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AIM: Over the last decades, the shift in age distribution towards older ages and the progressive ageing which has occurred in most populations have been paralleled by a global epidemic of obesity and its related metabolic disorders, primarily, type 2 diabetes (T2D). Dysfunction of the adipose tissue (AT) is widely recognized as a significant hallmark of the ageing process that, in turn, results in systemic metabolic alterations. These include insulin resistance, accumulation of ectopic lipids and chronic inflammation, which are responsible for an elevated risk of obesity and T2D onset associated to ageing. On the other hand, obesity and T2D, the paradigms of AT dysfunction, share many physiological characteristics with the ageing process, such as an increased burden of senescent cells and epigenetic alterations. Thus, these chronic metabolic disorders may represent a state of accelerated ageing. MATERIALS AND METHODS: A more precise explanation of the fundamental ageing mechanisms that occur in AT and a deeper understanding of their role in the interplay between accelerated ageing and AT dysfunction can be a fundamental leap towards novel therapies that address the causes, not just the symptoms, of obesity and T2D, utilizing strategies that target either senescent cells or DNA methylation. RESULTS: In this review, we summarize the current knowledge of the pathways that lead to AT dysfunction in the chronological ageing process as well as the pathophysiology of obesity and T2D, emphasizing the critical role of cellular senescence and DNA methylation. CONCLUSION: Finally, we highlight the need for further research focused on targeting these mechanisms.
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Tejido Adiposo/fisiopatología , Envejecimiento/fisiología , Enfermedades Metabólicas , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/patología , Senescencia Celular/genética , Enfermedad Crónica , Metilación de ADN/fisiología , Humanos , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , Persona de Mediana Edad , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Platelet-derived factors are biomaterials that might accelerate healing process in oral, maxillofacial, and several other applications. Release of specific factors by platelet concentrates is critical to achieving a successful outcome. Here, we have shown that platelet-rich fibrin (PRF) clots were beneficial sources of leukocytes, which may directly affect the release of chemokines and growth factors. When compared with the standard leukocyte-PRF (L-PRF), the experimental low-force modified procedure [defined as advanced-PRF (A-PRF)] entrapped the same content of viable leukocytes, released a similar amount of inflammatory cytokines, but secreted 3-, 1.6-, 3-, and 1.2-fold higher levels of Eotaxin, CCL5, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), respectively. A leukocyte-free scaffold, such as plasma rich in growth factors (PRGF), released only platelet-specific factors and, in particular, the F3 fraction, the richest in growth factors, secreted higher amount of CCL5 and PDGF compared to F1 and F2 fractions. In conclusion, different procedures and leukocyte content affect cytokine, chemokines, and growth factor release from platelet derivatives, which may be helpful in different clinical settings.
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Plaquetas/metabolismo , Quimiocinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucocitos/metabolismo , Adulto , Femenino , Humanos , Masculino , Plasma Rico en Plaquetas/metabolismoRESUMEN
Type 2 diabetes (T2D) and obesity are the major public health problems. Substantial efforts have been made to define loci and variants contributing to the individual risk of these disorders. However, the overall risk explained by genetic variation is very modest. Epigenetics is one of the fastest growing research areas in biomedicine as changes in the epigenome are involved in many biological processes, impact on the risk for several complex diseases including diabetes and may explain susceptibility. In this review, we focus on the role of DNA methylation in contributing to the risk of T2D and obesity.
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Diabetes Mellitus Tipo 2/genética , Epigénesis Genética/genética , Obesidad/genética , HumanosRESUMEN
BACKGROUND/OBJECTIVES: The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes. SUBJECTS/METHODS: We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox (Hox) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed. RESULTS: Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance (P<0.001) and insulin sensitivity (P<0.05). MeDIP-seq data analysis revealed a uniform distribution of differentially methylated regions (DMR) through the entire adipocyte genome, with a higher number of hypermethylated regions in HFD mice (P<0.005). This different methylation profile was accompanied by increased expression of the Dnmt3a DNA methyltransferase (Dnmt; P<0.05) and the methyl-CpG-binding domain protein Mbd3 (P<0.05) genes in HFD mice. Gene ontology analysis revealed that, in the HFD-treated mice, the Hox family of development genes was highly enriched in differentially methylated genes (P=0.008). To validate this finding, Hoxa5, which is implicated in fat tissue differentiation and remodeling, has been selected and analyzed by bisulphite sequencing, confirming hypermethylation in the adipose tissue from the HFD mice. Hoxa5 hypermethylation was associated with downregulation of Hoxa5 mRNA and protein expression. Feeding animals previously exposed to the HFD with a standard chow diet for two further months improved the metabolic phenotype of the animals, accompanied by return of Hoxa5 methylation and expression levels (P<0.05) to values similar to those of the control mice maintained under standard chow. CONCLUSIONS: HFD induces adipose tissue abnormalities accompanied by epigenetic changes at the Hoxa5 adipose tissue remodeling gene.
