RESUMEN
OBJECTIVE: The lactation-suckling period is critical for white adipose tissue (WAT) development. Early postnatal nutrition influences later obesity risk but underlying mechanisms remain elusive. Here, we tested whether altered postnatal nutrition specifically during suckling impacts epigenetic regulation of key metabolic genes in WAT and alter long-term adiposity set point. METHODS: We analyzed the effects of maternal high-fat (HF) feeding in rats exclusively during lactation-suckling on breast milk composition and its impact on male offspring visceral epidydimal (eWAT) and subcutaneous inguinal (iWAT) depots during suckling and in adulthood. RESULTS: Maternal HF feeding during lactation had no effect on mothers' body weight (BW) or global breast milk composition, but induced qualitative changes in breast milk fatty acid (FA) composition (high n-6/n-3 polyunsaturated FA ratio and low medium-chain FA content). During suckling, HF neonates showed increased BW and mass of both eWAT and iWAT depot but only eWAT displayed an enhanced adipogenic transcriptional signature. In adulthood, HF offspring were predisposed to weight gain and showed increased hyperplastic growth only in eWAT. This specific eWAT expansion was associated with increased expression and activity of stearoyl-CoA desaturase-1 (SCD1), a key enzyme of FA metabolism. SCD1 converts saturated FAs, e.g. palmitate and stearate, to monounsaturated FAs, palmitoleate and oleate, which are the predominant substrates for triglyceride synthesis. Scd1 upregulation in eWAT was associated with reduced DNA methylation in Scd1 promoter surrounding a PPARγ-binding region. Conversely, changes in SCD1 levels and methylation were not observed in iWAT, coherent with a depot-specific programming. CONCLUSIONS: Our data reveal that maternal HF feeding during suckling programs long-term eWAT expansion in part by SCD1 epigenetic reprogramming. This programming events occurred with drastic changes in breast milk FA composition, suggesting that dietary FAs are key metabolic programming factors in the early postnatal period.
Asunto(s)
Tejido Adiposo Blanco , Dieta Alta en Grasa , Epigénesis Genética/genética , Lactancia/genética , Estearoil-CoA Desaturasa , Tejido Adiposo Blanco/química , Tejido Adiposo Blanco/enzimología , Tejido Adiposo Blanco/metabolismo , Animales , Animales Recién Nacidos , Peso Corporal/genética , Femenino , Grasa Intraabdominal/química , Grasa Intraabdominal/enzimología , Grasa Intraabdominal/metabolismo , Masculino , Leche/química , Ratas Wistar , Estearoil-CoA Desaturasa/análisis , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismoRESUMEN
According to the Developmental Origin of Health and Disease (DOHaD) concept, maternal obesity and accelerated growth in neonates program obesity later in life. White adipose tissue (WAT) has been the focus of developmental programming events, although underlying mechanisms remain elusive. In rodents, WAT development primarily occurs during lactation. We previously reported that adult rat offspring from dams fed a high-fat (HF) diet exhibited fat accumulation and decreased peroxisome proliferator-activated receptor γ (PPARγ) mRNA levels in WAT. We hypothesized that PPARγ down-regulation occurs via epigenetic malprogramming which takes place during adipogenesis. We therefore examined epigenetic modifications in the PPARγ1 and PPARγ2 promoters in perirenal (pWAT) and inguinal fat pads of HF offspring at weaning (postnatal d 21) and in adulthood. Postnatal d 21 is a period characterized by active epigenomic remodeling in the PPARγ2 promoter (DNA hypermethylation and depletion in active histone modification H3ac and H3K4me3) in pWAT, consistent with increased DNA methyltransferase and DNA methylation activities. Adult HF offspring exhibited sustained hypermethylation and histone modification H3ac of the PPARγ2 promoter in both deposits, correlated with persistent decreased PPARγ2 mRNA levels. Consistent with the DOHaD hypothesis, retained epigenetic marks provide a mechanistic basis for the cellular memory linking maternal obesity to a predisposition for later adiposity.-Lecoutre, S., Pourpe, C., Butruille, L., Marousez, L., Laborie, C., Guinez, C., Lesage, J., Vieau, D., Eeckhoute, J., Gabory, A., Oger, F., Eberlé, D., Breton, C. Reduced PPARγ2 expression in adipose tissue of male rat offspring from obese dams is associated with epigenetic modifications.
