 94.6 ± 33.8; Pr = 82.9 ± 22.8; Tr = 92.5 ± 29.5 N; P < 0.05, Sham vs Pr). Thus, CCLD had a significant impact on bone histomorphometric parameters and pamidronate was highly effective in increasing bone mass in CCLD; however, preventive therapy with pamidronate has no advantage regarding bone fragility.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application.