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Tejido Adiposo/metabolismo , Metilación de ADN , Dieta Alta en Grasa , Regulación hacia Abajo , Proteínas de Homeodominio/genética , Fosfoproteínas/genética , Transcripción Genética , Animales , Modelos Animales de Enfermedad , Epigénesis Genética , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , Factores de TranscripciónRESUMEN
In the last decades, many reports have focused the attention on deleterious effects of novel environmental chemical compounds, including bisphenol A (BPA), on human health. BPA, a common and widely chemical contaminant acting as endocrine disruptor, accumulates in adipose tissue and may affect adipocyte metabolic and inflammatory functions. BPA, at low chronic doses, is now considered as an obesogen compound, and might contribute to the rise of metabolic syndrome, visceral adiposity and diabetes epidemics. The BPA worldwide presence in the environment is responsible for chronic exposure during vulnerable periods, such as foetal and neonatal life. The BPA source of contamination can occur via food, beverage, wastewater, air, dust and soil. BPA, as lipophilic compound, may accumulate into the adipose tissue already during foetal life and may affect adulthood health, through adverse effects on the growth and development of organs and tissues. Thus, based on several studies, it would be crucial to consider further actions aimed to refine risk assessment at least in vulnerable population, such as foetuses, infants and young children, to prevent metabolic diseases and obesity.
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Contaminantes Ocupacionales del Aire/efectos adversos , Compuestos de Bencidrilo/efectos adversos , Exposición a Riesgos Ambientales/efectos adversos , Síndrome Metabólico/etiología , Fenoles/efectos adversos , Humanos , Síndrome Metabólico/epidemiología , Medición de Riesgo , Poblaciones VulnerablesRESUMEN
The nuclear receptor PPAR γ is a key regulator of adipogenesis, and alterations of its function are associated with different pathological processes related to metabolic syndrome. We recently identified two PPARG transcripts encoding dominant negative PPAR γ isoforms. The existence of different PPARG variants suggests that alternative splicing is crucial to modulate PPAR γ function, underlying some underestimated aspects of its regulation. Here we investigate PPARG expression in different tissues and cells affected in metabolic syndrome and, in particular, during adipocyte differentiation of human mesenchymal stem cells. We defined the transcript-specific expression pattern of PPARG variants encoding both canonical and dominant negative isoforms and identified a novel PPARG transcript, γ 1ORF4. Our analysis indicated that, during adipogenesis, the transcription of alternative PPARG variants is regulated in a time-specific manner through differential usage of distinct promoters. In addition, our analysis describes-for the first time-the differential contribution of three ORF4 variants to this process, suggesting a still unexplored role for these dominant negative isoforms during adipogenesis. Therefore, our results highlight crucial aspects of PPARG regulation, suggesting the need of further investigation to rule out the differential impact of all PPARG transcripts in both physiologic and pathologic conditions, such as metabolism-related disorders.