Asunto(s)
Tejido Adiposo/metabolismo , Metilación de ADN , Epigénesis Genética , Obesidad/metabolismo , PPAR gamma/biosíntesis , Regiones Promotoras Genéticas , Tejido Adiposo/patología , Adiposidad/genética , Animales , Femenino , Histonas/genética , Histonas/metabolismo , Masculino , Obesidad/genética , PPAR gamma/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
OBJECTIVE: According to the Developmental Origin of Health and Disease (DOHaD) concept, maternal obesity and accelerated growth in neonates predispose offspring to white adipose tissue (WAT) accumulation. In rodents, adipogenesis mainly develops during lactation. The mechanisms underlying the phenomenon known as developmental programming remain elusive. We previously reported that adult rat offspring from high-fat diet-fed dams (called HF) exhibited hypertrophic adipocyte, hyperleptinemia and increased leptin mRNA levels in a depot-specific manner. We hypothesized that leptin upregulation occurs via epigenetic malprogramming, which takes place early during development of WAT. METHODS: As a first step, we identified in silico two potential enhancers located upstream and downstream of the leptin transcription start site that exhibit strong dynamic epigenomic remodeling during adipocyte differentiation. We then focused on epigenetic modifications (methylation, hydroxymethylation, and histone modifications) of the promoter and the two potential enhancers regulating leptin gene expression in perirenal (pWAT) and inguinal (iWAT) fat pads of HF offspring during lactation (postnatal days 12 (PND12) and 21 (PND21)) and in adulthood. RESULTS: PND12 is an active period for epigenomic remodeling in both deposits especially in the upstream enhancer, consistent with leptin gene induction during adipogenesis. Unlike iWAT, some of these epigenetic marks were still observable in pWAT of weaned HF offspring. Retained marks were only visible in pWAT of 9-month-old HF rats that showed a persistent "expandable" phenotype. CONCLUSIONS: Consistent with the DOHaD hypothesis, persistent epigenetic remodeling occurs at regulatory regions especially within intergenic sequences, linked to higher leptin gene expression in adult HF offspring in a depot-specific manner.
Asunto(s)
Epigénesis Genética , Leptina/genética , Obesidad/genética , Complicaciones del Embarazo/genética , Tejido Adiposo Blanco/metabolismo , Animales , Metilación de ADN , Femenino , Código de Histonas , Leptina/metabolismo , Masculino , Embarazo , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
Blood glucose fluctuates with the fasting-feeding cycle. One of the liver's functions is to maintain blood glucose concentrations within a physiological range. Glucokinase (GCK) or hexokinase IV, is the main enzyme that regulates the flux and the use of glucose in the liver leading to a compensation of hyperglycemia. In hepatocytes, GCK catalyzes the phosphorylation of glucose into glucose-6-phosphate. This critical enzymatic reaction is determinant for the metabolism of glucose in the liver which includes glycogen synthesis, glycolysis, lipogenesis and gluconeogenesis. In liver, simultaneous increase of glucose and insulin enhances GCK activity and gene expression, changes its subcellular location and interaction with regulatory proteins. The post-translational O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) acts as a glucose-sensitive modification and is believed to take part in hepatic glucose sensing by modifying key regulatory proteins. Therefore, we aimed to determine whether GCK is modified by O-GlcNAcylation in the liver of mice and investigated the role that this modification plays in regulating GCK protein expression. We demonstrated that endogenous GCK expression correlated with O-GlcNAc levels in the pathophysiological model ob/ob mice. More specifically, in response to the pharmacological inhibition of O-GlcNAcase (OGA) contents of GCK increased. Using the GlcNAc specific lectin succinylated-WGA and click chemistry labeling approaches, we demonstrated that GCK is modified by O-GlcNAcylation. Further, we demonstrated that siRNA-mediated Ogt knock-down not only decreases O-GlcNAc content but also GCK protein level. Altogether, our in vivo and in vitro results demonstrate that GCK expression is regulated by nutrient-sensing O-GlcNAc cycling in liver.
Asunto(s)
Acetilglucosamina/metabolismo , Glucoquinasa/metabolismo , Glucosa/farmacología , Animales , Estabilidad de Enzimas , Ayuno , Glicosilación/efectos de los fármacos , Células Hep G2 , Humanos , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Biológicos , N-Acetilglucosaminiltransferasas/metabolismo , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Liver Fatty Acid Synthase (FAS) is pivotal for de novo lipogenesis. Loss of control of this metabolic pathway contributes to the development of liver pathologies ranging from steatosis to nonalcoholic steatohepatitis (NASH) which can lead to cirrhosis and, less frequently, to hepatocellular carcinoma. Therefore, deciphering the molecular mechanisms governing the expression and function of key enzymes such as FAS is crucial. Herein, we link the availability of this lipogenic enzyme to the nutrient-dependent post-translational modification O-GlcNAc that is thought to be deregulated in metabolic diseases (diabetes, obesity, and metabolic syndrome). We demonstrate that expression and activity of liver FAS correlate with O-GlcNAcylation contents in ob/ob mice and in mice fed with a high-carbohydrate diet both in a transcription-dependent and -independent manner. More importantly, inhibiting the removal of O-GlcNAc residues in mice intraperitoneally injected with the selective and potent O-GlcNAcase (OGA) inhibitor Thiamet-G increases FAS expression. FAS and O-GlcNAc transferase (OGT) physically interact, and FAS is O-GlcNAc modified. Treatment of a liver cell line with drugs or nutrients that elevate the O-GlcNAcylation interferes with FAS expression. Inhibition of OGA increases the interaction between FAS and the deubiquitinase Ubiquitin-specific protease-2a (USP2A) in vivo and ex vivo, providing mechanistic insights into the control of FAS expression through O-GlcNAcylation. Together, these results reveal a new type of regulation of FAS, linked to O-GlcNAcylation status, and advance our knowledge on deregulation of lipogenesis in diverse forms of liver diseases.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Hígado/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Línea Celular , Alimentos , Lipogénesis/fisiología , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
According to the Developmental Origin of Health and Disease (DOHaD) concept, alterations of nutrient supply in the fetus or neonate result in long-term programming of individual body weight (BW) setpoint. In particular, maternal obesity, excessive nutrition, and accelerated growth in neonates have been shown to sensitize offspring to obesity. The white adipose tissue may represent a prime target of metabolic programming induced by maternal obesity. In order to unravel the underlying mechanisms, we have developed a rat model of maternal obesity using a high-fat (HF) diet (containing 60% lipids) before and during gestation and lactation. At birth, newborns from obese dams (called HF) were normotrophs. However, HF neonates exhibited a rapid weight gain during lactation, a key period of adipose tissue development in rodents. In males, increased BW at weaning (+30%) persists until 3months of age. Nine-month-old HF male offspring was normoglycemic but showed mild glucose intolerance, hyperinsulinemia, and hypercorticosteronemia. Despite no difference in BW and energy intake, HF adult male offspring was predisposed to fat accumulation showing increased visceral (gonadal and perirenal) depots weights and hyperleptinemia. However, only perirenal adipose tissue depot exhibited marked adipocyte hypertrophy and hyperplasia with elevated lipogenic (i.e. sterol-regulated element binding protein 1 (Srebp1), fatty acid synthase (Fas), and leptin) and diminished adipogenic (i.e. peroxisome proliferator-activated receptor gamma (Pparγ), 11ß-hydroxysteroid dehydrogenase type 1 (11ß-Hds1)) mRNA levels. By contrast, very few metabolic variations were observed in HF female offspring. Thus, maternal obesity and accelerated growth during lactation program offspring for higher adiposity via transcriptional alterations of visceral adipose tissue in a depot- and sex-specific manner.