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Abstract Platelet derivatives are commonly used in wound healing and tissue regeneration. Different procedures of platelet preparation may differentially affect growth factor release and cell growth. Preparation of platelet-rich fibrin (PRF) is accompanied by release of growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor ß1 (TGFß1), and several cytokines. When compared with the standard procedure for platelet-rich plasma (PRP), PRF released 2-fold less PDGF, but >15-fold and >2-fold VEGF and TGFß1, respectively. Also, the release of several cytokines (IL-4, IL-6, IL-8, IL-10, IFNγ, MIP-1α, MIP-1ß and TNFα) was significantly increased in PRF-conditioned medium (CM), compared to PRP-CM. Incubation of both human skin fibroblasts and human umbilical vein endothelial cells (HUVECs) with PRF-derived membrane (mPRF) or with PRF-CM enhanced cell proliferation by >2-fold (p<0.05). Interestingly, PRP elicited fibroblast growth at a higher extent compared to PRF. At variance, PRF effect on HUVEC growth was significantly greater than that of PRP, consistent with a higher concentration of VEGF in the PRF-CM. Thus, the procedure of PRP preparation leads to a larger release of PDGF, as a possible result of platelet degranulation, while PRF enhances the release of proangiogenic factors.
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Plaquetas/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Plasma Rico en Plaquetas , Adulto , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Adulto JovenRESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes and obesity are associated with increased risk of site-specific cancers. We have investigated whether metabolic alterations at the level of adipose-derived differentiating cells may affect specific phenotypes of breast cancer cells. METHODS: Growth profiles of breast cancer cell lines were evaluated in co-cultures with differentiated adipocytes or their precursor cells and upon treatment with adipocyte conditioned media. Production and release of cytokines and growth factors were assessed by real-time RT-PCR and multiplex-based ELISA assays. RESULTS: Co-cultures with either differentiated mouse 3T3-L1 or human mammary adipocytes increased viability of MCF-7 cells to a greater extent, when compared with their undifferentiated precursors. Adipocytes cultured in 25 mmol/l glucose were twofold more effective in promoting cell growth, compared with those grown in 5.5 mmol/l glucose, and activated mitogenic pathways in MCF-7 cells. Growth-promoting action was also enhanced when adipocytes were incubated in the presence of palmitate or oleate. Interestingly, 3T3-L1 and human adipocytes released higher amounts of keratinocyte-derived chemokine/IL-8, the protein 'regulated upon activation, normally T expressed, and secreted' (RANTES), and IGF-1, compared with their precursor cells. Their levels were reduced upon incubation with low glucose and enhanced by fatty acids. Moreover, both undifferentiated cells and differentiated adipocytes from obese individuals displayed about twofold higher IGF-1 release and MCF-7 cell growth induction than lean individuals. Finally, inhibition of the IGF-1 pathway almost completely prevented the growth-promoting effect of adipocytes on breast cancer cells. CONCLUSIONS/INTERPRETATION: IGF-1 release by adipocytes is regulated by glucose and fatty acids and may contribute to the control of cancer cell growth in obese individuals.
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Adipocitos/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Glucosa/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ácido Oléico/farmacología , Palmitatos/farmacología , Adenocarcinoma/patología , Adipocitos/efectos de los fármacos , Adipocitos/patología , Adulto , Anciano , Comunicación Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Quimiocina CCL5/metabolismo , Técnicas de Cocultivo , Femenino , Humanos , Interleucina-8/metabolismo , Células MCF-7 , Persona de Mediana Edad , Obesidad/metabolismo , Obesidad/patología , Transducción de Señal/fisiologíaRESUMEN
AMP-activated protein kinase (AMPK) serves as a major regulator of energy homeostasis and is activated by different glucose-lowering agents. Indeed, AMPK has been identified as an attractive target for the development of innovative molecules to treat type 2 diabetes. In this issue of Diabetologia (doi: 10.1007/s00125-011-2366-3 ), Huang and co-workers report that arctigenin activates muscle uptake of glucose and inhibits hepatocyte gluconeogenesis and lipogenesis by reducing mitochondrial respiration and inducing AMPK activity. Importantly, it is reported that arctigenin improves glucose and lipid metabolism in ob/ob mice. Based on this evidence, Huang and co-workers suggest that arctigenin may represent a valuable lead compound for developing innovative glucose-lowering molecules. While these findings are not entirely novel and mechanistic investigations are needed, the results strongly support the concept that arctigenin deserves to be further considered because of its several potentially beneficial in vivo effects. In particular, the authors conclude that further mechanistic studies on arctigenin might provide novel insight and opportunities for selective modulation of subcutaneous and visceral fat mass.
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Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Furanos/farmacología , Trastornos del Metabolismo de la Glucosa/tratamiento farmacológico , Lignanos/farmacología , Mitocondrias Musculares/efectos de los fármacos , Animales , MasculinoRESUMEN
TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.
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Autofagia/efectos de los fármacos , Músculo Esquelético/citología , Fosfoproteínas/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Proteínas Reguladoras de la Apoptosis , Astrocitos/citología , Astrocitos/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Línea Celular , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
AIMS/HYPOTHESIS: Beta cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (decline of glucose-stimulated insulin secretion, downregulation of specific gene expression). Apoptosis and dysfunction are caused, at least in part, by lipoglucotoxicity. The mechanisms implicated are oxidative stress, increase in the hexosamine biosynthetic pathway (HBP) flux and endoplasmic reticulum (ER) stress. Oxidative stress plays a role in glucotoxicity-induced beta cell dedifferentiation, while glucotoxicity-induced ER stress has been mostly linked to beta cell apoptosis. We sought to clarify whether ER stress caused by increased HBP flux participates in a dedifferentiating response of beta cells, in the absence of relevant apoptosis. METHODS: We used INS-1E cells and murine islets. We analysed the unfolded protein response and the expression profile of beta cells by real-time RT-PCR and western blot. The signal transmission pathway elicited by ER stress was investigated by real-time RT-PCR and immunofluorescence. RESULTS: Glucosamine and high glucose induced ER stress, but did not decrease cell viability in INS-1E cells. ER stress caused dedifferentiation of beta cells, as shown by downregulation of beta cell markers and of the transcription factor, pancreatic and duodenal homeobox 1. Glucose-stimulated insulin secretion was inhibited. These effects were prevented by the chemical chaperone, 4-phenyl butyric acid. The extracellular signal-regulated kinase (ERK) signal transmission pathway was implicated, since its inhibition prevented the effects induced by glucosamine and high glucose. CONCLUSIONS/INTERPRETATION: Glucotoxic ER stress dedifferentiates beta cells, in the absence of apoptosis, through a transcriptional response. These effects are mediated by the activation of ERK1/2.
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Desdiferenciación Celular , Glucosamina/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Sistema de Señalización de MAP Quinasas , Animales , Desdiferenciación Celular/efectos de los fármacos , Línea Celular , Células Clonales , Regulación hacia Abajo/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Insulina/genética , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fenilbutiratos/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Transactivadores/genética , Transactivadores/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
The endoplasmic reticulum (ER) is a complex and multifunctional organelle. It is the intracellular compartment of protein folding, a complex task, both facilitated and monitored by ER folding enzymes and molecular chaperones. The ER is also a stress-sensing organelle. It senses stress caused by disequilibrium between ER load and folding capacity and responds by activating signal transduction pathways, known as unfolded protein response (UPR). Three major classes of transducer are known, inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK), which sense with their endoluminal domain the state of protein folding, although the exact mechanism(s) involved is not entirely clear. Depending on whether the homeostatic response of the UPR is successful in restoring an equilibrium between ER load and protein folding or not, the two possible outcomes of the UPR so far considered have been life or death. Indeed, recent efforts have been devoted to understand the life/death switch mechanisms. However, recent data suggest that what appears to be a pure binary decision may in fact be more complex, and survival may be achieved at the expenses of luxury cell functions, such as expression of differentiation genes.