Asunto(s)
Tejido Adiposo/metabolismo , Lactancia/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos/fisiología , Obesidad/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Aumento de Peso/fisiología , Animales , Peso Corporal , Corticosterona/sangre , Femenino , Intolerancia a la Glucosa/metabolismo , Hiperinsulinismo/metabolismo , Masculino , PPAR gamma/genética , PPAR gamma/metabolismo , Embarazo , Ratas , Factores Sexuales , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
O-GlcNAcylation (O-linked beta-N-acetylglucosaminylation) is a widespread PTM confined within the nuclear, the cytosolic, and the mitochondrial compartments of eukaryotes. Recently, O-GlcNAcylation has been also detected in the close vicinity of plasma membranes particularly in lipid microdomains. The detection of this PTM can be easily done if appropriate controls and precautions are taken using a wide variety of tools including lectins, antibodies, or click-chemistry-based methods. In contrast, the identification of the proteins bearing O-GlcNAc moieties and the localization of the precise sites of O-GlcNAcylation remain challenging. This is due to the lability of the glycosidic bond between hydroxyl group of serine or threonine and N-acetylglucosamine using conventional fragmentation techniques such as CID. To tentatively overcome this technical limitation, electron-capture dissociation, or electron-transfer dissociation MS/MS are now used. Thanks to these breakthroughs, a large number of O-GlcNAc sites have been identified to date but these methodologies remain far from being used in routine.
Asunto(s)
Acetilglucosamina , Glicoproteínas , Proteómica/métodos , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Animales , Línea Celular , Glicoproteínas/análisis , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Humanos , Ratones , Procesamiento Proteico-Postraduccional , Ratas , Espectrometría de Masas en Tándem/métodosRESUMEN
Dysfunctions in Wnt signaling increase ß-catenin stability and are associated with cancers, including colorectal cancer. In addition, ß-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of ß-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of ß-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of ß-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of ß-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for ß-catenin stability. Analyses of ß-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the ß-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the ß-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of ß-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of ß-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.
Asunto(s)
Acetilglucosamina/metabolismo , Procesamiento Proteico-Postraduccional , Treonina/química , beta Catenina/química , Adenocarcinoma/etiología , Adenocarcinoma/metabolismo , Uniones Adherentes/metabolismo , Uniones Adherentes/patología , Secuencia de Aminoácidos , Animales , Colon/metabolismo , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Carbohidratos de la Dieta/metabolismo , Carbohidratos de la Dieta/toxicidad , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Glicosilación , Células HEK293 , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Mucosa Intestinal/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/fisiología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/farmacología , Vía de Señalización Wnt , alfa Catenina/metabolismo , beta Catenina/metabolismo , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/fisiologíaRESUMEN
The short half-life protooncogene ß-catenin acquires a remarkable stability in a large subset of cancers, mainly from mutations affecting its proteasomal degradation. In this sense, colorectal cancers (CRC) form a group of pathologies in which early steps of development are characterized by an aberrant expression of ß-catenin and an uncontrolled proliferation of epithelial cells. Diet has long been described as an influence in the emergence of CRC, but the molecular events that link metabolic disorders and CRC remain elusive. Part of the explanation may reside in hexosamine biosynthetic pathway (HBP) flux. We found that fasted mice being force-fed with glucose or glucosamine leads to an increase of ß-catenin and O-GlcNAcylation levels in the colon. MCF7 cells possessing intact Wnt/ß-catenin signaling heavily expressed ß-catenin when cultured in high glucose; this was reversed by the HBP inhibitor azaserine. HBP inhibition also decreased the expression of ß-catenin in HT29 and, to a lesser extent, HCT116 cells. The same observation was made with regard to the transcriptional activity of ß-catenin in HEK293 cells. Inhibition of HBP also blocked the glucose-mediated proliferation capacity of MCF7 cells, demonstrating that glucose affects both ß-catenin expression and cell proliferation through the HBP. The ultimate element conducting these events is the dynamic posttranslational modification O-GlcNAcylation, which is intimately linked to HBP; the modulation of its level affected the expression of ß-catenin and cell proliferation. In accordance with our findings, we propose that metabolic disorders correlate to CRC via an upregulation of HBP that reverberates on high O-GlcNAcylation levels including modification of ß-catenin.