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Apoptosis , Retículo Endoplásmico/metabolismo , Estrés Fisiológico , Respuesta de Proteína Desplegada , Animales , Desdiferenciación Celular , Retículo Endoplásmico/patología , Humanos , Recuperación de la Función , Transducción de SeñalRESUMEN
AIMS/HYPOTHESIS: Chronic hyperglycaemia aggravates insulin resistance, at least in part, by increasing the formation of advanced glycation end-products (AGEs). Methylglyoxal (MGO) is the most reactive AGE precursor and its abnormal accumulation participates in damage in various tissues and organs. Here we investigated the ability of MGO to interfere with insulin signalling and to affect beta cell functions in the INS-1E beta cell line. METHODS: INS-1E cells were incubated with MGO and then exposed to insulin or to glucose. Western blotting was used to study signalling pathways, and real-time PCR to analyse gene expression; insulin levels were determined by radioimmunoassay. RESULTS: Non-cytotoxic MGO concentrations inhibited insulin-induced IRS tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway activation independently from reactive oxygen species (ROS) production. Concomitantly, formation of AGE adducts on immunoprecipitated IRS was observed. Aminoguanidine reversed MGO inhibitory effects and the formation of AGE adducts on IRS. Further, the insulin- and glucose-induced expression of Ins1, Gck and Pdx1 mRNA was abolished by MGO. Finally, MGO blocked glucose-induced insulin secretion and PI3K/PKB pathway activation. These MGO effects were abolished by LiCl, which inhibits glycogen synthase kinase-3 (GSK-3). CONCLUSIONS/INTERPRETATION: MGO exerted major damaging effects on INS-1E cells impairing both insulin action and secretion. An important actor in these noxious MGO effects appears to be GSK-3. In conclusion, MGO participates not only in the pathogenesis of the debilitating complications of type 2 diabetes, but also in worsening of the diabetic state by favouring beta cell failure.
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Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Piruvaldehído/metabolismo , Vías Secretoras , Transducción de Señal , Animales , Transporte Biológico , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Glucoquinasa/genética , Glucoquinasa/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Insulina/genética , Proteínas Sustrato del Receptor de Insulina , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Vías Secretoras/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
AIMS/HYPOTHESIS: We determined the contribution to insulin resistance of the PH domain leucine-rich repeat protein phosphatase (PHLPP), which dephosphorylates Akt at Ser473, inhibiting its activity. We measured the abundance of PHLPP in fat and skeletal muscle from obese participants. To study the effect of PHLPP on insulin signalling, PHLPP (also known as PHLPP1) was overexpressed in HepG2 and L6 cells. METHODS: Subcutaneous fat samples were obtained from 82 morbidly obese and ten non-obese participants. Skeletal muscle samples were obtained from 12 obese and eight non-obese participants. Quantification of PHLPP-1 in human tissues was performed by immunoblotting. The functional consequences of recombinant PHLPP1 overexpression in hepatoma HepG2 cells and L6 myoblasts were investigated. RESULTS: Of the 82 obese participants, 31 had normal fasting glucose, 33 impaired fasting glucose and 18 type 2 diabetes. PHLPP-1 abundance was twofold higher in the three obese groups than in non-obese participants (p = 0.004). No differences were observed between obese participants with normal fasting glucose, impaired fasting glucose or type 2 diabetes. PHLPP-1 abundance was correlated with basal Akt Ser473 phosphorylation (r = -0.48; p = 0.001), BMI (r = 0.44; p < 0.0001), insulin (r = 0.35; p < 0.0001) and HOMA (r = 0.38; p < 0.0001). PHLPP-1 abundance was twofold higher in the skeletal muscle of 12 obese participants than in that of eight non-obese participants (p < 0.0001). Insulin treatment of HepG2 cells resulted in a dose- and time-dependent upregulation of PHLPP-1. Overexpression of PHLPP1 in HepG2 cells and L6 myoblasts resulted in impaired insulin signalling involving Akt/glycogen synthase kinase 3, glycogen synthesis and glucose transport. CONCLUSIONS/INTERPRETATION: Increased abundance of PHLPP-1, production of which is regulated by insulin, may represent a new molecular defect in insulin-resistant states such as obesity.
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Resistencia a la Insulina/fisiología , Proteínas Nucleares/metabolismo , Obesidad/metabolismo , Obesidad/fisiopatología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adolescente , Adulto , Anciano , Animales , Western Blotting , Línea Celular , Femenino , Células Hep G2 , Humanos , Técnicas In Vitro , Resistencia a la Insulina/genética , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Obesidad/genética , Fosfoproteínas Fosfatasas/genética , Ratas , Adulto JovenRESUMEN
We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.
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Citotoxicidad Inmunológica , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Células Asesinas Naturales/inmunología , Glándula Tiroides/citología , Línea Celular , Regulación hacia Abajo , Retículo Endoplásmico/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/inmunología , Desplegamiento Proteico , Tapsigargina/farmacología , Células Tumorales Cultivadas , Tunicamicina/farmacologíaRESUMEN
BACKGROUND: The cellular abundance of the phosphoprotein enriched in diabetes (PED/PEA-15), a 15 kDa protein related to insulin resistance (IR), is increased in women with polycystic ovary syndrome (PCOS). AIM: To investigate whether metformin (MET) has additive effects on PED/PEA-15 protein levels. MATERIAL/SUBJECTS AND METHODS: This is an open label, prospective clinical study over 6 months. Ten hyperandrogenic obese PCOS women [age: 24.6+/-1.6 yr; body mass index (BMI): 30.7+/-1.2 kg/m(2)] were treated with MET (1250 mg/day). Ten age- and BMI-matched normo-androgenic women were used as controls. Outcome measures are: PED/PEA-15 protein levels, fasting plasma glucose and insulin (FPI), reciprocal index of homeostasis model assessment of insulin resistance (1/HOMA-IR); quantitative insulin sensitivity check index (QUICKI); wholebody insulin sensitivity index (ISI); SHBG; total testosterone; free androgen index (FAI). RESULTS: At baseline FPI and PED/PEA- 15 protein levels were higher, while 1/HOMA-IR, QUICKI, and ISI were lower (p<0.001) in MET group than in controls. After treatment, independently of body weight and hyperandrogenism, FPI, and PED/PEA-15 protein levels decreased (p=0.001 and 0.004, respectively), while, 1/HOMA-IR, QUICKI, and ISI increased (p<0.001). PED/PEA-15 protein levels correlated significantly with ISI either before (r=0.636; p=0.048), and after treatment (r=0.758; p=0.011). CONCLUSIONS: PED/PEA-15 protein levels reduced after a short course of treatment with MET in a group hyperandrogenic obese PCOS women. This effect was independent of body weight and hyperandrogenism, and correlated with ISI, thus adding a further benefit to obese PCOS women.
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Resistencia a la Insulina/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metformina/uso terapéutico , Fosfoproteínas/metabolismo , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Adulto , Andrógenos/sangre , Proteínas Reguladoras de la Apoptosis , Glucemia/metabolismo , Femenino , Humanos , Insulina/sangre , Obesidad/sangre , Síndrome del Ovario Poliquístico/fisiopatología , Globulina de Unión a Hormona Sexual/metabolismoAsunto(s)
Proteínas de Ciclo Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Diabetes Mellitus Tipo 2/genética , Humanos , Resistencia a la Insulina/genética , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
AIMS/HYPOTHESIS: Overexpression of PED (also known as PEA15) determines insulin resistance and impaired insulin secretion and may contribute to progression toward type 2 diabetes. Recently, we found that the transcription factor hepatocyte nuclear factor (HNF)-4alpha binds to PED promoter and represses its transcription. However, the molecular details responsible for regulation of PED gene remain unclear. METHODS: Here we used gain and loss of function approaches to investigate the hypothesis that HNF-4alpha controls chromatin remodelling at the PED promoter in human cell lines. RESULTS: HNF-4alpha production and binding induce chromatin remodelling at the -250 to 50 region of PED, indicating that remodelling is limited to two nucleosomes located at the proximal promoter. Chromatin immunoprecipitation assays also revealed concomitant HNF-4alpha-induced deacetylation of histone H3 at Lys9 and Lys14, and increased dimethylation of histone H3 at Lys9. The latter was followed by reduction of histone H3 Lys4 dimethylation. HNF-4alpha was also shown to target the histone deacetylase complex associated with silencing mediator of retinoic acid and thyroid hormone receptor, both at the PED promoter, and at GRB14 and USP21 regulatory regions, leading to a reduction of mRNA levels. Moreover, HNF-4alpha silencing and PED overexpression were accompanied by a significant reduction of hepatic glycogen content. CONCLUSIONS/INTERPRETATION: These results show that HNF-4alpha serves as a scaffold protein for histone deacetylase activities, thereby inhibiting liver expression of genes including PED. Dysregulation of these mechanisms may lead to upregulation of the PED gene in type 2 diabetes.