Asunto(s)
Glucosamina/metabolismo , beta Catenina/biosíntesis , Acilación , Animales , Antimetabolitos Antineoplásicos/farmacología , Azaserina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colon/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Ayuno/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Procesamiento Proteico-Postraduccional , Regulación hacia Arriba , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
OBJECTIVE: Carbohydrate-responsive element-binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked ß-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction. RESEARCH DESIGN AND METHODS: O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBP(OG) in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice. RESULTS: Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBP(OG) in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBP(OG) levels were elevated compared with controls. Interestingly, reducing ChREBP(OG) levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice. CONCLUSIONS: Taken together, our results reveal that O-GlcNAcylation represents an important novel regulation of ChREBP activity in the liver under both physiological and pathophysiological conditions.
Asunto(s)
Hígado Graso/metabolismo , Hígado/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Hígado Graso/enzimología , Hígado Graso/genética , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , N-Acetilglucosaminiltransferasas/genética , Proteínas Nucleares/genética , Unión Proteica , Factores de Transcripción/genética , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The members of the 70kDa-heat shock proteins (HSP70) family play numerous fundamental functions in the cell such as promoting the assembly of multimeric complexes or helping the correct folding of nascent proteins to take place. In numerous previous studies we demonstrated that Hsp70 and its constitutive isoform Hsc70 are endowed of a GlcNAc-binding activity. The molecular modeling of the substrate binding domain of Hsc70 and in silico docking experiments using Ser/Thr-O-GlcNAc motifs allowed to define the potential carbohydrate-recognition region and to point out the crucial position of Arg469 as an amino-acid directly interacting with the sugar moiety. We cloned a flagged Hsc70 in a pCMV.SPORT6 vector and we showed that the mutation R469A decreased the GlcNAc-binding property of the chaperone of around 70%. This is the first work reporting the localization of the GlcNAc-binding domain of a member of the HSP70 family.
Asunto(s)
Acetilglucosamina/metabolismo , Arginina/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Acetilglucosamina/química , Animales , Arginina/química , Arginina/genética , Sitios de Unión , Células COS , Chlorocebus aethiops , Proteínas del Choque Térmico HSC70/química , Proteínas del Choque Térmico HSC70/genética , Humanos , Mutación , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
O-GlcNAcylation is widespread within the cytosolic and nuclear compartments of cells. This post-translational modification is likely an indicator of good health since its intracellular level correlates with the availability of extracellular glucose. Apart from its status as a nutrient sensor, O-GlcNAcylation may also act as a stress sensor since it exerts its fundamental effects in response to stress. Several studies report that the cell quickly responds to an insult by elevating O-GlcNAcylation levels and by unmasking a newly described Hsp70-GlcNAc binding property. From a more practical point of view, it has been shown that O-GlcNAcylation impairments contribute to the etiology of cardiovascular diseases, type-2 diabetes and Alzheimer's disease (AD), three illnesses common in occidental societies. Many studies have demonstrated that O-GlcNAcylation operates as a powerful cardioprotector and that by raising O-GlcNAcylation levels, the organism more successfully resists trauma-hemorrhage and ischemia/reperfusion injury. Recent data have also shown that insulin resistance and, more broadly, type-2 diabetes can be controlled by O-GlcNAcylation of the insulin pathway and O-GlcNAcylation of the gluconeogenesis transcription factors FoxO1 and CRCT2. Lastly, the finding that AD may correspond to a type-3 diabetes offers new perspectives into the knowledge of the neuropathology and into the search for new therapeutic avenues.