Asunto(s)
Epigénesis Genética/fisiología , Factor Nuclear 4 del Hepatocito/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Acetilación , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Inmunoprecipitación de Cromatina , Epigénesis Genética/genética , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Transgénicos , Nucleosomas/genética , Fosfoproteínas/genética , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS/HYPOTHESIS: Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells. METHODS: Real-time RT-PCR analysis, 2-deoxy-D: -glucose (2-DG) uptake and western blot analysis were carried out in rat and human muscle cell lines. RESULTS: In both rat and human myotubes, glucosamine treatment caused a significant increase in the expression of the ER stress markers immunoglobulin heavy chain-binding protein/glucose-regulated protein 78 kDa (BIP/GRP78 [also known as HSPA5]), X-box binding protein-1 (XBP1) and activating transcription factor 6 (ATF6). In addition, glucosamine impaired insulin-stimulated 2-DG uptake in both rat and human myotubes. Interestingly, pretreatment of both rat and human myotubes with the chemical chaperones 4-phenylbutyric acid (PBA) or tauroursodeoxycholic acid (TUDCA), completely prevented the effect of glucosamine on both ER stress induction and insulin-induced glucose uptake. In both rat and human myotubes, glucosamine treatment reduced mRNA and protein levels of the gene encoding GLUT4 and mRNA levels of the main regulators of the gene encoding GLUT4 (myocyte enhancer factor 2 a [MEF2A] and peroxisome proliferator-activated receptor-gamma coactivator 1alpha [PGC1alpha]). Again, PBA or TUDCA pretreatment prevented glucosamine-induced inhibition of GLUT4 (also known as SLC2A4), MEF2A and PGC1alpha (also known as PPARGC1A). Finally, we showed that overproduction of ATF6 is sufficient to inhibit the expression of genes GLUT4, MEF2A and PGC1alpha and that ATF6 silencing with a specific small interfering RNA is sufficient to completely prevent glucosamine-induced inhibition of GLUT4, MEF2A and PGC1alpha in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: In this work we show that glucosamine-induced ER stress causes insulin resistance in both human and rat myotubes and impairs GLUT4 production and insulin-induced glucose uptake via an ATF6-dependent decrease of the GLUT4 regulators MEF2A and PGC1alpha.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Retículo Endoplásmico/metabolismo , Glucosamina/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Factor de Transcripción Activador 6/genética , Análisis de Varianza , Animales , Western Blotting , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Glucosamina/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Transportador de Glucosa de Tipo 4/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacología , Resistencia a la Insulina/fisiología , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción MEF2 , Persona de Mediana Edad , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
AIMS/HYPOTHESIS: Overproduction of phosphoprotein enriched in diabetes (PED, also known as phosphoprotein enriched in astrocytes-15 [PEA-15]) is a common feature of type 2 diabetes and impairs insulin action in cultured cells and in mice. Nevertheless, the potential role of PED in diabetic complications is still unknown. METHODS: We studied the effect of PED overproduction and depletion on kidney function in animal and cellular models. RESULTS: Transgenic mice overexpressing PED (PEDTg) featured age-dependent increases of plasma creatinine levels and urinary volume, accompanied by expansion of the mesangial area, compared with wild-type littermates. Serum and kidney levels of TGF-beta1 were also higher in 6- and 9-month-old PEDTg. Overexpression of PED in human kidney 2 cells significantly increased TGF-beta1 levels, SMAD family members (SMAD)2/3 phosphorylation and fibronectin production. Opposite results were obtained following genetic silencing of PED in human kidney 2 cells by antisense oligonucleotides. Inhibition of phospholipase D and protein kinase C-beta by 2-butanol and LY373196 respectively reduced TGF-beta1, SMAD2/3 phosphorylation and fibronectin production. Moreover, inhibition of TGF-beta1 receptor activity and SMAD2/3 production by SB431542 and antisense oligonucleotides respectively reduced fibronectin secretion by about 50%. TGF-beta1 circulating levels were significantly reduced in Ped knockout mice and positively correlated with PED content in peripheral blood leucocytes of type 2 diabetic patients. CONCLUSIONS/INTERPRETATION: These data indicate that PED regulates fibronectin production via phospholipase D/protein kinase C-beta and TGF-beta1/SMAD pathways in kidney cells. Raised PED levels may therefore contribute to the abnormal accumulation of extracellular matrix and renal dysfunction in diabetes.