Asunto(s)
Enfermedad de Alzheimer/etiología , Enfermedades Cardiovasculares/etiología , Diabetes Mellitus Tipo 2/etiología , N-Acetilglucosaminiltransferasas/metabolismo , Acetilglucosamina/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Encéfalo/metabolismo , Enfermedades Cardiovasculares/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/fisiología , Glucosa/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Homeodominio/fisiología , Humanos , Resistencia a la Insulina/fisiología , Daño por Reperfusión Miocárdica/prevención & control , Complejo de la Endopetidasa Proteasomal/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Ubiquitinas/fisiologíaRESUMEN
During the past two decades, O-GlcNAc modification of cytosolic and nuclear proteins has been intensively studied. Nevertheless, the function of this post-translational modification remains unclear. It has been recently speculated that O-GlcNAc could act as a protective signal against proteasomal degradation, both by modifying target substrates and/or by inhibiting the proteasome itself. In this work, we have investigated the putative relation between O-GlcNAc and the ubiquitin pathway. First, we showed that the level of both modifications increased rapidly after thermal stress but, unlike ubiquitinated proteins, O-GlcNAc-modified proteins failed to be stabilized by inhibiting proteasome function. Increasing O-GlcNAc levels, using glucosamine or PUGNAc, enhanced ubiquitination. Inversely, when O-GlcNAc levels were reduced, using forskolin or glucose deprivation, ubiquitination decreased. Targeted-RNA interference of O-GlcNAc transferase also reduced ubiquitination and moreover halved cell thermotolerance. Finally, we demonstrated that the ubiquitin-activating enzyme E1 was O-GlcNAc modified and that its glycosylation and its interaction with Hsp70 varied according to the conditions of cell culture. Altogether, these results show that O-GlcNAc and ubiquitin are not strictly antagonistic post-translational modifications, but rather that the former might regulate the latter, and also suggest that E1 could be one of the common links between the two pathways.
Asunto(s)
Acetilglucosamina/metabolismo , Glicosilación , Proteínas/química , Proteínas/metabolismo , Ubiquitinación , Secuencia de Bases , Línea Celular , Supervivencia Celular , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Respuesta al Choque Térmico , Humanos , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño/genética , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against injuries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG(2) cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even if differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations suggesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property.
Asunto(s)
Acetilglucosamina/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Peróxido de Hidrógeno/farmacología , Inhibidores de Proteasoma , Pliegue de Proteína , Cloruro de Sodio/farmacología , Ácido Azetidinocarboxílico/química , Ácido Azetidinocarboxílico/farmacología , Cromatografía por Intercambio Iónico , Etanol/farmacología , Células HeLa , Humanos , Lectinas/metabolismo , Leupeptinas/farmacología , Prolina/química , Unión Proteica/efectos de los fármacos , Termodinámica , Uridina Difosfato N-Acetilglucosamina/aislamiento & purificación , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
It is well-accepted that protein quality control (occurring either after protein synthesis or after cell damage) is mainly ensured by HSP, but the mechanism by which HSP decides whether the protein will be degraded or not is poorly understood. Within this framework, it has been hypothesized that O-GlcNAc, a cytosolic and nuclear-specific glycosylation whose functions remain unclear, could take a part in the protection of proteins against degradation by modifying both the proteins themselves and the proteasome. Because the synthesis of O-GlcNAc is tightly correlated to glucose metabolism and Hsp70 was endowed with GlcNAc-binding property, we studied the relationship between GlcNAc-binding activity of both Hsp70 and Hsc70 (the nucleocytoplasmic forms of HSP70 family) and glucose availability and utilization. We thus demonstrated that low glucose concentration, inhibition of glucose utilization with 2DG, or inhibition of glucose transport with CytB led to an increase of Hsp70 and Hsc70 lectin activities. Interestingly, the response of Hsp70 and Hsc70 lectin activities toward variations of glucose concentration appeared different: Hsp70 lost its lectin activity when glucose concentration was >5 mM (i.e., physiological glucose concentration) in contrast to Hsc70 that exhibited a maximal lectin activity for glucose concentration approximately 5 mM and at high glucose concentrations. This work also demonstrates that HSP70 does not regulate its GlcNAc-binding properties through its own O-GlcNAc glycosylation.
Asunto(s)
Núcleo Celular/metabolismo , Glucosa/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Modificación Traduccional de las Proteínas/fisiología , Receptores N-Acetilglucosamina/metabolismo , Línea Celular Tumoral , Glucosa/farmacología , Glicosilación/efectos de los fármacos , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Modificación Traduccional de las Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
There are several lines of evidence that the modification of proteins by cytosolic- and nuclear-specific O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is closely related to neuropathologies, particularly Alzheimer's disease. Several neuronal proteins have been identified as being modified with O-GlcNAc; these proteins could form part of the inclusion bodies found, for example, in the most frequently observed neurologic disorder (i.e., Alzheimer's disease; Tau protein and beta-amyloid peptide are the well known aggregated proteins). O-GlcNAc proteins are also implicated in synaptosomal transport (e.g., synapsins and clathrin-assembly proteins). Inclusion bodies are partly characterized by a deficiency in the ubiquitin-proteasome system, avoiding the degradation of aggregated proteins. From this perspective, it appears interesting that substrate proteins could be protected against proteasomal degradation by being covalently modified with single N-acetylglucosamine on serine or threonine, and that the proteasome itself is modified and regulated by O-GlcNAc (in this case the turnover of neuronal proteins correlates with extracellular glucose). Interestingly, glucose uptake and metabolism are impaired in neuronal disorders, and this phenomenon is linked to increased phosphorylation. In view of the existence of the dynamic interplay between O-GlcNAc and phosphorylation, it is tempting to draw a parallel between the use of glucose, O-GlcNAc glycosylation and phosphorylation. Lastly, the two enzymes responsible for O-GlcNAc dynamism (i.e., O-GlcNAc transferase and glucosaminidase) are both enriched in the brain and genes that encode the two enzymes are located in two regions that are found to be frequently mutated in neurologic disorders. The data presented in this review strongly suggest that O-GlcNAc could play an active role in neurodegenerative diseases.
Asunto(s)
Acetilglucosamina/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Acetilglucosaminidasa/fisiología , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Glucosa/metabolismo , Glicosilación , Humanos , N-Acetilglucosaminiltransferasas/fisiología , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/fisiología , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Proteínas de Transporte Vesicular/metabolismo , Proteínas tau/metabolismoRESUMEN
Year 2004 marks the 20th anniversary of the discovery of O-linked N-acetylglucosamine (O-GlcNAc) by Gerald W. Hart. Despite interest for O-GlcNAc, the functions played by this single monosaccharide remain poorly understood, though numerous roles have been suggested, among which is the involvement of O-GlcNAc in the nuclear transport of cytosolic proteins. This idea was first sustained by studies on bovine serum albumin that showed that the protein could be actively carried to the nucleus when it was modified with sugars. In this paper, we will review data on this puzzling problem. We will first describe the well-established nuclear localisation signal (NLS)-dependent nuclear transport by presenting the different factors involved, and then, we will examine where and how O-GlcNAc could be involved in nuclear transport. Whereas it has been suggested that O-GlcNAc could interfere at two levels in the nuclear transport both by modifying proteins to be translocated to the nucleus and by modifying the nucleoporins of the nuclear pore complex, according to us, this second idea seems unlikely. Part of this study will also be dedicated to a relatively new concept in the nuclear transport: the role of the 70-kDa heat shock proteins (HSP70). The action of the chaperone in nuclear translocation was put forward 10 years ago, but new findings suggest that this mechanism could be linked to O-GlcNAc glycosylation.
Asunto(s)
Acetilglucosamina/metabolismo , Citosol/metabolismo , Glicosilación , Transporte de ProteínasRESUMEN
Numerous works demonstrated that the dynamic O-GlcNAc glycosylation could protect against the proteasomal degradation by modifying the target proteins and the proteasome itself. Considering that Hsp70 is a crucial component in the quality control of protein conformation in the proteasomal pathway, we investigated the possibility that Hsp70 physically interacts with O-GlcNAc proteins through a lectinic activity. First, we demonstrate that in HepG2 cells, Hsp70 can specifically bind to O-GlcNAc residues but also is itself modified by O-GlcNAc. Second, when cells were deprived of glucose (nutrient stress), Hsp70 lectinic activity markedly increased whereas its glycosylation dramatically decreased. On the other hand, a 42 degrees C thermic stress did not affect any of these features. Lastly, the nature of O-GlcNAc modified proteins co-immunoprecipitating with Hsp70 was similar for cells submitted to the thermic and to nutrient stress. These results strongly suggest that O-GlcNAc influences protein stability through specific interaction with 70-kDa-heat shock protein members.